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Sample records for spla2 enzymatic activity

  1. Interactions of pharmacologically active snake venom sPLA2 with different cell lines

    Science.gov (United States)

    Doumanov, Jordan; Mladenova, Kirilka; Aleksandrov, Radoslav; Danovski, Georgi; Petrova, Svetla

    2014-01-01

    Secreted Phospholipases A2 (sPLA2s) represent a large family of structurally related enzymes, which target different tissues and organs and induce numerous pharmacological effects based on their catalytic specificity – hydrolysis of the sn-2 ester bond of glycerophospholipids. The neurotoxin vipoxin, isolated from the venom of Vipera ammodytes meriodionalis, is a heterodimeric postsynaptic ionic complex composed of two protein subunits – a basic and toxic His48 sPLA2 enzyme and an acidic, enzymatically inactive and non-toxic component. In this paper, for the first time, we demonstrate that vipoxin sPLA2 enzyme affects cell integrity and viability of four cell types and causes different cell responses. The most dramatic local tissue effects were observed with RPE-1 (retinal pigment epithelial) cells followed by A549 (adenocarcinomic human alveolar epithelial) cells and MDCK (Madin-Darby Canine Kidney epithelial) cells. Products of the enzymatic reaction, lysophospholipids and unsaturated free fatty acids, act as lipid mediators that can induce membrane damaging or can stimulate cell proliferation. Our preliminary results on the cytotoxic effect of vipoxin sPLA2 on A549 cells are promising in searching of its eventual anticancer potential. PMID:26019578

  2. Hydrolysis of lipoproteins by sPLA2's enhances mitogenesis and eicosanoid release from vascular smooth muscle cells: Diverse activity of sPLA2's IIA, V and X.

    Science.gov (United States)

    Pruzanski, Waldemar; Kopilov, Julia; Kuksis, Arnis

    2016-01-01

    Mitogenesis of Vascular Smooth Muscle Cells (VSMC) plays an important role in atherogenesis. Until recently, the effect of lipid subfractions has not been clarified. Secretory phospholipases A2 (sPLA2's) hydrolyse glycerophospholipids and release pro-inflammatory lyso-lipids, oxidized and non-oxidized fatty acids and isoprostanes. They localize in the vascular wall. We hypothesized that structurally similar sPLA2's may exert different impact on VSMC. The influence of sPLA2's, IIA, V, X, HDL, LDL, and hydrolysis products was tested on mitogenesis of VSMC, i.e., the early effect on the cell membrane phospholipids, and on PGE2 and LTB4 release, i.e., late effect of Cyclooxygenase and 5-lipooxygenase activity in VSMC. Mitogenesis was significantly enhanced by HDL and LDL, and by products of sPLA2 hydrolysis. Hydrolysis of HDL or LDL enhanced mitogenic activity in order V>X>IIA. The release of PGE2 was enhanced by group X sPLA2 and by HDL hydrolyzed by groups V and X. LDL and its hydrolysis products enhanced the release of PGE2 in order X>V>IIA. The release of LTB4 was markedly increased by LDL and HDL, and by hydrolytic products of group V and X, but not group IIA sPLA2. Our study demonstrates a diverse interaction of pro-inflammatory sPLA2's with HDL and LDL affecting both mitogenesis and eicosanoid release from VSMC, therefore potentially enhancing their pro-atherogenic activity. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. sPLA2-IIA

    Indian Academy of Sciences (India)

    67

    Recent research showed that maslinic acid interacts with sPLA2-IIA ... Further analysis revealed that sPLA2-IIA only induced modest LDL ..... MDA/mg protein) compared to native LDL (2.043 nmol MDA/mg protein) while .... to modify extracellular non-cellular lipid components such as lipoproteins, ... The main pathway for.

  4. The anti-inflammatory activity of standard aqueous stem bark extract of Mangifera indica L. as evident in inhibition of Group IA sPLA2.

    Science.gov (United States)

    Dhananjaya, Bhadrapura Lakkappa; Shivalingaiah, Sudharshan

    2016-03-01

    The standard aqueous stem bark extract is consumed as herbal drink and used in the pharmaceutical formulations to treat patients suffering from various disease conditions in Cuba. This study was carried out to evaluate the modulatory effect of standard aqueous bark extract of M. indica on Group IA sPLA2. M. indica extract, dose dependently inhibited the GIA sPLA2 (NN-XIa-PLA2) activity with an IC50 value 8.1 µg/ml. M. indica extract effectively inhibited the indirect hemolytic activity up to 98% at ~40 µg/ml concentration and at various concentrations (0-50 µg/ml), it dose dependently inhibited the edema formation. When examined as a function of increased substrate and calcium concentration, there was no relieve of inhibitory effect on the GIA sPLA2. Furthermore, the inhibition was irreversible as evidenced from binding studies. It is observed that the aqueous extract ofM. indica effectively inhibits sPLA2 and it is associated inflammatory activities, which substantiate their anti-inflammatory properties. The mode of inhibition could be due to direct interaction of components present in the extract, with sPLA2 enzyme. Further studies on understanding the principal constituents, responsible for the anti-inflammatory activity would be interesting to develop this into potent anti-inflammatory agent.

  5. Novel genetic approach to investigate the role of plasma secretory phospholipase A2 (sPLA2)-V isoenzyme in coronary heart disease: modified Mendelian randomization analysis using PLA2G5 expression levels.

    Science.gov (United States)

    Holmes, Michael V; Exeter, Holly J; Folkersen, Lasse; Nelson, Christopher P; Guardiola, Montse; Cooper, Jackie A; Sofat, Reecha; Boekholdt, S Matthijs; Khaw, Kay-Tee; Li, Ka-Wah; Smith, Andrew J P; Van't Hooft, Ferdinand; Eriksson, Per; Franco-Cereceda, Anders; Asselbergs, Folkert W; Boer, Jolanda M A; Onland-Moret, N Charlotte; Hofker, Marten; Erdmann, Jeanette; Kivimaki, Mika; Kumari, Meena; Reiner, Alex P; Keating, Brendan J; Humphries, Steve E; Hingorani, Aroon D; Mallat, Ziad; Samani, Nilesh J; Talmud, Philippa J

    2014-04-01

    Secretory phospholipase A2 (sPLA2) enzymes are considered to play a role in atherosclerosis. sPLA2 activity encompasses several sPLA2 isoenzymes, including sPLA2-V. Although observational studies show a strong association between elevated sPLA2 activity and CHD, no assay to measure sPLA2-V levels exists, and the only evidence linking the sPLA2-V isoform to atherosclerosis progression comes from animal studies. In the absence of an assay that directly quantifies sPLA2-V levels, we used PLA2G5 mRNA levels in a novel, modified Mendelian randomization approach to investigate the hypothesized causal role of sPLA2-V in coronary heart disease (CHD) pathogenesis. Using data from the Advanced Study of Aortic Pathology, we identified the single-nucleotide polymorphism in PLA2G5 showing the strongest association with PLA2G5 mRNA expression levels as a proxy for sPLA2-V levels. We tested the association of this SNP with sPLA2 activity and CHD events in 4 prospective and 14 case-control studies with 27 230 events and 70 500 controls. rs525380C>A showed the strongest association with PLA2G5 mRNA expression (P=5.1×10(-6)). There was no association of rs525380C>A with plasma sPLA2 activity (difference in geometric mean of sPLA2 activity per rs525380 A-allele 0.4% (95% confidence intervals [-0.9%, 1.6%]; P=0.56). In meta-analyses, the odds ratio for CHD per A-allele was 1.02 (95% confidence intervals [0.99, 1.04]; P=0.20). This novel approach for single-nucleotide polymorphism selection for this modified Mendelian randomization analysis showed no association between rs525380 (the lead single-nucleotide polymorphism for PLA2G5 expression, a surrogate for sPLA2-V levels) and CHD events. The evidence does not support a causal role for sPLA2-V in CHD.

  6. Increased expression and activity of group IIA and X secretory phospholipase A2 in peritumoral versus central colon carcinoma tissue

    DEFF Research Database (Denmark)

    Tribler, Line; Jensen, Lotte T.; Jørgensen, Kent

    2007-01-01

    Secretory phospholipase A2 (sPLA2) type IIA and X was analyzed in tumors from 22 patients with colon adenocarcinomas in order to determine the involvement and activity of sPLA2 in colon cancer. Evaluation of immunoreactive sPLA2 IIA by Western blotting showed a significantly higher level...... in the periphery of the tumors, compared to central tumor regions. Increased levels of sPLA2 IIA protein correlated with a two-fold increase in sPLA2 enzymatic activity in the peripheral regions compared to central regions. Nineteen out of 22 tumors showed high levels of sPLA2 IIA, whereas 7 out of the 22 tumors...... showed sPLA2 type X. These data demonstrate that both sPLA2 type IIA and X are present in human colon cancer and suggest a role for sPLA2 in colon cancer tumor immunology and tumorigenesis....

  7. Cooption of secretory phospholipase (SPLA2) for different aspects of gravity receptor-associated mineralization in vertebrate phylogeny

    Science.gov (United States)

    Thalmann, R.; Lu, W.

    2009-04-01

    Vertebrate gravity-associated minerals consists of either a single large stone (otolith), or an assembly of minute biomineral particles, otoconia ("ear dust"). Otoliths and both, amphibian and reptilian otoconia, consist of aragonite, whereas avian and mammalian otoconia consist of calcite. Vertebrate gravity-associated minerals are the product of site-directed biologically-controlled mineralization. Insoluble frame work molecules specify sites of nucleation and direction of crystal growth. Soluble matrix proteins modulate growth kinetics and crystal morphology. It is most remarkable that the principal insoluble frame work protein, otolin, is the same for both, otolith and otoconia. Otolin is a novel type of collagen, homologous to the network-forming collagen type X prevalent in mature chondrocytes. The principal soluble matrix proteins of calcitic, aragonitic, and most likely also of vateritic otoconia are all homologs of SPLA2, which is most prevalent in pancreatic secretion and snake venoms. Otonin90 (OC90), the principal soluble matrix protein of calcitic otoconia consists of two SPLA-like (SPLAL) domains, which are connected by a sizeable linker segment and contain significant terminal extensions. The MW of the protein backbone amounts to approximately 50 kDa. The molecule contains, in addition massive post-translational modifications, 80% of which are accounted for by sulfated GAGs, resulting in a total MW of 100 KDa. The protein backbone is moderately acidic, pI 4.4, but the pI of the whole molecule is 2.9, indicating a substantial acidity of the GAG component. In adapting SPLA2 for mineral modulation the enzymatic site is modified and presumed nonfunctional. The seven SH- bonds are rigorously conserved in both, OC90 and otoconin22 (OC22). It appears that the SH-bonds of the parent SPLA2 are intended to stabilize the molecule to ensure continued enzymatic activity in the hostile environment of the gut. It therefore seems logical that SPLA2 was coopted for

  8. A dangerous liaison: Leptin and sPLA2-IIA join forces to induce proliferation and migration of astrocytoma cells.

    Directory of Open Access Journals (Sweden)

    Rubén Martín

    Full Text Available Glioblastoma, the most aggressive type of primary brain tumour, shows worse prognosis linked to diabetes or obesity persistence. These pathologies are chronic inflammatory conditions characterized by altered profiles of inflammatory mediators, including leptin and secreted phospholipase A2-IIA (sPLA2-IIA. Both proteins, in turn, display diverse pro-cancer properties in different cell types, including astrocytes. Herein, to understand the underlying relationship between obesity and brain tumors, we investigated the effect of leptin, alone or in combination with sPLA2-IIA on astrocytoma cell functions. sPLA2-IIA induced up-regulation of leptin receptors in 1321N1 human astrocytoma cells. Leptin, as well as sPLA2-IIA, increased growth and migration in these cells, through activation/phosphorylation of key proteins of survival cascades. Leptin, at concentrations with minimal or no activating effects on astrocytoma cells, enhanced growth and migration promoted by low doses of sPLA2-IIA. sPLA2-IIA alone induced a transient phosphorylation pattern in the Src/ERK/Akt/mTOR/p70S6K/rS6 pathway through EGFR transactivation, and co-addition of leptin resulted in a sustained phosphorylation of these signaling regulators. Mechanistically, EGFR transactivation and tyrosine- and serine/threonine-protein phosphatases revealed a key role in this leptin-sPLA2-IIA cross-talk. This cooperative partnership between both proteins was also found in primary astrocytes. These findings thus indicate that the adipokine leptin, by increasing the susceptibility of cells to inflammatory mediators, could contribute to worsen the prognosis of tumoral and neurodegenerative processes, being a potential mediator of some obesity-related medical complications.

  9. Revisiting the use of sPLA2-sensitive liposomes in cancer therapy

    DEFF Research Database (Denmark)

    Pourhassan, Houman; Clergeaud Veiga, Gael; Hansen, Anders Elias

    2017-01-01

    The first developed secretory phospholipase A2 (sPLA2) sensitive liposomal cisplatin formulation (LiPlaCis®) is currently undergoing clinical evaluation. In the present study we revisit and evaluate critical preclinical parameters important for the therapeutic potential and safety of platinum drugs......, here oxaliplatin (L-OHP), formulated in sPLA2 sensitive liposomes. We show the mole percentage of negatively charged phospholipid needed to obtain enzyme-sensitivity for saturated systems is ≥ 25% for 16-carbon chain lipid membranes, and > 40% for 18-chain lipid membranes, which was surprising as 25......% is used clinically in LiPlaCis®. Efficient sPLA2-dependent growth inhibition of colorectal cancer cells was demonstrated in vitro, where cell membrane degradation and cytolysis depends on the sensitivity of the formulation towards the enzyme and is governed by the amount of lysolipids generated...

  10. Mechanistic Study of the sPLA2 Mediated Hydrolysis of a Thio-ester Pro Anticancer Ether Lipid

    DEFF Research Database (Denmark)

    Linderoth, Lars; Fristrup, Peter; Hansen, Martin

    2009-01-01

    Secretory phospholipase A2 (sPLA2) is an interesting enzyme for triggered liposomal drug delivery to tumor tissue due the overexpression of sPLA2 in cancerous tissue. A drug delivery system based on the triggered release of therapeutics from sPLA2-sensitive liposomes constituted of pro anticancer...... ether lipids, which become cytotoxic upon sPLA2-catalyzed hydrolysis has previously been established. To optimize the hydrolysis rate of the lipids and thereby optimizing the release profile of the drugs from the liposomes, we have synthesized a thio-ester pro anticancer ether lipid. Liposomes...... constituted of this lipid showed an altered rate of hydrolysis by sPLA2. We have tested the cytotoxicity of the thio-ester pro anticancer ether lipids toward cancer cells, and the results showed that the cytotoxicity is indeed maintained upon sPLA2 exposure. To further understand the origin for the observed...

  11. Maslinic acid modulates secreted phospholipase A2-IIA (sPLA2-IIA ...

    Indian Academy of Sciences (India)

    Wei Hsum Yap

    2018-03-29

    Mar 29, 2018 ... Further analysis revealed that sPLA2-IIA only induced modest LDL oxidation and that inhibitory .... COX-2, was also reduced in primary human chondrocyte, primary rat .... oxidation (4.34 nmol MDA/mg protein) compared to native ..... rheumatoid fibroblast-like synoviocyte arachidonic acid meta- bolism.

  12. Phospholipids and their degrading enzyme in the tears of soft contact lens wearers.

    Science.gov (United States)

    Yamada, Masakazu; Mochizuki, Hiroshi; Kawashima, Motoko; Hata, Seiichiro

    2006-12-01

    Low tear phospholipids levels are associated with tear film instability in soft contact lens wearers. We assayed levels of phospholipids and their degrading enzyme secretory phospholipase A2 (sPLA2) both in tears and deposited on contact lenses composed of 2 hydrophilic materials after 1 day of routine use. Polymacon (Medalist; FDA group 1, low water/nonionic) and Etafilcon A (One Day Acuvue; group 4, high water/ionic) contact lenses were worn for 12 hours by 16 experienced contact lens wearers. Phospholipids in tear fluids and deposited on contact lenses were estimated by phosphorus determination with ammonium molybdate through enzymatic digestion. Double-antibody sandwich ELISA was used to determine group IIa sPLA2 concentrations, and sPLA2 activity was assayed using 1,2-diheptanoyl thio-phosphatidylcholine as substrate. Phospholipids concentrations in tears with Polymacon and Etafilcon A were 186 +/- 39 and 162 +/- 33 microg/mL, respectively. The latter concentration was significantly lower than that observed in the same subjects when not wearing contact lenses (P = 0.0023). In tears, both group IIa sPLA2 concentrations and enzymatic activity remained unchanged, regardless of lens wearing. However, Etafilcon A (0.57 +/- 0.09 microg/lens) showed more group IIa sPLA2 deposition than Polymacon (0.01 +/- 0.01 microg/lens; P < 0.001). Furthermore, group IIa sPLA2 deposited on Etafilcon A but not on Polymacon lenses retained its enzymatic activity. Significant differences of group IIa sPLA2 deposition were found in the 2 lenses tested. Such deposition might induce phospholipid hydrolysis in tears and thereby promote tear film instability in hydrophilic contact lens wearers.

  13. Distinct enzymatic and cellular characteristics of two secretory phospholipases A2 in the filamentous fungus Aspergillus oryzae.

    Science.gov (United States)

    Nakahama, Tomoyuki; Nakanishi, Yoshito; Viscomi, Arturo R; Takaya, Kohei; Kitamoto, Katsuhiko; Ottonello, Simone; Arioka, Manabu

    2010-04-01

    Microbial secretory phospholipases A(2) (sPLA(2)s) are among the last discovered and least known members of this functionally diverse family of enzymes. We analyzed here two sPLA(2)s, named sPlaA and sPlaB, of the filamentous ascomycete Aspergillus oryzae. sPlaA and sPlaB consist of 222 and 160 amino acids, respectively, and share the conserved Cys and catalytic His-Asp residues typical of microbial sPLA(2)s. Two sPLA(2)s differ in pH optimum, Ca(2+) requirement and expression profile. The splaA mRNA was strongly upregulated in response to carbon starvation, oxidative stress and during conidiation, while splaB was constitutively expressed at low levels and was weakly upregulated by heat shock. Experiments with sPLA(2) overexpressing strains demonstrated that two enzymes produce subtly different phospholipid composition variations and also differ in their subcellular localization: sPlaA is most abundant in hyphal tips and secreted to the medium, whereas sPlaB predominantly localizes to the ER-like intracellular compartment. Both sPLA(2) overexpressing strains were defective in conidiation, which was more pronounced for sPlaB overexpressors. Although no major morphological abnormality was detected in either DeltasplaA or DeltasplaB mutants, hyphal growth of DeltasplaB, but not that of DeltasplaA, displayed increased sensitivity to H(2)O(2) treatment. These data indicate that two A. oryzae sPLA(2) enzymes display distinct, presumably non-redundant, physiological functions.

  14. Ex vivo effect of varespladib on secretory phospholipase A2 alveolar activity in infants with ARDS.

    Directory of Open Access Journals (Sweden)

    Daniele De Luca

    Full Text Available Secretory phospholipase A2 (sPLA2 plays a pivotal role in acute respiratory distress syndrome (ARDS. This enzyme seems an interesting target to reduce surfactant catabolism and lung tissue inflammation. Varespladib is a specifically designed indolic sPLA2 inhibitor, which has shown promising results in animals and adults. No specific data in pediatric ARDS patients are yet available.We studied varespladib in broncho-alveolar lavage (BAL fluids obtained ex vivo from pediatric ARDS patients. Clinical data and worst gas exchange values during the ARDS course were recorded. Samples were treated with saline or 10-40-100 µM varespladib and incubated at 37°C. Total sPLA2 activity was measured by non-radioactive method. BAL samples were subjected to western blotting to identify the main sPLA isotypes with different sensitivity to varespladib. Results was corrected for lavage dilution using the serum-to-BAL urea ratio and for varespladib absorbance.Varespladib reduces sPLA2 activity (p<0.0001 at 10,40 and 100 µM; both sPLA2 activity reduction and its ratio to total proteins significantly raise with increasing varespladib concentrations (p<0.001. IC(50 was 80 µM. Western blotting revealed the presence of sPLA2-IIA and -IB isotypes in BAL samples. Significant correlations exist between the sPLA2 activity reduction/proteins ratio and PaO(2 (rho = 0.63;p<0.001, PaO(2/FiO(2 (rho = 0.7; p<0.001, oxygenation (rho = -0.6; p<0.001 and ventilation (rho = -0.4;p = 0.038 indexes.Varespladib significantly inhibits sPLA2 in BAL of infants affected by post-neonatal ARDS. Inhibition seems to be inversely related to the severity of gas exchange impairment.

  15. Lactadherin inhibits secretory phospholipase A2 activity on pre-apoptotic leukemia cells.

    Directory of Open Access Journals (Sweden)

    Steffen Nyegaard

    Full Text Available Secretory phospholipase A2 (sPLA2 is a critical component of insect and snake venoms and is secreted by mammalian leukocytes during inflammation. Elevated secretory PLA2 concentrations are associated with autoimmune diseases and septic shock. Many sPLA2's do not bind to plasma membranes of quiescent cells but bind and digest phospholipids on the membranes of stimulated or apoptotic cells. The capacity of these phospholipases to digest membranes of stimulated or apoptotic cells correlates to the exposure of phosphatidylserine. In the present study, the ability of the phosphatidyl-L-serine-binding protein, lactadherin to inhibit phospholipase enzyme activity has been assessed. Inhibition of human secretory phospholipase A2-V on phospholipid vesicles exceeded 90%, whereas inhibition of Naja mossambica sPLA2 plateaued at 50-60%. Lactadherin inhibited 45% of activity of Naja mossambica sPLA2 and >70% of human secretory phospholipase A2-V on the membranes of human NB4 leukemia cells treated with calcium ionophore A23187. The data indicate that lactadherin may decrease inflammation by inhibiting sPLA2.

  16. Dimethyl ester of bilirubin exhibits anti-inflammatory activity through inhibition of secretory phospholipase A2, lipoxygenase and cyclooxygenase.

    Science.gov (United States)

    Joshi, Vikram; Umashankara, M; Ramakrishnan, Chandrasekaran; Nanjaraj Urs, Ankanahalli N; Suvilesh, Kanve Nagaraj; Velmurugan, Devadasan; Rangappa, Kanchugarakoppal S; Vishwanath, Bannikuppe Sannanaik

    2016-05-15

    Overproduction of arachidonic acid (AA) mediated by secretory phospholipase A2 group IIA (sPLA2IIA) is a hallmark of many inflammatory disorders. AA is subsequently converted into pro-inflammatory eicosanoids through 5-lipoxygenase (5-LOX) and cyclooxygenase-1/2 (COX-1/2) activities. Hence, inhibition of sPLA2IIA, 5-LOX and COX-1/2 activities is critical in regulating inflammation. We have previously reported unconjugated bilirubin (UCB), an endogenous antioxidant, as sPLA2IIA inhibitor. However, lipophilic UCB gets conjugated in liver with glucuronic acid into hydrophilic conjugated bilirubin (CB). Since hydrophobicity is pre-requisite for sPLA2IIA inhibition, conjugation reduces the efficacy of UCB. In this regard, UCB was chemically modified and derivatives were evaluated for sPLA2IIA, 5-LOX and COX-1/2 inhibition. Among the derivatives, BD1 (dimethyl ester of bilirubin) exhibited ∼ 3 fold greater inhibitory potency towards sPLA2IIA compared to UCB. Both UCB and BD1 inhibited human 5-LOX and COX-2 activities; however only BD1 inhibited AA induced platelet aggregation. Molecular docking studies demonstrated BD1 as better inhibitor of aforesaid enzymes than UCB and other endogenous antioxidants. These data suggest that BD1 exhibits strong anti-inflammatory activity through inhibition of AA cascade enzymes which is of great therapeutic importance. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Key role of group v secreted phospholipase A2 in Th2 cytokine and dendritic cell-driven airway hyperresponsiveness and remodeling.

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    William R Henderson

    Full Text Available Previous work has shown that disruption of the gene for group X secreted phospholipase A2 (sPLA2-X markedly diminishes airway hyperresponsiveness and remodeling in a mouse asthma model. With the large number of additional sPLA2s in the mammalian genome, the involvement of other sPLA2s in the asthma model is possible - in particular, the group V sPLA2 (sPLA2-V that like sPLA2-X is highly active at hydrolyzing membranes of mammalian cells.The allergen-driven asthma phenotype was significantly reduced in sPLA2-V-deficient mice but to a lesser extent than observed previously in sPLA2-X-deficient mice. The most striking difference observed between the sPLA2-V and sPLA2-X knockouts was the significant impairment of the primary immune response to the allergen ovalbumin (OVA in the sPLA2-V(-/- mice. The impairment in eicosanoid generation and dendritic cell activation in sPLA2-V(-/- mice diminishes Th2 cytokine responses in the airways.This paper illustrates the diverse roles of sPLA2s in the immunopathogenesis of the asthma phenotype and directs attention to developing specific inhibitors of sPLA2-V as a potential new therapy to treat asthma and other allergic disorders.

  18. Identification and functional analysis of the S-layer protein SplA of Paenibacillus larvae, the causative agent of American Foulbrood of honey bees.

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    Lena Poppinga

    Full Text Available The gram-positive, spore-forming bacterium Paenibacillus larvae is the etiological agent of American Foulbrood (AFB, a globally occurring, deathly epizootic of honey bee brood. AFB outbreaks are predominantly caused by two genotypes of P. larvae, ERIC I and ERIC II, with P. larvae ERIC II being the more virulent genotype on larval level. Recently, comparative proteome analyses have revealed that P. larvae ERIC II but not ERIC I might harbour a functional S-layer protein, named SplA. We here determine the genomic sequence of splA in both genotypes and demonstrate by in vitro self-assembly studies of recombinant and purified SplA protein in combination with electron-microscopy that SplA is a true S-layer protein self-assembling into a square 2D lattice. The existence of a functional S-layer protein is novel for this bacterial species. For elucidating the biological function of P. larvae SplA, a genetic system for disruption of gene expression in this important honey bee pathogen was developed. Subsequent analyses of in vivo biological functions of SplA were based on comparing a wild-type strain of P. larvae ERIC II with the newly constructed splA-knockout mutant of this strain. Differences in cell and colony morphology suggest that SplA is a shape-determining factor. Marked differences between P. larvae ERIC II wild-type and mutant cells with regard to (i adhesion to primary pupal midgut cells and (ii larval mortality as measured in exposure bioassays corroborate the assumption that the S-layer of P. larvae ERIC II is an important virulence factor. Since SplA is the first functionally proven virulence factor for this species, our data extend the knowledge of the molecular differences between these two genotypes of P. larvae and contribute to explaining the observed differences in virulence. These results present an immense advancement in our understanding of P. larvae pathogenesis.

  19. Activated platelets contribute to oxidized low-density lipoproteins and dysfunctional high-density lipoproteins through a phospholipase A2-dependent mechanism

    NARCIS (Netherlands)

    Blache, Denis; Gautier, Thomas; Tietge, Uwe J. F.; Lagrost, Laurent

    Plasma activity of secretory phospholipase A2 (sPLA2) increases in patients with cardiovascular disease. The present study investigated whether platelet-released sPLA2 induces low-density lipoprotein (LDL) and high-density lipoprotein (HDL) modifications that translate into changes in lipoprotein

  20. The secretory phospholipase A2 group IIA: a missing link between inflammation, activated renin-angiotensin system, and atherogenesis?

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    Dimitar Divchev

    2008-06-01

    Full Text Available Dimitar Divchev, Bernhard SchiefferDepartment of Cardiology and Angiology, Medizinische Hochschule Hannover, GermanyAbstract: Inflammation, lipid peroxidation and chronic activation of the renin–angiotensin system (RAS are hallmarks of the development of atherosclerosis. Recent studies have suggested the involvement of the pro-inflammatory secretory phospholipase A2 (sPLA2-IIA in atherogenesis. This enzyme is produced by different cell types through stimulation by proinflammatory cytokines. It is detectable in the intima and in media smooth muscle cells, not only in atherosclerotic lesions but also in the very early stages of atherogenesis. sPLA2-IIA can hydrolyse the phospholipid monolayers of low density lipoproteins (LDL. Such modified LDL show increased affinity to proteoglycans. The modified particles have a greater tendency to aggregate and an enhanced ability to insert cholesterol into cells. This modification may promote macrophage LDL uptake leading to the formation of foam cells. Furthermore, sPLA2-IIA is not only a mediator for localized inflammation but may be also used as an independent predictor of adverse outcomes in patients with stable coronary artery disease or acute coronary syndromes. An interaction between activated RAS and phospholipases has been indicated by observations showing that inhibitors of sPLA2 decrease angiotensin (Ang II-induced macrophage lipid peroxidation. Meanwhile, various interactions between Ang II and oxLDL have been demonstrated suggesting a central role of sPLA2-IIA in these processes and offering a possible target for treatment. The role of sPLA2-IIA in the perpetuation of atherosclerosis appears to be the missing link between inflammation, activated RAS and lipidperoxidation.Keywords: secretory phospholipase A2, lipoproteins, renin-angiotensin system, inflammation, atherosclerosis

  1. Secretory Phospholipase A2 Hydrolysis Phospholipid Analogs is Dependent on Water Accessibility to the Active Site

    DEFF Research Database (Denmark)

    Peters, Günther H.J.; Møller, Martin S.; Jørgensen, Kent

    2007-01-01

    A new and unnatural type of phospholipids with the head group attached to the 2-position of the glycerol backbone has been synthesized and shown to be a good substrate for secretory phospholipase A2 (sPLA2). To investigate the unexpected sPLA2 activity, we have compared three different phospholip...

  2. Secreted phospholipase A2 of Clonorchis sinensis activates hepatic stellate cells through a pathway involving JNK signalling.

    Science.gov (United States)

    Wu, Yinjuan; Li, Ye; Shang, Mei; Jian, Yu; Wang, Caiqin; Bardeesi, Adham Sameer A; Li, Zhaolei; Chen, Tingjin; Zhao, Lu; Zhou, Lina; He, Ai; Huang, Yan; Lv, Zhiyue; Yu, Xinbing; Li, Xuerong

    2017-03-16

    Secreted phospholipase A2 (sPLA2) is a protein secreted by Clonorchis sinensis and is a component of excretory and secretory products (CsESPs). Phospholipase A2 is well known for its role in liver fibrosis and inhibition of tumour cells. The JNK signalling pathway is involved in hepatic stellate cells (HSCs) activation. Blocking JNK activity with SP600125 inhibits HSCs activation. In a previous study, the protein CssPLA2 was expressed in insoluble inclusion bodies. Therefore, it's necessary to express CssPLA2 in water-soluble form and determine whether the enzymatic activity of CssPLA2 or cell signalling pathways is involved in liver fibrosis caused by clonorchiasis. Balb/C mice were given an abdominal injection of MBP-CssPLA2. Liver sections with HE and Masson staining were observed to detect accumulation of collagen. Western blot of mouse liver was done to detect the activation of JNK signalling pathway. In vitro, HSCs were incubated with MBP-CssPLA2 to detect the activation of HSCs as well as the activation of JNK signalling pathway. The mutant of MBP-CssPLA2 without enzymatic activity was constructed and was also incubated with HSCs to check whether activation of the HSCs was related to the enzymatic activity of MBP-CssPLA2. The recombinant protein MBP-CssPLA2 was expressed soluble and of good enzymatic activity. A mutant of CssPLA2, without enzymatic activity, was also constructed. In vivo liver sections of Balb/C mice that were given an abdominal injection of 50 μg/ml MBP-CssPLA2 showed an obvious accumulation of collagen and a clear band of P-JNK1 could be seen by western blot of the liver tissue. In vitro, MBP-CssPLA2, as well as the mutant, was incubated with HSCs and it was proved that activation of HSCs was related to activation of the JNK signalling pathway instead of the enzymatic activity of MBP-CssPLA2. Activation of HSCs by CssPLA2 is related to the activation of the JNK signalling pathway instead of the enzymatic activity of CssPLA2. This finding

  3. Correlation of secretory phospholipase-A2 activity and fatty acids in cerebrospinal fluid with liver enzymes tests

    Directory of Open Access Journals (Sweden)

    Sepideh Ghodoosifar

    2016-02-01

    Full Text Available Introduction: The aim was to determine whether secretory phospholipase-A2 (sPLA2 activity and fatty acids in cerebrospinal fluid (CSF are correlated with liver enzymes tests. Methods: CSF and serum samples were collected from 49 patients (age 18-65 as part of routine diagnostic testing. Along with serum liver enzymes aspartate aminotransferase (AST, alanine aminotransferase (ALT and alkaline phosphatase (ALP, the fatty acid composition of CSF was measured by gas liquid chromatography. CSF enzyme activities of sPLA2 were measured using the standard assay with diheptanoyl thio-phosphatidylcholin as substrate. Results: The saturated fatty acids (SFAs including palmitic acid and stearic acid were positively, and the unsaturated fatty acids including oleic acid and linoleic acid were negatively correlated with liver enzymes tests. In regression analysis with adjustment for body mass index (BMI, the elevated liver enzymes tests were positively associated with activity of sPLA2 (β > 0.31, P 0.38, P < 0.010 and negatively with total monounsaturated fatty acids (MUFAs (β < -0.40, P < 0.001 contents of CSF. Conclusion: CSF activity of sPLA2 and fatty acids may be linked to peripheral markers of liver function, suggesting an indirect impact of central fatty acids on hepatocytes function and metabolism.

  4. Inhibition of Group IIA Secretory Phospholipase A2 and its Inflammatory Reactions in Mice by Ethanolic Extract of Andrographis paniculata, a Well-known Medicinal Food

    Science.gov (United States)

    Kishore, V.; Yarla, N. S.; Zameer, F.; Nagendra Prasad, M. N.; Santosh, M. S.; More, S. S.; Rao, D. G.; Dhananjaya, Bhadrapura Lakkappa

    2016-01-01

    Andrographis paniculata Nees is an important medicinal plant found in the tropical regions of the world, which has been traditionally used in Indian and Chinese medicinal systems. It is also used as medicinal food. A. paniculata is found to exhibit anti-inflammatory activities; however, its inhibitory potential on inflammatory Group IIA phospholipases A2 (PLA2) and its associated inflammatory reactions are not clearly understood. The aim of the present study is to evaluate the inhibitory/neutralizing potential of ethanolic extract of A. paniculata on the isolated inflammatory PLA2 (VRV-PL-VIIIa) from Daboii rusellii pulchella (belonging to Group IIA inflammatory secretory PLA2 [sPLA2]) and its associated edema-induced activities in Swiss albino mice. A. paniculata extract dose dependently inhibited the Group IIA sPLA2 enzymatic activity with an IC50 value of 10.3 ± 0.5 μg/ml. Further, the extract dose dependently inhibited the edema formation, when co-injected with enzyme indicating that a strong correlation exists between lipolytic and pro-inflammatory activities of the enzyme. In conclusion, results of this study shows that the ethanolic extract of A. paniculata effectively inhibits Group IIA sPLA2 and its associated inflammatory activities, which substantiate its anti-inflammatory properties. The results of the present study warranted further studies to develop bioactive compound (s) in ethanolic extract of A. paniculata as potent therapeutic agent (s) for inflammatory diseases. SUMMARY This study emphasis the anti-inflammatory effect of A. paniculata by inhibiting the inflammatory Group IIA sPLA2 and its associated inflammatory activities such as edema. It was found that there is a strong correlation between lipolytic activity and pro-inflammatory activity inhibition. Therefore, the study suggests that the extract processes potent anti-inflammatory agents, which could be developed as a potential therapeutic agent against inflammatory and related diseases

  5. Endogenous secreted phospholipase A2 group X regulates cysteinyl leukotrienes synthesis by human eosinophils.

    Science.gov (United States)

    Hallstrand, Teal S; Lai, Ying; Hooper, Kathryn A; Oslund, Rob C; Altemeier, William A; Matute-Bello, Gustavo; Gelb, Michael H

    2016-01-01

    Phospholipase A2s mediate the rate-limiting step in the formation of eicosanoids such as cysteinyl leukotrienes (CysLTs). Group IVA cytosolic PLA2α (cPLA2α) is thought to be the dominant PLA2 in eosinophils; however, eosinophils also have secreted PLA2 (sPLA2) activity that has not been fully defined. To examine the expression of sPLA2 group X (sPLA2-X) in eosinophils, the participation of sPLA2-X in the formation of CysLTs, and the mechanism by which sPLA2-X initiates the synthesis of CysLTs in eosinophils. Peripheral blood eosinophils were obtained from volunteers with asthma and/or allergy. A rabbit polyclonal anti-sPLA2-X antibody identified sPLA2-X by Western blot. We used confocal microscopy to colocalize the sPLA2-X to intracellular structures. An inhibitor of sPLA2-X (ROC-0929) that does not inhibit other mammalian sPLA2s, as well as inhibitors of the mitogen-activated kinase cascade (MAPK) and cPLA2α, was used to examine the mechanism of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-mediated formation of CysLT. Eosinophils express the mammalian sPLA2-X gene (PLA2G10). The sPLA2-X protein is located in the endoplasmic reticulum, golgi, and granules of eosinophils and moves to the granules and lipid bodies during fMLP-mediated activation. Selective sPLA2-X inhibition attenuated the fMLP-mediated release of arachidonic acid and CysLT formation by eosinophils. Inhibitors of p38, extracellular-signal-regulated kinases 1/2 (p44/42 MAPK), c-Jun N-terminal kinase, and cPLA2α also attenuated the fMLP-mediated formation of CysLT. The sPLA2-X inhibitor reduced the phosphorylation of p38 and extracellular-signal-regulated kinases 1/2 (p44/42 MAPK) as well as cPLA2α during cellular activation, indicating that sPLA2-X is involved in activating the MAPK cascade leading to the formation of CysLT via cPLA2α. We further demonstrate that sPLA2-X is activated before secretion from the cell during activation. Short-term priming with IL-13 and TNF/IL-1β increased the

  6. Secretory Phospholipase A(2) Activity toward Diverse Substrates

    DEFF Research Database (Denmark)

    Madsen, Jesper Jonasson; Linderoth, Lars; Subramanian, Arun Kumar

    2011-01-01

    We have studied secretory phospholipase A(2)-IIA (sPLA(2)) activity toward different phospholipid analogues by performing biophysical 1 characterizations and molecular dynamics simulations. The phospholipids were natural substrates, triple alkyl phospholipids, a prodrug anticancer etherlipid, and...

  7. An Amperometric Biosensor for the Determination of Bacterial Sepsis Biomarker, Secretory Phospholipase Group 2-IIA Using a Tri-Enzyme System

    Directory of Open Access Journals (Sweden)

    Nik Nurhanan Nik Mansor

    2018-02-01

    Full Text Available A tri-enzyme system consisting of choline kinase/choline oxidase/horseradish peroxidase was used in the rapid and specific determination of the biomarker for bacterial sepsis infection, secretory phospholipase Group 2-IIA (sPLA2-IIA. These enzymes were individually immobilized onto the acrylic microspheres via succinimide groups for the preparation of an electrochemical biosensor. The reaction of sPLA2-IIA with its substrate initiated a cascading enzymatic reaction in the tri-enzyme system that led to the final production of hydrogen peroxide, which presence was indicated by the redox characteristics of potassium ferricyanide, K3Fe(CN6. An amperometric biosensor based on enzyme conjugated acrylic microspheres and gold nanoparticles composite coated onto a carbon-paste screen printed electrode (SPE was fabricated and the current measurement was performed at a low potential of 0.20 V. This enzymatic biosensor gave a linear range 0.01–100 ng/mL (R2 = 0.98304 with a detection limit recorded at 5 × 10−3 ng/mL towards sPLA2-IIA. Moreover, the biosensor showed good reproducibility (relative standard deviation (RSD of 3.04% (n = 5. The biosensor response was reliable up to 25 days of storage at 4 °C. Analysis of human serum samples for sPLA2-IIA indicated that the biosensor has potential for rapid bacterial sepsis diagnosis in hospital emergency department.

  8. Secretory phospholipase A2 in dromedary tears: a host defense against staphylococci and other gram-positive bacteria.

    Science.gov (United States)

    Ben Bacha, Abir; Abid, Islem

    2013-03-01

    The best known physiologic function of secreted phospholipase A2 (sPLA2) group IIA (sPLA2-IIA) is defense against bacterial infection through hydrolytic degradation of bacterial membrane phospholipids. In fact, sPLA2-IIA effectively kills Gram-positive bacteria and to a lesser extent Gram-negative bacteria and is considered a major component of the eye's innate immune defense system. The antibacterial properties of sPLA2 have been demonstrated in rabbit and human tears. In this report, we have analyzed the bactericidal activity of dromedary tears and the subsequently purified sPLA2 on several Gram-positive bacteria. Our results showed that the sPLA2 displays a potent bactericidal activity against all the tested bacteria particularly against the Staphylococcus strains when tested in the ionic environment of tears. There is a synergic action of the sPLA2 with lysozyme when added to the bacteria culture prior to sPLA2. Interestingly, lysozyme purified from dromedary tears showed a significant bactericidal activity against Listeria monocytogene and Staphylococcus epidermidis, whereas the one purified from human tears displayed no activity against these two strains. We have also demonstrated that Ca(2+) is crucial for the activity of dromedary tear sPLA2 and to a less extent Mg(2+) ions. Given the presence of sPLA2 in tears and intestinal secretions, this enzyme may play a substantial role in innate mucosal and systemic bactericidal defenses against Gram-positive bacteria.

  9. Secretory Phospholipase A(2)-IIA and Cardiovascular Disease

    NARCIS (Netherlands)

    Holmes, Michael V.; Simon, Tabassome; Exeter, Holly J.; Folkersen, Lasse; Asselbergs, Folkert W.; Guardiola, Montse; Cooper, Jackie A.; Palmen, Jutta; Hubacek, Jaroslav A.; Carruthers, Kathryn F.; Horne, Benjamin D.; Brunisholz, Kimberly D.; Mega, Jessica L.; Van Iperen, Erik P. A.; Li, Mingyao; Leusink, Maarten; Trompet, Stella; Verschuren, Jeffrey J. W.; Hovingh, G. Kees; Dehghan, Abbas; Nelson, Christopher P.; Kotti, Salma; Danchin, Nicolas; Scholz, Markus; Haase, Christiane L.; Rothenbacher, Dietrich; Swerdlow, Daniel I.; Kuchenbaecker, Karoline B.; Staines-Urias, Eleonora; Goel, Anuj; van 't Hooft, Ferdinand; Gertow, Karl; de Faire, Ulf; Panayiotou, Andrie G.; Tremoli, Elena; Baldassarre, Damiano; Veglia, Fabrizio; Holdt, Lesca M.; Beutner, Frank; Gansevoort, Ron T.; Navis, Gerjan J.; Mateo Leach, Irene; Breitling, Lutz P.; Brenner, Hermann; Thiery, Joachim; Dallmeier, Dhayana; Franco-Cereceda, Anders; Boer, Jolanda M. A.; Stephens, Jeffrey W.; Hofker, Marten H.; Tedgui, Alain; Hofman, Albert; Uitterlinden, Andre G.; Adamkova, Vera; Pitha, Jan; Onland-Moret, N. Charlotte; Cramer, Maarten J.; Nathoe, Hendrik M.; Spiering, Wilko; Klungel, Olaf H.; Kumari, Meena; Whincup, Peter H.; Morrow, David A.; Braund, Peter S.; Hall, Alistair S.; Olsson, Anders G.; Doevendans, Pieter A.; Trip, Mieke D.; Tobin, Martin D.; Hamsten, Anders; Watkins, Hugh; Koenig, Wolfgang; Nicolaides, Andrew N.; Teupser, Daniel; Day, Ian N. M.; Carlquist, John F.; Gaunt, Tom R.; Ford, Ian; Sattar, Naveed; Tsimikas, Sotirios; Schwartz, Gregory G.; Lawlor, Debbie A.; Morris, Richard W.; Sandhu, Manjinder S.; Poledne, Rudolf; Maitland-van der Zee, Anke H.; Khaw, Kay-Tee; Keating, Brendan J.; van der Harst, Pim; Price, Jackie F.; Mehta, Shamir R.; Yusuf, Salim; Witteman, Jaqueline C. M.; Franco, Oscar H.; Jukema, J. Wouter; de Knijff, Peter; Tybjaerg-Hansen, Anne; Rader, Daniel J.; Farrall, Martin; Samani, Nilesh J.; Kivimaki, Mika; Fox, Keith A. A.; Humphries, Steve E.; Anderson, Jeffrey L.; Boekholdt, S. Matthijs; Palmer, Tom M.; Eriksson, Per; Pare, Guillaume; Hingorani, Aroon D.; Sabatine, Marc S.; Mallat, Ziad; Casas, Juan P.; Talmud, Philippa J.

    2013-01-01

    Objectives This study sought to investigate the role of secretory phospholipase A(2) (sPLA(2))-IIA in cardiovascular disease. Background Higher circulating levels of sPLA(2)-IIA mass or sPLA(2) enzyme activity have been associated with increased risk of cardiovascular events. However, it is not

  10. Single lipid vesicle assay for characterizing single-enzyme kinetics of phospholipid hydrolysis in a complex biological fluid.

    Science.gov (United States)

    Tabaei, Seyed R; Rabe, Michael; Zetterberg, Henrik; Zhdanov, Vladimir P; Höök, Fredrik

    2013-09-25

    Imaging of individual lipid vesicles is used to track single-enzyme kinetics of phospholipid hydrolysis. The method is employed to quantify the catalytic activity of phospholipase A2 (PLA2) in both pure and complex biological fluids. The measurements are demonstrated to offer a subpicomolar limit of detection (LOD) of human secretory PLA2 (sPLA2) in up to 1000-fold-diluted cerebrospinal fluid (CSF). An additional new feature provided by the single-enzyme sensitivity is that information about both relative concentration variations of active sPLA2 in CSF and the specific enzymatic activity can be simultaneously obtained. When CSF samples from healthy controls and individuals diagnosed with Alzheimer's disease (AD) are analyzed, the specific enzymatic activity is found to be preserved within 7% in the different CSF samples whereas the enzyme concentration differs by up to 56%. This suggests that the previously reported difference in PLA2 activity in CSF samples from healthy and AD individuals originates from differences in the PLA2 expression level rather than from the enzyme activity. Conventional ensemble averaging methods used to probe sPLA2 activity do not allow one to obtain such information. Together with an improvement in the LOD of at least 1 order of magnitude compared to that of conventional assays, this suggests that the method will become useful in furthering our understanding of the role of PLA2 in health and disease and in detecting the pharmacodynamic effects of PLA2-targeting drug candidates.

  11. Secretory Phospholipase A2-IIA and Cardiovascular Disease: A Mendelian Randomization Study

    NARCIS (Netherlands)

    Holmes, Michael V.; Simon, Tabassome; Exeter, Holly J.; Folkersen, Lasse; Asselbergs, Folkert W.; Guardiola, Montse; Cooper, Jackie A.; Palmen, Jutta; Hubacek, Jaroslav A.; Carruthers, Kathryn F.; Horne, Benjamin D.; Brunisholz, Kimberly D.; Mega, Jessica L.; van Iperen, Erik P. A.; Li, Mingyao; Leusink, Maarten; Trompet, Stella; Verschuren, Jeffrey J. W.; Hovingh, G. Kees; Dehghan, Abbas; Nelson, Christopher P.; Kotti, Salma; Danchin, Nicolas; Scholz, Markus; Haase, Christiane L.; Rothenbacher, Dietrich; Swerdlow, Daniel I.; Kuchenbaecker, Karoline B.; Staines-Urias, Eleonora; Goel, Anuj; van 't Hooft, Ferdinand; Gertow, Karl; de Faire, Ulf; Panayiotou, Andrie G.; Tremoli, Elena; Baldassarre, Damiano; Veglia, Fabrizio; Holdt, Lesca M.; Beutner, Frank; Gansevoort, Ron T.; Navis, Gerjan J.; Mateo Leach, Irene; Breitling, Lutz P.; Brenner, Hermann; Thiery, Joachim; Dallmeier, Dhayana; Franco-Cereceda, Anders; Boer, Jolanda M. A.; Stephens, Jeffrey W.; Hofker, Marten H.; Tedgui, Alain; Hofman, Albert; Uitterlinden, André G.; Adamkova, Vera; Pitha, Jan; Onland-Moret, N. Charlotte; Cramer, Maarten J.; Nathoe, Hendrik M.; Spiering, Wilko; Klungel, Olaf H.; Kumari, Meena; Whincup, Peter H.; Morrow, David A.; Braund, Peter S.; Hall, Alistair S.; Olsson, Anders G.; Doevendans, Pieter A.; Trip, Mieke D.; Tobin, Martin D.; Hamsten, Anders; Watkins, Hugh; Koenig, Wolfgang; Nicolaides, Andrew N.; Teupser, Daniel; Day, Ian N. M.; Carlquist, John F.; Gaunt, Tom R.; Ford, Ian; Sattar, Naveed; Tsimikas, Sotirios; Schwartz, Gregory G.; Lawlor, Debbie A.; Morris, Richard W.; Sandhu, Manjinder S.; Poledne, Rudolf; Maitland-van der Zee, Anke H.; Khaw, Kay-Tee; Keating, Brendan J.; van der Harst, Pim; Price, Jackie F.; Mehta, Shamir R.; Yusuf, Salim; Witteman, Jaqueline C. M.; Franco, Oscar H.; Jukema, J. Wouter; de Knijff, Peter; Tybjaerg-Hansen, Anne; Rader, Daniel J.; Farrall, Martin; Samani, Nilesh J.; Kivimaki, Mika; Fox, Keith A. A.; Humphries, Steve E.; Anderson, Jeffrey L.; Boekholdt, S. Matthijs; Palmer, Tom M.; Eriksson, Per; Paré, Guillaume; Hingorani, Aroon D.; Sabatine, Marc S.; Mallat, Ziad; Casas, Juan P.; Talmud, Philippa J.

    2013-01-01

    This study sought to investigate the role of secretory phospholipase A2 (sPLA2)-IIA in cardiovascular disease. Higher circulating levels of sPLA2-IIA mass or sPLA2 enzyme activity have been associated with increased risk of cardiovascular events. However, it is not clear if this association is

  12. Molecular details of secretory phospholipase A2 from flax (Linum usitatissimum L.) provide insight into its structure and function.

    Science.gov (United States)

    Gupta, Payal; Dash, Prasanta K

    2017-09-11

    Secretory phospholipase A 2 (sPLA 2 ) are low molecular weight proteins (12-18 kDa) involved in a suite of plant cellular processes imparting growth and development. With myriad roles in physiological and biochemical processes in plants, detailed analysis of sPLA 2 in flax/linseed is meagre. The present work, first in flax, embodies cloning, expression, purification and molecular characterisation of two distinct sPLA 2 s (I and II) from flax. PLA 2 activity of the cloned sPLA 2 s were biochemically assayed authenticating them as bona fide phospholipase A 2 . Physiochemical properties of both the sPLA 2 s revealed they are thermostable proteins requiring di-valent cations for optimum activity.While, structural analysis of both the proteins revealed deviations in the amino acid sequence at C- & N-terminal regions; hydropathic study revealed LusPLA 2 I as a hydrophobic protein and LusPLA 2 II as a hydrophilic protein. Structural analysis of flax sPLA 2 s revealed that secondary structure of both the proteins are dominated by α-helix followed by random coils. Modular superimposition of LusPLA 2 isoforms with rice sPLA 2 confirmed monomeric structural preservation among plant phospholipase A 2 and provided insight into structure of folded flax sPLA 2 s.

  13. Deposition of lipid, protein, and secretory phospholipase A2 on hydrophilic contact lenses.

    Science.gov (United States)

    Mochizuki, Hiroshi; Yamada, Masakazu; Hatou, Shin; Kawashima, Motoko; Hata, Seiichiro

    2008-01-01

    Recent studies have shown that low tear phospholipid levels are associated with tear film instability in hydrophilic contact lens wearers. The concentration of secretory phospholipase A2 (sPLA2), the enzyme that hydrolyzes phospholipids, in tears is known to exceed the levels found in serum by four orders of magnitude. This study was performed to determine the levels of sPLA2 from the deposition on two different frequent-replacement contact lens materials. Polymacon and etafilcon A contact lenses worn for 2 weeks by 16 experienced contact lens wearers were used for the analysis. Total lipids were determined by the sulfo-phospho-vanillin reaction. Phospholipids in lipid extracts were estimated by phosphorus determination with ammonium molybdate through enzymatic digestion. Total protein was measured by bicinchoninic acid analysis. Double-antibody sandwich enzyme-linked immunosorbent assay was used to determine sPLA2 concentrations. Total lipid deposition was found to be greater in the polymacon group (66.3+/-16.3 microg/lens) than in the etafilcon A group, although phospholipids were not detected in either group. The etafilcon A group had greater deposition of protein (3.7+/-0.7 mg/lens) than the polymacon group had. The etafilcon A group deposited statistically significantly more group IIa sPLA2 (1.1+/-0.3 microg/lens) than the polymacon group (0.07+/-0.04 microg/lens) did (P<0.001). There was a significant difference in the lipid and protein deposition profiles in the two lenses tested. A significant amount of sPLA2 in the deposition on contact lenses may play a role in tear film instability in hydrophilic contact lens wearers.

  14. Glutamate signalling and secretory phospholipase A2 modulate the release of arachidonic acid from neuronal membranes

    DEFF Research Database (Denmark)

    Rodriguez De Turco, Elena B; Jackson, Fannie R; DeCoster, Mark A

    2002-01-01

    The lipid mediators generated by phospholipases A(2) (PLA(2)), free arachidonic acid (AA), eicosanoids, and platelet-activating factor, modulate neuronal activity; when overproduced, some of them become potent neurotoxins. We have shown, using primary cortical neuron cultures, that glutamate...... and secretory PLA(2) (sPLA(2)) from bee venom (bv sPLA(2)) and Taipan snake venom (OS2) elicit synergy in inducing neuronal cell death. Low concentrations of sPLA(2) are selective ligands of cell-surface sPLA(2) receptors. We investigated which neuronal arachidonoyl phospholipids are targeted by glutamate......) and in minor changes in other phospholipids. A similar profile, although of greater magnitude, was observed 20 hr posttreatment. Glutamate (80 microM) induced much less mobilization of (3)H-AA than did sPLA(2) and resulted in a threefold greater degradation of (3)H-AA PE than of (3)H-AA PC by 20 hr...

  15. The enzymatic activity of the VEGFR2 receptor for the biosynthesis of dinucleoside polyphosphates.

    Science.gov (United States)

    Jankowski, Vera; Schulz, Anna; Kretschmer, Axel; Mischak, Harald; Boehringer, Falko; van der Giet, Markus; Janke, Doreen; Schuchardt, Mirjam; Herwig, Ralf; Zidek, Walter; Jankowski, Joachim

    2013-09-01

    The group of dinucleoside polyphosphates encompasses a large number of molecules consisting of two nucleosides which are connected by a phosphate chain of variable length. While the receptors activated by dinucleoside polyphosphates as well as their degradation have been studied in detail, its biosynthesis has not been elucidated so far. Since endothelial cells released the dinucleoside polyphosphate uridine adenosine tetraphosphate (Up4A), we tested cytosolic proteins of human endothelial cells obtained from dermal vessels elicited for enzymatic activity. When incubated with ADP and UDP, these cells showed increasing concentrations of Up4A. The underlying enzyme was isolated by chromatography and the mass spectrometric analysis revealed that the enzymatic activity was caused by the vascular endothelial growth factor receptor 2 (VEGFR2). Since VEGFR2 but neither VEGFR1 nor VEGFR3 were capable to synthesise dinucleoside polyphosphates, Tyr-1175 of VEGFR2 is most likely essential for the enzymatic activity of interest. Further, VEGFR2-containing cells like HepG2, THP-1 and RAW264.7 were capable of synthesising dinucleoside polyphosphates. VEGFR2-transfected HEK 293T/17 but not native HEK 293T/17 cells synthesised dinucleoside polyphosphates in vivo too. The simultaneous biosynthesis of dinucleoside polyphosphates could amplify the response to VEGF, since dinucleoside polyphosphates induce cellular growth via P2Y purinergic receptors. Thus the biosynthesis of dinucleoside polyphosphates by VEGFR2 may enhance the proliferative response to VEGF. Given that VEGFR2 is primarily expressed in endothelial cells, the biosynthesis of dinucleoside polyphosphates is mainly located in the vascular system. Since the vasculature is also the main site of action of dinucleoside polyphosphates, activating vascular purinoceptors, blood vessels appear as an autocrine system with respect to dinucleoside polyphosphates. We conclude that VEGFR2 receptor is capable of synthesising

  16. Lactadherin inhibits secretory phospholipase A2 activity on pre-apoptotic leukemia cells

    DEFF Research Database (Denmark)

    Nyegaard, Steffen; Novakovic, Valerie A.; Rasmussen, Jan Trige

    2013-01-01

    Secretory phospholipase A2 (sPLA2) is a critical component of insect and snake venoms and is secreted by mammalian leukocytes during inflammation. Elevated secretory PLA2 concentrations are associated with autoimmune diseases and septic shock. Many sPLA2’s do not bind to plasma membranes of quies...

  17. A snake venom group IIA PLA2 with immunomodulatory activity induces formation of lipid droplets containing 15-d-PGJ2 in macrophages.

    Science.gov (United States)

    Giannotti, Karina Cristina; Leiguez, Elbio; Carvalho, Ana Eduarda Zulim de; Nascimento, Neide Galvão; Matsubara, Márcio Hideki; Fortes-Dias, Consuelo Latorre; Moreira, Vanessa; Teixeira, Catarina

    2017-06-22

    Crotoxin B (CB) is a catalytically active group IIA sPLA 2 from Crotalus durissus terrificus snake venom. In contrast to most GIIA sPLA 2 s, CB exhibits anti-inflammatory effects, including the ability to inhibit leukocyte functions. Lipid droplets (LDs) are lipid-rich organelles associated with inflammation and recognized as a site for the synthesis of inflammatory lipid mediators. Here, the ability of CB to induce formation of LDs and the mechanisms involved in this effect were investigated in isolated macrophages. The profile of CB-induced 15-d-PGJ 2 (15-Deoxy-Delta-12,14-prostaglandin J 2 ) production and involvement of LDs in 15-d-PGJ 2 biosynthesis were also investigated. Stimulation of murine macrophages with CB induced increased number of LDs and release of 15-d-PGJ 2 . LDs induced by CB were associated to PLIN2 recruitment and expression and required activation of PKC, PI3K, MEK1/2, JNK, iPLA 2 and PLD. Both 15-d-PGJ 2 and COX-1 were found in CB-induced LDs indicating that LDs contribute to the inhibitory effects of CB by acting as platform for synthesis of 15-d-PGJ 2 , a pro-resolving lipid mediator. Together, our data indicate that an immunomodulatory GIIA sPLA 2 can directly induce LD formation and production of a pro-resolving mediator in an inflammatory cell and afford new insights into the roles of LDs in resolution of inflammatory processes.

  18. AMPK Signaling Involvement for the Repression of the IL-1β-Induced Group IIA Secretory Phospholipase A2 Expression in VSMCs.

    Directory of Open Access Journals (Sweden)

    Khadija El Hadri

    Full Text Available Secretory Phospholipase A2 of type IIA (sPLA2 IIA plays a crucial role in the production of lipid mediators by amplifying the neointimal inflammatory context of the vascular smooth muscle cells (VSMCs, especially during atherogenesis. Phenformin, a biguanide family member, by its anti-inflammatory properties presents potential for promoting beneficial effects upon vascular cells, however its impact upon the IL-1β-induced sPLA2 gene expression has not been deeply investigated so far. The present study was designed to determine the relationship between phenformin coupling AMP-activated protein kinase (AMPK function and the molecular mechanism by which the sPLA2 IIA expression was modulated in VSMCs. Here we find that 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR treatment strongly repressed IL-1β-induced sPLA2 expression at least at the transcriptional level. Our study reveals that phenformin elicited a dose-dependent inhibition of the sPLA2 IIA expression and transient overexpression experiments of constitutively active AMPK demonstrate clearly that AMPK signaling is involved in the transcriptional inhibition of sPLA2-IIA gene expression. Furthermore, although the expression of the transcriptional repressor B-cell lymphoma-6 protein (BCL-6 was markedly enhanced by phenformin and AICAR, the repression of sPLA2 gene occurs through a mechanism independent of BCL-6 DNA binding site. In addition we show that activation of AMPK limits IL-1β-induced NF-κB pathway activation. Our results indicate that BCL-6, once activated by AMPK, functions as a competitor of the IL-1β induced NF-κB transcription complex. Our findings provide insights on a new anti-inflammatory pathway linking phenformin, AMPK and molecular control of sPLA2 IIA gene expression in VSMCs.

  19. Point of care testing of phospholipase A2 group IIA for serological diagnosis of rheumatoid arthritis

    Science.gov (United States)

    Liu, Nathan J.; Chapman, Robert; Lin, Yiyang; Mmesi, Jonas; Bentham, Andrew; Tyreman, Matthew; Abraham, Sonya; Stevens, Molly M.

    2016-02-01

    Secretory phospholipase A2 group IIA (sPLA2-IIA) was examined as a point of care marker for determining disease activity in rheumatoid (RA) and psoriatic (PsA) arthritis. Serum concentration and activity of sPLA2-IIA were measured using in-house antibodies and a novel point of care lateral flow device assay in patients diagnosed with varying severities of RA (n = 30) and PsA (n = 25) and found to correlate strongly with C-reactive protein (CRP). Levels of all markers were elevated in patients with active RA over those with inactive RA as well as both active and inactive PsA, indicating that sPLA2-IIA can be used as an analogue to CRP for RA diagnosis at point of care.Secretory phospholipase A2 group IIA (sPLA2-IIA) was examined as a point of care marker for determining disease activity in rheumatoid (RA) and psoriatic (PsA) arthritis. Serum concentration and activity of sPLA2-IIA were measured using in-house antibodies and a novel point of care lateral flow device assay in patients diagnosed with varying severities of RA (n = 30) and PsA (n = 25) and found to correlate strongly with C-reactive protein (CRP). Levels of all markers were elevated in patients with active RA over those with inactive RA as well as both active and inactive PsA, indicating that sPLA2-IIA can be used as an analogue to CRP for RA diagnosis at point of care. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr08423g

  20. Purification and biochemical characterization of pancreatic phospholipase A2 from the common stingray Dasyatis pastinaca

    Directory of Open Access Journals (Sweden)

    Gargouri Youssef

    2011-02-01

    Full Text Available Abstract Background Mammalian sPLA2-IB are well characterized. In contrast, much less is known about aquatic ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes. Results A marine stingray phospholipase A2 (SPLA2 was purified from delipidated pancreas. Purified SPLA2, which is not glycosylated protein, was found to be monomeric protein with a molecular mass of 14 kDa. A specific activity of 750 U/mg for purified SPLA2 was measured at optimal conditions (pH 8.5 and 40 °C in the presence of 4 mM NaTDC and 8 mM CaCl2 using PC as substrate. The sequence of the first twenty first amino-acid residues at the N-terminal extremity of SPLA2 was determined and shows a close similarity with known mammal and bird pancreatic secreted phospholipases A2. SPLA2 stability in the presence of organic solvents, as well as in acidic and alkaline pH and at high temperature makes it a good candidate for its application in food industry. Conclusions SPLA2 has several advantageous features for industrial applications. Stability of SPLA2 in the presence of organic solvents, and its tolerance to high temperatures, basic and acidic pH, makes it a good candidate for application in food industry to treat phospholipid-rich industrial effluents, or to synthesize useful chemical compounds.

  1. Pressure Modulation of the Enzymatic Activity of Phospholipase A2, A Putative Membrane-Associated Pressure Sensor.

    Science.gov (United States)

    Suladze, Saba; Cinar, Suleyman; Sperlich, Benjamin; Winter, Roland

    2015-10-07

    Phospholipases A2 (PLA2) catalyze the hydrolysis reaction of sn-2 fatty acids of membrane phospholipids and are also involved in receptor signaling and transcriptional pathways. Here, we used pressure modulation of the PLA2 activity and of the membrane's physical-chemical properties to reveal new mechanistic information about the membrane association and subsequent enzymatic reaction of PLA2. Although the effect of high hydrostatic pressure (HHP) on aqueous soluble and integral membrane proteins has been investigated to some extent, its effect on enzymatic reactions operating at the water/lipid interface has not been explored, yet. This study focuses on the effect of HHP on the structure, membrane binding and enzymatic activity of membrane-associated bee venom PLA2, covering a pressure range up to 2 kbar. To this end, high-pressure Fourier-transform infrared and high-pressure stopped-flow fluorescence spectroscopies were applied. The results show that PLA2 binding to model biomembranes is not significantly affected by pressure and occurs in at least two kinetically distinct steps. Followed by fast initial membrane association, structural reorganization of α-helical segments of PLA2 takes place at the lipid water interface. FRET-based activity measurements reveal that pressure has a marked inhibitory effect on the lipid hydrolysis rate, which decreases by 75% upon compression up to 2 kbar. Lipid hydrolysis under extreme environmental conditions, such as those encountered in the deep sea where pressures up to the kbar-level are encountered, is hence markedly affected by HHP, rendering PLA2, next to being a primary osmosensor, a good candidate for a sensitive pressure sensor in vivo.

  2. Enzymatic activity of the cellulolytic complex produced by trichoderma reesei. Enzymatic hydrolysis of cellulose

    International Nuclear Information System (INIS)

    Alfonsel Jaen, M.; Negro, M.J.; Saez, R.; Martin Moreno, C.

    1986-01-01

    The enzymatic activity characterization of the cellulolytic complex obtained from Trichoderma reese QM 9414 and the influence of the enzymatic hydrolysis conditions on the hydrolysis yield are studied. Pure cellulose and native or alkali pretreated biomass from Onopordum nervosum have been used as substrates. The values of pH, temperature, substrate concentration and enzyme-substrate ratio for the optimum activity of that complex, evaluated as glucose and reducing sugars productions, have been selected. Previous studies on enzymatic hydrolysis of O. nervosum have shown a remarkable effect of the alkaline pretreatments on the final hydrolysis yield. (author). 10 figs.; 10 refs

  3. Enzymatic activity of the cellulolytic complex produced by Trichoderma reesei. Enzymatic hydrolysis of cellulose

    International Nuclear Information System (INIS)

    Alfonsel J, M.; Negro A, M. J.; Saez A, R.; Martin M, C.

    1986-01-01

    The enzymatic activity characterization of the cellulolytic complex obtained from Trichoderma reesei QM 9414 and the influence of the enzymatic hydrolysis conditions on the hydrolysis yield are studied. Pure cellulose and native or alkali pretreated biomass Onopordum nervosum have been used as substrates. The values of pH, temperature, substrate concentration and enzyme-substrate ratio for the optimum activity of that complex, evaluated as glucose and reducing sugars production, have been selected. Previous studies on enzymatic hydrolysis of 0. nervosum have shown a remarkable effect of the alkaline pretreatments on the final hydrolysis yield. (Author) 10 refs

  4. Enzymatic Activity of Free-Prostate-Specific Antigen (f-PSA) Is Not Required for Some of its Physiological Activities

    Science.gov (United States)

    Chadha, Kailash C.; Nair, Bindukumar B.; Chakravarthi, Srikant; Zhou, Rita; Godoy, Alejandro; Mohler, James L.; Aalinkeel, Ravikumar; Schwartz, Stanley A.; Smith, Gary J.

    2015-01-01

    BACKGROUND Prostate specific antigen (PSA) is a well known biomarker for early diagnosis and management of prostate cancer. Furthermore, PSA has been documented to have anti-angiogenic and anti-tumorigenic activities in both in vitro and in vivo studies. However, little is known about the molecular mechanism(s) involved in regulation of these processes, in particular the role of the serine-protease enzymatic activity of PSA. METHODS Enzymatic activity of PSA isolated directly from seminal plasma was inhibited specifically (>95%) by incubation with zinc2+. Human umbilical vein endothelial cells (HUVEC) were utilized to compare/contrast the physiological effects of enzymatically active versus inactive PSA. RESULTS Equimolar concentrations of enzymatically active PSA and PSA enzymatically inactivated by incubation with Zn2+ had similar physiological effects on HUVEC, including inhibiting the gene expression of pro-angiogenic growth factors, like VEGF and bFGF, and up-regulation of expression of the anti-angiogenic growth factor IFN-γ; suppression of mRNA expression for markers of blood vessel development, like FAK, FLT, KDR, TWIST-1; P-38; inhibition of endothelial tube formation in the in vitro Matrigel Tube Formation Assay; and inhibition of endothelial cell invasion and migration properties. DISCUSSION Our data provides compelling evidence that the transcriptional regulatory and the anti-angiogenic activities of human PSA are independent of the innate enzymatic activity PMID:21446007

  5. Modulation of the pharmacological effects of enzymatically-active PLA2 by BTL-2, an isolectin isolated from the Bryothamnion triquetrum red alga

    Directory of Open Access Journals (Sweden)

    Nagano Celso S

    2008-06-01

    Full Text Available Abstract Background An interaction between lectins from marine algae and PLA2 from rattlesnake was suggested some years ago. We, herein, studied the effects elicited by a small isolectin (BTL-2, isolated from Bryothamnion triquetrum, on the pharmacological and biological activities of a PLA2 isolated from rattlesnake venom (Crotalus durissus cascavella, to better understand the enzymatic and pharmacological mechanisms of the PLA2 and its complex. Results This PLA2 consisted of 122 amino acids (approximate molecular mass of 14 kDa, its pI was estimated to be 8.3, and its amino acid sequence shared a high degree of similarity with that of other neurotoxic and enzymatically-active PLA2s. BTL-2 had a molecular mass estimated in approximately 9 kDa and was characterized as a basic protein. In addition, BTL-2 did not exhibit any enzymatic activity. The PLA2 and BTL-2 formed a stable heterodimer with a molecular mass of approximately 24–26 kDa, estimated by molecular exclusion HPLC. In the presence of BTL-2, we observed a significant increase in PLA2 activity, 23% higher than that of PLA2 alone. BTL-2 demonstrated an inhibition of 98% in the growth of the Gram-positive bacterial strain, Clavibacter michiganensis michiganensis (Cmm, but only 9.8% inhibition of the Gram-negative bacterial strain, Xanthomonas axonopodis pv passiflorae (Xap. PLA2 decreased bacterial growth by 27.3% and 98.5% for Xap and Cmm, respectively, while incubating these two proteins with PLA2-BTL-2 inhibited their growths by 36.2% for Xap and 98.5% for Cmm. PLA2 significantly induced platelet aggregation in washed platelets, whereas BTL-2 did not induce significant platelet aggregation in any assay. However, BTL-2 significantly inhibited platelet aggregation induced by PLA2. In addition, PLA2 exhibited strong oedematogenic activity, which was decreased in the presence of BTL-2. BTL-2 alone did not induce oedema and did not decrease or abolish the oedema induced by the 48

  6. Three enzymatically active neurotoxins of Clostridium botulinum strain Af84: BoNT/A2, /F4, and /F5.

    Science.gov (United States)

    Kalb, Suzanne R; Baudys, Jakub; Smith, Theresa J; Smith, Leonard A; Barr, John R

    2014-04-01

    Botulinum neurotoxins (BoNTs) are produced by various species of clostridia and are potent neurotoxins which cause the disease botulism, by cleaving proteins needed for successful nerve transmission. There are currently seven confirmed serotypes of BoNTs, labeled A-G, and toxin-producing clostridia typically only produce one serotype of BoNT. There are a few strains (bivalent strains) which are known to produce more than one serotype of BoNT, producing either both BoNT/A and /B, BoNT/A and /F, or BoNT/B and /F, designated as Ab, Ba, Af, or Bf. Recently, it was reported that Clostridium botulinum strain Af84 has three neurotoxin gene clusters: bont/A2, bont/F4, and bont/F5. This was the first report of a clostridial organism containing more than two neurotoxin gene clusters. Using a mass spectrometry based proteomics approach, we report here that all three neurotoxins, BoNT/A2, /F4, and /F5, are produced by C. botulinum Af84. Label free MS(E) quantification of the three toxins indicated that toxin composition is 88% BoNT/A2, 1% BoNT/F4, and 11% BoNT/F5. The enzymatic activity of all three neurotoxins was assessed by examining the enzymatic activity of the neurotoxins upon peptide substrates, which mimic the toxins' natural targets, and monitoring cleavage of the substrates by mass spectrometry. We determined that all three neurotoxins are enzymatically active. This is the first report of three enzymatically active neurotoxins produced in a single strain of Clostridium botulinum.

  7. Enzymatic activities of Azotobacter chroococcum and survival in ...

    African Journals Online (AJOL)

    Enzymatic activities of Azotobacter chroococcum and survival in schloropyrifos amended sterile and non-sterile. M Shukla, V Kumar, RL Thakur, N Narula. Abstract. No Abstract. Cameroon Journal of Experimental Biology Vol. 2 (2) 2006: pp. 88-94. AJOL African Journals Online. HOW TO USE AJOL... for Researchers · for ...

  8. Variations in Enzymatic Activities of Shoots and Roots as an Indicator for Irradiated Seeds

    International Nuclear Information System (INIS)

    Abdelbbaary, N.A.; Elagamay, M.R.

    2005-01-01

    Germinated seedlings from oil seeds (sesame and sunflower) and legumes (Trigonella, Haricot, broad bean and cow pea) were irradiated with gamma rays at doses of 0, 0.2, 0.4, 0.8 and 1 kGy and the data were collected from shoots and roots. Enzymatic activities appeared to be correlated with gamma irradiation dose. The enzymatic activities of irradiated seeds understudy were significantly higher than controls. The peroxidase activities were nearly similar in both roots and shoots, while acid phosphatase activities in roots were higher than in shoots. Also protein contents were higher in roots. The peroxidase and acid phosphatase specific activities in roots were similar. Shoots peroxidase enzymatic activity increased with increased gamma doses. The seedling under study showed two different levels of peroxidase activity, higher as sesame, Trigonella and Sunflower, and lower such as all other legumes understudy. Similar tendency have been also noticed in roots-enzymatic activity, positive correlation between gamma doses treatment and peroxidase enzymatic activity, again two groups higher activity cow pea, broad bean, bean and Trigonella lower such as sesame, such as sesame, sunflower and haircut

  9. Clonaje y caracterización molecular in silico de un transcrito de fosfolipasa A2 aislado del veneno de la serpiente peruana Lachesis muta Molecular cloning and characterization in silico of phospholipase A2 transcripto isolated from Lachesis muta peruvian snake venom

    Directory of Open Access Journals (Sweden)

    Karim L. Jimenez

    2010-12-01

    subsequent sequencing. By bioinformatic analysis identified an open reading frame of 414 nucleotides that encoded 138 amino acids including a signal peptide of 16 aminoacids, molecular weight and pI were 13 976 kDa and 5.66 respectively. Results. The aminoacid sequence was called Lm-PLA2-Peru, contains an aspartate at position 49, this aminoacid in conjunction with other conserved residues such as Tyr-28, Gly-30, Gly-32, His-48, Tyr52, Asp99 are important for enzymatic activity. The comparison with the amino acid sequence data banks showed of similarity between PLA2 from Lachesis stenophrys (93% and other PLA2 snake venoms and over 80% of other sPLA2 family Viperidae venoms. A phylogenetic analysis showed that Lm-PLA2-Peru grouped with other acidic [Asp49] sPLA2 previously isolated from Bothriechis schlegelii venom showing 89 % nucleotide sequence identity. Finally, the computer modeling indicated that enzyme had the characteristic structure of sPLA2 group II that consisted of three α-helices, a β-wing, a short helix and a calcium-binding loop. Conclusion. The nucleotide sequence corresponding to the first transcript of gene from PLA2 cloned of Lachesis muta venom, snake from the Peruvian rainforest.

  10. Liposome based radiosensitizer cancer therapy

    DEFF Research Database (Denmark)

    Pourhassan, Houman

    Liposome-encapsulated chemotherapeutics have been used in the treatment of a variety of cancers and are feasible for use as mono-therapeutics as well as for combination therapy in conjunction with other modalities. Despite widespread use of liposomal drugs in cancer patient care, insufficient drug...... biomolecules. By modulating the liposomal membrane, liposomes can become sensitive towards enzymatically-driven destabilization and/or functionalization, thereby allowing control of the release of encapsulated therapeutics within the diseased tissue upon intrinsic stimulation from tumor-associated enzymes...... in tumor-bearing mice.The safety and efficacy of sPLA2-sensitive liposomal L-OHP was assessed in sPLA2-deficient FaDu hypopharyngeal squamous cell carcinoma and sPLA2-expressing Colo205 colorectal adenocarcinoma. Also, the feasibility of multimodal cancer therapy employing L-OHP encapsulated in MMP...

  11. Malondialdehyde level and some enzymatic activities in subclinical ...

    African Journals Online (AJOL)

    The purpose of this study was to evaluate the changes occurring in milk malondialdehyde (MDA) level and some enzymatic activities as a result of subclinical mastitis (SCM) in dairy cows. A total of 124 milk samples were collected from 124 lactating cows from the same herd in the period between the 2nd week after calving ...

  12. The standard aqueous stem bark extract of Mangifera indica L. inhibits toxic PLA2 - NN-XIb-PLA2 of Indian cobra venom.

    Science.gov (United States)

    Dhananjaya, Bhadrapura Lakkappa; Sudarshan, Shivalingaiah; Dongol, Yashad; More, Sunil S

    2016-05-01

    The aqueous extract of Mangifera indica is known to possess diverse medicinal properties, which also includes anti-snake venom activities. However, its inhibitory potency and mechanism of action on multi-toxic snake venom phospholipases A2s are still unknown. Therefore, the objective of this study was to evaluate the modulatory effect of standard aqueous bark extract of M. indica on NN-XIb-PLA2 of Indian cobra venom. The in vitro sPLA2, in situ hemolytic and in vivo edema inhibition effect were carried out as described. Also the effect of substrate and calcium concentration was carried out. M. indica extract dose dependently inhibited the GIA sPLA2 (NN-XIb-PLA2) activity with an IC50 value of 7.6 μg/ml. M. indica extract effectively inhibited the indirect hemolytic activity up to 98% at ∼40 μg/ml concentration. Further, M. indica extract (0-50 μg/ml) inhibited the edema formed in a dose dependent manner. When examined as a function of increased substrate and calcium concentration, there was no relieve of inhibitory effect of M. indica extract on the NN-XIb-PLA2. Further, the inhibition was irreversible as evident from binding studies. The in vitro inhibition is well correlated with in situ and in vivo edema inhibiting activities of M. indica. As the inhibition is independent of substrate and calcium and was irreversible, it can be concluded that M. indica extract mode of inhibition could be due to direct interaction of components present in the extract with the PLA2 enzyme. The aqueous extract of M. indica effectively inhibits svPLA2 enzymatic and its associated toxic activities, which substantiate their anti-snake venom properties. Further in-depth studies on the role and mechanism of the principal constituents present in the extract, responsible for the anti-PLA2 activity will be interesting to develop them into potent antisnake component and also as an anti-inflammatory agent.

  13. Cultural Considerations for Security Cooperation Operations in South Sudan: Understanding the Sudan People’s Liberation Army (SPLA)

    Science.gov (United States)

    2012-04-01

    relationships among people, living and dead, animals, plants, and natural and supernatural phenomena. “Fatalism, the belief that life is destined...thoughts persist today. According to quotes from militiamen in one of 14 Nenad Marinkovic’s articles , “Almost all South Sudan militias claim...foreign threats. He utilized these very arms to wage war against the SPLA and supply the government backed militias.63 In an article in the Small War

  14. Bee Venom Phospholipase A2: Yesterday's Enemy Becomes Today's Friend.

    Science.gov (United States)

    Lee, Gihyun; Bae, Hyunsu

    2016-02-22

    Bee venom therapy has been used to treat immune-related diseases such as arthritis for a long time. Recently, it has revealed that group III secretory phospholipase A2 from bee venom (bee venom group III sPLA2) has in vitro and in vivo immunomodulatory effects. A growing number of reports have demonstrated the therapeutic effects of bee venom group III sPLA2. Notably, new experimental data have shown protective immune responses of bee venom group III sPLA2 against a wide range of diseases including asthma, Parkinson's disease, and drug-induced organ inflammation. It is critical to evaluate the beneficial and adverse effects of bee venom group III sPLA2 because this enzyme is known to be the major allergen of bee venom that can cause anaphylactic shock. For many decades, efforts have been made to avoid its adverse effects. At high concentrations, exposure to bee venom group III sPLA2 can result in damage to cellular membranes and necrotic cell death. In this review, we summarized the current knowledge about the therapeutic effects of bee venom group III sPLA2 on several immunological diseases and described the detailed mechanisms of bee venom group III sPLA2 in regulating various immune responses and physiopathological changes.

  15. Detection of extracellular enzymatic activity in microorganisms ...

    African Journals Online (AJOL)

    Detection of extracellular enzymatic activity in microorganisms isolated from waste vegetable oil contaminated soil using plate methodologies. Eugenia G. Ortiz Lechuga, Isela Quintero Zapata, Katiushka Arévalo Niño ...

  16. Detection of extracellular enzymatic activity in microorganisms ...

    African Journals Online (AJOL)

    sunny t

    2015-09-18

    Sep 18, 2015 ... microorganisms with all three enzymatic activities, thereby establishing these techniques as ... supplemented at 1% with vegetable oils, including olive (OLI) ..... cepacia lipase for biodiesel fuel production from soybean oil.

  17. Secreted Phospholipases A2 from Animal Venoms in Pain and Analgesia

    Science.gov (United States)

    Zambelli, Vanessa O.; Picolo, Gisele; Fernandes, Carlos A. H.

    2017-01-01

    Animal venoms comprise a complex mixture of components that affect several biological systems. Based on the high selectivity for their molecular targets, these components are also a rich source of potential therapeutic agents. Among the main components of animal venoms are the secreted phospholipases A2 (sPLA2s). These PLA2 belong to distinct PLA2s groups. For example, snake venom sPLA2s from Elapidae and Viperidae families, the most important families when considering envenomation, belong, respectively, to the IA and IIA/IIB groups, whereas bee venom PLA2 belongs to group III of sPLA2s. It is well known that PLA2, due to its hydrolytic activity on phospholipids, takes part in many pathophysiological processes, including inflammation and pain. Therefore, secreted PLA2s obtained from animal venoms have been widely used as tools to (a) modulate inflammation and pain, uncovering molecular targets that are implicated in the control of inflammatory (including painful) and neurodegenerative diseases; (b) shed light on the pathophysiology of inflammation and pain observed in human envenomation by poisonous animals; and, (c) characterize molecular mechanisms involved in inflammatory diseases. The present review summarizes the knowledge on the nociceptive and antinociceptive actions of sPLA2s from animal venoms, particularly snake venoms. PMID:29311537

  18. An Asp49 Phospholipase A2 from Snake Venom Induces Cyclooxygenase-2 Expression and Prostaglandin E2 Production via Activation of NF-κB, p38MAPK, and PKC in Macrophages

    Directory of Open Access Journals (Sweden)

    Vanessa Moreira

    2014-01-01

    Full Text Available Phospholipases A2 (PLA2 are key enzymes for production of lipid mediators. We previously demonstrated that a snake venom sPLA2 named MT-III leads to prostaglandin (PGE2 biosynthesis in macrophages by inducing the expression of cyclooxygenase-2 (COX-2. Herein, we explored the molecular mechanisms and signaling pathways leading to these MT-III-induced effects. Results demonstrated that MT-III induced activation of the transcription factor NF-κB in isolated macrophages. By using NF-κB selective inhibitors, the involvement of this factor in MT-III-induced COX-2 expression and PGE2 production was demonstrated. Moreover, MT-III-induced COX-2 protein expression and PGE2 release were attenuated by pretreatment of macrophages with SB202190, and Ly294002, and H-7-dihydro compounds, indicating the involvement of p38MAPK, PI3K, and PKC pathways, respectively. Consistent with this, MT-III triggered early phosphorylation of p38MAPK, PI3K, and PKC. Furthermore, SB202190, H-7-dihydro, but not Ly294002 treatment, abrogated activation of NF-κB induced by MT-III. Altogether, these results show for the first time that the induction of COX-2 protein expression and PGE2 release, which occur via NF-κB activation induced by the sPLA2-MT-III in macrophages, are modulated by p38MAPK and PKC, but not by PI3K signaling proteins.

  19. Testing the applicability of rapid on-site enzymatic activity detection for surface water monitoring

    Science.gov (United States)

    Stadler, Philipp; Vogl, Wolfgang; Juri, Koschelnik; Markus, Epp; Maximilian, Lackner; Markus, Oismüller; Monika, Kumpan; Peter, Strauss; Regina, Sommer; Gabriela, Ryzinska-Paier; Farnleitner Andreas, H.; Matthias, Zessner

    2015-04-01

    On-site detection of enzymatic activities has been suggested as a rapid surrogate for microbiological pollution monitoring of water resources (e.g. using glucuronidases, galactosidases, esterases). Due to the possible short measuring intervals enzymatic methods have high potential as near-real time water quality monitoring tools. This presentation describes results from a long termed field test. For twelve months, two ColiMinder devices (Vienna Water Monitoring, Austria) for on-site determination of enzymatic activity were tested for stream water monitoring at the experimental catchment HOAL (Hydrological Open Air Laboratory, Center for Water Resource Systems, Vienna University of Technology). The devices were overall able to follow and reflect the diverse hydrological and microbiological conditions of the monitored stream during the test period. Continuous data in high temporal resolution captured the course of enzymatic activity in stream water during diverse rainfall events. The method also proofed sensitive enough to determine diurnal fluctuations of enzymatic activity in stream water during dry periods. The method was able to capture a seasonal trend of enzymatic activity in stream water that matches the results gained from Colilert18 analysis for E. coli and coliform bacteria of monthly grab samples. Furthermore the comparison of ColiMinder data with measurements gained at the same test site with devices using the same method but having different construction design (BACTcontrol, microLAN) showed consistent measuring results. Comparative analysis showed significant differences between measured enzymatic activity (modified fishman units and pmol/min/100ml) and cultivation based analyses (most probable number, colony forming unit). Methods of enzymatic activity measures are capable to detect ideally the enzymatic activity caused by all active target bacteria members, including VBNC (viable but nonculturable) while cultivation based methods cannot detect VBNC

  20. PARTIAL CHARACTERIZATION OF ENZYMATIC ACTIVITIES PRODUCED BY A WILD STRAIN OF A. NIGER

    Directory of Open Access Journals (Sweden)

    María Martos

    2012-12-01

    Full Text Available Aspergillus niger, isolated from decay citrus peels in the province of Misiones, was able to produce pectinases by submerged fermentation. The enzymatic extract exhibited polygalacturonase, pectinesterase and lyase activities. Others enzymes capable of degrading cell wall polymers were also detected in the enzymatic extract such as cellulases and xylanases. Polygalacturonase was an endo-polygalacturonase. The enzyme exhibited a maximal activity at pH range between 4.5 to 5.0, was stable in the pH range from 2.5 to 5.5 and remained unchanged when was incubated at temperatures lower than 50 ºC. The fungi produced three PG isoenzymes. The enzymatic extract was able to clarify apple juice. The results observed make the pectinolytic enzymes produced by A. niger appropriate for future application in fruit juice processing industries.

  1. Quantitation of Na+, K+-atpase Enzymatic Activity in Tissues of the Mammalian Vestibular System

    Science.gov (United States)

    Kerr, T. P.

    1985-01-01

    In order to quantify vestibular Na(+), K(+)-ATPase, a microassay technique was developed which is sufficiently sensitive to measure the enzymatic activity in tissue from a single animal. The assay was used to characterize ATPase in he vestibular apparatus of the Mongolian gerbil. The quantitative procedure employs NPP (5 mM) as synthetic enzyme substrate. The assay relies upon spectrophotometric measurement (410 nm) of nitrophenol (NP) released by enzymatic hydrolysis of the substrate. Product formation in the absence of ouabain reflects both specific (Na(+), K(+)-ATPase) and non-specific (Mg(++)-ATPase) enzymatic activity. By measuring the accumulation of reaction product (NP) at three-minute intervals during the course of incubation, it is found that the overall enzymatic reaction proceeds linearly for at least 45 minutes. It is therefore possible to determine two separate reaction rates from a single set of tissues. Initial results indicate that total activity amounts to 53.3 + or - 11.2 (S.E.M.) nmol/hr/mg dry tissue, of which approximately 20% is ouabain-sensitive.

  2. PARP1 Val762Ala polymorphism reduces enzymatic activity

    International Nuclear Information System (INIS)

    Wang Xiaogan; Wang Zhaoqi; Tong Weimin; Shen Yan

    2007-01-01

    Poly(ADP-ribose) polymerase 1 (PARP1) modifies a variety of nuclear proteins by poly(ADP-ribosyl)ation, and plays diverse roles in molecular and cellular processes. A common PARP1 single nucleotide polymorphism (SNP) at codon 762, resulting in the substitution of alanine (Ala) for valine (Val) in the catalytic domain has been implicated in susceptibility to cancer. To characterize the functional effect of this polymorphism on PARP1, we performed in vitro enzymatic analysis on PARP1-Ala762 and PARP1-Val762. We found that PARP1-Ala762 displayed 57.2% of the activity of PARP1-Val762 for auto-poly(ADP-ribosyl)ation and 61.9% of the activity of PARP1-Val762 for trans-poly(ADP-ribosyl)ation of histone H1. The kinetic characterization revealed that the K m of PARP1-Ala762 was increased to a 1.2-fold of the K m of PARP1-Val762 for trans-poly(ADP-ribosyl)ation. Thus, the PARP1 Val762Ala polymorphism reduces the enzymatic activity of PARP1 by increasing K m . This finding suggests that different levels of poly(ADP-ribosyl)ation by PARP1 might aid in understanding Cancer risk of carriers of the PARP1 Val762Ala polymorphism

  3. Synthesis of sn-1 functionalized phospholipids as substrates for secretory phospholipase A2

    DEFF Research Database (Denmark)

    Linderoth, Lars; Peters, Günther H.J.; Jørgensen, K.

    2007-01-01

    Secretory phospholipase A2 (sPLA2) represents a family of small water-soluble enzymes that catalyze the hydrolysis of phospholipids in the sn-2 position liberating free fatty acids and lysophospholipids. Herein we report the synthesis of two new phospholipids (1 and 2) with bulky allyl-substituen......Secretory phospholipase A2 (sPLA2) represents a family of small water-soluble enzymes that catalyze the hydrolysis of phospholipids in the sn-2 position liberating free fatty acids and lysophospholipids. Herein we report the synthesis of two new phospholipids (1 and 2) with bulky allyl...... of the allyl-substituents by a zinc mediated allylation. Small unilamellar liposomes composed of phospholipids 1 and 2 were subjected to sPLA2 activity measurements. Our results show that only phospholipid 1 is hydrolyzed by the enzyme. Molecular dynamics simulations revealed that the lack of hydrolysis...

  4. Bee Venom Phospholipase A2: Yesterday’s Enemy Becomes Today’s Friend

    Science.gov (United States)

    Lee, Gihyun; Bae, Hyunsu

    2016-01-01

    Bee venom therapy has been used to treat immune-related diseases such as arthritis for a long time. Recently, it has revealed that group III secretory phospholipase A2 from bee venom (bee venom group III sPLA2) has in vitro and in vivo immunomodulatory effects. A growing number of reports have demonstrated the therapeutic effects of bee venom group III sPLA2. Notably, new experimental data have shown protective immune responses of bee venom group III sPLA2 against a wide range of diseases including asthma, Parkinson’s disease, and drug-induced organ inflammation. It is critical to evaluate the beneficial and adverse effects of bee venom group III sPLA2 because this enzyme is known to be the major allergen of bee venom that can cause anaphylactic shock. For many decades, efforts have been made to avoid its adverse effects. At high concentrations, exposure to bee venom group III sPLA2 can result in damage to cellular membranes and necrotic cell death. In this review, we summarized the current knowledge about the therapeutic effects of bee venom group III sPLA2 on several immunological diseases and described the detailed mechanisms of bee venom group III sPLA2 in regulating various immune responses and physiopathological changes. PMID:26907347

  5. Heavy metal pollution and soil enzymatic activity

    Energy Technology Data Exchange (ETDEWEB)

    Tyler, G

    1974-01-01

    The activity of hydrolytic soil enzymes was studied on spruce mor, polluted with Cu and Zn from a brass foundry in Sweden. Approximately straight regression lines were obtained between enzymatic activity or respiration rate and log Cu + Zn concentration, with highly significant negative regression coefficients for urease and acid phosphatase activity as well as respiration rate, whereas US -glucosidase activity was not measurably lower at high concentrations of Cu + Zn. 17 references, 5 figures.

  6. Synergy by secretory phospholipase A2 and glutamate on inducing cell death and sustained arachidonic acid metabolic changes in primary cortical neuronal cultures

    DEFF Research Database (Denmark)

    Kolko, M; DeCoster, M A; de Turco, E B

    1996-01-01

    glutamate and sPLA2 from bee venom. sPLA2, at concentrations eliciting low neurotoxicity (acid into triacylglycerols. Free [3H]arachidonic acid accumulated at higher enzyme concentrations......, from Taipan snake venom. The NMDA receptor antagonist MK-801 blocked glutamate effects and partially inhibited sPLA2 OS2 but not sPLA2 from bee venom-induced arachidonic acid release. Thus, the synergy with glutamate and very low concentrations of exogenously added sPLA2 suggests a potential role......Secretory and cytosolic phospholipases A2 (sPLA2 and cPLA2) may contribute to the release of arachidonic acid and other bioactive lipids, which are modulators of synaptic function. In primary cortical neuron cultures, neurotoxic cell death and [3H]arachidonate metabolism was studied after adding...

  7. Rapid estimation of compost enzymatic activity by spectral analysis method combined with machine learning.

    Science.gov (United States)

    Chakraborty, Somsubhra; Das, Bhabani S; Ali, Md Nasim; Li, Bin; Sarathjith, M C; Majumdar, K; Ray, D P

    2014-03-01

    The aim of this study was to investigate the feasibility of using visible near-infrared (VisNIR) diffuse reflectance spectroscopy (DRS) as an easy, inexpensive, and rapid method to predict compost enzymatic activity, which traditionally measured by fluorescein diacetate hydrolysis (FDA-HR) assay. Compost samples representative of five different compost facilities were scanned by DRS, and the raw reflectance spectra were preprocessed using seven spectral transformations for predicting compost FDA-HR with six multivariate algorithms. Although principal component analysis for all spectral pretreatments satisfactorily identified the clusters by compost types, it could not separate different FDA contents. Furthermore, the artificial neural network multilayer perceptron (residual prediction deviation=3.2, validation r(2)=0.91 and RMSE=13.38 μg g(-1) h(-1)) outperformed other multivariate models to capture the highly non-linear relationships between compost enzymatic activity and VisNIR reflectance spectra after Savitzky-Golay first derivative pretreatment. This work demonstrates the efficiency of VisNIR DRS for predicting compost enzymatic as well as microbial activity. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Secretory Phospholipase A2-IIA and Cardiovascular Disease

    DEFF Research Database (Denmark)

    Holmes, Michael V; Simon, Tabassome; Exeter, Holly J

    2013-01-01

    This study sought to investigate the role of secretory phospholipase A2 (sPLA2)-IIA in cardiovascular disease.......This study sought to investigate the role of secretory phospholipase A2 (sPLA2)-IIA in cardiovascular disease....

  9. Enzymatic activity measured by microcalorimetry in soil amended with organic residues

    Directory of Open Access Journals (Sweden)

    Karina Cenciani

    2011-08-01

    Full Text Available Enzymatic activity is an important property for soil quality evaluation. Two sequences of experiments were carried out in order to evaluate the enzymatic activity in a soil (Rhodic Eutrudox amended with cattle manure, earthworm casts, or sewage sludges from the municipalities of Barueri and Franca. The activity of commercial enzymes was measured by microcalorimetry in the same soil samples after sterilization. In the first experiment, the enzyme activities of cellulase, protease, and urease were determined in the soil samples during a three month period. In the second sequence of experiments, the thermal effect of the commercial enzymes cellulase, protease, and urease on sterilized soil samples under the same tretaments was monitored for a period of 46 days. The experimental design was randomized and arranged as factorial scheme in five treatments x seven samplings with five replications. The treatment effects were statistically evaluated by one-way analysis of variance. Tukey´s test was used to compare means at p < 0.05. The presence of different sources of organic residues increased the enzymatic activity in the sampling period. Cattle manure induced the highest enzymatic activity, followed by municipal sewage sludge, whereas earthworm casts induced the lowest activity, but differed from control treatment. The thermal effect on the enzyme activity of commercial cellulase, protease, and urease showed a variety of time peaks. These values probably oscillated due to soil physical-chemical factors affecting the enzyme activity on the residues.

  10. Altered enzymatic activity and allele frequency of OMI/HTRA2 in Alzheimer's disease

    Science.gov (United States)

    Westerlund, Marie; Behbahani, Homira; Gellhaar, Sandra; Forsell, Charlotte; Belin, Andrea Carmine; Anvret, Anna; Zettergren, Anna; Nissbrandt, Hans; Lind, Charlotta; Sydow, Olof; Graff, Caroline; Olson, Lars; Ankarcrona, Maria; Galter, Dagmar

    2011-01-01

    The serine-protease OMI/HTRA2, required for several cellular processes, including mitochondrial function, autophagy, chaperone activity, and apoptosis, has been implicated in the pathogenesis of both Alzheimer's disease (AD) and Parkinson's disease (PD). Western blot quantification of OMI/HTRA2 in frontal cortex of patients with AD (n=10) and control subjects (n=10) in two separate materials indicated reduced processed (active, 35 kDa) OMI/HTRA2 levels, whereas unprocessed (50 kDa) enzyme levels were not significantly different between the groups. Interestingly, the specific protease activity of OMI/HTRA2 was found to be significantly increased in patients with AD (n=10) compared to matched control subjects (n=10) in frontal cortex in two separate materials. Comparison of OMI/HTRA2 mRNA levels in frontal cortex and hippocampus, two brain areas particularly affected by AD, indicated similar levels in patients with AD (n=10) and matched control subjects (n=10). In addition, we analyzed the occurrence of the OMI/HTRA2 variants A141S and G399S in Swedish case-control materials for AD and PD and found a weak association of A141S with AD, but not with PD. In conclusion, our genetic, histological, and biochemical findings give further support to an involvement of OMI/HTRA2 in the pathology of AD; however, further studies are needed to clarify the role of this gene in neurodegeneration.—Westerlund, M., Behbahani, H., Gellhaar, S., Forsell, C., Carmine Belin, A., Anvret, A., Zettergren, A., Nissbrandt, H., Lind, C., Sydow, O., Graff, C., Olson, L., Ankarcrona, M., Galter, D. Altered enzymatic activity and allele frequency of OMI/HTRA2 in Alzheimer's disease. PMID:21163861

  11. Enzymatically active 2',5'-oligoadenylate synthetases are widely distributed among Metazoa, including protostome lineage.

    Science.gov (United States)

    Päri, Mailis; Kuusksalu, Anne; Lopp, Annika; Kjaer, Karina Hansen; Justesen, Just; Kelve, Merike

    2014-02-01

    2',5'-Oligoadenylate synthetases (OASs) belong to the nucleotidyl transferase family together with poly(A) polymerases, CCA-adding enzymes and the recently discovered cyclic-GMP-AMP synthase (cGAS). Mammalian OASs have been thoroughly characterized as components of the interferon-induced antiviral system. The OAS activity and the respective genes were also discovered in marine sponges where the interferon system is absent. In this study the recombinant OASs from several multicellular animals and their closest unicellular relative, a choanoflagellate, were expressed in a bacterial expression system and their enzymatic activities were examined. We demonstrated 2-5A synthesizing activities of OASs from the marine sponge Tedania ignis, a representative of the phylogenetically oldest metazoan phylum (Porifera), from an invertebrate of the protostome lineage, the mollusk Mytilus californianus (Mollusca), and from a vertebrate species, a cartilaginous fish Leucoraja erinacea (Chordata). However, the expressed proteins from an amphibian, the salamander Ambystoma mexicanum (Chordata), and from a protozoan, the marine choanoflagellate Monosiga brevicollis (Choanozoa), did not show 2-5A synthesizing activity. Differently from other studied OASs, OAS from the marine sponge T. ignis was able to catalyze the formation of oligomers having both 2',5'- and 3',5'-phosphodiester linkages. Our data suggest that OASs from sponges and evolutionarily higher animals have similar activation mechanisms which still include different affinities and possibly different structural requirements for the activating RNAs. Considering their 2'- and 3'-specificities, sponge OASs could represent a link between evolutionarily earlier nucleotidyl transferases and 2'-specific OASs from higher animals. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  12. Edema toxin impairs anthracidal phospholipase A2 expression by alveolar macrophages.

    Directory of Open Access Journals (Sweden)

    Benoit Raymond

    2007-12-01

    Full Text Available Bacillus anthracis, the etiological agent of anthrax, is a spore-forming gram-positive bacterium. Infection with this pathogen results in multisystem dysfunction and death. The pathogenicity of B. anthracis is due to the production of virulence factors, including edema toxin (ET. Recently, we established the protective role of type-IIA secreted phospholipase A2 (sPLA2-IIA against B. anthracis. A component of innate immunity produced by alveolar macrophages (AMs, sPLA2-IIA is found in human and animal bronchoalveolar lavages at sufficient levels to kill B. anthracis. However, pulmonary anthrax is almost always fatal, suggesting the potential impairment of sPLA2-IIA synthesis and/or action by B. anthracis factors. We investigated the effect of purified ET and ET-deficient B. anthracis strains on sPLA2-IIA expression in primary guinea pig AMs. We report that ET inhibits sPLA2-IIA expression in AMs at the transcriptional level via a cAMP/protein kinase A-dependent process. Moreover, we show that live B. anthracis strains expressing functional ET inhibit sPLA2-IIA expression, whereas ET-deficient strains induced this expression. This stimulatory effect, mediated partly by the cell wall peptidoglycan, can be counterbalanced by ET. We conclude that B. anthracis down-regulates sPLA2-IIA expression in AMs through a process involving ET. Our study, therefore, describes a new molecular mechanism implemented by B. anthracis to escape innate host defense. These pioneering data will provide new molecular targets for future intervention against this deadly pathogen.

  13. Antimicrobial and enzymatic activity of anemophilous fungi of a public university in Brazil

    Directory of Open Access Journals (Sweden)

    LAUREANA V. SOBRAL

    2017-10-01

    Full Text Available ABSTRACT To the fungal microbiota the UFPE and biotechnological potential enzymatic and antimicrobial production. Air conditioned environments were sampled using a passive sedimentation technique, the air I ratio and the presence of aflatoxigenic strains evaluated for ANVISA. Icelles were to determine the enzymatic activity of lipase, amylase and protease metabolic liquids to determine antimicrobial activity. Diversity was observed in all CAV environments, CFU/m3 ranged from 14 to 290 and I/E ratio from 0.1 to 1.5. The of the fungal genera were: Aspergillus (50%, Penicillium (21%, Talaromyces (14%, Curvularia and Paecilomyces (7% each. Aspergillus sydowii (Bainier & Sartory Thom & Church presented enzymatic activity and the Talaromyces purpureogenus Samson, Yilmaz, Houbraken, Spierenb., Seifert, Peterson, Varga & Frisvad presented antibacterial activity against all bacteria that all environments present fungal species biodiversity no toxigenic or pathogenic fungi were found, according to ANVISA legislation for conditioned environments and airborne filamentous fungi present potential for enzymatic and antimicrobial activity.

  14. The variations of enzymatic activity of pepsin preparation by γ-irradiation

    International Nuclear Information System (INIS)

    Kimura, Syojiro; Taimatsu, Meiko; Kanbashi, Toshitaka; Okamoto, Shinichi; Ohnishi, Tokuhiro.

    1993-01-01

    Effect of γ-irradiation on the enzymatic activity of raw pepsin and some saccharated pepsin preparations were studied in the dose range from 0 to 300 kGy. As a result, the apparent reduction rate of saccharated pepsin preparations is less than of raw pepsin. K values of raw and saccharated pepsins were 0.014 and 0.0040-0.0061, and G values of raw and saccharated pepsins were 3.98 and 1.13-1.73, respectively. The lower K and G values of saccharated pepsin than those of raw pepsin seem to be due to radiolytic products of lactose in the preparations as an excipient. Retention rates of enzymatic activity of irradiated preparations at the dose of 25 kGy, which is a complete sterilization dose of pharmaceutical materials, were estimated to be 83% for raw pepsin, and 86% and 93% for saccharated pepsin preparations. At the dose of 10 kGy suggested for food irradiation the retention rates were more than 93% for all pepsins. Therefore, this method is applicable considering the stability of the enzymatic activity after irradiation in the proper range of dose. However, it is necessary to consider the fact that radiolytic products of lactose affect the measurement of enzymatic activity. (author)

  15. Expression and location of mRNAs encoding multiple forms of secretory phospholipase A2 in the rat retina

    DEFF Research Database (Denmark)

    Kolko, Miriam; Christoffersen, Nanna R; Barreiro, Sebastian G

    2004-01-01

    of sPLA(2)s in neuronal signaling and survival [Kolko et al. (1996) J. Biol. Chem. 271: 32722-32728]. To date, no retinal sPLA(2)s have been cloned or characterized. We evaluated the existence and abundance of sPLA(2) subtypes in rat retina and explored their possible involvement in light...

  16. Protective effects against H2O2-induced damage by enzymatic hydrolysates of an edible brown seaweed, sea tangle (Laminaria japonica).

    Science.gov (United States)

    Park, Pyo-Jam; Kim, Eun-Kyung; Lee, Seung-Jae; Park, Sun-Young; Kang, Dong-Soo; Jung, Bok-Mi; Kim, Kui-Shik; Je, Jae-Young; Ahn, Chang-Bum

    2009-02-01

    Enzymatic hydrolysates of Laminaria japonica were evaluated for antioxidative activities using hydroxyl radical scavenging activity and protective effects against H(2)O(2)-induced DNA and cell damage. In addition, activities of antioxidative enzymes, including catalase, glutathione peroxidase, and glutathione S-transferase, of the enzymatic hydrolysates from L. japonica were also estimated. L. japonica was first enzymatically hydrolyzed by seven carbohydrases (Dextrozyme, AMG, Promozyme, Maltogenase, Termamyl, Viscozyme, and Celluclast [all from Novo Co., Novozyme Nordisk, Bagsvaerd, Denmark]) and five proteinases (Flavourzyme, Neutrase, Protamex, Alcalase [all from Novo Co.], and pancreatic trypsin). The hydroxyl radical scavenging activities of Promozyme and pancreatic trypsin hydrolysates from L. japonica were the highest as compared to those of the other carbohydrases and proteinases, and their 50% inhibitory concentration values were 1.67 and 317.49 mug/mL, respectively. The pancreatic trypsin hydrolysates of L. japonica exerted a protective effect on H(2)O(2)-induced DNA damage. We also evaluated the protective effect on hydroxyl radical-induced oxidative damage in PC12 cells via propidium iodide staining using a flow cytometer. The AMG and pancreatic trypsin hydrolysates of L. japonica dose-dependently protected PC12 cells against cell death caused by hydroxyl radical-induced oxidative damage. Additionally, we analyzed the activity of antioxidative enzymes such as catalase, glutathione peroxidase, and the phase II biotransformation enzyme glutathione S-transferase in L. japonica-treated cells. The activity of all antioxidative enzymes was higher in L. japonica-treated cells compared with the nontreated cells. These results indicate that enzymatic hydrolysates of L. japonica possess antioxidative activity.

  17. The effects of xylitol and sorbitol on lysozyme- and peroxidase-related enzymatic and candidacidal activities.

    Science.gov (United States)

    Kim, Bum-Soo; Chang, Ji-Youn; Kim, Yoon-Young; Kho, Hong-Seop

    2015-07-01

    To investigate whether xylitol and sorbitol affect enzymatic and candidacidal activities of lysozyme, the peroxidase system, and the glucose oxidase-mediated peroxidase system. Xylitol and sorbitol were added to hen egg-white lysozyme, bovine lactoperoxidase, glucose oxidase-mediated peroxidase, and whole saliva in solution and on hydroxyapatite surfaces. The enzymatic activities of lysozyme, peroxidase, and glucose oxidase-mediated peroxidase were determined by the turbidimetric method, the NbsSCN assay, and production of oxidized o-dianisidine, respectively. Candidacidal activities were determined by comparing colony forming units using Candida albicans ATCC strains 10231, 11006, and 18804. While xylitol and sorbitol did not affect the enzymatic activity of hen egg-white lysozyme both in solution and on hydroxyapatite surfaces, they did inhibit the enzymatic activity of salivary lysozyme significantly in solution, but not on the surfaces. Xylitol and sorbitol enhanced the enzymatic activities of both bovine lactoperoxidase and salivary peroxidase significantly in a dose-dependent manner in solution, but not on the surfaces. Sorbitol, but not xylitol, inhibited the enzymatic activity of glucose oxidase-mediated peroxidase significantly. Both xylitol and sorbitol did not affect candidacidal activities of hen egg-white lysozyme, the bovine lactoperoxidase system, or the glucose oxidase-mediated bovine lactoperoxidase system. Xylitol and sorbitol inhibited salivary lysozyme activity, but enhanced both bovine lactoperoxidase and salivary peroxidase activities significantly in solution. Xylitol and sorbitol did not augment lysozyme- and peroxidase-related candidacidal activities. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Enzymatic browning control in cut apples (Red delicious through a system of active packaging

    Directory of Open Access Journals (Sweden)

    Felipe Jadán Piedra

    2017-03-01

    Full Text Available Enzymatic browning is one of the most relevant mechanisms of deterioration that take place in fresh-cut fruit and vegetables, as a consequence of the activity of the polyphenol oxidase enzyme on the phenolic compounds release after cellular lysis . This work is focused on the reduction of these enzymatic activity by an active packaging technology, which make use of a material that incorporates antioxidant active agents. Thus, films of ethylene-vynil alcohol copolymer (EVOH containing a typical food antioxidant, such as ascorbic acid and a polyphenol oxidase-inhibiting agent, the 4-hexylresorcinol have been developed and used to wrap apple slices. The evolution of color, the enzymatic activity and the kinetic of agents release to food simulants were monitored. The results showed an improvement of apple slice color stability and a reduction of the enzymatic activity. The film with 10 % of agents in 3/1 ratio (4-hexylresorcinol/ascorbic acid provided the best results.

  19. First results on enzymatic activities in two salt marsh soils under different hydromorphic level and vegetation

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    Carmen Trasar-Cepeda

    2015-12-01

    Full Text Available Salt-marsh soils are soils characterized by non-permanent hydric saturation that, depending on factors like duration of submersion periods, are dominated by different salt-tolerant plant species. The composition of microbial communities is an essential component in trophic dynamics and biogeochemical processes in salt marshes, and determines the level of enzymatic activities, which catalyze the conversion of complex molecules into simpler ones. Despite of this, the enzymatic activities in marsh-soils has not yet been investigated. The aim of this study was to analyze the enzymatic activities in two soil profiles of marsh-soils under different water saturation level and dominated by different plant species [Juncus maritimus Lam and Spartina maritima (Curtis Fernald (Sp]. In both soils, the enzymatic activities were much lower than the levels typically found in terrestrial ecosystems. The enzymatic activities were measured both in air-dried and in re-moistened and incubated soil samples. In air-dried samples, the enzymatic activities were higher in Juncus than in Spartina soil and tended to decrease with depth, being sharper the decrease in Juncus than in Spartina soil. Re-moistened and pre-incubated soils showed a general increase in all the enzymatic activities and throughout the whole soil profile, especially in Spartina soils. Hydrolase activities showed a strong and positive relationship with organic matter content both in air-dried and in re-moistened soil samples, higher in these latter. In general, oxidoreductase activities only showed this relationship in re-moistened soil samples. More studies, preferably using freshly collected soil samples, are needed to understand the relationship between enzymatic activities and these environmental conditions.

  20. Effect of topical application of fluoride gel NaF 2% on enzymatic and non-enzymatic antioxidant parameters of saliva.

    Science.gov (United States)

    Leite, Mariana Ferreira; Ferreira, Nayara Ferraz D'Assumpção; Shitsuka, Caleb David Willy Moreira; Lima, Amanda Martins; Masuyama, Mônica Miyuki; Sant'Anna, Giselle Rodrigues; Yamaguti, Paula Mochidome; Polotow, Tatiana G; de Barros, Marcelo Paes

    2012-06-01

    The aim of the study was to evaluate the effect of topical fluoride gel NaF 2% application on antioxidant parameters of whole saliva from children. The saliva mechanically stimulated with parafilm was collected from 25 children (6-12 years) attending the Clinic of Paediatric Dentistry of Universidade Cruzeiro do Sul, São Paulo, Brazil, before (control group) and immediately after application of neutral fluoride gel NaF 2% (fluoride-gel group), according to the Standards for Research Using Human Subjects, Resolution 196/96 of the USA National Health Council of 10/10/1996. Afterwards, pre-post ferric-reducing antioxidant power (FRAP), trolox-equivalent antioxidant capacity (TEAC), uric acid, reduced/oxidised glutathione content (GSH/GSSG) and total peroxidase activity (TPO) were evaluated in whole saliva of both groups. All non-enzymatic antioxidant parameters were augmented by fluoride-gel NaF 2% application, whereas a notable reduction (31%) of peroxidase activity was concomitantly observed in the children's saliva (p ≤ 0.05). Nevertheless, the reducing power of saliva was kept unaltered under these circumstances (p ≤ 0.05). Despite the reduced activity of peroxidase (an important antimicrobial and antioxidant enzyme), the topical fluoride gel NaF 2% favourably stimulated the release of non-enzymatic antioxidant components of saliva, sustaining the reducing power of saliva and the natural defences of the oral cavity. Copyright © 2011 Elsevier Ltd. All rights reserved.

  1. Enzymatic activity of fungi isolated from crops

    Directory of Open Access Journals (Sweden)

    Wioletta A. Żukiewicz-Sobczak

    2016-12-01

    Full Text Available Aim: To detect and assess the activity of extracellular hydrolytic enzymes and to find differences in enzymograms between fungi isolated from wheat and rye samples and grown on Czapek-Dox Broth and Sabouraud Dextrose Broth enriched with cereal (wheat or rye. Isolated strains were also classified in the scale of biosafety levels (BSL. Material and methods: The study used 23 strains of fungi cultured from samples of wheat and rye (grain, grain dust obtained during threshing and soil collected in the Lublin region (eastern Poland. API ZYM test (bioMérieux was carried out according to the manufacturer’s instructions. Classification of BSL (Biosafety levels was based on the current literature. Results : High enzymatic activity was found in strains cultured in media containing 1% of wheat grain ( Bipolaris holmi, Penicillium decumbens and with an addition of 1% of rye grain ( Cladosporium herbarum, Aspergillus versicolor, Alternaria alternata . The total number of enzymes varied depending on the type of media, and in most cases it was higher in the culture where an addition of cereal grains was used. Conclusions : Isolated strains of fungi reveal differences in the profiles of the enzyme assay. It can be assumed that the substrate enriched in grains stimulate the higher activity of mold enzymes. Key words: enzymatic activity, mold fungi, zymogram, biohazards.

  2. Effect of restricted motion in high temperature on enzymatic activity of the pancreas

    Science.gov (United States)

    Abdusattarov, A.; Smirnova, G. I.

    1980-01-01

    Effects of 30 day hypodynamia coupled with high temperature (35-36 C) on enzymatic activity of the pancreas of male adult rats were studied. The test animals were divided into four groups. Group one served as controls (freedom of movement and a temperature of 25-26 C, considered optimal). The remaining animals were divided into three additional groups: Group two freedom of movement but high temperature (35-36 C); group three hypodynamia but an optimal temperature; group four hypodynamia and 35-36 C. Considerable change in the enzymatic activity in the pancreas of the four groups is observed in three experimental groups (two, three, and four) as compared to the control (group one). The results indicate that adaption of the organism to the thermal factor and restricted movement is accompanied by a change in the enzymatic spectrum of the pancreas. With the combined effect of these two stresses under conditions of the adaption of the organism especially sharp shifts occur in the enzymatic activity.

  3. Bee Venom Phospholipase A2: Yesterday’s Enemy Becomes Today’s Friend

    OpenAIRE

    Gihyun Lee; Hyunsu Bae

    2016-01-01

    Bee venom therapy has been used to treat immune-related diseases such as arthritis for a long time. Recently, it has revealed that group III secretory phospholipase A2 from bee venom (bee venom group III sPLA2) has in vitro and in vivo immunomodulatory effects. A growing number of reports have demonstrated the therapeutic effects of bee venom group III sPLA2. Notably, new experimental data have shown protective immune responses of bee venom group III sPLA2 against a wide range of diseases inc...

  4. Effects of Polyelectrolyte Complex Micelles and Their Components on the Enzymatic Activity of Lipase

    NARCIS (Netherlands)

    Lindhoud, Saskia; Norde, Willem; Stuart, Martien Cohen

    2010-01-01

    The enzymatic activity of Hi-lipase embedded in complexes of poly-2-methylvinylpyridinium-co-poly(ethylene oxide) (P2MVP(41)-PEG(205)) and poly(acrylic acid)(PAA(139)) is studied as a function of the PAA(139) + P2MVP(41) - PEO(205) complex composition. The measurements revealed that there are

  5. Impact of herbaceous vegetation on the enzymatic activity of coal mining wastes

    Energy Technology Data Exchange (ETDEWEB)

    Osmanczyk, D

    1980-01-01

    Differences in the enzymatic activity of reclaimed and crude dump wastes after coal mining were investigated. Due to the increased activity of six investigated enzymes (dehydrogenase, catalase, saccharase, BETA-glucosidase, urease and asparaginase), a favourable impact of herbaceous vegetation on the biological activation of the breeding-ground was noticed. Particularly in the case of sacharase and BETA-glucosidase, an increase of the enzymatic activity at a rate of several times or even more than ten times speaks not only for an adequate increase of the metabolic rate of carbohydrates but also for specific properties of the habitat which favours an adsorption of these enzymes. (6 refs.) (In Polish)

  6. Effect of wheat and Miscanthus straw biochars on soil enzymatic activity, ecotoxicity, and plant yield

    Science.gov (United States)

    Mierzwa-Hersztek, Monika; Gondek, Krzysztof; Klimkowicz-Pawlas, Agnieszka; Baran, Agnieszka

    2017-07-01

    The variety of technological conditions and raw materials from which biochar is produced is the reason why its soil application may have different effects on soil properties and plant growth. The aim of this study was to evaluate the effect of the addition of wheat straw and Miscanthus giganteus straw (5 t DM ha-1) and biochar obtained from this materials in doses of 2.25 and 5 t DM ha-1 on soil enzymatic activity, soil ecotoxicity, and plant yield (perennial grass mixture with red clover). The research was carried out under field conditions on soil with the granulometric composition of loamy sand. No significant effect of biochar amendment on soil enzymatic activity was observed. The biochar-amended soil was toxic to Vibrio fischeri and exhibited low toxicity to Heterocypris incongruens. Application of wheat straw biochar and M. giganteus straw biochar in a dose of 5 t DM ha-1 contributed to an increase in plant biomass production by 2 and 14%, respectively, compared to the soil with mineral fertilisation. Biochars had a more adverse effect on soil enzymatic activity and soil ecotoxicity to H. incongruens and V. fischeri than non-converted wheat straw and M. giganteus straw, but significantly increased the grass crop yield.

  7. An Investigation of the Changes in Enzymatic and Non-Enzymatic Salivary Antioxidants Caused by Exhausting Aerobic Activity in Non-Athletic Men

    Directory of Open Access Journals (Sweden)

    Yazgaldi Nazari

    2016-12-01

    Full Text Available Background and Objectives: In the present study, the effect of acute aerobic exercise on enzymatic and non-enzymatic salivary antioxidants variations in non-athlete men, was investigated. Methods: In this experimental study, 25 male non-athlete collegiates (age, 21.2±1.6 years; weight, 68.62±10.1kg; body fat, 16.75±2.9%; and Vo2 max, 37.54±2.4ml/kg/min participated voluntarily in this study. Saliva samples were collected in three phases (before, immediately, and 1 hour after running on treadmill according to Astrand test. The activity of peroxidase and catalase, and concentration of uric acid were measured by laboratory methods. Then, to assess the obtained changes, repeated measures statistical test, and in case of significance, post-hoc Bonferroni test were used for pairwise comparing of the measuring phases at the significance level of p≤0.05 used.  Results: The activity of peroxidase significantly increased immediately and 1 hour after exercise compared to the baseline; Also, the concentration of uric acid significantly increased after aerobic exercise, but catalase enzyme activity significantly decreased after aerobic exercise (p<0.05. No significant change was observed in saliva flow rate after exercise. Conclusion: According to the findings of this study, aerobic exercise causes the production of free radicals, and salivary antioxidant system increases as the body biological response to neutralize and counteract the damaging effects of free radicals.

  8. PSA-alpha-2-macroglobulin complex is enzymatically active in the serum of patients with advanced prostate cancer and can degrade circulating peptide hormones.

    Science.gov (United States)

    Kostova, Maya B; Brennen, William Nathaniel; Lopez, David; Anthony, Lizamma; Wang, Hao; Platz, Elizabeth; Denmeade, Samuel R

    2018-08-01

    Prostate cancer cells produce high levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the tumor microenvironment but is presumed to be enzymatically inactive in the blood due to complex formation with serum protease inhibitors α-1-antichymotrypsin and α-2-macroglobulin (A2M). PSA-A2M complexes cannot be measured by standard ELISA assays and are also rapidly cleared from the circulation. Thus the exact magnitude of PSA production by prostate cancer cells is not easily measured. The PSA complexed to A2M is unable to cleave proteins but maintains the ability to cleave small peptide substrates. Thus, in advanced prostate cancer, sufficient PSA-A2M may be in circulation to effect total A2M levels, levels of cytokines bound to A2M and hydrolyze small circulating peptide hormones. Total A2M levels in men with advanced prostate cancer and PSA levels above 1000 ng/mL were measured by ELISA and compared to controls. Additional ELISA assays were used to measure levels of IL-6 and TGF-beta which can bind to A2M. The ability of PSA-A2M complexes to hydrolyze protein and peptide substrates was analyzed ± PSA inhibitor. Enzymatic activity of PSA-A2M in serum of men with high PSA levels was also assayed. Serum A2M levels are inversely correlated with PSA levels in men with advanced prostate cancer. Il-6 Levels are significantly elevated in men with PSA >1000 ng/mL compared to controls with PSA PSA-A2M complex in serum of men with PSA levels >1000 ng/mL can hydrolyze small fluorescently labeled peptide substrates but not large proteins that are PSA substrates. PSA can hydrolyze small peptide hormones like PTHrP and osteocalcin. PSA complexed to A2M retains the ability to degrade PTHrP. In advanced prostate cancer with PSA levels >1000 ng/mL, sufficient PSA-A2M is present in circulation to produce enzymatic activity against circulating small peptide hormones. Sufficient PSA is produced in advanced prostate cancer to alter

  9. Effects of Human Parvovirus B19 and Bocavirus VP1 Unique Region on Tight Junction of Human Airway Epithelial A549 Cells

    Science.gov (United States)

    Chiu, Chun-Ching; Shi, Ya-Fang; Yang, Jiann-Jou; Hsiao, Yuan-Chao; Tzang, Bor-Show; Hsu, Tsai-Ching

    2014-01-01

    As is widely recognized, human parvovirus B19 (B19) and human bocavirus (HBoV) are important human pathogens. Obviously, both VP1 unique region (VP1u) of B19 and HBoV exhibit the secreted phospholipase A2 (sPLA2)-like enzymatic activity and are recognized to participate in the pathogenesis of lower respiratory tract illnesses. However, exactly how, both VP1u from B19 and HBoV affect tight junction has seldom been addressed. Therefore, this study investigates how B19-VP1u and HBoV-VP1u may affect the tight junction of the airway epithelial A549 cells by examining phospholipase A2 activity and transepithelial electrical resistance (TEER) as well as performing immunoblotting analyses. Experimental results indicate that TEER is more significantly decreased in A549 cells by treatment with TNF-α (10 ng), two dosages of B19-VP1u and BoV-VP1u (400 ng and 4000 ng) or bee venom PLA2 (10 ng) than that of the control. Accordingly, more significantly increased claudin-1 and decreased occludin are detected in A549 cells by treatment with TNF-α or both dosages of HBoV-VP1u than that of the control. Additionally, more significantly decreased Na+/K+ ATPase is observed in A549 cells by treatment with TNF-α, high dosage of B19-VP1u or both dosages of BoV-VP1u than that of the control. Above findings suggest that HBoV-VP1u rather than B19 VP1u likely plays more important roles in the disruption of tight junction in the airway tract. Meanwhile, this discrepancy appears not to be associated with the secreted phospholipase A2 (sPLA2)-like enzymatic activity. PMID:25268969

  10. Effects of Polyelectrolyte Complex Micelles and Their Components on the Enzymatic Activity of Lipase

    NARCIS (Netherlands)

    Lindhoud, Saskia; Norde, Willem; Cohen Stuart, Martinus Abraham

    2010-01-01

    The enzymatic activity of Hl-lipase embedded in complexes of poly-2-methylvinylpyridinium-co-poly(ethylene oxide) (P2MVP41−PEO205) and poly(acrylic acid)(PAA139) is studied as a function of the PAA139 + P2MVP41−PEO205 complex composition. The measurements revealed that there are several factors that

  11. Magnetic Fe3S4 nanoparticles with peroxidase-like activity, and their use in a photometric enzymatic glucose assay

    International Nuclear Information System (INIS)

    Ding, Caiping; Yan, Yinghan; Zhang, Cuiling; Xian, Yuezhong; Xiang, Dongshan

    2016-01-01

    Greigite magnetic nanoparticles (Fe 3 S 4 -MNPs) were prepared and reveal a peroxidase-like activity. Kinetic studies revealed a pseudo-enzymatic activity that is much higher than that of other magnetic nanomaterial-based enzyme mimetics. This finding was exploited to design a photometric enzymatic glucose assay based on the formation of H 2 O 2 during enzymatic oxidation of glucose by glucose oxidase, and the formation of a blue product from an enzyme substrate that is catalytically oxidized by H 2 O 2 in the presence of Fe 3 S 4 -MNPs. Glucose can be detected in the 2 to 100 μM concentration range, and the low detection limit is 0.16 μM. The method was applied to quantify glucose in human serum. In our perception, this enzyme mimetic has a large potential in that it may be used in other oxidase based assays, but also in ELISAs. (author)

  12. Structural and enzymatic characterization of the phosphotriesterase OPHC2 from Pseudomonas pseudoalcaligenes.

    Directory of Open Access Journals (Sweden)

    Guillaume Gotthard

    Full Text Available BACKGROUND: Organophosphates (OPs are neurotoxic compounds for which current methods of elimination are unsatisfactory; thus bio-remediation is considered as a promising alternative. Here we provide the structural and enzymatic characterization of the recently identified enzyme isolated from Pseudomonas pseudoalcaligenes dubbed OPHC2. OPHC2 belongs to the metallo-β-lactamase superfamily and exhibits an unusual thermal resistance and some OP degrading abilities. PRINCIPAL FINDINGS: The X-ray structure of OPHC2 has been solved at 2.1 Å resolution. The enzyme is roughly globular exhibiting a αβ/βα topology typical of the metallo-β-lactamase superfamily. Several structural determinants, such as an extended dimerization surface and an intramolecular disulfide bridge, common features in thermostable enzymes, are consistent with its high Tm (97.8°C. Additionally, we provide the enzymatic characterization of OPHC2 against a wide range of OPs, esters and lactones. SIGNIFICANCE: OPHC2 possesses a broad substrate activity spectrum, since it hydrolyzes various phosphotriesters, esters, and a lactone. Because of its organophosphorus hydrolase activity, and given its intrinsic thermostability, OPHC2 is an interesting candidate for the development of an OPs bio-decontaminant. Its X-ray structure shed light on its active site, and provides key information for the understanding of the substrate binding mode and catalysis.

  13. Differentiation of enzymatic activity of yeasts and yeast-like microorganisms isolated from various environments

    Directory of Open Access Journals (Sweden)

    Elżbieta Bogusławska-Wąs

    2014-08-01

    Full Text Available The aim of study was to determinate enzymatic activity of yeast-like organisms - Candida lipolytica, Rhodotorula rubra, Trichosporon beigelii, Zygosaccharomyces sp. - isolated from the Szczecin Lagoon and herring salads. We have shown that lipolytic activity was higher than protcolytic for every strain tested. The lowest activity level was found out for amylolytic hydrolases. The results also demonstrated that yeast-like organisms isolated from the Szczecin Lagoon revealed much higher average enzymatic activity compared to tbe same species isolated from herring salads, excepting C. lipolytica.

  14. Genetic ontogeny of pancreatic enzymes in Labrus bergylta larvae and the effect of feed type on enzyme activities and gene expression.

    Science.gov (United States)

    Hansen, Truls Wergeland; Folkvord, Arild; Grøtan, Espen; Sæle, Øystein

    2013-03-01

    A newly cultivated wrasse species, Labrus bergylta, have shown great potential for use in Atlantic salmon (Salmo salar) farms in the battle against sea lice (Lepeoptheirus salmonis) infections. Hatchery reared L. bergylta were studied from 2 to 55 DPH to examine the molecular basis of digestive ontogeny related to the pancreas. An isolated feeding trial was performed on 27-34 DPH larvae to compare the effect of diet on enzyme activity and the possible exogenous contribution by live feed. The following genes coding for key pancreatic enzymes were analyzed by qPCR: trypsin, Cyp7 A1, BAL, sPLA(2) 1B, amylase and pancreatic chitinase. Enzyme activity was measured on trypsin, neutral lipase, sPLA(2), amylase and chitinase in fed and unfed larvae. We did not observe any effects of the formulated diet v.s. rotifers on enzyme activities of neutral lipase, chitinase and sPLA(2). However, a probable feed-dependency was observed at a transcriptional level, where rotifers seem to stimulate upregulation. The regulation of BAL was the only exception, where an upregulation was observed after weaning both in the ontogeny series and the experimental part. Our data on pancreatic chitinase and amylase mRNA levels suggest the importance of carbohydrates in the diet of early larval and juvenile L. bergylta. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. Extracellular enzymatic activities and physiological profiles of yeasts colonizing fruit trees.

    Science.gov (United States)

    Molnárová, Jana; Vadkertiová, Renáta; Stratilová, Eva

    2014-07-01

    Yeasts form a significant and diverse part of the phyllosphere microbiota. Some yeasts that inhabit plants have been found to exhibit extracellular enzymatic activities. The aim of the present study was to investigate the ability of yeasts isolated from leaves, fruits, and blossoms of fruit trees cultivated in Southwest Slovakia to produce extracellular enzymes, and to discover whether the yeasts originating from these plant organs differ from each other in their physiological properties. In total, 92 strains belonging to 29 different species were tested for: extracellular protease, β-glucosidase, lipase, and polygalacturonase activities; fermentation abilities; the assimilation of xylose, saccharose and alcohols (methanol, ethanol, glycerol); and for growth in a medium with 33% glucose. The black yeast Aureobasidium pullulans showed the largest spectrum of activities of all the species tested. Almost 70% of the strains tested demonstrated some enzymatic activity, and more than 90% utilized one of the carbon compounds tested. Intraspecies variations were found for the species of the genera Cryptococcus and Pseudozyma. Interspecies differences of strains exhibiting some enzymatic activities and utilizing alcohols were also noted. The largest proportion of the yeasts exhibited β-glucosidase activity and assimilated alcohols independently of their origin. The highest number of strains positive for all activities tested was found among the yeasts associated with leaves. Yeasts isolated from blossoms assimilated saccharose and D-xylose the most frequently of all the yeasts tested. The majority of the fruit-inhabiting yeasts grew in the medium with higher osmotic pressure. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Enzymatic Activity of Candida spp. from Oral Cavity and Urine in Children with Nephrotic Syndrome.

    Science.gov (United States)

    Olczak-Kowalczyk, Dorota; Roszkowska-Blaim, Maria; Dąbkowska, Maria; Swoboda-Kopeć, Ewa; Gozdowski, Dariusz; Mizerska-Wasiak, Małgorzata; Demkow, Urszula; Pańczyk-Tomaszewska, Małgorzata

    2017-01-01

    Oral colonization with Candida spp. is not synonymous with a systemic active infection. The aim of the study was to evaluate enzymatic activity of Candida strains isolated from the oral cavity in patients with nephrotic syndrome (NS) and to compare it with the activity determined in urine. We studied 32 children with NS and 26 control healthy children. Children with NS were treated with glucocorticosteroids, cyclosporin A, mycophenolate mofetil or azathioprine. In all children, API-ZYM enzymatic tests were performed to evaluate hydrolytic enzymes of Candida isolated from the oral cavity and in urine. Candida spp. were isolated from the oral cavity in 11 patients with NS (34.4%), all receiving immunosuppressive treatment. All strains produced valine arylamidase, 9 alpha-glucosidase (E16), and 9 N-acetyl-beta-glucosaminidase (E18). A positive correlation between the presence of Candida in the oral cavity and E16 and E18 enzymatic activity in both oral cavity and urine was found. A dose of cyclosporin A had an effect on the enzymatic activity (p Candida invasion. The results of this study suggest that oral candida infection should be monitored in children with nephrotic syndrome, particularly those treated with immunosuppressive agents.

  17. PX-18 Protects Human Saphenous Vein Endothelial Cells under Arterial Blood Pressure.

    Science.gov (United States)

    Kupreishvili, Koba; Stooker, Wim; Emmens, Reindert W; Vonk, Alexander B A; Sipkens, Jessica A; van Dijk, Annemieke; Eijsman, Leon; Quax, Paul H; van Hinsbergh, Victor W M; Krijnen, Paul A J; Niessen, Hans W M

    2017-07-01

    Arterial blood pressure-induced shear stress causes endothelial cell apoptosis and inflammation in vein grafts after coronary artery bypass grafting. As the inflammatory protein type IIA secretory phospholipase A 2 (sPLA 2 -IIA) has been shown to progress atherosclerosis, we hypothesized a role for sPLA 2 -IIA herein. The effects of PX-18, an inhibitor of both sPLA 2 -IIA and apoptosis, on residual endothelium and the presence of sPLA 2 -IIA were studied in human saphenous vein segments (n = 6) perfused at arterial blood pressure with autologous blood for 6 hrs. The presence of PX-18 in the perfusion blood induced a significant 20% reduction in endothelial cell loss compared to veins perfused without PX18, coinciding with significantly reduced sPLA 2 -IIA levels in the media of the vein graft wall. In addition, PX-18 significantly attenuated caspase-3 activation in human umbilical vein endothelial cells subjected to shear stress via mechanical stretch independent of sPLA 2 -IIA. In conclusion, PX-18 protects saphenous vein endothelial cells from arterial blood pressure-induced death, possibly also independent of sPLA 2 -IIA inhibition. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Impact of Heavy Metals in Enzymatic Activity of Soils from Hidalgo, Mexico

    International Nuclear Information System (INIS)

    Reyes-Ortigoza, A. L.; Reyes-Solis, I. E.; Galicia-Palacios, M. S.; Montiel-Arteaga, S.

    2009-01-01

    The soils from Valle of Mezquital, Hidalgo, Mexico have been irrigated with waste waters from Mexico City for more than 88 years. the present investigation was made in order to know the relationship between heavy metal contents and time of irrigation with waste waters and production of CO 2 and enzymatic activity in soils from Valle Mezquital for knowing the disponibility of nutrients and degradation of soils. (Author)

  19. The dual effects of Maillard reaction and enzymatic hydrolysis on the antioxidant activity of milk proteins.

    Science.gov (United States)

    Oh, N S; Lee, H A; Lee, J Y; Joung, J Y; Lee, K B; Kim, Y; Lee, K W; Kim, S H

    2013-08-01

    The objective of this study was to determine the enhanced effects on the biological characteristics and antioxidant activity of milk proteins by the combination of the Maillard reaction and enzymatic hydrolysis. Maillard reaction products were obtained from milk protein preparations, such as whey protein concentrates and sodium caseinate with lactose, by heating at 55°C for 7 d in sodium phosphate buffer (pH 7.4). The Maillard reaction products, along with untreated milk proteins as controls, were hydrolyzed for 0 to 3h with commercial proteases Alcalase, Neutrase, Protamex, and Flavorzyme (Novozymes, Bagsværd, Denmark). The antioxidant activity of hydrolyzed Maillard reaction products was determined by reaction with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, their 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, and the ability to reduce ferric ions. Further characteristics were evaluated by the o-phthaldialdehyde method and sodium dodecyl sulfate-PAGE. The degree of hydrolysis gradually increased in a time-dependent manner, with the Alcalase-treated Maillard reaction products being the most highly hydrolyzed. Radical scavenging activities and reducing ability of hydrolyzed Maillard reaction products increased with increasing hydrolysis time. The combined products of enzymatic hydrolysis and Maillard reaction showed significantly greater antioxidant activity than did hydrolysates or Maillard reaction products alone. The hydrolyzed Maillard reaction products generated by Alcalase showed significantly higher antioxidant activity when compared with the other protease products and the antioxidant activity was higher for the whey protein concentrate groups than for the sodium caseinate groups. These findings indicate that Maillard reaction products, coupled with enzymatic hydrolysis, could act as potential antioxidants in the pharmaceutical, food, and dairy industries. Copyright © 2013 American Dairy Science Association

  20. Rapid tryptic mapping using enzymatically active mass spectrometer probe tips

    Energy Technology Data Exchange (ETDEWEB)

    Dogruel, D.; Williams, P.; Nelson, R.W. [Arizona State Univ., Tempe, AZ (United States)

    1995-12-01

    A method has been developed for rapid, sensitive, and accurate tryptic mapping of polypeptides using matrix-assisted laser desorption/ionization time-of-flight mass analysis. The technique utilizes mass spectrometer probe tips which have been activated through the covalent immobilization of trypsin. The enzymatically active probe tips were used for the tryptic mapping of chicken egg lysozyme and the results compared with those obtained using either free trypsin or agarose-immobilized trypsin. A significant increase in the overall sensitivity of the process was observed using the active probe tips, as well as the production of more characteristic proteolytic fragments and the elimination of background signals due to the autolysis of the trypsin. Further, probe tip digestions were found to be rapid and convenient. 19 refs., 6 figs., 2 tabs.

  1. Quantitative Collection and Enzymatic Activity of Glucose Oxidase Nanotubes Fabricated by Templated Layer-by-Layer Assembly.

    Science.gov (United States)

    Zhang, Shouwei; Demoustier-Champagne, Sophie; Jonas, Alain M

    2015-08-10

    We report on the fabrication of enzyme nanotubes in nanoporous polycarbonate membranes via the layer-by-layer (LbL) alternate assembly of polyethylenimine (PEI) and glucose oxidase (GOX), followed by dissolution of the sacrificial template in CH2Cl2, collection, and final dispersion in water. An adjuvant-assisted filtration methodology is exploited to extract quantitatively the nanotubes without loss of activity and morphology. Different water-soluble CH2Cl2-insoluble adjuvants are tested for maximal enzyme activity and nanotube stability; whereas NaCl disrupts the tubes by screening electrostatic interactions, the high osmotic pressure created by fructose also contributes to loosening the nanotubular structures. These issues are solved when using neutral, high molar mass dextran. The enzymatic activity of intact free nanotubes in water is then quantitatively compared to membrane-embedded nanotubes, showing that the liberated nanotubes have a higher catalytic activity in proportion to their larger exposed surface. Our study thus discloses a robust and general methodology for the fabrication and quantitative collection of enzymatic nanotubes and shows that LbL assembly provides access to efficient enzyme carriers for use as catalytic swarming agents.

  2. Origin and evolution of group XI secretory phospholipase A2 from flax (Linum usitatissimum) based on phylogenetic analysis of conserved domains.

    Science.gov (United States)

    Gupta, Payal; Saini, Raman; Dash, Prasanta K

    2017-07-01

    Phospholipase A 2 (PLA 2 ) belongs to class of lipolytic enzymes (EC 3.1.1.4). Lysophosphatidic acid (LPA) and free fatty acids (FFAs) are the products of PLA 2 catalyzed hydrolysis of phosphoglycerides at sn-2 position. LPA and FFA that act as second mediators involved in the development and maturation of plants and animals. Mining of flax genome identified two phospholipase A 2 encoding genes, viz., LusPLA 2 I and LusPLA 2 II (Linum usitatissimum secretory phospholipase A 2 ). Molecular simulation of LusPLA 2 s with already characterized plant sPLA 2 s revealed the presence of conserved motifs and signature domains necessary to classify them as secretory phospholipase A 2 . Phylogenetic analysis of flax sPLA 2 with representative sPLA 2 s from other organisms revealed that they evolved rapidly via gene duplication/deletion events and shares a common ancestor. Our study is the first report of detailed phylogenetic analysis for secretory phospholipase A 2 in flax. Comparative genomic analysis of two LusPLA 2 s with earlier reported plant sPLA 2 s, based on their gene architectures, sequence similarities, and domain structures are presented elucidating the uniqueness of flax sPLA 2 .

  3. Detection and quantification of microparticles from different cellular lineages using flow cytometry. Evaluation of the impact of secreted phospholipase A2 on microparticle assessment.

    Science.gov (United States)

    Rousseau, Matthieu; Belleannee, Clemence; Duchez, Anne-Claire; Cloutier, Nathalie; Levesque, Tania; Jacques, Frederic; Perron, Jean; Nigrovic, Peter A; Dieude, Melanie; Hebert, Marie-Josee; Gelb, Michael H; Boilard, Eric

    2015-01-01

    Microparticles, also called microvesicles, are submicron extracellular vesicles produced by plasma membrane budding and shedding recognized as key actors in numerous physio(patho)logical processes. Since they can be released by virtually any cell lineages and are retrieved in biological fluids, microparticles appear as potent biomarkers. However, the small dimensions of microparticles and soluble factors present in body fluids can considerably impede their quantification. Here, flow cytometry with improved methodology for microparticle resolution was used to detect microparticles of human and mouse species generated from platelets, red blood cells, endothelial cells, apoptotic thymocytes and cells from the male reproductive tract. A family of soluble proteins, the secreted phospholipases A2 (sPLA2), comprises enzymes concomitantly expressed with microparticles in biological fluids and that catalyze the hydrolysis of membrane phospholipids. As sPLA2 can hydrolyze phosphatidylserine, a phospholipid frequently used to assess microparticles, and might even clear microparticles, we further considered the impact of relevant sPLA2 enzymes, sPLA2 group IIA, V and X, on microparticle quantification. We observed that if enriched in fluids, certain sPLA2 enzymes impair the quantification of microparticles depending on the species studied, the source of microparticles and the means of detection employed (surface phosphatidylserine or protein antigen detection). This study provides analytical considerations for appropriate interpretation of microparticle cytofluorometric measurements in biological samples containing sPLA2 enzymes.

  4. Enzymatic activity of the cellulolytic complex produced by Trichoderma reesei. Enzymatic hydrolysis of cellulose; Actividad enzimatica del complejo celulolitico producido por Trichoderma reesei. Hidrolisis enzimatica de la celulosa

    Energy Technology Data Exchange (ETDEWEB)

    Alfonsel, M; Negro, M J; Saez, R; Martin, C

    1986-07-01

    The enzymatic activity characterization of the cellulolytic complex obtained from Trichoderma reesei QM 9414 and the influence of the enzymatic hydrolysis conditions on the hydrolysis yield are studied. Pure cellulose and native or alkali pretreated biomass Onopordum nervosum have been used as substrates. The values of pH, temperature, substrate concentration and enzyme-substrate ratio for the optimum activity of that complex, evaluated as glucose and reducing sugars production, have been selected. Previous studies on enzymatic hydrolysis of 0. nervosum have shown a remarkable effect of the alkaline pretreatments on the final hydrolysis yield. (Author) 10 refs.

  5. Effect of aflatoxin B1 on growth and enzymatic activity of a native strain of Bacillus sp

    Directory of Open Access Journals (Sweden)

    Alex Sáez Vega

    2004-01-01

    Full Text Available The effect of different aflatoxin B1 (AFAB1 concentrations on alkaline protease growth and enzymatic activity was evaluated; a native strain of alkalophilic Bacillus sp cultivated in CSL (Corn Steep Liquor was used. It was found that the effect of AFAB1 on the strain inhibited its growth and enzymatic activity to 1 ppm, showing that the strain is highly sensible to AFAB1, meaning that medium obtained f rom Colombian corn contaminated with this mycotoxin cannot be easily used. Concentrations less than 0.1 ppm did not affect growth and enzymatic activity. Key words: Bacillus, aflatoxin, alkaline proteases.

  6. Cross-talk between oxysterols and glucocorticoids: differential regulation of secreted phopholipase A2 and impact on oligodendrocyte death.

    Directory of Open Access Journals (Sweden)

    Amalia Trousson

    Full Text Available BACKGROUND: Oxysterols are oxidized forms of cholesterol. They have been shown to be implicated in cholesterol turnover, inflammation and in neurodegenerative diseases such as Alzheimer's disease and multiple sclerosis. Glial cells are targets of oxysterols: they inhibit astrocyte proliferation after brain injury, and we have previously shown that 25-hydroxycholesterol (25OH provokes oligodendrocyte apoptosis and stimulates the expression of sPLA2 type IIA (sPLA2-IIA, which has a protective effect. METHODOLOGY/PRINCIPAL FINDINGS: As glucocorticoids are well-known for their anti-inflammatory effects, our aim was to understand their direct effects on oxysterol-induced responses in oligodendrocytes (sPLA2-IIA stimulation and apoptosis. We demonstrate that the synthetic glucocorticoid dexamethasone (Dex abolishes the stimulation of sPLA2-IIA by 25-hydroxycholesterol (25-OH. This inhibition is mediated by the glucocorticoid receptor (GR, which decreases the expression of the oxysterol receptor Pregnane X Receptor (PXR and interferes with oxysterol signaling by recruiting a common limiting coactivator PGC1alpha. Consistent with the finding that sPLA2-IIA can partially protect oligodendrocytes against oxysterol-triggered apoptosis, we demonstrate here that the inhibition of sPLA2-IIA by Dex accelerates the apoptotic phenomenon, leading to a shift towards necrosis. We have shown by atomic force microscopy and electron microscopy that 25-OH and Dex alters oligodendrocyte shape and disorganizes the cytoplasm. CONCLUSIONS/SIGNIFICANCE: Our results provide a new understanding of the cross-talk between oxysterol and glucocorticoid signaling pathways and their respective roles in apoptosis and oligodendrocyte functions.

  7. Enzymatic Hydrolysis of Oleuropein from Olea europea (Olive Leaf Extract and Antioxidant Activities

    Directory of Open Access Journals (Sweden)

    Jiao-Jiao Yuan

    2015-02-01

    Full Text Available Oleuropein (OE, the main polyphenol in olive leaf extract, is likely to decompose into hydroxytyrosol (HT and elenolic acid under the action of light, acid, base, high temperature. In the enzymatic process, the content of OE in olive leaf extract and enzyme are key factors that affect the yield of HT. A selective enzyme was screened from among 10 enzymes with a high OE degradation rate. A single factor (pH, temperature, time, enzyme quantity optimization process and a Box-Behnken design were studied for the enzymatic hydrolysis of 81.04% OE olive leaf extract. Additionally, enzymatic hydrolysis results with different substrates (38.6% and 81.04% OE were compared and the DPPH antioxidant properties were also evaluated. The result showed that the performance of hydrolysis treatments was best using hemicellulase as a bio-catalyst, and the high purity of OE in olive extract was beneficial to biotransform OE into HT. The optimal enzymatic conditions for achieving a maximal yield of HT content obtained by the regression were as follows: pH 5, temperature 55 °C and enzyme quantity 55 mg. The experimental result was 11.31% ± 0.15%, and the degradation rate of OE was 98.54%. From the present investigation of the antioxidant activity determined by the DPPH method, the phenol content and radical scavenging effect were both decreased after enzymatic hydrolysis by hemicellulase. However, a high antioxidant activity of the ethyl acetate extract enzymatic hydrolysate (IC50 = 41.82 μg/mL was demonstated. The results presented in this work suggested that hemicellulase has promising and attractive properties for industrial production of HT, and indicated that HT might be a valuable biological component for use in pharmaceutical products and functional foods.

  8. Development of a cell-based bioassay for phospholipase A2-triggered liposomal drug release

    DEFF Research Database (Denmark)

    Arouri, Ahmad; Trojnar, Jakub; Schmidt, Steffen

    2015-01-01

    models, the pattern of sPLA2-assisted drug release is unknown due to the lack of a suitable bio-relevant model. We report here on the development of a novel bioluminescence living-cell-based luciferase assay for the monitoring of sPLA2-triggered release of luciferin from liposomes. To this end, we...

  9. Enzymatic activities produced by mixed Saccharomyces and non-Saccharomyces cultures: relationship with wine volatile composition.

    Science.gov (United States)

    Maturano, Yolanda Paola; Assof, Mariela; Fabani, María Paula; Nally, María Cristina; Jofré, Viviana; Rodríguez Assaf, Leticia Anahí; Toro, María Eugenia; Castellanos de Figueroa, Lucía Inés; Vazquez, Fabio

    2015-11-01

    During certain wine fermentation processes, yeasts, and mainly non-Saccharomyces strains, produce and secrete enzymes such as β-glucosidases, proteases, pectinases, xylanases and amylases. The effects of enzyme activity on the aromatic quality of wines during grape juice fermentation, using different co-inoculation strategies of non-Saccharomyces and Saccharomyces cerevisiae yeasts, were assessed in the current study. Three strains with appropriate enological performance and high enzymatic activities, BSc562 (S. cerevisiae), BDv566 (Debaryomyces vanrijiae) and BCs403 (Candida sake), were assayed in pure and mixed Saccharomyces/non-Saccharomyces cultures. β-Glucosidase, pectinase, protease, xylanase and amylase activities were quantified during fermentations. The aromatic profile of pure and mixed cultures was determined at the end of each fermentation. In mixed cultures, non-Saccharomyces species were detected until day 4-5 of the fermentation process, and highest populations were observed in MSD2 (10% S. cerevisiae/90% D. vanrijiae) and MSC1 (1% S. cerevisiae/99% C. sake). According to correlation and multivariate analysis, MSD2 presented the highest concentrations of terpenes and higher alcohols which were associated with pectinase, amylase and xylanase activities. On the other hand, MSC1 high levels of β-glucosidase, proteolytic and xylanolytic activities were correlated to esters and fatty acids. Our study contributes to a better understanding of the effect of enzymatic activities by yeasts on compound transformations that occur during wine fermentation.

  10. Biochemical characterization of Arabidopsis thaliana starch branching enzyme 2.2 reveals an enzymatic positive cooperativity.

    Science.gov (United States)

    Wychowski, A; Bompard, C; Grimaud, F; Potocki-Véronèse, G; D'Hulst, C; Wattebled, F; Roussel, X

    2017-09-01

    Starch Branching Enzymes (SBE) catalyze the formation of α(1 → 6) branching points on starch polymers: amylopectin and amylose. SBEs are classified in two groups named type 1 and 2. Both types are present in the entire plant kingdom except in some species such as Arabidopsis thaliana that expresses two type 2 SBEs: BE2.1 and BE2.2. The present work describes in vitro enzymatic characterization of the recombinant BE2.2. The function of recombinant BE2.2 was characterized in vitro using spectrophotometry assay, native PAGE and HPAEC-PAD analysis. Size Exclusion Chromatography separation and SAXS experiments were used to identify the oligomeric state and for structural analysis of this enzyme. Optimal pH and temperature for BE2.2 activity were determined to be pH 7 and 25 °C. A glucosyl donor of at least 12 residues is required for BE2.2 activity. The reaction results in the transfer in an α(1 → 6) position of a glucan preferentially composed of 6 glucosyl units. In addition, BE2.2, which has been shown to be monomeric in absence of substrate, is able to adopt different active forms in presence of branched substrates, which affect the kinetic parameters. BE2.2 has substrate specificity similar to those of the other type-2 BEs. We propose that the different conformations of the enzyme displaying more or less affinity toward its substrates would explain the adjustment of the kinetic data to the Hill equation. This work describes the enzymatic parameters of Arabidopsis BE2.2. It reveals for the first time conformational changes for a branching enzyme, leading to a positive cooperative binding process of this enzyme. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  11. PLA2 - a major regulator of volume-sensitive taurine release in NIH3T3 fibroblasts

    DEFF Research Database (Denmark)

    Lambert, I. H.

    2006-01-01

    -lipoxygenase (5-LO) system is prevented by the 5-LO inhibitor ETH 615-139 and is reduced under hypertonic conditions. Exposure to the amphiphilic bee venom peptide melittin, which has no effect on the kinetic properties of PLA2 but promotes substrate replenishment, induces release of arachidonic acid...... conditions but has only a minor effect on the melittin-induced taurine efflux under hypertonic conditions. Bromoenol lactone and manoalide, known inhibitors of Ca2+-independent phospholipase A2 (iPLA2) and secretory phospholipase A2 (sPLA2), respectively, reduce arachidonic acid and taurine release from NIH3......T3 cells under hypotonic conditions and following addition of melittin. It is suggested that iPLA2/sPLA2 activity is responsible for the volume-sensitivity of taurine release in NIH3T3 mouse fibroblasts....

  12. Enzyme-immobilized SiO2-Si electrode: Fast interfacial electron transfer with preserved enzymatic activity

    Science.gov (United States)

    Wang, Gang; Yau, Siu-Tung

    2005-12-01

    The enzyme, glucose oxidase (GOx), is immobilized using electrostatic interaction on the native oxide of heavily doped n-type silicon. Voltammetric measurement shows that the immobilized GOx gives rise to a very fast enzyme-silicon interfacial electron transfer rate constant of 7.9s-1. The measurement also suggests that the enzyme retains its native conformation when immobilized on the silicon surface. The preserved native conformation of GOx is further confirmed by testing the enzymatic activity of the immobilized GOx using glucose. The GOx-immobilized silicon is shown to behave as a glucose sensor that detects glucose with concentrations as low as 50μM.

  13. Role of secretory phospholipase A(2) in rhythmic contraction of pulmonary arteries of rats with monocrotaline-induced pulmonary arterial hypertension.

    Science.gov (United States)

    Tanabe, Yoshiyuki; Saito-Tanji, Maki; Morikawa, Yuki; Kamataki, Akihisa; Sawai, Takashi; Nakayama, Koichi

    2012-01-01

    Excessive stretching of the vascular wall in accordance with pulmonary arterial hypertension (PAH) induces a variety of pathogenic cellular events in the pulmonary arteries. We previously reported that indoxam, a selective inhibitor for secretory phospholipase A(2) (sPLA(2)), blocked the stretch-induced contraction of rabbit pulmonary arteries by inhibition of untransformed prostaglandin H(2) (PGH(2)) production. The present study was undertaken to investigate involvement of sPLA(2) and untransformed PGH(2) in the enhanced contractility of pulmonary arteries of experimental PAH in rats. Among all the known isoforms of sPLA(2), sPLA(2)-X transcript was most significantly augmented in the pulmonary arteries of rats with monocrotaline-induced pulmonary hypertension (MCT-PHR). The pulmonary arteries of MCT-PHR frequently showed two types of spontaneous contraction in response to stretch; 27% showed rhythmic contraction, which was sensitive to indoxam and SC-560 (selective COX-1 inhibitor), but less sensitive to NS-398 (selective COX-2 inhibitor); and 47% showed sustained incremental tension (tonic contraction), which was insensitive to indoxam and SC-560, but sensitive to NS-398 and was attenuated to 45% of the control. Only the rhythmically contracting pulmonary arteries of MCT-PHR produced a substantial amount of untransformed PGH(2), which was abolished by indoxam. These results suggest that sPLA(2)-mediated PGH(2) synthesis plays an important role in the rhythmic contraction of pulmonary arteries of MCT-PHR.

  14. Binding of Nickel to Testicular Glutamate–Ammonia Ligase Inhibits Its Enzymatic Activity

    Science.gov (United States)

    SUN, YINGBIAO; OU, YOUNG; CHENG, MIN; RUAN, YIBING; VAN DER HOORN, FRANS A.

    2016-01-01

    SUMMARY Exposure to nickel has been shown to cause damage to the testis in several animal models. It is not known if the testis expresses protein(s) that can bind nickel. To test this, we used a nickel-binding assay to isolate testicular nickel-binding proteins. We identified glutamate–ammonia ligase (GLUL) as a prominent nickel-binding protein by mass spectrometry. Protein analysis and reverse transcriptase polymerase chain reaction showed that GLUL is expressed in the testis, predominantly in interstitial cells. We determined that GLUL has a higher affinity for nickel than for its regular co-factor manganese. We produced an enzymatically active, recombinant GLUL protein. Upon binding, nickel interferes with the manganese-catalyzed enzymatic activity of recombinant GLUL protein. We also determined that GLUL activity in testes of animals exposed to nickel sulfate is reduced. Our results identify testicular GLUL as the first testicular protein shown to be affected by nickel exposure. PMID:21254280

  15. Immobilization of inorganic pyrophosphatase on nanodiamond particles retaining its high enzymatic activity.

    Science.gov (United States)

    Rodina, Elena V; Valueva, Anastasiya V; Yakovlev, Ruslan Yu; Vorobyeva, Nataliya N; Kulakova, Inna I; Lisichkin, Georgy V; Leonidov, Nikolay B

    2015-12-21

    Nanodiamond (ND) particles are popular platforms for the immobilization of molecular species. In the present research, enzyme Escherichia coli inorganic pyrophosphatase (PPase) was immobilized on detonation ND through covalent or noncovalent bonding and its enzymatic activity was characterized. Factors affecting adsorption of PPase such as ND size and surface chemistry were studied. The obtained material is a submicron size association of ND particles and protein molecules in approximately equal amounts. Both covalently and noncovalently immobilized PPase retains a significant enzymatic activity (up to 95% of its soluble form) as well as thermostability. The obtained hybrid material has a very high enzyme loading capacity (∼1 mg mg(-1)) and may be considered as a promising delivery system of biologically active proteinaceous substances, particularly in the treatment of diseases such as calcium pyrophosphate crystal deposition disease and related pathologies. They can also be used as recoverable heterogeneous catalysts in the traditional uses of PPase.

  16. Comparison of the role that entropy has played in processes of non-enzymatic and enzymatic catalysis

    International Nuclear Information System (INIS)

    Dixon Pineda, Manuel Tomas

    2012-01-01

    The function that entropy has played is compared in processes of non-enzymatic and enzymatic catalysis. The processes followed are showed: the kinetics of the acid hydrolysis of 3-pentyl acetate and cyclopentyl acetate catalyzed by hydrochloric acid and enzymatic hydrolysis of ethyl acetate and γ-butyrolactone catalyzed by pig liver esterase. The activation parameters of Eyring were determined for each process and interpreted the contribution of the entropy of activation for catalysis in this type of model reactions. (author) [es

  17. Direct electrocatalytic reduction of coenzyme NAD{sup +} to enzymatically-active 1,4-NADH employing an iridium/ruthenium-oxide electrode

    Energy Technology Data Exchange (ETDEWEB)

    Ullah, Nehar, E-mail: nehar.ullah@mail.mcgill.ca; Ali, Irshad; Omanovic, Sasha

    2015-01-15

    A thermally prepared iridium/ruthenium-oxide coating (Ir{sub 0.8}Ru{sub 0.2}-oxide) formed on a titanium substrate was investigated as a possible electrode for direct electrochemical regeneration of enzymatically-active 1,4-NADH from its oxidized form NAD{sup +}, at various electrode potentials, in a batch electrochemical reactor. The coating surface was characterized by ‘cracked mud’ morphology, yielding a high surface roughness. The NADH regeneration results showed that the percentage of enzymatically-active 1,4-NADH present in the product mixture (i.e. recovery) is strongly dependent on the electrode potential, reaching a maximum (88%) at −1.70 V vs. MSE. The relatively high recovery was explained on the basis of availability of adsorbed ‘active’ hydrogen (H{sub ads}) on the Ir/Ru-oxide surface, i.e. on the basis of electrochemical hydrogenation. - Highlights: • Ir{sub 0.8}Ru{sub 0.2}-oxide coating was formed thermally on a Ti substrate. • Electrochemical regeneration of enzymatically-active 1,4-NADH was investigated. • The 1,4-NADH recovery percentage is strongly dependent on the electrode potential. • A highest recovery, 88%, was obtained at −1.70 V vs. MSE. • The NADH regeneration process involved electrochemical hydrogenation.

  18. Constraints imposed by transmembrane domains affect enzymatic activity of membrane-associated human CD39/NTPDase1 mutants.

    Science.gov (United States)

    Musi, Elgilda; Islam, Naziba; Drosopoulos, Joan H F

    2007-05-01

    Human CD39/NTPDase1 is an endothelial cell membrane-associated nucleotidase. Its large extracellular domain rapidly metabolizes nucleotides, especially ADP released from activated platelets, inhibiting further platelet activation/recruitment. Previous studies using our recombinant soluble CD39 demonstrated the importance of residues S57, D54, and D213 for enzymatic/biological activity. We now report effects of S57A, D54A, and D213A mutations on full-length (FL)CD39 function. Enzymatic activity of alanine modified FLCD39s was less than wild-type, contrasting the enhanced activity of their soluble counterparts. Furthermore, conservative substitutions D54E and D213E led to enzymes with activities greater than the alanine modified FLCD39s, but less than wild-type. Reductions in mutant activities were primarily associated with reduced catalytic rates. Differences in enzymatic activity were not attributable to gross changes in the nucleotide binding pocket or the enzyme's ability to multimerize. Thus, composition of the active site of wild-type CD39 appears optimized for ADPase function in the context of the transmembrane domains.

  19. Functional analysis of two PLA2G2A variants associated with secretory phospholipase A2-IIA levels.

    Directory of Open Access Journals (Sweden)

    Holly J Exeter

    Full Text Available Secretory phospholipase A2 group IIA (sPLA2-IIA has been identified as a biomarker of atherosclerosis in observational and animal studies. The protein is encoded by the PLA2G2A gene and the aim of this study was to test the functionality of two PLA2G2A non-coding SNPs, rs11573156 C>G and rs3767221 T>G where the rare alleles have been previously associated with higher and lower sPLA2-IIA levels respectively.Luciferase assays, electrophoretic mobility shift assays (EMSA, and RNA expression by RT-PCR were used to examine allelic differences. For rs3767221 the G allele showed ∼55% lower luciferase activity compared to the T allele (T = 62.1 (95% CI 59.1 to 65.1 G = 27.8 (95% CI 25.0 to 30.6, p = 1.22×10⁻³⁵, and stronger EMSA binding of a nuclear protein compared to the T-allele. For rs11573156 C >G there were no luciferase or EMSA allelic differences seen. In lymphocyte cell RNA, from individuals of known rs11573156 genotype, there was no allelic RNA expression difference for exons 5 and 6, but G allele carriers (n = 7 showed a trend to lower exon 1-2 expression compared to CC individuals. To take this further, in the ASAP study (n = 223, an rs11573156 proxy (r² = 0.91 showed ∼25% higher liver expression of PLA2G2A (1.67×10⁻¹⁷ associated with the G allele. However, considering exon specific expression, the association was greatly reduced for exon 2 (4.5×10⁻⁵ compared to exons 3-6 (10⁻¹⁰ to 10⁻²⁰, suggesting rs11573156 G allele-specific exon 2 skipping.Both SNPs are functional and provide useful tools for Mendelian Randomisation to determine whether the relationship between sPLA2-IIA and coronary heart disease is causal.

  20. Measure of enzymatic activity coincident with 2450 MHz microwave exposure

    Energy Technology Data Exchange (ETDEWEB)

    Ward, T R; Allis, J W; Elder, J A

    1975-09-01

    Enzyme preparations were exposed to microwave radiation at 2450 MHz and enzymatic activity was simultaneously monitored spectrophotometrically with a crossed-beam exposure detection system. Enzymes studied were glucose 6-phosphate dehydrogenase from human red blood cells and yeast, adenylate kinase from rat liver mitochondria and rabbit muscle, and rat liver microsomal NADPH cytochrome c reductase. No difference was found between the specific activity at 25/sup 0/C of unirradiated controls and enzyme preparations irradiated at an absorbed dose rate of 42 W/kg.

  1. Activation of Hsp90 Enzymatic Activity and Conformational Dynamics through Rationally Designed Allosteric Ligands.

    Science.gov (United States)

    Sattin, Sara; Tao, Jiahui; Vettoretti, Gerolamo; Moroni, Elisabetta; Pennati, Marzia; Lopergolo, Alessia; Morelli, Laura; Bugatti, Antonella; Zuehlke, Abbey; Moses, Mike; Prince, Thomas; Kijima, Toshiki; Beebe, Kristin; Rusnati, Marco; Neckers, Len; Zaffaroni, Nadia; Agard, David A; Bernardi, Anna; Colombo, Giorgio

    2015-09-21

    Hsp90 is a molecular chaperone of pivotal importance for multiple cell pathways. ATP-regulated internal dynamics are critical for its function and current pharmacological approaches block the chaperone with ATP-competitive inhibitors. Herein, a general approach to perturb Hsp90 through design of new allosteric ligands aimed at modulating its functional dynamics is proposed. Based on the characterization of a first set of 2-phenylbenzofurans showing stimulatory effects on Hsp90 ATPase and conformational dynamics, new ligands were developed that activate Hsp90 by targeting an allosteric site, located 65 Å from the active site. Specifically, analysis of protein responses to first-generation activators was exploited to guide the design of novel derivatives with improved ability to stimulate ATP hydrolysis. The molecules' effects on Hsp90 enzymatic, conformational, co-chaperone and client-binding properties were characterized through biochemical, biophysical and cellular approaches. These designed probes act as allosteric activators of the chaperone and affect the viability of cancer cell lines for which proper functioning of Hsp90 is necessary. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Lipid droplets induced by secreted phospholipase A2 and unsaturated fatty acids protect breast cancer cells from nutrient and lipotoxic stress.

    Science.gov (United States)

    Jarc, Eva; Kump, Ana; Malavašič, Petra; Eichmann, Thomas O; Zimmermann, Robert; Petan, Toni

    2018-03-01

    Cancer cells driven by the Ras oncogene scavenge unsaturated fatty acids (FAs) from their environment to counter nutrient stress. The human group X secreted phospholipase A 2 (hGX sPLA 2 ) releases FAs from membrane phospholipids, stimulates lipid droplet (LD) biogenesis in Ras-driven triple-negative breast cancer (TNBC) cells and enables their survival during starvation. Here we examined the role of LDs, induced by hGX sPLA 2 and unsaturated FAs, in protection of TNBC cells against nutrient stress. We found that hGX sPLA 2 releases a mixture of unsaturated FAs, including ω-3 and ω-6 polyunsaturated FAs (PUFAs), from TNBC cells. Starvation-induced breakdown of LDs induced by low micromolar concentrations of unsaturated FAs, including PUFAs, was associated with protection from cell death. Interestingly, adipose triglyceride lipase (ATGL) contributed to LD breakdown during starvation, but it was not required for the pro-survival effects of hGX sPLA 2 and unsaturated FAs. High micromolar concentrations of PUFAs, but not OA, induced oxidative stress-dependent cell death in TNBC cells. Inhibition of triacylglycerol (TAG) synthesis suppressed LD biogenesis and potentiated PUFA-induced cell damage. On the contrary, stimulation of LD biogenesis by hGX sPLA 2 and suppression of LD breakdown by ATGL depletion reduced PUFA-induced oxidative stress and cell death. Finally, lipidomic analyses revealed that sequestration of PUFAs in LDs by sPLA 2 -induced TAG remodelling and retention of PUFAs in LDs by inhibition of ATGL-mediated TAG lipolysis protect from PUFA lipotoxicity. LDs are thus antioxidant and pro-survival organelles that guard TNBC cells against nutrient and lipotoxic stress and emerge as attractive targets for novel therapeutic interventions. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. ASPECTS CONCERNING THE ENZYMATIC ACTIVITY IN SEVERAL THERMOACTINOMYCETE STRAINS

    Directory of Open Access Journals (Sweden)

    Simona Dunca

    2003-08-01

    Full Text Available In the thermoactinomycete strains subjected to examination the values of their recorded enzymatic activities (i.e. α-amy lase, protease, exo-β-1,4 – glucanase, endo -β-1,4 – glucanase and β-glucosidase were lower in the stationary cultures as compared to the stirred ones. The strain Thermomonospora fusca BB255 was found to be highly cellulase- producing and at the same time able to synthesize α-amy lases and proteases.

  4. Enzymatic hydrolysis of esters from 2-carboxy-6- methoxy-2,3 ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-11-02

    Nov 2, 2009 ... Full Length Research Paper. Enzymatic hydrolysis .... ml), followed by water (2 × 15 ml) and dried with anhydrous sodium .... dOptical purity of the acid was determined as the methyl ester by HPLC. eE values ..... Harada I, Hirose Y, Nakaozaki M (1968). ... Nicholas WR, Yang CG, Shi Z, He C (2006). Gold(I)- ...

  5. Respiration and enzymatic activities as indicators of stabilization of sewage sludge composting.

    Science.gov (United States)

    Nikaeen, Mahnaz; Nafez, Amir Hossein; Bina, Bijan; Nabavi, BiBi Fatemeh; Hassanzadeh, Akbar

    2015-05-01

    The objective of this work was to study the evolution of physico-chemical and microbial parameters in the composting process of sewage sludge (SS) with pruning wastes (PW) in order to compare these parameters with respect to their applicability in the evaluation of organic matter (OM) stabilization. To evaluate the composting process and organic matter stability, different microbial activities were compared during composting of anaerobically digested SS with two volumetric ratios, 1:1 and 3:1 of PW:SS and two aeration techniques including aerated static piles (ASP) and turned windrows (TW). Dehydrogenase activity, fluorescein diacetate hydrolysis, and specific oxygen uptake rate (SOUR) were used as microbial activity indices. These indices were compared with traditional parameters, including temperature, pH, moisture content, organic matter, and C/N ratio. The results showed that the TW method and 3:1 (PW:SS) proportion was superior to the ASP method and 1:1 proportion, since the former accelerate the composting process by catalyzing the OM stabilization. Enzymatic activities and SOUR, which reflect microbial activity, correlated well with temperature fluctuations. Based on these results it appears that SOUR and the enzymatic activities are useful parameters to monitor the stabilization of SS compost. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Self-Assembly of Spider Silk-Fusion Proteins Comprising Enzymatic and Fluorescence Activity.

    Science.gov (United States)

    Humenik, Martin; Mohrand, Madeleine; Scheibel, Thomas

    2018-04-18

    The recombinant spider silk protein eADF4(C16) was genetically fused either with esterase 2 (EST2) or green fluorescent protein (GFP). The fusions EST-eADF4(C16) and GFP-eADF4(C16) were spectroscopically investigated and showed native structures of EST and GFP. The structural integrity was confirmed by the enzymatic activity of EST and the fluorescence of GFP. The spider silk moiety retained its intrinsically unstructured conformation in solution and the self-assembly into either nanofibrils or nanoparticles could be controlled by the concentration of phosphate. Particles, however, showed significantly lower activity of the EST and GFP domains likely caused by a steric hindrance. However, upon self-assembly of EST-eADF4(C16) and GFP-eADF4(C16) into fibrils the protein activities were retained. In general, the fusion of globular enzymes with the spider silk domain allows the generation of fibrous biomaterials with catalytic or light emitting properties.

  7. Optimization of enzymatic clarification of green asparagus juice using response surface methodology.

    Science.gov (United States)

    Chen, Xuehong; Xu, Feng; Qin, Weidong; Ma, Lihua; Zheng, Yonghua

    2012-06-01

    Enzymatic clarification conditions for green asparagus juice were optimized by using response surface methodology (RSM). The asparagus juice was treated with pectinase at different temperatures (35 °C-45 °C), pH values (4.00-5.00), and enzyme concentrations (0.6-1.8 v/v%). The effects of enzymatic treatment on juice clarity and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging capacity were investigated by employing a 3-factor central composite design coupled with RSM. According to response surface analysis, the optimal enzymatic treatment condition was pectinase concentration of 1.45%, incubation temperature of 40.56 °C and pH of 4.43. The clarity, juice yield, and soluble solid contents in asparagus juice were significantly increased by enzymatic treatment at the optimal conditions. DPPH radical-scavenging capacity was maintained at the level close to that of raw asparagus juice. These results indicated that enzymatic treatment could be a useful technique for producing green asparagus juice with high clarity and high-antioxidant activity. Treatment with 1.45% pectinase at 40.56 ° C, pH 4.43, significantly increased the clarity and yield of asparagus juice. In addition, enzymatic treatment maintained antioxidant activity. Thus, enzymatic treatment has the potential for industrial asparagus juice clarification. © 2012 Institute of Food Technologists®

  8. [Purification of human goose-type lysozyme 2 (HLysG2) from human seminal plasma and analysis of its enzymatic properties].

    Science.gov (United States)

    Huang, Peng; Yang, Zhifang; Bao, Jianying; Zhang, Ning; Li, Wenshu

    2017-03-01

    Objective To purify human goose-type lysozyme 2 (HLysG2) from human seminal plasma by chromatography and analyze its enzymatic properties. Methods The distribution of HLysG2 in semen was analyzed by Western blot analysis. Seminal plasma was subjected to the separation of target protein using cation-exchange chromatography, chitin affinity chromatography and size-exclusion chromatography. The purified product was identified by Western blot analysis and mass spectrometry (MS).The purity was analyzed by high performance liquid chromatography (HPLC). Then, the optimum pH, ion concentration and temperature of HLysG2 and its standard activity were determined by the turbidimetric assay. The bactericidal activity of HLysG2 was assessed by the colony-forming assay. Results The existence of HLysG2 in seminal plasma was confirmed by Western blot analysis. A protein of about 21.5 kDa was purified from seminal plasma by the three kinds of chromatography and identified as HLysG2 by Western blot analysis and MS. The final purity of the purified product was above 99.0% and the peak enzymatic activity reached 13 800 U/mg under the condition of pH 6.4, 0.09 mol/L Na + , 30DegreesCelsius. In vitro assay indicated that HLysG2 had a significant killing effect on Micrococcus lysodeikticus, Bacillus subtilis and Staphylococcus aureus, but not on Pseudomonas aeruginosa and Escherichia coli. Conclusion Native HLysG2 can be obtained from seminal plasma by chromatography. It has in vitro bactericidal activity against Gram-positive bacteria, suggesting that it might play a role in innate immunity of the male reproductive system.

  9. Enzymatic Conversion of CO2 to Bicarbonate in Functionalized Mesoporous Silica

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Yuehua; Chen, Baowei; Qi, Wen N.; Li, Xiaolin; Shin, Yongsoon; Lei, Chenghong; Liu, Jun

    2012-05-01

    We report here that carbonic anhydrase (CA), the fastest enzyme that can covert carbon dioxide to bicarbonate, can be spontaneously entrapped in functionalized mesoporous silica (FMS) with super-high loading density (up to 0.5 mg of protein/mg of FMS) due to the dominant electrostatic interaction. The binding of CA to HOOC-FMS can result in the protein’s conformational change comparing to the enzyme free in solution, but can be overcome with increased protein loading density. The higher the protein loading density, the less conformational change, hence the higher enzymatic activity and the higher enzyme immobilization efficiency. The electrostatically bound CA can be released by changing pH. The released enzyme still displayed the native conformational structure and the same high enzymatic activity as that prior to the enzyme entrapment. This work opens up a new approach converting carbon dioxide to biocarbonate in a biomimetic nanoconfiguration that can be integrated with the other part of biosynthesis process for the assimilation of carbon dioxide.

  10. Glycosylation site-targeted PEGylation of glucose oxidase retains native enzymatic activity.

    Science.gov (United States)

    Ritter, Dustin W; Roberts, Jason R; McShane, Michael J

    2013-04-10

    Targeted PEGylation of glucose oxidase at its glycosylation sites was investigated to determine the effect on enzymatic activity, as well as the bioconjugate's potential in an optical biosensing assay. Methoxy-poly(ethylene glycol)-hydrazide (4.5kDa) was covalently coupled to periodate-oxidized glycosylation sites of glucose oxidase from Aspergillus niger. The bioconjugate was characterized using gel electrophoresis, liquid chromatography, mass spectrometry, and dynamic light scattering. Gel electrophoresis data showed that the PEGylation protocol resulted in a drastic increase (ca. 100kDa) in the apparent molecular mass of the protein subunit, with complete conversion to the bioconjugate; liquid chromatography data corroborated this large increase in molecular size. Mass spectrometry data proved that the extent of PEGylation was six poly(ethylene glycol) chains per glucose oxidase dimer. Dynamic light scattering data indicated the absence of higher-order oligomers in the PEGylated GOx sample. To assess stability, enzymatic activity assays were performed in triplicate at multiple time points over the course of 29 days in the absence of glucose, as well as before and after exposure to 5% w/v glucose for 24h. At a confidence level of 95%, the bioconjugate's performance was statistically equivalent to native glucose oxidase in terms of activity retention over the 29 day time period, as well as following the 24h glucose exposure. Finally, the bioconjugate was entrapped within a poly(2-hydroxyethyl methacrylate) hydrogel containing an oxygen-sensitive phosphor, and the construct was shown to respond approximately linearly with a 220±73% signal change (n=4, 95% confidence interval) over the physiologically-relevant glucose range (i.e., 0-400mg/dL); to our knowledge, this represents the first demonstration of PEGylated glucose oxidase incorporated into an optical biosensing assay. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. Ship-borne measurements of microbial enzymatic activity: A rapid biochemical indicator for microbial water quality monitoring

    Science.gov (United States)

    Stadler, Philipp; Loken, Luke; Crawford, John; Schramm, Paul; Sorsa, Kirsti; Kuhn, Catherine; Savio, Domenico; Striegl, Rob; Butman, David; Stanley, Emily; Farnleitner, Andreas H.; Zessner, Matthias

    2017-04-01

    Contamination of aquatic ecosystems by human and animal wastes is a global concern for water quality. Disclosing fate and transport processes of fecal indicator organism (FIO) in large water bodies is a big challenge due to material intensive and time consuming methods used in microbiological water quality monitoring. In respect of utilization of large surface water resources there is a dearth of rapid microbiological methods that allow a near-real time health related water quality monitoring to be implemented into early warning systems. The detection of enzymatic activities has been proposed as a rapid surrogate for microbiological pollution monitoring of water and water resources (Cabral, 2010; Farnleitner et al., 2001, 2002). Methods such as the beta-D-Glucuronidase assay (GLUC), targeting FIO such as E. coli, were established. New automated enzymatic assays have been implemented during the last years into on-site monitoring stations, ranging from ground- to surface waters (Ryzinska-Paier et al., 2014; Stadler et al., 2017, 2016). While these automated enzymatic methods cannot completely replace assays for culture-based FIO enumeration, they yielded significant information on pollution events and temporal dynamics on a catchment specific basis, but were restricted to stationary measurements. For the first time we conducted ship-borne and automated measurements of enzymatic GLUC activity on large fresh water bodies, including the Columbia River, the Mississippi River and Lake Mendota. Not only are automated enzymatic assays technically feasible from a mobile vessel, but also can be used to localize point sources of potential microbial fecal contamination, such as tributaries or storm drainages. Spatial and temporal patterns of enzymatic activity were disclosed and the habitat specific correlation with microbiological standard assays for FIO determined due to reference samples. The integration of rapid and automated enzymatic assays into well-established systems

  12. DNA-Based Sensor for Real-Time Measurement of the Enzymatic Activity of Human Topoisomerase I

    DEFF Research Database (Denmark)

    Marcussen, Lærke Bay; Jepsen, Morten Leth; Kristoffersen, Emil Laust

    2013-01-01

    Sensors capable of quantitative real-time measurements may present the easiest and most accurate way to study enzyme activities. Here we present a novel DNA-based sensor for specific and quantitative real-time measurement of the enzymatic activity of the essential human enzyme, topoisomerase I....... The basic design of the sensor relies on two DNA strands that hybridize to form a hairpin structure with a fluorophore-quencher pair. The quencher moiety is released from the sensor upon reaction with human topoisomerase I thus enabling real-time optical measurement of enzymatic activity. The sensor....... The cytotoxic effect of camptothecins correlates directly with the intracellular topoisomerase I activity. We therefore envision that the presented sensor may find use for the prediction of cellular drug response. Moreover, inhibition of topoisomerase I by camptothecin is readily detectable using the presented...

  13. Gamma radiation effect on biological activity and enzymatic properties of snake venoms

    International Nuclear Information System (INIS)

    Herrera, E.; Yarleque, A.; Campos, S.; Zavaleta, A.

    1986-01-01

    The effect of gamma radiation, from Co-60, on the biological activity and on some enzymatic activities, present in the venoms of Lachesis muta and Bothrops atrox, using samples of dried venom that had been irradiated at a dose of 0.1, 0.5 and 1.0 Mrad have been studied. Variations in the degree of hemorrhage and local necrosis were observed in albino mice injected subcutaneously with venoms of both types. The reduction of the biological activity was greater for the local hemorrhagic effect and was dependent on the doses of irradiation. The specific activity of various enzymes, present in both venoms, is affected by the gamma radiation, at a dose of 0.1 Mrad the order of increasing inactivation being: exonuclease (4%), phospholipase (24%), caseinolytic enzyme (20%), tamesterase (33%), a thrombine-like enzyme (40%), fibrinolytic enzyme (41%), 5'-nucleotidase (50%) and endonuclease (55%). The enzymatic inactivation was augmented by 0.5 and 1.0 Mrad, without maintaining an arithmetic relation. The enzyme of major resistance to the radiation was exonuclease, whereas 5'-nucleotidase and endonuclease were the most sensitive. No significant changes were observed in the spectrum of UV absorbtion (range 260 to 290 nm) nor in the contents of L-tyrosine in the irradiated venoms

  14. ENZYMATIC AND NON-ENZYMATIC ANTIOXIDANT DEFENSE WITH ALZHEIMER DISEASE1

    Directory of Open Access Journals (Sweden)

    A. Vaisi-Raygani

    2007-07-01

    Full Text Available The etiopathogenesis of Alzheimer's disease (AD is still unclear.  However, long-term oxidative stress is believed to be one of the major contributing factors in progression of neuronal degeneration and decline of cognitive function in AD. In order to assess the presence of oxidative stress in AD, we examined the enzymatic activities of the erythrocyte Cu-Zn superoxide dismutase (Cu-Zn SOD, glutathione peroxidase (GSH-Px, catalase (CAT, and plasma level of total antioxidant status (TAS in AD and control groups (age and sex-matched. The results showed that the Cu-Zn SOD activity was significantly higher and the level of GSH-Px and TAS activities were significantly lower in AD subjects than that in the control group (2111 ± 324 U/grHb, 43.7 ± 11.6 U/grHb, and 1.17 ± 0.23 mmol/l compared with 1371 ± 211 U/grHb; t= -2.17, P = 0.036, 56.3 ± 9.5 U/grHb; t=3.8, P = 0.014, and 1.54±0.2 mmol/l; t=11.18, P < 0.001, respectively.  While, the erythrocyte CAT activity was lower in AD subjects compared to the control group, the difference was not statistically significant (t = 1.3, P = 0.15. These findings support the idea that the oxidative stress plays an important role in the pathogenesis underlying AD neurodegeneration. In addition, the enzymatic activity of the erythrocyte Cu-Zn SOD and GSH-Px and the plasma level of TAS can be used as a measure of the oxidative stress and a marker for pathological changes in the brain of patients with AD. 

  15. ASSOCIATION BETWEEN ENZYMATIC AND NON-ENZYMATIC ANTIOXIDANT DEFENSE WITH ALZHEIMER DISEASE

    Directory of Open Access Journals (Sweden)

    A. Vaisi-Raygani

    2008-04-01

    Full Text Available The etiopathogenesis of dementia in Alzheimer's disease (AD is still unclear. However, long-term oxidative stress is believed to be one of the major contributing factors in progression of neuronal degeneration and decline of cognitive function in AD. In order to assess the presence of oxidative stress in AD, we examined the enzymatic activities of the erythrocyte Cu-Zn superoxide dismutase (Cu-Zn SOD, glutathione peroxidase (GSH-Px, catalase (CAT, and plasma level of total antioxidant status (TAS in AD and control groups (age and sex-matched. The results showed that the Cu-Zn SOD activity was significantly higher and the level of GSH-Px and TAS activities were significantly lower in AD subjects than that in the control group (2111±324 U/grHb, 43.7±11.6 U/grHb, and 1.17 ±0.23 mmol/L compared with 1371±211 U/gHb; t= -2.17, p=0.036, 56.3±9.5 U/gHb; t=3.8, p=0.014, and 1.54±0.2 mmol/L; t=11.18, P<0.001, respectively. While, the erythrocyte CAT activity was lower in AD subjects compared to the control group, the difference was not statistically significant (t=1.3, P=0.15. These findings support the idea that the oxidative stress plays an important role in the pathogenesis underlying AD neurodegeneration. In addition, the enzymatic activity of the erythrocyte Cu-Zn SOD and GSH-Px and the plasma level of TAS can be used as a measure of the oxidative stress and a marker for pathological changes in the brain of patients with AD.

  16. Highly efficient and enzymatic regioselective undecylenoylation of gastrodin in 2-methyltetrahydrofuran-containing systems.

    Science.gov (United States)

    Yang, Rongling; Liu, Xueming; Chen, Zhiyi; Yang, Chunying; Lin, Yaosheng; Wang, Siyuan

    2014-01-01

    Highly efficient and regioselective acylation of pharmacologically interesting gastrodin with vinyl undecylenic acid has been firstly performed through an enzymatic approach. The highest catalytic activity and regioselectivity towards the acylation of 7'-hydroxyl of gastrodin was obtained with Pseudomonas cepacia lipase. In addition, it was observed the lipase displayed higher activity in the eco-friendly solvent 2-methyltetrahydrofuran-containing systems than in other organic solvents. In the co-solvent mixture of tetrahydrofuran and 2-methyltetrahydrofuran (3/1, v/v), the reaction rate was 60.6 mM/h, substrate conversion exceeded 99%, and 7'-regioselectivity was 93%. It was also interesting that the lipase-catalyzed acylation couldn't be influenced by the benzylic alcohol in gastrodin. However, pseudomonas cepacia lipase displayed different regioselectivity towards gastrodin and arbutin.

  17. Oculocutaneous albinism type 1: link between mutations, tyrosinase conformational stability, and enzymatic activity.

    Science.gov (United States)

    Dolinska, Monika B; Kus, Nicole J; Farney, S Katie; Wingfield, Paul T; Brooks, Brian P; Sergeev, Yuri V

    2017-01-01

    Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase gene. Two subtypes of OCA1 have been described: severe OCA1A with complete absence of tyrosinase activity and less severe OCA1B with residual tyrosinase activity. Here, we characterize the recombinant human tyrosinase intramelanosomal domain and mutant variants, which mimic genetic changes in both subtypes of OCA1 patients. Proteins were prepared using site-directed mutagenesis, expressed in insect larvae, purified by chromatography, and characterized by enzymatic activities, tryptophan fluorescence, and Gibbs free energy changes. The OCA1A mutants showed very low protein expression and protein yield and are enzymatically inactive. Mutants mimicking OCA1B were biochemically similar to the wild type, but exhibited lower specific activities and protein stabilities. The results are consistent with clinical data, which indicates that OCA1A mutations inactivate tyrosinase and result in severe phenotype, while OCA1B mutations partially inactivate tyrosinase and result in OCA1B albinism. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

  18. Chemical Characterization, Antioxidant and Enzymatic Activity of Brines from Scandinavian Marinated Herring Products

    DEFF Research Database (Denmark)

    Gringer, Nina; Osman, Ali; Nielsen, Henrik Hauch

    2014-01-01

    Brines generated during the last marination step in the production of marinated herring (Clupea harengus) were chemically characterized and analyzed for antioxidant and enzyme activities. The end-products were vinegar cured, spice cured and traditional barrel-salted herring with either salt...... or spices. The chemical characterization encompassed pH, dry matter, ash, salt, fatty acids, protein, polypeptide pattern, iron and nitrogen. The antioxidant activity was tested with three assays measuring: iron chelation, reducing power and radical scavenging activity. The enzymatic activity for peroxidase...

  19. The diversity, extracellular enzymatic activities and photoprotective compounds of yeasts isolated in Antarctica

    Directory of Open Access Journals (Sweden)

    Aline B. M Vaz

    2011-09-01

    Full Text Available The diversity of yeasts collected from different sites in Antarctica (Admiralty Bay, King George Island and Port Foster Bay and Deception Island and their ability to produce extracellular enzymes and mycosporines were studied. Samples were collected during the austral summer season, between November 2006 and January 2007, from the rhizosphere of Deschampsia antarctica, ornithogenic (penguin guano soil, soil, marine and lake sediments, marine water and freshwater from lakes. A total of 89 isolates belonging to the following genera were recovered: Bensingtonia, Candida, Cryptococcus, Debaryomyces, Dioszegia, Exophiala, Filobasidium, Issatchenkia (Pichia, Kodamaea, Leucosporidium, Leucosporidiella, Metschnikowia, Nadsonia, Pichia, Rhodotorula, and Sporidiobolus, and the yeast-like fungi Aureobasidium, Leuconeurospora and Microglossum. Cryptococcus victoriae was the most frequently identified species. Several species isolated in our study have been previously reported to be Antarctic psychophilic yeasts, including Cr. antarcticus, Cr. victoriae, Dioszegia hungarica and Leucosporidium scottii. The cosmopolitan yeast species A. pullulans, C. zeylanoides, D. hansenii, I. orientalis, K. ohmeri, P. guilliermondii, Rh. mucilaginosa, and S. salmonicolor were also isolated. Five possible new species were identified. Sixty percent of the yeasts had at least one detectable extracellular enzymatic activity. Cryptococcus antarcticus, D. aurantiaca, D. crocea, D. hungarica, Dioszegia sp., E. xenobiotica, Rh. glaciales, Rh. laryngis, Microglossum sp. 1 and Microglossum sp. 2 produced mycosporines. Of the yeast isolates, 41.7% produced pigments and/or mycosporines and could be considered adapted to survive in Antarctica. Most of the yeasts had extracellular enzymatic activities at 4ºC and 20ºC, indicating that they could be metabolically active in the sampled substrates.

  20. Changes in the amino acid profiles and free radical scavenging activities of Tenebrio molitor larvae following enzymatic hydrolysis.

    Science.gov (United States)

    Tang, Yujiao; Debnath, Trishna; Choi, Eun-Ju; Kim, Young Wook; Ryu, Jung Pyo; Jang, Sejin; Chung, Sang Uk; Choi, Young-Jin; Kim, Eun-Kyung

    2018-01-01

    Tenebrio molitor (T. molitor) larvae provide food at low environmental cost and contribute positively to livelihoods. In this research, we compared the amino acids compositions and antioxidant activities of various extracts of T. molitor to enhance their quality as food. For the comparison, distilled water extracts, enzymatic hydrolysates, and condensed enzymatic hydrolysates of T. molitor larvae were prepared. Their amino acids (AAs) profiles and antioxidant activities, including ferric-reducing antioxidant power, oxygen radical absorption capacity, and DPPH, hydroxyl radical, and hydrogen peroxide radical scavenging properties assay were analyzed. DW extracts had the lowest AAs contents and antioxidant activity compared with enzymatic extracts. Condensed hydrolysates with a combination of alcalase and flavourzyme (C-A+F) exhibited the highest levels of total free AAs (11.1759 g/100 g). C-A+F produced higher total hydrolyzed AAs (32.5292 g/100 g) compared with the other groups. The C-A+F possessed the strongest antioxidant activity. Notably, the antioxidant activities of the hydrolysates and the total hydrolyzed AAs amount were correlated. Taken together, our findings showed that C-A+F was a promising technique for obtaining extracts of T. molitor larvae with antioxidant activity as potential nutritious functional food.

  1. Effect of enzymatic mash treatment and storage on phenolic composition, antioxidant activity, and turbidity of cloudy apple juice.

    Science.gov (United States)

    Oszmiański, Jan; Wojdylo, Aneta; Kolniak, Joanna

    2009-08-12

    The effects of different commercial enzymatic mash treatments on yield, turbidity, color, and polyphenolic and sediment of procyanidins content of cloudy apple juice were studied. Addition of pectolytic enzymes to mash treatment had positive effect on the production of cloud apple juices by improving polyphenolic contents, especially procyanidins and juice yields (68.3% in control samples to 77% after Pectinex Yield Mash). As summary of the effect of enzymatic mash treatment, polyphenol contents in cloudy apple juices significantly increased after Pectinex Yield Mash, Pectinex Smash XXL, and Pectinex XXL maceration were applied but no effect was observed after Pectinex Ultra-SPL I Panzym XXL use, compared to the control samples. The content of polymeric procyanidins represented 50-70% of total polyphenols, but in the present study, polymeric procyanidins were significantly lower in juices than in fruits and also affected by enzymatic treatment (Pectinex AFP L-4 and Panzym Yield Mash) compared to the control samples. The enzymatic treatment decreased procyanidin content in most sediment with the exception of Pectinex Smash XXL and Pectinex AFP L-4. Generally in samples that were treated by pectinase, radical scavenging activity of cloudy apple juices was increased compared to the untreated reference samples. The highest radical scavenging activity was associated with Pectinex Yield Mash, Pectinex Smash XXL, and Pectinex XXL enzyme and the lowest activity with Pectinex Ultra SP-L and Pectinex APFL-4. However, in the case of enzymatic mash treatment cloudy apple juices showed instability of turbidity and low viscosity. These results must be ascribed to the much higher hydrolysis of pectin by enzymatic preparation which is responsible for viscosity. During 6 months of storage at 4 degrees C small changes in analyzed parameters of apple juices were observed.

  2. Modelling the Effects of Ageing Time of Starch on the Enzymatic Activity of Three Amylolytic Enzymes

    Science.gov (United States)

    Guerra, Nelson P.; Pastrana Castro, Lorenzo

    2012-01-01

    The effect of increasing ageing time (t) of starch on the activity of three amylolytic enzymes (Termamyl, San Super, and BAN) was investigated. Although all the enzymatic reactions follow michaelian kinetics, v max decreased significantly (P enzymes and the release of the reaction products to the medium. A similar effect was observed when the enzymatic reactions were carried out with unaged starches supplemented with different concentrations of gelatine [G]. The inhibition in the amylolytic activities was best mathematically described by using three modified forms of the Michaelis-Menten model, which included a term to consider, respectively, the linear, exponential, and hyperbolic inhibitory effects of t and [G]. PMID:22666116

  3. Destruction of enzymatic activities of corn and soybean leaves exposed to ozone

    Energy Technology Data Exchange (ETDEWEB)

    Leffler, H R; Cherry, J H

    1974-01-01

    Experiments were conducted to determine the effects of a single ozone exposure on selected enzymatic activities and chlorophyll contents of corn and soybean seedlings. Both nitrite reductase activity and chlorophyll content of the seedlings were found to be quite sensitive to ozonation and were seen to decrease as much as 50% after exposure to 80 parts per hundred million (pphm) ozone. After exposure to lower levels of ozone, less-pronounced decreases were observed. Nitrate reductase activity was reduced only after exposure to seedling leaf tissues to high concentrations of ozone. These results are discussed in relation to the concept of a two-phase response to oxidant exposure. The first phase is at the chloroplast level and is quite sensitive to the low as well as the high concentrations of ozone; the second is at the cellular level and is relatively resistant to all but the highest ozone concentrations. 27 references, 2 tables.

  4. Specific inflammatory response of Anemonia sulcata (Cnidaria) after bacterial injection causes tissue reaction and enzymatic activity alteration.

    Science.gov (United States)

    Trapani, M R; Parisi, M G; Parrinello, D; Sanfratello, M A; Benenati, G; Palla, F; Cammarata, M

    2016-03-01

    The evolution of multicellular organisms was marked by adaptations to protect against pathogens. The mechanisms for discriminating the ''self'' from ''non-self" have evolved into a long history of cellular and molecular strategies, from damage repair to the co-evolution of host-pathogen interactions. We investigated the inflammatory response in Anemonia sulcata (Cnidaria: Anthozoa) following injection of substances that varied in type and dimension, and observed clear, strong and specific reactions, especially after injection of Escherichia coli and Vibrio alginolyticus. Moreover, we analyzed enzymatic activity of protease, phosphatase and esterase, showing how the injection of different bacterial strains alters the expression of these enzymes and suggesting a correlation between the appearance of the inflammatory reaction and the modification of enzymatic activities. Our study shows for the first time, a specific reaction and enzymatic responses following injection of bacteria in a cnidarian. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Biosensing strategies based on enzymatic reactions and nanoparticles.

    Science.gov (United States)

    Díez-Buitrago, Beatriz; Briz, Nerea; Liz-Marzán, Luis M; Pavlov, Valeri

    2018-04-16

    Enzymes are pivotal elements in bioanalysis due to their specificity and extremely high catalytic activity. The sensitivity of bioanalytical assays depends mainly on the capacity of an observer to detect the product(s) of a biocatalytic reaction. Both natural and artificial compounds have been traditionally used to evaluate enzymatic activities. The drawbacks of chromogenic and fluorogenic organic enzymatic substrates are their high cost and low stability, resulting in high background signals. We review here state of the art assays in the detection of enzymatic activities using recent advances in nanoscience. Novel methods based on the use of nanoparticles lead to increased sensitivity and decreased costs for bioanalysis based on enzymes as recognition elements and signal amplifiers in Enzyme-Linked Immunosorbent Assays (ELISA). Novel approaches toward the detection of enzymatic activities are based on biocatalytic synthesis, modulation, etching, and aggregation of nanoparticles under physiological conditions.

  6. Highly efficient and enzymatic regioselective undecylenoylation of gastrodin in 2-methyltetrahydrofuran-containing systems.

    Directory of Open Access Journals (Sweden)

    Rongling Yang

    Full Text Available Highly efficient and regioselective acylation of pharmacologically interesting gastrodin with vinyl undecylenic acid has been firstly performed through an enzymatic approach. The highest catalytic activity and regioselectivity towards the acylation of 7'-hydroxyl of gastrodin was obtained with Pseudomonas cepacia lipase. In addition, it was observed the lipase displayed higher activity in the eco-friendly solvent 2-methyltetrahydrofuran-containing systems than in other organic solvents. In the co-solvent mixture of tetrahydrofuran and 2-methyltetrahydrofuran (3/1, v/v, the reaction rate was 60.6 mM/h, substrate conversion exceeded 99%, and 7'-regioselectivity was 93%. It was also interesting that the lipase-catalyzed acylation couldn't be influenced by the benzylic alcohol in gastrodin. However, pseudomonas cepacia lipase displayed different regioselectivity towards gastrodin and arbutin.

  7. Recent advances in the elucidation of enzymatic function in natural product biosynthesis [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Gao-Yi Tan

    2016-02-01

    Full Text Available With the successful production of artemisinic acid in yeast, the promising potential of synthetic biology for natural product biosynthesis is now being realized. The recent total biosynthesis of opioids in microbes is considered to be another landmark in this field. The importance and significance of enzymes in natural product biosynthetic pathways have been re-emphasized by these advancements. Therefore, the characterization and elucidation of enzymatic function in natural product biosynthesis are undoubtedly fundamental for the development of new drugs and the heterologous biosynthesis of active natural products. Here, discoveries regarding enzymatic function in natural product biosynthesis over the past year are briefly reviewed.

  8. Ebselen inhibits QSOX1 enzymatic activity and suppresses invasion of pancreatic and renal cancer cell lines.

    Science.gov (United States)

    Hanavan, Paul D; Borges, Chad R; Katchman, Benjamin A; Faigel, Douglas O; Ho, Thai H; Ma, Chen-Ting; Sergienko, Eduard A; Meurice, Nathalie; Petit, Joachim L; Lake, Douglas F

    2015-07-30

    Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that is overexpressed in diverse tumor types. Its enzymatic activity promotes the growth and invasion of tumor cells and alters extracellular matrix composition. In a nude mouse-human tumor xenograft model, tumors containing shRNA for QSOX1 grew significantly more slowly than controls, suggesting that QSOX1 supports a proliferative phenotype in vivo. High throughput screening experiments identified ebselen as an in vitro inhibitor of QSOX1 enzymatic activity. Ebselen treatment of pancreatic and renal cancer cell lines stalled tumor growth and inhibited invasion through Matrigel in vitro. Daily oral treatment with ebselen resulted in a 58% reduction in tumor growth in mice bearing human pancreatic tumor xenografts compared to controls. Mass spectrometric analysis of ebselen-treated QSOX1 mechanistically revealed that C165 and C237 of QSOX1 covalently bound to ebselen. This report details the anti-neoplastic properties of ebselen in pancreatic and renal cancer cell lines. The results here offer a "proof-of-principle" that enzymatic inhibition of QSOX1 may have clinical relevancy.

  9. Enzymatic in-situ generation of H2O2 for decolorization of Acid Blue 113 by fenton process

    Directory of Open Access Journals (Sweden)

    Karimi Afzal

    2012-01-01

    Full Text Available Decolorization of Acid Blue 113 in an aqueous medium by bio-Fenton process has been investigated in this research. Enzymatic oxidation of glucose was performed to in-situ generation of H2O2 which was employed to react with Fe2+ for producing hydroxyl radicals. The effect of various parameters include concentrations of 113, glucose, and FeSO4, activity of glucose oxidase (GOx and the effect of pH were assessed. The highest decolorization of AB 113 were achieved at Fe2+ concentration of 0.2 mmol/L, pH =4.0, glucose concentration of 0.018 mol/L, and glucose oxidase activity of 2500 U/L in the constant temperature (23 ±0.1ºC and constant shaking rate (160 r/min, while the concentration of 113 was 40 mg/L. In these conditions, 113 decolorization efficiency after 60 min was obtained about 95%.

  10. pCO2 and enzymatic activity in a river floodplain system of the Danube under different hydrological settings.

    Science.gov (United States)

    Sieczko, Anna; Demeter, Katalin; Mayr, Magdalena; Meisterl, Karin; Peduzzi, Peter

    2014-05-01

    Surface waters may serve as either sinks or sources of CO2. In contrast to rivers, which are typically sources of CO2 to the atmosphere, the role of fringing floodplains in CO2 flux is largely understudied. This study was conducted in a river-floodplain system near Vienna (Austria). The sampling focused on changing hydrological situations, particularly on two distinct flood events: a typical 1-year flood in 2012 and an extraordinary 100-year flood in 2013. One objective was to determine partial pressure of CO2 (pCO2) in floodplain lakes with different degree of connectivity to the main channel, and compare the impact of these two types of floods. Another aim was to decipher which fraction of the dissolved organic matter (DOM) pool contributed to pCO2 by linking pCO2 with optical properties of DOM and extracellular enzymatic activity (EEA) of microbes. The EEA is a valuable tool, especially for assessing the non-chromophoric but rapidly utilized DOM-fraction during floods. In 2012 and 2013, the floodplain lakes were dominated by supersaturated pCO2 conditions, which indicates that they served as CO2 sources. Surprisingly, there were no significant differences in pCO2 between the two types of flood. Our findings imply that the extent of the flood had minor impact on pCO2, but the general occurrence of a flood appears to be important. During the flood in 2013 significantly more dissolved organic carbon (DOC) (pcarbohydrates.

  11. Effect of Different Calcium Sources Application on Antioxidant, Enzymatic Activity and Qualitative Characteristics of Apple (Malus domestic)

    OpenAIRE

    MirHassan Rasouli-Sadaghiani; Mohammad Moghaddas Gerani; Sanaz Ashrafi Saeidlou; Ebrahim Sepehr

    2017-01-01

    In order to determine the effect of different Ca sources, in improving enzymatic activity and some qualitative properties of red apple (Malusdomestica) an experiment was carried out in a completely randomized design. Apple trees were sprayed 5 times with CaCl2, CaO, Ca-EDTA and Ca(NO3)2 salts with an interval of 20 days from late June until early October. After harvesting fruits were kept in cold storage at standard condition for 150 days. Nitrogen, phosphorus, potassium, calcium, magnesium, ...

  12. Quercetin modulates activities of Taiwan cobra phospholipase A2 ...

    Indian Academy of Sciences (India)

    2012-04-25

    Apr 25, 2012 ... Quercetin incorporation led to a reduction in PLA2 enzymatic activity and membrane-damaging activity ... membrane physical properties such as membrane fluidity or ..... eral blood mononuclear cells from diabetes patients.

  13. Enzymatic synthesis of tritium-labelled prostaglandin D2 and its conversion to other prostaglandins

    International Nuclear Information System (INIS)

    Shram, S.I.; Lazurkina, T.Yu.; Shevchenko, V.P.; Nagaev, I.Yu.; Myasoedov, N.F.

    1994-01-01

    The one-stage enzymatic synthesis of tritium-labelled prostaglandin D 2 from labelled arachidonic acid was performed by using the enzyme system PGH-synthetase/PGH-PGD-isomerase. By enzymatic and chemical transformation of [ 3 H]PGD 2 the following compounds were obtained: 15-keto-13,14-dihydro-[ 3 H]PGD 2 , 9α,11β-[ 3 H]PGF 2 , 9-deoxy-Δ 9 -[ 3 H]-PGD 2 ([ 3 H]PGJ 2 ) and Δ 12 -13,14-dihydro-[ 3 H]PGJ 2 . It was found that L-selectride is a more effective reducing agent than sodium borohydride in the synthesis of 9α, 11β-[ 3 H]PGF 2 . (Author)

  14. Determination of myoglobin based on its enzymatic activity by stopped-flow spectrophotometry

    Science.gov (United States)

    Zheng, Qi; Liu, Zhihong; Cai, Ruxiu

    2005-04-01

    A new method has been developed for the determination of myoglobin (Mb) based on its enzymatic activity for the oxidation of o-phenylenediamine (OPDA) with hydrogen peroxide. Stopped-flow spectrophotometry was used to study the kinetic behavior of the oxidation reaction. The catalytic activity of Mb was compared to other three kinds of catalyst. The time dependent absorbance of the reaction product, 2,3-diamimophenazine (DAPN), at a wavelength of 426 nm was recorded. The initial reaction rate obtained at 40 °C was found to be proportional to the concentration of Mb in the range of 1.0 × 10 -6 to 4.0 × 10 -9 mol L -1. The detection limit of Mb was found to be 9.93 × 10 -10 mol L -1. The relative standard deviations were within 5% for the determination of different concentrations of Mb. Excess of bovine serum albumin (BSA), Ca(II), Mg(II), Cu(II), glucose, caffeine, lactose and uric acid did not interfere.

  15. Common HEXB polymorphisms reduce serum HexA and HexB enzymatic activities, potentially masking Tay-Sachs disease carrier identification.

    Science.gov (United States)

    Vallance, Hilary; Morris, Tara J; Coulter-Mackie, Marion; Lim-Steele, Joyce; Kaback, Michael

    2006-02-01

    A DNA-proven Tay-Sachs disease (TSD) carrier and his brother were found to have serum percent Hexosaminidase A (%HexA) enzymatic activities in the non-carrier range, while the leukocyte %HexA profiles clearly identified them as TSD heterozygotes. Both their serum HexA and HexB enzymatic activities were below reference range, suggesting inheritance of mutations in both the HEXA (alpha-subunit) and HEXB (beta-subunit) genes. DNA sequencing revealed that both individuals, carried the common HEXA 1277_1278insTATC mutation, and two common HEXB polymorphisms: [619A>G (+) delTG]. To determine if these HEXB polymorphisms reduce HexA and HexB enzymatic activities, 69 DNA samples from subjects previously screened enzymatically in both serum and leukocytes for TSD carrier status were selected for either high, mid-range or low serum Total Hex (defined as the sum of HexA and HexB) activities and were tested for the HEXB mutations. Further, three additional TSD carriers ascertained by the atypical pattern of normal serum %HexA but carrier leukocyte %HexA, were found to have the [delTG (+) 619A>G] genotype. In addition, the frequency of the [delTG (+) 619A>G] genotype was significantly higher (P G] haplotype in the Ashkenazi Jewish population (approximately 10%), up to 10% of TSD carriers may have normal serum %HexA values with low total Hex. Accordingly, serum %HexA should not be the sole criterion used for carrier status determination. Where total Hex activity is reduced, further testing with leukocyte Hex profiles is indicated.

  16. Enzymatic degradation of cyclic 2,3-diphosphoglycerate to 2,3-diphosphoglycerate in Methanobacterium thermoautotrophicum

    International Nuclear Information System (INIS)

    Krishna Sastry, M.V.; Robertson, D.E.; Moynihan, J.A.; Roberts, M.F.

    1992-01-01

    2,3-Diphosphoglycerate (2,3-DPG) has been found to be the product of the enzymatic degradation of cyclic 2,3-diphosphoglycerate (cDPG) in the archaebacterium Methanobacterium thermoautotrophicum ΔH. Although 2,3-DPG has not previously been detected as a major soluble component of M. thermoautotrophicum, large pools accumulated at an incubation temperature of 50C. Under these conditions, cellular activity was significantly decreased; a return of the culture to the optimum growth temperature restored the 2,3-DPG pool back to original low levels and caused steady-state cDPG levels to increase again. While 13 CO 2 -pulse/ 12 CO 2 -chase experiments at 50C showed that the cDPG turned over, the appearance of 2,3-DPG at NMR-visible concentrations required at least 10 h. Production of 2,3-DPG in vivo was prevented by exposure of the cells to O 2 . the enzyme responsible for this hydrolysis of cDPG was purified by affinity chromatography and appears to be a 33-kDa protein. Activity was detected in the presence of oxygen and was enhanced by a solution of 1 M KCl, 25 mM MgCl 2 , and dithiothreitol. Both K m and V max have been determined at 37C; kinetics also indicate that in vitro the product, 2,3-DPG, is an inhibitor of cDPG hydrolysis. These findings are discussed in view of a proposed role for cDPG in methanogens

  17. Influence of season, environment and feeding habits on the enzymatic activity of peptidase and β-glucosidase in the gastrointestinal tract of two Siluriformes fishes (Teleostei

    Directory of Open Access Journals (Sweden)

    Silvana Duarte

    2013-06-01

    Full Text Available The enzymatic activities involved in the digestion of proteins and carbohydrates were compared among three organs of the digestive track of two Siluriformes fish species, and between two areas: a reservoir, and an area downriver of it. Our aim was to test the hypothesis that the digestive organs of species with varied feeding habits have different enzymatic activities, and that the enzymatic activity differs among seasons and environmental conditions. The iliophagous/herbivorous species Hypostomus auroguttatus Kner, 1854 had higher trypsin-like, chymotrypsin-like peptidase and β-glucosidase activity in the intestine when compared with the omnivorous species Pimelodus maculatus Lacepède, 1803, whereas the latter had more hepatic trypsin-like activity than the former. The peak of activity of the three enzymes in H. auroguttatus was recorded in the winter and spring. On the other hand, P. maculatus tended to have the more prominent peptidase and β-glucosidase activity in the summer, and the smallest in the winter. The intestine of H. auroguttatus had higher enzymatic (trypsin, chymotrypsin and β-glucosidase activity than the stomach and the liver. For P. maculatus, the highest β-glucosidase activity was found in the liver. The enzymatic activity of H. aurogutattus did not differ between lotic and lentic systems, whereas P. maculatus had comparatively higher stomach and hepatic trypsin levels and hepatic chymotrypsin-like activities in the reservoir than down in the river. These findings indicate that, in H. auroguttatus, most digestive activity occurs in the intestine, which is long and adapted for the digestion of bottom-river vegetable matter and detritus. The seasons and the type of the system (lentic vs. lotic seem to affect the enzymatic activity for these two species differently, a likely consequence of their different lifestyles.

  18. Biphasically Modulating the Activity of Carboxypeptidase G2 with Ultrasound

    Directory of Open Access Journals (Sweden)

    Wanying Ma

    2017-07-01

    Full Text Available Background/Aims: Carboxypeptidase G2 (CPG2 has been used for cancer prodrug therapy to realize the targeted release of active drugs, but there yet lacks a means to modulate the CPG2 activity. Here ultrasound was used to modulate the CPG2 activity. Methods: The activity of insonated CPG2 was determined, and then underlying biochemical (i.e., monomer, dimer and conformation and ultrasonic (i.e., heat and cavitation mechanisms were explored. Results: Ultrasound (1.0 MHz increased or decreased the enzymatic activity; the activity decreased as zero- or first-order kinetics, depending on the intensity. L1 (10 W/cm2 for 200 s improved the activity via increasing the specific activity. L2 or L3 (20 W/cm2 for 1200 or 3000 s decreased the activity via disassembling the dimer, degrading the monomer, inducing glycosylation, transforming conformation and decreasing the specific activity. An increase or a slight decrease of activity attributable to 10 W/cm2 was reversible, but the activity decrease due to 20 W/cm2 was irreversible. The enzymatic modulation was realized via cavitation. Conclusion: Ultrasound can biphasically modulate the CPG2 activity, and can be employed in the CPG2-prodrug therapy to adjust the release and moles of active drugs.

  19. Site-Specific Bioconjugation of an Organometallic Electron Mediator to an Enzyme with Retained Photocatalytic Cofactor Regenerating Capacity and Enzymatic Activity

    Directory of Open Access Journals (Sweden)

    Sung In Lim

    2015-04-01

    Full Text Available Photosynthesis consists of a series of reactions catalyzed by redox enzymes to synthesize carbohydrates using solar energy. In order to take the advantage of solar energy, many researchers have investigated artificial photosynthesis systems mimicking the natural photosynthetic enzymatic redox reactions. These redox reactions usually require cofactors, which due to their high cost become a key issue when constructing an artificial photosynthesis system. Combining a photosensitizer and an Rh-based electron mediator (RhM has been shown to photocatalytically regenerate cofactors. However, maintaining the high concentration of cofactors available for efficient enzymatic reactions requires a high concentration of the expensive RhM; making this process cost prohibitive. We hypothesized that conjugation of an electron mediator to a redox enzyme will reduce the amount of electron mediators necessary for efficient enzymatic reactions. This is due to photocatalytically regenerated NAD(PH being readily available to a redox enzyme, when the local NAD(PH concentration near the enzyme becomes higher. However, conventional random conjugation of RhM to a redox enzyme will likely lead to a substantial loss of cofactor regenerating capacity and enzymatic activity. In order to avoid this issue, we investigated whether bioconjugation of RhM to a permissive site of a redox enzyme retains cofactor regenerating capacity and enzymatic activity. As a model system, a RhM was conjugated to a redox enzyme, formate dehydrogenase obtained from Thiobacillus sp. KNK65MA (TsFDH. A RhM-containing azide group was site-specifically conjugated to p-azidophenylalanine introduced to a permissive site of TsFDH via a bioorthogonal strain-promoted azide-alkyne cycloaddition and an appropriate linker. The TsFDH-RhM conjugate exhibited retained cofactor regenerating capacity and enzymatic activity.

  20. EPSPS variability, gene expression, and enzymatic activity in glyphosate-resistant biotypes of Digitaria insularis.

    Science.gov (United States)

    Galeano, E; Barroso, A A M; Vasconcelos, T S; López-Rubio, A; Albrecht, A J P; Victoria Filho, R; Carrer, H

    2016-08-12

    Weed resistance to herbicides is a natural phenomenon that exerts selection on individuals in a population. In Brazil, glyphosate resistance was recently detected in Digitaria insularis. The objective of this study was to elucidate mechanisms of weed resistance in this plant, including genetic variability, allelism, amino acid substitutions, gene expression, and enzymatic activity levels. Most of these have not previously been studied in this species. D. insularis DNA sequences were used to analyze genetic variability. cDNA from resistant and susceptible plants was used to identify mutations, alleles, and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) expression, using real-time quantitative reverse transcription-polymerase chain reaction. In addition, EPSPS activity was measured. We found a decrease in genetic variability between populations related to glyphosate application. Substitutions from proline to threonine and tyrosine to cysteine led to a decrease in EPSPS affinity for the glyphosate. In addition, the EPSPS enzymatic activity was slightly higher in resistant plants, whereas EPSPS gene expression was almost identical in both biotypes, suggesting feedback regulation at different levels. To conclude, our results suggest new molecular mechanisms used by D. insularis to increase glyphosate resistance.

  1. Roles of phospholipase A2 isoforms in swelling- and melittin-induced arachidonic acid release and taurine efflux in NIH3T3 fibroblasts

    DEFF Research Database (Denmark)

    Pedersen, Stine Helene Falsig; Poulsen, Kristian Arild; Lambert, Ian H.

    2006-01-01

    Osmotic swelling of NIH3T3 mouse fibroblasts activates a bromoenol lactone (BEL)-sensitive taurine efflux, pointing to the involvement of a Ca2+-independent phospholipase A2 (iPLA2) (Lambert IH. J Membr Biol 192: 19-32, 2003). We report that taurine efflux from NIH3T3 cells was not only increased...... by cell swelling but also decreased by cell shrinkage. Arachidonic acid release to the cell exterior was similarly decreased by shrinkage yet not detectably increased by swelling. NIH3T3 cells were found to express cytosolic calcium-dependent cPLA2-IVA, cPLA2-IVB, cPLA2-IVC, iPLA2-VIA, iPLA2-VIB......, and secretory sPLA2-V. Arachidonic acid release from swollen cells was partially inhibited by BEL and by the sPLA2-inhibitor manoalide. Cell swelling elicited BEL-sensitive arachidonic acid release from the nucleus, to which iPLA2-VIA localized. Exposure to the bee venom peptide melittin, to increase PLA2...

  2. Combined enzymatic hydrolysis and fermentation of hemicellulose to 2,3-butanediol

    Energy Technology Data Exchange (ETDEWEB)

    Yu, E.K.C.; Deschatelets, L.; Saddler, J.N.

    1984-06-01

    Hemicellulose-rich fractions from several agricultural residues were converted to 2,3-butanediol by a combined enzymatic hydrolysis and fermentation process. Culture filtrates from Trichoderma harzianum E58 were used to hydrolyze the substrates while Klebsiella pneumoniae fermented the liberated sugars to 2,3-butanediol. Approximately 50-60% of a 5% (w/v) xylan preparation could be hydrolyzed and quantitatively converted to 2,3-butanediol using this procedure. Although enzymatic hydrolysis was optimal at pH 5.0 and 50/sup 0/C, the combined hydrolysis and fermentation was most efficient at pH 6.5 and 30/sup 0/C. Combined hydrolysis and fermentation resulted in butanediol levels that were 20-40% higher than could be obtained with a separate hydrolysis and fermentation process. The hemicellulose-rich water-soluble fractions obtained from a variety of steam-exploded agricultural residues could be readily used by the combined hydrolysis and fermentation approach resulting in butanediol yields of 0.4-0.5 g/g of reducing sugar utilized.

  3. Methods for determining enzymatic activity comprising heating and agitation of closed volumes

    Science.gov (United States)

    Thompson, David Neil; Henriksen, Emily DeCrescenzo; Reed, David William; Jensen, Jill Renee

    2016-03-15

    Methods for determining thermophilic enzymatic activity include heating a substrate solution in a plurality of closed volumes to a predetermined reaction temperature. Without opening the closed volumes, at least one enzyme is added, substantially simultaneously, to the closed volumes. At the predetermined reaction temperature, the closed volumes are agitated and then the activity of the at least one enzyme is determined. The methods are conducive for characterizing enzymes of high-temperature reactions, with insoluble substrates, with substrates and enzymes that do not readily intermix, and with low volumes of substrate and enzyme. Systems for characterizing the enzymes are also disclosed.

  4. Effects of lead contamination on soil enzymatic activities, microbial biomass, and rice physiological indices in soil-lead-rice (Oryza sativa L.) system.

    Science.gov (United States)

    Zeng, Lu S; Liao, Min; Chen, Cheng L; Huang, Chang Y

    2007-05-01

    The effect of lead (Pb) treatment on the soil enzymatic activities, soil microbial biomass, rice physiological indices and rice biomass were studied in a greenhouse pot experiment. Six levels of Pb viz. 0(CK), 100, 300, 500, 700, 900 mg/kg soil were applied in two types of paddy soils. The results showed that Pb treatment had a stimulating effect on soil enzymatic activities and microbial biomass carbon (Cmic) at low concentration and an inhibitory influence at higher concentration. The degree of influence on enzymatic activities and Cmic by Pb was related to the clay and organic matter contents of the soils. When the Pb treatment was raised to the level of 500 mg/kg, ecological risk appeared both to soil microorganisms and plants. The results also revealed a consistent trend of increased chlorophyll contents and rice biomass initially, maximum at a certain Pb treatment, and then decreased gradually with the increase in Pb concentration. Pb was effective in inducing proline accumulation and its toxicity causes oxidative stress in rice plants. Therefore, it was concluded that soil enzymatic activities, Cmic and rice physiological indices, could be sensitive indicators to reflect environmental stress in soil-lead-rice system.

  5. Hibiscus sabdariffa L. and Its Bioactive Constituents Exhibit Antiviral Activity against HSV-2 and Anti-enzymatic Properties against Urease by an ESI-MS Based Assay.

    Science.gov (United States)

    Hassan, Sherif T S; Švajdlenka, Emil; Berchová-Bímová, Kateřina

    2017-04-30

    For decades, Hibiscus sabdariffa L. and its phytochemicals have been shown to possess a wide range of pharmacologic properties. In this study, aqueous extract of Hibiscus sabdariffa (AEHS) and its bioactive constituent protocatechuic acid (PCA), have been evaluated in vitro for their antiviral activity against HSV-2 clinical isolates and anti-enzymatic activity against urease. Antiherpetic activity was evaluated by the titer reduction assay in infected Vero cells, and cytotoxicity was evaluated by the neutral red dye-uptake method. Anti-urease activity was determined by a developed Electrospray Ionization-Mass Spectrometry (ESI-MS)-based assay. PCA showed potent anti-HSV-2 activity compared with that of acyclovir, with EC 50 values of 0.92 and 1.43 µg∙mL -1 , respectively, and selectivity indices > 217 and > 140, respectively. For the first time, AEHS was shown to exert anti-urease inhibition activity, with an IC 50 value of 82.4 µg∙mL -1 . This, combined with its safety, could facilitate its use in practical applications as a natural urease inhibitor. Our results present Hibiscus sabdariffa L. and its bioactive compound PCA as potential therapeutic agents in the treatment of HSV-2 infection and the treatment of diseases caused by urease-producing bacteria.

  6. Hibiscus sabdariffa L. and Its Bioactive Constituents Exhibit Antiviral Activity against HSV-2 and Anti-enzymatic Properties against Urease by an ESI-MS Based Assay

    Directory of Open Access Journals (Sweden)

    Sherif T. S. Hassan

    2017-04-01

    Full Text Available For decades, Hibiscus sabdariffa L. and its phytochemicals have been shown to possess a wide range of pharmacologic properties. In this study, aqueous extract of Hibiscus sabdariffa (AEHS and its bioactive constituent protocatechuic acid (PCA, have been evaluated in vitro for their antiviral activity against HSV-2 clinical isolates and anti-enzymatic activity against urease. Antiherpetic activity was evaluated by the titer reduction assay in infected Vero cells, and cytotoxicity was evaluated by the neutral red dye-uptake method. Anti-urease activity was determined by a developed Electrospray Ionization-Mass Spectrometry (ESI-MS-based assay. PCA showed potent anti-HSV-2 activity compared with that of acyclovir, with EC50 values of 0.92 and 1.43 µg∙mL−1, respectively, and selectivity indices > 217 and > 140, respectively. For the first time, AEHS was shown to exert anti-urease inhibition activity, with an IC50 value of 82.4 µg∙mL−1. This, combined with its safety, could facilitate its use in practical applications as a natural urease inhibitor. Our results present Hibiscus sabdariffa L. and its bioactive compound PCA as potential therapeutic agents in the treatment of HSV-2 infection and the treatment of diseases caused by urease-producing bacteria.

  7. Production of xylooligosaccharide from wheat bran by microwave assisted enzymatic hydrolysis.

    Science.gov (United States)

    Wang, Tseng-Hsing; Lu, Shin

    2013-06-01

    The effective production of xylooligosaccharides (XOS) from wheat bran was investigated. Wheat bran contains rich hemicellulose which can be hydrolyzed by enzyme; the XOS were obtained by microwave assisted enzymatic hydrolysis. To improve the productivity of XOS, repeated microwave assisted enzymatic hydrolysis and activated carbon adsorption method was chosen to eliminate macromolecules in the XOS. On the basis of experimental data, an industrial XOS production process consisting of pretreatment, repeated microwave assisted enzymatic treatment and purification was designed. Using the designed process, 3.2g dry of purified XOS was produced from 50 g dry wheat bran powder. Copyright © 2012 Elsevier Ltd. All rights reserved.

  8. Blood parameters and enzymatic and oxidative activity in the liver of chickens fed with calcium anacardate

    Directory of Open Access Journals (Sweden)

    Carlos Eduardo Braga Cruz

    Full Text Available ABSTRACT The aim of this research was to evaluate the inclusion of calcium anacardate (CAC as a source of anacardic acid in the diet of broiler chickens on blood parameters, and enzymatic and oxidative activity in the liver. A total of 840 male chicks, one day old, were kept in a completely randomised experimental design, with six treatments and seven replications of 20 birds, totalling 140 birds per treatment. The treatments consisted of feed without the addition of growth promoter (GP, feed with GP, and feed with no GP and the addition of CAC at levels of 0.25, 0.50, 0.75 and 1%. The biochemical blood variables to be analysed were uric acid, total cholesterol, HDL, LDL, creatinine, AST, ALT, triglycerides, total erythrocytes, haemoglobin, haematocrit, mean corpuscular volume, corpuscular haemoglobin concentration, total plasma protein, total leukocytes, heterophils, lymphocytes, platelets and heterophil/lymphocyte ratio. The concentrations of superoxide dismutase, glutathione peroxidase and malondialdehyde were analysed for the enzymatic and oxidative parameters in the liver. There were no significant differences between treatments in the blood parameters or the enzymatic and oxidative activity in the liver of the chickens, demonstrating that the use of calcium anacardate as a source of anacardic acid is non-toxic, and does not affect these parameters.

  9. Plasmin enzymatic activity in the presence of actin

    Directory of Open Access Journals (Sweden)

    Yusova E. I.

    2015-10-01

    Full Text Available Aim. To study the changes in the plasmin activity towards substrates with high and low molecular mass in the presence of actin. Methods. The proteins used for this investigation were obtained by affinity chromatography and gel-filtration. The plasmin enzymatic activity was determined by a turbidimetric assay and a chromogenic substrate-based assay. The enzyme linked immunosorbent assay and biotin-avidin-phosphatase system were used to study the interaction of plasminogen and its fragments with actin. Results. It was shown that G-actin causes 1.5-fold decrease in the rate of polymeric fibrin hydrolysis by plasmin and Glu-plasminogen activated by the tissue plasminogen activator. However, actin did not impede plasmin autolysis and had no influence on its amidase activity. We have studied an interaction of biotinylated Glu-plasminogen and its fragments (kringle 1-3, kringle 4 and mini-plasminogen with immobilized G-actin. Glu-plasminogen and kringle 4 had a high affinity towards actin (C50 is 113 and 117 nM correspondingly. Mini-plasminogen and kringe 4 did not bind to actin. A similar affinity of Glu-plasminogen and kringle 1-3 towards actin proves the involvement of the kringle 1-3 lysine-binding sites of the native plasminogen form in the actin interaction. Conclusions. Actin can modulate plasmin specificity towards high molecular mass substrates through its interaction with lysine-binding sites of the enzyme kringle domains. Actin inhibition of the fibrinolytic activity of plasmin is due to its competition with fibrin for thelysine binding sites of plasminogen/plasmin.

  10. Blocked Enzymatic Etching of Gold Nanorods: Application to Colorimetric Detection of Acetylcholinesterase Activity and Its Inhibitors.

    Science.gov (United States)

    Saa, Laura; Grinyte, Ruta; Sánchez-Iglesias, Ana; Liz-Marzán, Luis M; Pavlov, Valeri

    2016-05-04

    The anisotropic morphology of gold nanorods (AuNRs) has been shown to lead to nonuniform ligand distribution and preferential etching through their tips. We have recently demonstrated that this effect can be achieved by biocatalytic oxidation with hydrogen peroxide, catalyzed by the enzyme horseradish peroxidase (HRP). We report here that modification of AuNRs with thiol-containing organic molecules such as glutathione and thiocholine hinders enzymatic AuNR etching. Higher concentrations of thiol-containing molecules in the reaction mixture gradually decrease the rate of enzymatic etching, which can be monitored by UV-vis spectroscopy through changes in the AuNR longitudinal plasmon band. This effect can be applied to develop novel optical assays for acetylcholinesterase (AChE) activity. The biocatalytic hydrolysis of acetylthiocholine by AChE yields thiocholine, which prevents enzymatic AuNR etching in the presence of HRP. Additionally, the same bioassay can be used for the detection of nanomolar concentrations of AChE inhibitors such as paraoxon and galanthamine.

  11. A singular enzymatic megacomplex from Bacillus subtilis.

    Science.gov (United States)

    Straight, Paul D; Fischbach, Michael A; Walsh, Christopher T; Rudner, David Z; Kolter, Roberto

    2007-01-02

    Nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and hybrid NRPS/PKS are of particular interest, because they produce numerous therapeutic agents, have great potential for engineering novel compounds, and are the largest enzymes known. The predicted masses of known enzymatic assembly lines can reach almost 5 megadaltons, dwarfing even the ribosome (approximately 2.6 megadaltons). Despite their uniqueness and importance, little is known about the organization of these enzymes within the native producer cells. Here we report that an 80-kb gene cluster, which occupies approximately 2% of the Bacillus subtilis genome, encodes the subunits of approximately 2.5 megadalton active hybrid NRPS/PKS. Many copies of the NRPS/PKS assemble into a single organelle-like membrane-associated complex of tens to hundreds of megadaltons. Such an enzymatic megacomplex is unprecedented in bacterial subcellular organization and has important implications for engineering novel NRPS/PKSs.

  12. Enzymatic degradation of cyclic 2,3-diphosphoglycerate to 2,3-diphosphoglycerate in Methanobacterium thermoautotrophicum.

    Science.gov (United States)

    Sastry, M V; Robertson, D E; Moynihan, J A; Roberts, M F

    1992-03-24

    2,3-Diphosphoglycerate (2,3-DPG) has been found to be the product of the enzymatic degradation of cyclic 2,3-diphosphoglycerate (cDPG) in the archaebacterium Methanobacterium thermoautotrophicum delta H. Although 2,3-DPG has not previously been detected as a major soluble component of M. thermoautotrophicum, large pools accumulated at an incubation temperature of 50 degrees C (below the optimum growth temperature of 62 degrees C). Under these conditions, cellular activity was significantly decreased; a return of the culture to the optimum growth temperature restored the 2,3-DPG pool back to original low levels and caused steady-state cDPG levels to increase again. While 13CO2-pulse/12CO2-chase experiments at 50 degrees C showed that the cDPG turned over, the appearance of 2,3-DPG at NMR-visible concentrations required at least 10 h. Production of 2,3-DPG in vivo was prevented by exposure of the cells to O2. The enzyme responsible for this hydrolysis of cDPG was purified by affinity chromatography and appears to be a 33-kDa protein. Activity was detected in the presence of oxygen and was enhanced by a solution of 1 M KCl, 25 mM MgCl2, and dithiothreitol. Both Km and Vmax have been determined at 37 degrees C; kinetics also indicate that in vitro the product, 2,3-DPG, is an inhibitor of cDPG hydrolysis. These findings are discussed in view of a proposed role for cDPG in methanogens.

  13. Evaluation of Oil Removal Efficiency and Enzymatic Activity in Some fungal Strains for Bioremediation of Petroleum-Polluted Soils

    Directory of Open Access Journals (Sweden)

    Fariba Mohsenzadeh

    2012-12-01

    Full Text Available Background: Petroleum pollution is a global disaster and there are several soil cleaning methods including bioremediation.Methods: In a field study, fugal strains were isolated from oil-contaminated sites of Arak refinery (Iran and their growth ability was checked in potato dextrose agar (PDA media containing 0-10% v/v crude oil, the activity of three enzymes (Catalase, Peroxidase and Phenol Oxidase was evaluated in the fungal colonies and bioremediation ability of the fungi was checked in the experimental pots containing 3 kg sterilized soil and different concentrations of petroleum (0-10% w/w.Results: Four fungal strains, Acromonium sp., Alternaria sp., Aspergillus terreus and Penicillium sp., were selected asthe most resistant ones. They were able to growth in the subjected concentrations and Alternaria sp. showed thehighest growth ability in the petroleum containing media. The enzyme assay showed that the enzymatic activity was increased in the oil-contaminated media. Bioremediation results showed that the studied fungi were able to decrease petroleum pollution. The highest petroleum removing efficiency of Aspergillus terreus, Penicillium sp.,Alternaria sp. and Acromonium sp. was evaluated in the 10%, 8%, 8% and 2% petroleum pollution respectively.Conclusions: Fungi are important microorganisms in decreasing of petroleum pollution. They have bioremediation potency that is related to their enzymatic activities.

  14. Lactose hydrolysis in an enzymatic membrane reactor

    Energy Technology Data Exchange (ETDEWEB)

    Mertens, B; Huyghebaert, A

    1987-10-01

    The enzymatic hydrolysis of lactose in whey permeate with subsequent recuperation of Saccharomyces lactis lactase by means of ultrafiltration was investigated. In whey permeate, S. lactis lactase shows maximal activity at pH 6.5; the optimal temperature was found to be 45/sup 0/C and is limited by strong thermal inactivation beyond this temperature. High activity combined with acceptable thermal inactivation (< 10% after 5 h incubation) was established at 30/sup 0/C. S. lactis lactase also displays considerable activity at low temperature (5/sup 0/C). Enzyme stability is reduced drastically by demineralisation: addition of low concentrations of manganese ions (10/sup -3/ M) considerably enhances stability. Using a DDS Lab-Unit 35 fitted with GR61PP polysulphon membranes (cut-off: 20.000), pilot scale experiments were carried out (pH 6.5; 30/sup 0/C) in which whey permeate was hydrolyzed to a degree of hydrolysis of 82% minimum. Enzyme recuperation amounted to 96.5% per batch, all enzyme activity loss being due to thermal inactivation. Microbiological examination of the enzymatic membrane reactor showed that growth of mcicroorganisms can largely be suppressed by working at lower temperature (5/sup 0/C). Eventually, 50 ppm H/sub 2/O/sub 2/ or sterile filtration will adequately solve microbiological problems without affecting enzyme activity.

  15. Enzymatic digestive activity and absorption efficiency in Tagelus dombeii upon Alexandrium catenella exposure

    Science.gov (United States)

    Fernández-Reiriz, M. J.; Navarro, J. M.; Cisternas, B. A.; Babarro, J. M. F.; Labarta, U.

    2013-12-01

    We analyzed absorption efficiency (AE) and digestive enzyme activity (amylase, cellulase complex, and laminarinase) of the infaunal bivalve Tagelus dombeii originating from two geographic sites, Corral-Valdivia and Melinka-Aysén, which have different long-term paralytic shellfish poisoning (PSP) exposure rates. We report the effects of past feeding history (origin) on T. dombeii exposed to a mixed diet containing the toxic dinoflagellate Alexandrium catenella and another dinoflagellate-free control diet over a 12-day period in the laboratory. Absorption efficiency values of T. dombeii individuals that experienced PSP exposure in their habitat (Melinka-Aysén) remained unchanged during exposure to toxic food in the laboratory. In contrast, T. dombeii from a non-PSP exposure field site (Corral-Valdivia) showed a significant reduction in AE with toxic exposure time. This study established that the amylase and cellulase complexes were the most important enzymes in the digestive glands of Tagelus from both sites. The temporal evolution of enzymatic activity under toxic diet was fitted to exponential (amylase and cellulase) and to a logarithmic (laminarinase) models. In all fits, we found significant effect of origin in the model parameters. At the beginning of the experiment, higher enzymatic activity was observed for clams from Corral-Valdivia. The amylase activity decreased with time exposure for individuals from Corral and increased for individuals from Melinka. Cellulase activity did not vary over time for clams from Corral, but increased for individuals from Melinka and laminarinase activity decreased over time for individuals from Corral and remained unchanged over time for Melinka. A feeding history of exposure to the dinoflagellate A. catenella was reflected in the digestive responses of both T. dombeii populations.

  16. Phospholipase C produced by Clostridium botulinum types C and D: comparison of gene, enzymatic, and biological activities with those of Clostridium perfringens alpha-toxin.

    Science.gov (United States)

    Fatmawati, Ni Nengah Dwi; Sakaguchi, Yoshihiko; Suzuki, Tomonori; Oda, Masataka; Shimizu, Kenta; Yamamoto, Yumiko; Sakurai, Jun; Matsushita, Osamu; Oguma, Keiji

    2013-01-01

    Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs), the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival.

  17. Assessment of Napropamide Dissipation and its Effect on Soil Enzymatic Activity

    Directory of Open Access Journals (Sweden)

    Mirosław Onyszko

    2017-11-01

    Full Text Available This paper assesses the dissipation of napropamide and its impact on the activity of dehydrogenases, alkaline phosphatase, acid phosphatase, and urease in sandy clay loam. The experiment was carried out on soil samples with organic carbon content of 12.08 g·kg-1, total nitrogen content of 0.97 g·kg-1, and pH 5.24 with the following variable factors: (a dose of Devrinol 450 SC formation (containing 450 g of napropamide in dm3: 0 (control, 0.5, 1, 2, 4, 8, and 16-fold hold of field dose; (b day of experiment: 1, 7, 14, 28, 56, and 112. The half-life of napropamide ranged from 33.50 to 71.42 days. The use of napropamide at the dose recommended by the manufacturer and at the dose reduced by half appeared to exhibit low toxicity in relation to enzymes determined. In contrast, the application of elevated napropamide doses decreased the values of biochemical parameters of the soil in most cases. The Pearson correlation coefficients showed statistically significant negative correlation between the content of napropamide residues and the enzymatic activity of the soil.

  18. Conformational change results in loss of enzymatic activity of jack bean urease on its interaction with silver nanoparticle.

    Science.gov (United States)

    Ponnuvel, Shobana; Subramanian, Balakumar; Ponnuraj, Karthe

    2015-10-01

    Urease is an enzyme produced by microbes such as bacteria, yeast and fungi. Plants also produce this enzyme. Urease action splits urea into ammonia and carbamate. This action is having important implications in agro-chemical, medicinal and environment. Therefore there is always a constant search for new and novel compounds which could inhibit this enzyme. Here we have studied the interaction of jack bean urease (JBU) with silver nanoparticle to analyze the influence of the resultant protein corona formation on the catalytic property of JBU. Several techniques like UV-Vis, gel shift assay and CD spectroscopy have been used to characterize this interaction. Urease activity assay suggests that the protein corona formation inhibits the enzymatic action of JBU. The loss of enzymatic action could be either due to the nanoparticle blocking the active site of JBU or a conformational change in the protein. The CD spectra of JBU-AgNP complexes clearly revealed significant changes in the secondary structural composition of the JBU and this could be the reason for the loss of enzymatic activity of JBU. This study revealed an interesting observation, where the interaction of AgNP with JBU resulted destabilization of hexameric nature of JBU which is otherwise highly stable. The results of the present study could be useful in the development of nanoparticle based material for inhibiting the ureolytic activity of ureases in different fields.

  19. The effect of ultraviolet treatment on enzymatic activity and total phenolic content of minimally processed potato slices.

    Science.gov (United States)

    Teoh, Li Shing; Lasekan, Ola; Adzahan, Noranizan Mohd; Hashim, Norhashila

    2016-07-01

    In this work, potato slices were exposed to different doses of UV-C irradiation (i.e. 2.28, 6.84, 11.41, and 13.68 kJ m -2 ) with or without pretreatment [i.e. ascorbic acid and calcium chloride (AACCl) dip] and stored at 4 ± 1 °C. Changes in enzymatic activities of polyphenol oxidase (PPO), peroxidase (POD) and phenylalanine ammonia lyase (PAL), as well as total phenolic content (TPC) were investigated after 0, 3, 7 and 10 days of storage. Results showed that untreated and UV-C treated potato slices at 13.68 kJ m -2 dosage level showed significantly higher PPO, POD and PAL activities. Conversely, untreated potato slices showed the lowest TPC during storage period. Potato slices subjected to AACCl dip plus UV-C at 6.84 kJ m -2 produced lower PPO, POD and PAL activities, as well as maintained a high TPC during storage.

  20. Influence of Different Lignocellulose Sources on Endo-1,4-β-Glucanase Gene Expression and Enzymatic Activity of Bacillus amyloliquefaciens B31C

    Directory of Open Access Journals (Sweden)

    Rosangela Di Pasqua

    2014-01-01

    Full Text Available Conversion of cellulose into fermentable sugars for ethanol production is currently performed by enzymatic hydrolysis catalyzed by cellulases. The cellulases are produced by a wide variety of microorganisms, playing a major role in the recycling of biomass. The endo-1,4-β-glucanase (CelB31C from Bacillus amyloliquefaciens B31C, isolated from compost and previously selected on the basis of highest cellulase activity levels among Bacillus isolated, was characterized as being a potential candidate for a biocatalyst in lignocellulose conversion for second-generation bioethanol production. The aim of this work was to evaluate the changes in production of enzymatic activity of the endo-1,4-β-glucanase (CelB31C and the expression of its gene (bglC using a carboxymethylcellulase activity assay and qRT-PCR analysis, respectively, during growth of B. amyloliquefaciens B31C on different cellulose sources: carboxymethylcellulose (CMC, pure cellulose from Arundo donax, pretreated Arundo donax biomass (Chemtex, and microcrystalline cellulose (Avicel. The results showed that both the expression of bglC gene and the enzymatic activity production are related to the type of cellulose source. The strain showed a high enzymatic activity on lignocellulosic biomass and on microcrystalline cellulose. Furthermore, the highest gene expression occurred during the exponential phase of growth, except in the presence of Avicel.

  1. Secretory phospholipase A2-mediated neuronal cell death involves glutamate ionotropic receptors

    DEFF Research Database (Denmark)

    Kolko, Miriam; de Turco, Elena B; Diemer, Nils Henrik

    2002-01-01

    To define the significance of glutamate ionotropic receptors in sPLA -mediated neuronal cell death we used the NMDA receptor antagonist MK-801 and the AMPA receptor antagonist PNQX. In primary neuronal cell cultures both MK-801 and PNQX inhibited sPLA - and glutamate-induced neuronal death. [ H...

  2. Enzymatic synthesis of dimeric glycomimetic ligands of NK cell activation receptors

    Czech Academy of Sciences Publication Activity Database

    Drozdová, Anna; Bojarová, Pavla; Křenek, Karel; Weignerová, Lenka; Hensen, B.; Elling, L.; Christensen, H.; Jensen, H.H.; Pelantová, Helena; Kuzma, Marek; Bezouška, Karel; Krupová, Monika; Adámek, David; Slámová, Kristýna; Křen, Vladimír

    2011-01-01

    Roč. 346, č. 12 (2011), s. 1599-1609 ISSN 0008-6215 R&D Projects: GA ČR GP203/09/P024; GA ČR GD305/09/H008; GA ČR GA303/09/0477 Institutional research plan: CEZ:AV0Z50200510 Keywords : beta-N-Acetylhexosaminidase * alactosyltransferase * Enzymatic glycosylation Subject RIV: CE - Biochemistry Impact factor: 2.332, year: 2011

  3. The feasibility of single-port laparoscopic appendectomy using a solo approach: a comparative study.

    Science.gov (United States)

    Kim, Say-June; Choi, Byung-Jo; Jeong, Wonjun; Lee, Sang Chul

    2016-03-01

    To investigate the feasibility and safety of solo surgery with single-port laparoscopic appendectomy, which is termed herein solo-SPLA (solo-single-port laparoscopic appendectomy). This study prospectively collected and retrospectively analyzed data from patients who had undergone either non-solo-SPLA (n = 150) or solo-SPLA (n = 150). Several devices were utilized for complete, skin-to-skin solo-SPSA, including a Lone Star Retractor System and an adjustable mechanical camera holder. Operating times were not significantly different between solo- and non-solo-SPLA (45.0 ± 21.0 minutes vs. 46.7 ± 26.1 minutes, P = 0.646). Most postoperative variables were also comparable between groups, including the necessity for intravenous analgesics (0.7 ± 1.2 ampules [solo-SPLA] vs. 0.9 ± 1.5 ampules [non-solo-SPLA], P = 0.092), time interval to gas passing (1.3 ± 1.0 days vs. 1.4 ± 1.0 days, P = 0.182), and the incidence of postoperative complications (4.0% vs. 8.7%, P = 0.153). Moreover, solo-SPLA effectively lowered the operating cost by reducing surgical personnel expenses. Solo-SPLA economized staff numbers and thus lowered hospital costs without lengthening of operating time. Therefore, solo-SPLA could be considered a safe and feasible alternative to non-solo-SPLA.

  4. Metabolic regulation analysis of an ethanologenic Escherichia coli strain based on RT-PCR and enzymatic activities

    Directory of Open Access Journals (Sweden)

    Martinez Alfredo

    2008-05-01

    Full Text Available Abstract Background A metabolic regulation study was performed, based upon measurements of enzymatic activities, fermentation performance, and RT-PCR analysis of pathways related to central carbon metabolism, in an ethanologenic Escherichia coli strain (CCE14 derived from lineage C. In comparison with previous engineered strains, this E coli derivative has a higher ethanol production rate in mineral medium, as a result of the elevated heterologous expression of the chromosomally integrated genes encoding PDCZm and ADHZm (pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis. It is suggested that this behavior might be due to lineage differences between E. coli W and C. Results This study demonstrated that the glycolytic flux is controlled, in this case, by reactions outside glycolysis, i.e., the fermentative pathways. Changes in ethanol production rate in this ethanologenic strain result in low organic acid production rates, and high glycolytic and ethanologenic fluxes, that correlate with enhanced transcription and enzymatic activity levels of PDCZm and ADHZm. Furthermore, a higher ethanol yield (90% of the theoretical in glucose-mineral media was obtained with CCE14 in comparison with previous engineered E. coli strains, such as KO11, that produces a 70% yield under the same conditions. Conclusion Results suggest that a higher ethanol formation rate, caused by ahigher PDCZm and ADHZm activities induces a metabolic state that cells compensate through enhanced glucose transport, ATP synthesis, and NAD-NADH+H turnover rates. These results show that glycolytic enzymatic activities, present in E. coli W and C under fermentative conditions, are sufficient to contend with increases in glucose consumption and product formation rates.

  5. Some aspects of rat platelet and serum phospholipase A2 activities

    NARCIS (Netherlands)

    Aarsman, A.J.; Roosenboom, C.F.P.; Geffen, G.E.W. van; Bosch, H. van den

    1985-01-01

    Rat platelet lysate contained appreciable phospholipase A2 activity. In agreement with literature data this enzymatic activity eluted in the void volume of a Sephadex G-100 column. When the void volume peak was chromatographed over a Matrex gel blue A column, part of the phospholipase A2 activity

  6. Enzymatic activity of a novel halotolerant lipase from Haloarcula hispanica 2TK2

    Directory of Open Access Journals (Sweden)

    Ozgen Melis

    2016-06-01

    Full Text Available A strain of Haloarcula hispanica isolated from Tuzkoy salt mine, Turkey exhibited extracellular lipolytic activity. Important parameters such as carbon sources and salt concentration for lipase production were investigated. Optimal conditions for the enzyme production from Haloarcula hispanica 2TK2 were determined. It was observed that the lipolytic activity of Haloarcula hispanica was stimulated by some of the carbon sources. The high lipase acitivity values were obtained in the presence of 2% (v/v walnut oil (6.16 U/ml, 1% (v/v fish oil (5.07 U/ml, 1% (v/v olive oil (4.52 U/ml and 1% (w/v stearic acid (4.88 U/ml at 4M NaCl concentration. Lipase was partially purified by ammonium sulfate precipitation and ultrafiltration. Optimal temperature and pH values were determined as 45°C and 8.0, respectively. Lipase activity decreased with the increasing salt concentration, but 85% activity of the enzyme was maintained at 5M NaCl concentration. The enzyme preserved 41% of its relative activity at 90°C. The partially purified lipase maintained its activity in the presence of surfactants such as Triton X-100 and SDS. Therefore, the lipase which is an extremozyme may have potential applications especially in detergent industry.

  7. Preparation and Enzymatic Degradation of Porous Crosslinked Polylactides of Biomass Origin

    Directory of Open Access Journals (Sweden)

    Yuya Kido

    2014-06-01

    Full Text Available To understand the enzymatic degradation behavior of crosslinked polylactide (PLA, the preparation and enzymatic degradation of both thermoplastic (linear and crosslinked PLAs that have pore structures with different dimensions were carried out. The porous structures of the linear PLA samples were of micro and nanoporous nature, and prepared by batch foaming with supercritical CO2 and compared with the porous structures of crosslinked PLA (Lait-X created by the salt leaching method. The surface and cross-sectional morphologies of the porous structures were investigated by using scanning electron microscopy. The morphological analysis of porous Lait-X showed a rapid loss of physical features within 120 h of exposure to proteinase-K enzymatic degradation at 37 °C. Due to the higher affinity for water, enhanced enzymatic activity as compared to the linear PLA porous structures in the micro and nanoporous range was observed.

  8. Impact of the redox-cycling herbicide diquat on transcript expression and antioxidant enzymatic activities of the freshwater snail Lymnaea stagnalis

    Energy Technology Data Exchange (ETDEWEB)

    Bouetard, Anthony, E-mail: anthony.bouetard@rennes.inra.fr [INRA, UMR INRA-Agrocampus Ouest ESE 0985, Equipe Ecotoxicologie et Qualite des Milieux Aquatiques, 65 rue de Saint-Brieuc, 35042 Rennes cedex (France); Besnard, Anne-Laure; Vassaux, Daniele; Lagadic, Laurent; Coutellec, Marie-Agnes [INRA, UMR INRA-Agrocampus Ouest ESE 0985, Equipe Ecotoxicologie et Qualite des Milieux Aquatiques, 65 rue de Saint-Brieuc, 35042 Rennes cedex (France)

    2013-01-15

    The presence of pesticides in the environment results in potential unwanted effects on non-target species. Freshwater organisms inhabiting water bodies adjacent to agricultural areas, such as ditches, ponds and marshes, are good models to test such effects as various pesticides may reach these habitats through several ways, including aerial drift, run-off, and drainage. Diquat is a non-selective herbicide used for crop protection or for weed control in such water bodies. In this study, we investigated the effects of diquat on a widely spread aquatic invertebrate, the holarctic freshwater snail Lymnaea stagnalis. Due to the known redox-cycling properties of diquat, we studied transcript expression and enzymatic activities relative to oxidative and general stress in the haemolymph and gonado-digestive complex (GDC). As diquat is not persistent, snails were exposed for short times (5, 24, and 48 h) to ecologically relevant concentrations (22.2, 44.4, and 222.2 {mu}g l{sup -1}) of diquat dibromide. RT-qPCR was used to quantify the transcription of genes encoding catalase (cat), a cytosolic superoxide dismutase (Cu/Zn-sod), a selenium-dependent glutathione peroxidase (gpx), a glutathione reductase (gred), the retinoid X receptor (rxr), two heat shock proteins (hsp40 and hsp70), cortactin (cor) and the two ribosomal genes r18S and r28s. Enzymatic activities of SOD, Gpx, Gred and glutathione S-transferase (GST) were investigated in the GDC using spectrophoto/fluorometric methods. Opposite trends were obtained in the haemolymph depending on the herbicide concentration. At the lowest concentration, effects were mainly observed after 24 h of exposure, with over-transcription of cor, hsp40, rxr, and sod, whereas higher concentrations down-regulated the expression of most of the studied transcripts, especially after 48 h of exposure. In the GDC, earlier responses were observed and the fold-change magnitude was generally much higher: transcription of all target genes increased

  9. Impact of potato cultivation and cattle farming on physicochemical parameters and enzymatic activities of Neotropical high Andean Páramo ecosystem soils.

    Science.gov (United States)

    Avellaneda-Torres, Lizeth Manuela; León Sicard, Tomás Enrique; Torres Rojas, Esperanza

    2018-08-01

    The Andean Páramos are high mountain ecosystems whose soils are essential for the management of South American water resources, but research on anthropic impacts to these soils is currently minimal and insufficient. The objective of this study was to evaluate the impacts of potato (Solanum tuberosum) cultivation and livestock on the physicochemical parameters and enzymatic activities that determine the soil quality of the Neotropical high Andean Páramo ecosystem in the Nevados National Natural Park (Nevados NNP) in Colombia. It was hypothesised that sites with potato crops and livestock farming would exhibit significant changes in soil physicochemical parameters and enzymatic activities compared with Páramo sites that have been conserved without agriculture. Samples were collected from soils under potato cultivation, livestock and Páramo (subject to the lowest degree of human intervention possible), on three farms in the El Bosque District at three different altitudes (Buenos Aires, El Edén and La Secreta) during two seasons (dry and rainy). The results showed that none of the physical parameters under study presented statistically significant differences due to the type of use (livestock, potato crop or Páramo), season of sampling (dry or rainy season) or altitude (different farms). The chemical parameters that statistically significantly differed due to land use were organic carbon, cation exchange capacity, calcium, potassium, and ammonium and those that showed statistically significant differences associated with the sampling timing were organic carbon, nitrogen, cation exchange capacity, total carbon, C/N and nitrate. Additionally, there were differences in organic carbon due to the altitude of the farms. With respect to enzymatic activities, those of β-glucosidase, phosphodiesterase and urease significantly decreased in soils under potato cultivation and livestock relative to those of Páramo, but those of acid phosphatase and protease increased

  10. Tunable Enzymatic Activity and Enhanced Stability of Cellulase Immobilized in Biohybrid Nanogels.

    Science.gov (United States)

    Peng, Huan; Rübsam, Kristin; Jakob, Felix; Schwaneberg, Ulrich; Pich, Andrij

    2016-11-14

    the enzyme. The biohybrid nanogels demonstrated significantly improved stability in preserving enzymatic activity compared with free cellulase. The functional biohybrid nanogels with tunable enzymatic activity and improved stability are promising candidates for applications in biocatalysis, biomass conversion, or energy utilization fields.

  11. Enzymatic vegetable organic extracts as soil biochemical biostimulants and atrazine extenders.

    Science.gov (United States)

    García-Martínez, Ana María; Tejada, Manuel; Díaz, Ana Isabel; Rodríguez-Morgado, Bruno; Bautista, Juan; Parrado, Juan

    2010-09-08

    The purpose of this study was to gather information on the potential effects of organic biostimulants on soil activity and atrazine biodegradation. Carob germ enzymatic extract (CGEE) and wheat condensed distiller solubles enzymatic extract (WCDS-EE) have been obtained using an enzymatic process; their main organic components are soluble carbohydrates and proteins in the form of peptides and free amino acids. Their application to soil results in high biostimulation, rapidly increased dehydrogenase, phosphatase and glucosidase activities, and an observed atrazine extender capacity due to inhibition of its mineralization. The extender capacity of both extracts is proportional to the protein/carbohydrate ratio content. As a result, these enzymatic extracts are highly microbially available, leading to two independent phenomena, fertility and an atrazine persistence that is linked to increased soil activity.

  12. Chemical interaction of disulfiram with nitrosodimethylamine after in vitro enzymatic activation

    International Nuclear Information System (INIS)

    Tacchi, A.M.; Bertram, B.; Wiessler, M.

    1984-01-01

    The in vitro reaction between disulfiram (DSF) and N-nitroso[ 14 C]dimethylamine [( 14 C]NDMA) was studied. Incubations of DSF with [ 14 C]NDMA were carried out in the presence of rat liver microsomes, control 9000 g (S9) supernatant fraction and phenobarbital-induced S9 fraction. HPLC analysis and liquid scintillation measurement provided evidence for the formation of methyldiethyldithiocarbamate (MeDDTC) as a product of the reaction between diethyldithiocarbamate (DDTC), the main active metabolite of DSF and the 'methyl-cation' released by NDMA after enzymatic activation. The amount of MeDDTC found here was consistent with the rate of oxidation of NDMA to formaldehyde. Scintillation counting confirmed that other radioactive peaks, not due to MeDDTC, were unrelated to the methylation of L-cysteine by [ 14 C]NDMA

  13. Enzymatic synthesis of tritium-labelled prostaglandin D[sub 2] and its conversion to other prostaglandins

    Energy Technology Data Exchange (ETDEWEB)

    Shram, S.I.; Lazurkina, T.Yu.; Shevchenko, V.P.; Nagaev, I.Yu.; Myasoedov, N.F. (AN SSSR, Moscow (Russian Federation). Inst. Molekulyarnoj Genetiki)

    1994-04-01

    The one-stage enzymatic synthesis of tritium-labelled prostaglandin D[sub 2] from labelled arachidonic acid was performed by using the enzyme system PGH-synthetase/PGH-PGD-isomerase. By enzymatic and chemical transformation of [[sup 3]H]PGD[sub 2] the following compounds were obtained: 15-keto-13,14-dihydro-[[sup 3]H]PGD[sub 2], 9[alpha],11[beta]-[[sup 3]H]PGF[sub 2], 9-deoxy-[Delta][sup 9]-[[sup 3]H]-PGD[sub 2] ([[sup 3]H]PGJ[sub 2]) and [Delta][sup 12]-13,14-dihydro-[[sup 3]H]PGJ[sub 2]. It was found that L-selectride is a more effective reducing agent than sodium borohydride in the synthesis of 9[alpha], 11[beta]-[[sup 3]H]PGF[sub 2]. (Author).

  14. Enzymatic Saccharification of Lignocelluloses Should be Conducted at Elevated pH 5.2-6.2

    Science.gov (United States)

    T.Q. Lan; Hongming Lou; J.Y. Zhu

    2013-01-01

    This study revealed that cellulose enzymatic saccharification response curves of lignocellulosic substrates were very different from those of pure cellulosic substrates in terms of optimal pH and pH operating window. The maximal enzymatic cellulose saccharification of lignocellulosic substrates occurs at substrate suspension

  15. Varespladib (LY315920 Appears to Be a Potent, Broad-Spectrum, Inhibitor of Snake Venom Phospholipase A2 and a Possible Pre-Referral Treatment for Envenomation

    Directory of Open Access Journals (Sweden)

    Matthew Lewin

    2016-08-01

    Full Text Available Snakebite remains a neglected medical problem of the developing world with up to 125,000 deaths each year despite more than a century of calls to improve snakebite prevention and care. An estimated 75% of fatalities from snakebite occur outside the hospital setting. Because phospholipase A2 (PLA2 activity is an important component of venom toxicity, we sought candidate PLA2 inhibitors by directly testing drugs. Surprisingly, varespladib and its orally bioavailable prodrug, methyl-varespladib showed high-level secretory PLA2 (sPLA2 inhibition at nanomolar and picomolar concentrations against 28 medically important snake venoms from six continents. In vivo proof-of-concept studies with varespladib had striking survival benefit against lethal doses of Micrurus fulvius and Vipera berus venom, and suppressed venom-induced sPLA2 activity in rats challenged with 100% lethal doses of M. fulvius venom. Rapid development and deployment of a broad-spectrum PLA2 inhibitor alone or in combination with other small molecule inhibitors of snake toxins (e.g., metalloproteases could fill the critical therapeutic gap spanning pre-referral and hospital setting. Lower barriers for clinical testing of safety tested, repurposed small molecule therapeutics are a potentially economical and effective path forward to fill the pre-referral gap in the setting of snakebite.

  16. Antioxidative activities of hydrolysates from edible birds nest using enzymatic hydrolysis

    Science.gov (United States)

    Muhammad, Nurul Nadia; Babji, Abdul Salam; Ayub, Mohd Khan

    2015-09-01

    Edible bird's nest protein hydrolysates (EBN) were prepared via enzymatic hydrolysis to investigate its antioxidant activity. Two types of enzyme (alcalase and papain) were used in this study and EBN had been hydrolysed with different hydrolysis time (30, 60, 90 and 120 min). Antioxidant activities in EBN protein hydrolysate were measured using DPPH, ABTS+ and Reducing Power Assay. From this study, increased hydrolysis time from 30 min to 120 min contributed to higher DH, as shown by alcalase (40.59%) and papain (24.94%). For antioxidant assay, EBN hydrolysed with papain showed higher scavenging activity and reducing power ability compared to alcalase. The highest antioxidant activity for papain was at 120 min hydrolysis time with ABTS (54.245%), DPPH (49.78%) and Reducing Power (0.0680). Meanwhile for alcalase, the highest antioxidant activity was at 30 min hydrolysis time. Even though scavenging activity for EBN protein hydrolysates were high, the reducing power ability was quite low as compared to BHT and ascorbic Acid. This study showed that EBN protein hydrolysate with alcalase and papain treatments potentially exhibit high antioxidant activity which have not been reported before.

  17. Chemo‐enzymatic epoxidation–process options for improving biocatalytic productivity

    DEFF Research Database (Denmark)

    Hagström, Anna E. V.; Törnvall, Ulrika; Nordblad, Mathias

    2011-01-01

    The reactor choice is crucial when designing a process where inactivation of the biocatalyst is a problem. The main bottleneck for the chemo‐enzymatic epoxidation has been found to be enzyme inactivation by the hydrogen peroxide, H2O2, substrate. In the work reported here, the effect of reaction...... parameters on the reaction performance have been investigated and used to establish suitable operating strategies to minimize the inactivation of the enzyme, using rapeseed methyl ester (RME) as a substrate in a solvent‐free system. The use of a controlled fed‐batch reactor for maintaining H2O2 concentration...... at 1.5 M resulted in increased productivity, up to 76 grams of product per gram of biocatalyst with higher retention of enzyme activity. Further investigation included a multistage design that separated the enzymatic reaction and the saturation of the RME substrate with H2O2 into different vessels...

  18. Evaluation of oil removal efficiency and enzymatic activity in some fungal strains for bioremediation of petroleum-polluted soils

    Directory of Open Access Journals (Sweden)

    Mohsenzadeh Fariba

    2012-12-01

    Full Text Available Abstract Background Petroleum pollution is a global disaster and there are several soil cleaning methods including bioremediation. Methods In a field study, fugal strains were isolated from oil-contaminated sites of Arak refinery (Iran and their growth ability was checked in potato dextrose agar (PDA media containing 0-10% v/v crude oil, the activity of three enzymes (Catalase, Peroxidase and Phenol Oxidase was evaluated in the fungal colonies and bioremediation ability of the fungi was checked in the experimental pots containing 3 kg sterilized soil and different concentrations of petroleum (0-10% w/w. Results Four fungal strains, Acromonium sp., Alternaria sp., Aspergillus terreus and Penicillium sp., were selected as the most resistant ones. They were able to growth in the subjected concentrations and Alternaria sp. showed the highest growth ability in the petroleum containing media. The enzyme assay showed that the enzymatic activity was increased in the oil-contaminated media. Bioremediation results showed that the studied fungi were able to decrease petroleum pollution. The highest petroleum removing efficiency of Aspergillus terreus, Penicillium sp., Alternaria sp. and Acromonium sp. was evaluated in the 10%, 8%, 8% and 2% petroleum pollution respectively. Conclusions Fungi are important microorganisms in decreasing of petroleum pollution. They have bioremediation potency that is related to their enzymatic activities.

  19. Fluoranthene induced changes in photosynthetic pigments, biochemical compounds and enzymatic activities in two microalgal species: Chlorella vulgaris Beijerinck and Desmodesmus subspicatus Chodat

    Directory of Open Access Journals (Sweden)

    Miral Patel

    2014-02-01

    Full Text Available The photosynthetic pigments, biochemical and enzymatic activities in two freshwater microalgal species, Chlorella vulgaris and Desmodesmus subspicatus at different fluoranthene concentrations were compared with the control conditions. During 16-days of incubation period when treated with fluoranthene, both microalgal species exhibited variable amount of photosynthetic pigment, biochemical compounds and enzymatic activities. The addition of fluoranthene at concentrations ranged from 1.5 mg l-1; to 10 mg l-1; to microalgal cultures led to changes in all different metabolites but the patterns varied from species to species. Among the two species tested, pigment, biochemical and enzymatic contents were remarkably declined from 7 % to 95% in C. vulgaris. Moreover, all metabolites in D. subspicatus also diminishing significantly by 3% to 88% of fluoranthene doses (10ppm. These results suggest that fluoranthene-induced changes of pigments, biochemical and enzymatic variations in test microalgae, D. subspicatus and C. vulgaris, might reveal its resistance and ability to metabolize PAHs. At the same time, the PAH impact changes on different metabolic activities were higher at 12 and 16 days than at 4 and 8 days in treated microalgae. DOI: http://dx.doi.org/10.3126/ije.v3i1.9941 International Journal of Environment Vol.3(1 2014: 41-55

  20. Impacts of dissolved organic matter on aqueous behavior of nano/micron-titanium nitride and their induced enzymatic/non-enzymatic antioxidant activities in Scenedesmus obliquus.

    Science.gov (United States)

    Zhang, Xin; Wang, Zhuang; Wang, Se; Fang, Hao; Zhang, Fan; Wang, De-Gao

    2017-01-02

    Freshwater dispersion stability and ecotoxicological effects of titanium nitride (TiN) with particle size of 20 nm, 50 nm, and 2-10 μm in the presence of dissolved organic matter (DOM) at various concentrations were studied. The TiN particles that had a more negative zeta potential and smaller hydrodynamic size showed more stable dispersion in an aqueous medium when DOM was present than when DOM was absent. Biochemical assays indicated that relative to the control, the TiN particles in the presence of DOM alleviated to some extent the antioxidative stress enzyme activity in Scenedesmus obliquus. In addition, it was found that the TiN with a primary size of 50 nm at a high concentration presented a significant impact on non-enzymatic antioxidant defense in algal cells.

  1. Diverse regulation of retinal pigment epithelium phagocytosis of photoreceptor outer segments by calcium-independent phospholipase A₂, group VIA and secretory phospholipase A₂, group IB

    DEFF Research Database (Denmark)

    Zhan, Chen; Wang, Jinmei; Kolko, Miriam

    2012-01-01

    PURPOSE: To investigate the roles of the phospholipases A(2) (PLA(2)) subtypes, iPLA(2)-VIA and sPLA(2)-IB in retinal pigment epithelium (RPE) phagocytosis of photoreceptor outer segments (POS) and to explore a possible interaction between sPLA(2)-IB and iPLA(2)-VIA in the RPE. METHODS: To explore...... the role of iPLA(2)-VIA in RPE phagocytosis of POS, experiments with iPLA(2)-VIA vector transfection, iPLA(2)-VIA(-/-) knockout (KO) mice, and iPLA(2)-VIA inhibition by bromoenol lactone (BEL) were done. Exogenous addition of sPLA(2)-IB was used to investigate the role of sPLA(2)-IB in RPE phagocytosis....... A Luciferase Reporter Vector containing the iPLA(2)-VIA promoter was used to study the effects of sPLA(2)-IB on the iPLA(2)-VIA promoter. RESULTS: ARPE-19 and primary mouse RPE cells transfected with iPLA(2)-VIA showed increased phagocytosis. Phagocytosis was reduced in primary mouse RPE inhibited with BEL...

  2. Enzymatic Browning: a practical class

    Directory of Open Access Journals (Sweden)

    Maria Teresa Pedrosa Silva Clerici

    2014-10-01

    Full Text Available This paper presents a practical class about the enzymes polyphenol oxidases, which have been shown to be responsible for the enzymatic browning of fruits and vegetables. Vegetables samples were submitted to enzymatic inactivation process with chemical reagents, as well as by bleaching methods of applying heat by conventional oven and microwave oven. Process efficiency was assessed qualitatively by both observing the guaiacol peroxidase activity and after the storage period under refrigeration or freezing. The practical results obtained in this class allow exploring multidisciplinary knowledge in food science, with practical applications in everyday life.

  3. The development of orally administrable gemcitabine prodrugs with D-enantiomer amino acids: enhanced membrane permeability and enzymatic stability.

    Science.gov (United States)

    Tsume, Yasuhiro; Incecayir, Tuba; Song, Xueqin; Hilfinger, John M; Amidon, Gordon L

    2014-04-01

    Gemcitabine prodrugs with D- and L-configuration amino acids were synthesized and their chemical stability in buffers, resistance to glycosidic bond metabolism, enzymatic activation, permeability in Caco-2 cells and mouse intestinal membrane, anti-proliferation activity in cancer cell were determined and compared to that of parent drug, gemcitabine. Prodrugs containing D-configuration amino acids were enzymatically more stable than ones with L-configuration amino acids. The activation of all gemcitabine prodrugs was 1.3-17.6-fold faster in cancer cell homogenate than their hydrolysis in buffer, suggesting enzymatic action. The enzymatic activation of amino acid monoester prodrugs containing D-configuration amino acids in cell homogenates was 2.2-10.9-fold slower than one of amino acid monoester prodrugs with L-configuration amino acids. All prodrugs exhibited enhanced resistance to glycosidic bond metabolism by thymidine phosphorylase compared to parent gemcitabine. Gemcitabine prodrugs showed superior the effective permeability in mouse jejunum to gemcitabine. More importantly, the high plasma concentration of d-amino acid gemcitabine prodrugs was observed more than one of L-amino acid gemcitabine prodrugs. In general, the 5'-mono-amino acid monoester gemcitabine prodrugs exhibited higher permeability and uptake than their parent drug, gemcitabine. Cell proliferation assays in AsPC-1 pancreatic ductal cell line indicated that gemcitabine prodrugs were more potent than their parent drug, gemcitabine. The transport and enzymatic profiles of 5'-D-valyl-gemcitabine and 5'-D-phenylalanyl-gemcitabine suggest their potential for increased oral uptake and delayed enzymatic bioconversion as well as enhanced uptake and cytotoxic activity in cancer cells, would facilitate the development of oral dosage form for anti-cancer agents and, hence, improve the quality of life for the cancer patients. Copyright © 2014. Published by Elsevier B.V.

  4. E. coli-Derived L-Asparaginase Retains Enzymatic and Cytotoxic Activity In Vitro for Canine and Feline Lymphoma after Cold Storage

    Directory of Open Access Journals (Sweden)

    Jackie M. Wypij

    2013-01-01

    Full Text Available Background. L-asparaginase is effective in treating canine and feline lymphoma, however chemotherapy poses a significant financial cost to veterinary clients, limiting therapy for many pets. Single dose vials result in significant drug wastage, and drug shortages limit consistent availability for pets. Hypothesis. E. coli-derived asparaginase retains enzymatic and antineoplastic activity in canine and feline lymphoma cells after cold storage. Methods. E. coli-derived asparaginase was cold-stored: refrigeration (7–14 days and freezing (14 days–six months, one to three freeze/thaw cycles. Enzymatic activity of asparaginase was measured via a modified asparagine assay. Effects of cold-stored asparaginase on cell proliferation and cytotoxicity were measured in feline (MYA-1, F1B and canine (17–71, OSW lymphoma cells. Results. Cold-stored E. coli-derived asparaginase retains antineoplastic activity in all four cell lines tested. Cold-stored E. coli-derived L-asparaginase depletes asparagine and retains enzymatic activity. Duration of refrigeration, duration of freezing, and number of freeze-thaw cycles have minimal effect on asparaginase enzyme activity. Conclusions and Clinical Importance. This study establishes a scientific basis for long-term cold storage of reconstituted E. coli-derived asparaginase that may result in better utilization of limited drug resources and improve financial feasibility of E. coli-derived asparaginase as a therapeutic option for pets with lymphoma.

  5. Unraveling the mechanism responsible for the contrasting tolerance of Synechocystis and Synechococcus to Cr(VI): Enzymatic and non-enzymatic antioxidants

    Energy Technology Data Exchange (ETDEWEB)

    Gupta, Alka [Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085 (India); Ballal, Anand, E-mail: aballal@barc.gov.in [Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085 (India); Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai 40085 (India)

    2015-07-15

    Highlights: • Cr(VI) accumulation generates higher ROS in Synechocystis than in Synechococcus. • Synechococcus exhibits better photosynthetic activity in response to Cr(VI). • Synechococcus has higher enzymatic/non-enzymatic antioxidants than Synechocystis. • Synechococcus shows better tolerance to other oxidative stresses than Synechocystis. • Differential detoxification of ROS is responsible for the contrasting tolerance to Cr(VI) - Abstract: Two unicellular cyanobacteria, Synechocystis and Synechococcus, showed contrasting tolerance to Cr(VI); with Synechococcus being 12-fold more tolerant than Synechocystis to potassium dichromate. The mechanism responsible for this differential sensitivity to Cr(VI) was explored in this study. Total content of photosynthetic pigments as well as photosynthetic activity decreased at lower concentration of Cr(VI) in Synechocystis as compared to Synechococcus. Experiments with {sup 51}Cr showed Cr to accumulate intracellularly in both the cyanobacteria. At lower concentrations, Cr(VI) caused excessive ROS generation in Synechocystis as compared to that observed in Synechococcus. Intrinsic levels of enzymatic antioxidants, i.e., superoxide dismutase, catalase and 2-Cys-peroxiredoxin were considerably higher in Synechococcus than Synechocystis. Content of total thiols (both protein as well as non-protein) and reduced glutathione (GSH) was also higher in Synechococcus as compared to Synechocystis. This correlated well with higher content of carbonylated proteins observed in Synechocystis than Synechococcus. Additionally, in contrast to Synechocystis, Synechococcus exhibited better tolerance to other oxidative stresses like high intensity light and H{sub 2}O{sub 2}. The data indicate that the disparity in the ability to detoxify ROS could be the primary mechanism responsible for the differential tolerance of these cyanobacteria to Cr(VI)

  6. Efficient production of lactulose from whey powder by cellobiose 2-epimerase in an enzymatic membrane reactor.

    Science.gov (United States)

    Wu, Lingtian; Xu, Cen; Li, Sha; Liang, Jinfeng; Xu, Hong; Xu, Zheng

    2017-06-01

    In this study, the gene encoding cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) was successfully expressed in Bacillus subtilis WB800. After the fermentation medium optimization, the activity of recombinant strain was 4.5-fold higher than the original medium in a 7.5L fermentor. The optimal catalytic pH and temperature of crude CsCE were 7.0 and 80°C, respectively. An enzymatic synthesis of lactulose was developed using cheese-whey lactose as its substrate. The maximum conversion rate of whey powder obtained was 58.5% using 7.5 U/mL CsCE. The enzymatic membrane reactor system exhibited a great operational stability, confirmed with the higher lactose conversion (42.4%) after 10 batches. To our best knowledge, this is the first report of lactulose synthesis in food grade strain, which improve the food safety, and we not only realize the biological production of lactulose, but also make good use of industrial waste, which have positive impact on environment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Analysis of Enzymatic Activity of Matrix Metalloproteinase (MMP) by Collagen Zymography in Melanoma.

    Science.gov (United States)

    Walia, Vijay; Samuels, Yardena

    2018-01-01

    Protein zymography is the most commonly used technique to study the enzymatic activity of matrix metalloproteinases (MMPs) and their inhibitors. MMPs are proteolytic enzymes that promote extracellular matrix degradation. MMPs are frequently mutated in malignant melanomas as well as other cancers and are linked to increasing incidence of tumor metastasis. Substrate zymography characterizes MMP activity by their ability to degrade preferred substrates. Here we describe the collagen zymography technique to measure the active or latent form of MMPs using MMP-8 as an example, which is a frequently mutated MMP family member in malignant melanomas. The same technique can be used with the modification of substrate to detect metalloproteinase activity of other MMPs. Both wild-type and mutated forms of MMPs can be analyzed using a single gel using this method.

  8. Relationship between Porcine Sperm Motility and Sperm Enzymatic Activity using Paper-based Devices

    Science.gov (United States)

    Matsuura, Koji; Huang, Han-Wei; Chen, Ming-Cheng; Chen, Yu; Cheng, Chao-Min

    2017-04-01

    Mammalian sperm motility has traditionally been analyzed to determine fertility using computer-assisted semen analysis (CASA) systems. To develop low-cost and robust male fertility diagnostics, we created a paper-based MTT assay and used it to estimate motile sperm concentration. When porcine sperm motility was inhibited using sperm enzyme inhibitors for sperm enzymes related to mitochondrial activity and glycolysis, we simultaneously recorded sperm motility and enzymatic reactivity using a portable motility analysis system (iSperm) and a paper-based MTT assay, respectively. When using our paper-based MTT-assay, we calculated the area mean value signal intensity (AMV) to evaluate enzymatic reactivity. Both sperm motility and AMV decreased following treatment with iodoacetamide (IODO) and 3-bromopyruvic acid (3BP), both of which are inhibitors of glycolytic enzymes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a correlation between recorded motility using iSperm and AMV from our paper-based assay (P Based on this inhibitor study, sperm motility can be estimated using our paper-based MTT-assay.

  9. Enzymatic Activity of the Mycelium Compared with Oospore Development During Infection of Pea Roots by Aphanomyces euteiches

    DEFF Research Database (Denmark)

    Kjøller, Rasmus; Rosendahl, Søren

    1998-01-01

    To describe the disease cycle of the root pathogen Aphanomyces euteiches, enzymatic activity in the mycelium was compared with the development of oospores in pea roots. Plants were inoculated with two zoospore concentrations to achieve different disease levels. Hyphae were stained for fungal...

  10. Evaluation of the effects of a VEGFR-2 inhibitor compound on alanine aminotransferase gene expression and enzymatic activity in the rat liver.

    Science.gov (United States)

    Fuentealba, Carmen; Bera, Monali; Jessen, Bart; Sace, Fred; Stevens, Greg J; Trajkovic, Dusko; Yang, Amy H; Evering, Winston

    2011-08-17

    Traditional assessment of drug-induced hepatotoxicity includes morphological examination of the liver and evaluation of liver enzyme activity in serum. The objective of the study was to determine the origin of drug-related elevation in serum alanine aminotransferase (ALT) activity in the absence of morphologic changes in the liver by utilizing molecular and immunohistochemical techniques. Sixteen female Sprague-Dawley rats were divided into 2 groups (control and treated, n = 4 per group) and treated rats were dosed orally twice daily (400 mg/kg/day) for 7 days with a VEGFR-2 compound (AG28262), which in a previous study caused ALT elevation without morphological changes. Serum of both treated and control animals were evaluated on day 3 of treatment and at day 8. Three separate liver lobes (caudate, right medial, and left lateral) were examined for determination of ALT tissue activity, ALT gene expression and morphological changes. ALT activity was significantly (p < 0.01) elevated on day 3 and further increased on day 8. Histologic changes or increase in TUNEL and caspase3 positive cells were not observed in the liver lobes examined. ALT gene expression in the caudate lobe was significantly up-regulated by 63%. ALT expression in the left lateral lobe was not significantly affected. Statistically significant increased liver ALT enzymatic activity occurred in the caudate (96%) and right medial (41%) lobes but not in the left lateral lobe. AG28262, a VEFG-r2 inhibitor, causes an increase in serum ALT, due in part to both gene up-regulation. Differences between liver lobes may be attributable to differential distribution of blood from portal circulation. Incorporation of molecular data, such as gene and protein expression, and sampling multiple liver lobes may shed mechanistic insight to the evaluation of hepatotoxicity.

  11. Warming and organic matter sources impact the proportion of dissolved to total activities in marine extracellular enzymatic rates

    KAUST Repository

    Baltar, Federico; Moran, Xose Anxelu G.; Lø nborg, Christian

    2017-01-01

    Extracellular enzymatic activities (EEAs) are the rate-limiting step in the degradation of organic matter. Extracellular enzymes can be found associated to cells or dissolved in the surrounding water. The proportion of cell-free EEA constitutes

  12. Comprehensive analysis to explain reduced or increased SOD1 enzymatic activity in ALS patients and their relatives.

    Science.gov (United States)

    Keskin, Isil; Birve, Anna; Berdynski, Mariusz; Hjertkvist, Karin; Rofougaran, Reza; Nilsson, Torbjörn K; Glass, Jonathan D; Marklund, Stefan L; Andersen, Peter M

    2017-08-01

    To characterise stabilities in erythrocytes of mutant SOD1 proteins, compare SOD1 enzymatic activities between patients with different genetic causes of ALS and search for underlying causes of deviant SOD1 activities in individuals lacking SOD1 mutations. Blood samples from 4072 individuals, ALS patients with or without a SOD1 mutation, family members and controls were studied. Erythrocyte SOD1 enzymatic activities normalised to haemoglobin content were determined, and effects of haemoglobin disorders on dismutation assessed. Coding SOD1 sequences were analysed by Sanger sequencing, exon copy number variations by fragment length analysis and by TaqMan Assay. Of the 44 SOD1 mutations found, 75% caused severe destabilisation of the mutant protein but in 25% it was physically stable. Mutations producing structural changes caused halved erythrocyte SOD1 activities. There were no differences in SOD1 activities between patients without a SOD1 mutation and control individuals or carriers of TBK1 mutations and C9orf72 HRE . In the low and high SOD1 activity groups no deviations were found in exon copy numbers and intron gross structures. Thalassemias and iron deficiency were associated with increased SOD1 activity/haemoglobin ratios. Adjunct erythrocyte SOD1 activity analysis reliably signals destabilising SOD1 mutations including intronic mutations that are missed by exon sequencing.

  13. Modulation of Enzymatic Activities of Dual Functional Peroxiredoxin by Gamma Irradiation

    International Nuclear Information System (INIS)

    Hong, Sung Hyun; Lee, Seung Sik; Park, Chul Hong; Chung, Byung Yeoup

    2012-01-01

    Recently, enzymes have frequently been used as catalysts in various bio-industrial, commercial, and pharmaceutical applications, because they are more stable, more efficient, and less toxic than the synthetic catalysts. However, one of their major disadvantages is their low thermostability, which leads the researchers to develop new forms of industrially important enzymes with increased resistance to inactivation and aggregation. This study describes a strategy for modifying the molecular chaperone activity of peroxiredoxin (Prx) by using gamma irradiation. Prxs are a ubiquitous family of antioxidant enzymes. Upon oxidation of their peroxidatic Cys, the molecules undergo a structural conversion from a low-molecular-weight (LMW) species acting as a peroxidase to a high-molecular-weight (HMW) complex functioning as a chaperone. In the present study, we examined the effect of gamma irradiation on PP1084 with respect to its protein structure and enzymatic function. The use of gamma irradiation as a physical treatment can increase the cohesive strength of the protein by forming cross-links. The aims of the present work were (1) to improve the chaperone activity of PP1084 by gamma irradiation, (2) to identify the 'optimal' intensity of gamma irradiation, and (3) to investigate the influence of gamma irradiation on protein hydrophobicity as related to chaperone function. Following PP1084 treatment with 30 kGy gamma irradiation, the PP1084 chaperone activity enhanced by about 3-4-fold compared with nonirradiated PP1084, while the peroxidase activity decreased. Ongoing research efforts are addressing the physical modifications of PP1084 protein by gamma irradiation

  14. Ultrasonic-assisted enzymatic extraction of phenolics from broccoli (Brassica oleracea L. var. italica) inflorescences and evaluation of antioxidant activity in vitro.

    Science.gov (United States)

    Wu, Hao; Zhu, Junxiang; Yang, Long; Wang, Ran; Wang, Chengrong

    2015-06-01

    An efficient ultrasonic-assisted enzymatic extraction technique was applied to extracting phenolics from broccoli inflorescences without organic solvents. The synergistic model of enzymolysis and ultrasonication simultaneously was selected, and the enzyme combination was optimized by orthogonal test: cellulase 7.5 mg/g FW (fresh weight), pectinase 10 mg/g FW, and papain 1.0 mg/g FW. The operating parameters in ultrasonic-assisted enzymatic extraction were optimized with response surface methodology using Box-Behnken design. The optimal extraction conditions were as follows: ultrasonic power, 440 W; liquid to material ratio, 7.0:1 mL/g; pH value of 6.0 at 54.5 ℃ for 10 min. Under these conditions, the extraction yield of phenolics achieved 1.816 ± 0.0187 mg gallic acid equivalents/gram FW. The free radical scavenging activity of ultrasonic-assisted enzymatic extraction extracts was determined by 1,1-diphenyl-2-picrylhydrazyl·assay with EC50 values of 0.25, and total antioxidant activity was determined by ferric reducing antioxidant power assay with ferric reducing antioxidant power value of 0.998 mmol FeSO4/g compared with the referential ascorbic acid of 1.184 mmol FeSO4/g. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  15. Expression of eicosanoid biosynthetic and catabolic enzymes in peritoneal endometriosis.

    Science.gov (United States)

    Lousse, J-C; Defrère, S; Colette, S; Van Langendonckt, A; Donnez, J

    2010-03-01

    Increased peritoneal eicosanoid concentrations have been reported in endometriosis patients and might be important in disease-associated pain and inflammation. Here, we evaluated the expression of key biosynthetic and catabolic enzymes involved in this abnormal eicosanoid production in peritoneal macrophages and endometriotic lesions. Peritoneal macrophages, endometriotic lesions and matched eutopic endometrium were collected from endometriosis patients (n = 40). Peritoneal macrophages and eutopic endometrium samples were also collected from disease-free women (n = 25). Expression of type IIA secretory phospholipase A(2) (sPLA(2)-IIA), cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1), 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and 5-lipoxygenase (5-LO) was quantified by real-time PCR, and these five key enzymes were localized by immunohistochemistry. sPLA(2)-IIA, COX-2 and mPGES-1 mRNA was significantly increased in peritoneal macrophages of endometriosis patients compared with controls (P = 0.006, P = 0.016 and P = 0.025, respectively). In endometriosis patients, sPLA(2)-IIA, mPGES-1 and 15-PGDH mRNA was significantly enhanced in peritoneal lesions compared with matched eutopic endometrium (P endometriosis group compared with controls (P = 0.023). Finally, sPLA(2)-IIA, COX-2, mPGES-1 and 15-PGDH immunostaining was found mainly in endometrial glands, whereas 5-LO was distributed throughout the glands and stroma. Our study highlights an imbalance between eicosanoid biosynthesis and degradation in endometriosis patients. Both peritoneal macrophages and endometriotic lesions may be involved. Research into new molecules inhibiting biosynthetic enzymes (such as sPLA(2)-IIA and mPGES-1) and/or activating catabolic enzymes (such as 15-PGDH) may prove to be a major field of investigation in the development of targeted medical therapies.

  16. The effects of different uranium concentrations on soil microbial populations and enzymatic activities

    International Nuclear Information System (INIS)

    Bagherifam, S.; Lakziyan, A.; Ahmadi, S. J.; Fotovvat, A.; Rahimi, M. F.

    2010-01-01

    Uranium is an ubiquitous constituent of natural environment with an average concentration of 4 mg/kg in earth crust. However, in local areas it may exceed the normal concentration due to human activities resulting in radionuclide contamination in groundwater and surface soil. The effect of six levels of uranium concentration (0, 50, 100,250. 500 and 1000 mg kg -1 ) on soil phosphatase activities and microbial populations were studied in a completely randomized design as a factorial experiment with three replications. The results showed a significant decrease in phosphatase activity. The result of the experiment suggests that soil microbial populations (bacteria, funji and actinomycetes) decrease by increasing the uranium levels in the soil. Therefore, assessment of soil enzymatic activities and microbial populations can be helpful as a useful index for a better management of uranium and radioactive contaminated soils.

  17. Enzymatic Activity Detection via Electrochemistry for Enceladus

    Science.gov (United States)

    Studemeister, Lucy; Koehne, Jessica; Quinn, Richard

    2017-01-01

    Electrochemical detection of biological molecules is a pertinent topic and application in many fields such as medicine, environmental spills, and life detection in space. Proteases, a class of molecules of interest in the search for life, catalyze the hydrolysis of peptides. Trypsin, a specific protease, was chosen to investigate an optimized enzyme detection system using electrochemistry. This study aims at providing the ideal functionalization of an electrode that can reliably detect a signal indicative of an enzymatic reaction from an Enceladus sample.

  18. Enzymatic Degradation of Poly(ethylene 2,5-furanoate Powders and Amorphous Films

    Directory of Open Access Journals (Sweden)

    Simone Weinberger

    2017-10-01

    Full Text Available Poly(ethylene 2,5-furanoate (PEF is arousing great interest as a biobased alternative to plastics like poly(ethylene terephthalate (PET due to its wide range of potential applications, such as food and beverage packaging, clothing, and in the car industry. In the present study, the hydrolysis of PEF powders of different molecular masses (Mn = 55, Mw = 104 kg/mol and Mn = 18, Mw = 29 kg/mol and various particle sizes (180 < d and 180 < d < 425 µm using cutinase 1 from Thermobifida cellulosilytica (Thc_cut1 was studied. Thereby, the effects of molecular mass, particle size and crystallinity on enzymatic hydrolysis were investigated. The results show that particles with lower molecular mass are hydrolyzed faster than those with higher masses, and that the higher the molecular mass, the lower the influence of the particle size on the hydrolysis. Furthermore, cutinases from Humicola insolens (HiC and Thc_cut1 were compared with regard to their hydrolytic activity on amorphous PEF films (measured as release of 2,5-furandicarboxylic acid (FDCA and weight loss in different reaction media (1 M KPO pH 8, 0.1 M Tris-HCl pH 7 and at different temperatures (50 °C and 65 °C. A 100% hydrolysis of the PEF films was achieved after only 72 h of incubation with a HiC in 1 M KPO pH 8 at 65 °C. Moreover, the hydrolysis reaction was monitored by LC/TOF-MS analysis of the released reaction products and by Scanning Electron Microscopy (SEM examination of the polymer surfaces. Enzymatic hydrolysis of PEF with Thc_cut1 and HiC has potential for use in surface functionalization and recycling purposes.

  19. Improved enzymatic production of phenolated glycerides through alkyl phenolate intermediate

    DEFF Research Database (Denmark)

    Yang, Zhiyong; Feddern, Vivian; Glasius, Marianne

    2011-01-01

    This work reported a novel approach for synthesis of dihydrocaffoylated glycerides, consisting of 2 steps: enzymatic synthesis of octyl dihydrocaffeate (as a synthetic intermediate) from octanol and dihydrocaffeic acid (DHCA), and enzymatic interesterification of triglycerides with octyl dihydroc......This work reported a novel approach for synthesis of dihydrocaffoylated glycerides, consisting of 2 steps: enzymatic synthesis of octyl dihydrocaffeate (as a synthetic intermediate) from octanol and dihydrocaffeic acid (DHCA), and enzymatic interesterification of triglycerides with octyl...

  20. New eutectic ionic liquids for lipase activation and enzymatic preparation of biodiesel†

    Science.gov (United States)

    Zhao, Hua; Baker, Gary A.; Holmes, Shaletha

    2012-01-01

    The enzymatic preparation of biodiesel has been hampered by the lack of suitable solvents with desirable properties such as high lipase compatibility, low cost, low viscosity, high biodegradability, and ease of product separation. Recent interest in using ionic liquids (ILs) as advanced reaction media has led to fast reaction rates and high yields in the enzymatic synthesis of biodiesel. However, conventional (i.e., cation–anion paired) ILs based on imidazolium and other quaternary ammonium salts remain too expensive for wide application at industrial scales. In this study, we report on newly-synthesized eutectic ILs derived from choline acetate or choline chloride coupled with biocompatible hydrogen-bond donors, such as glycerol. These eutectic solvents have favorable properties including low viscosity, high biodegradability, and excellent compatibility with Novozym® 435, a commercial immobilized Candida antarctica lipase B. Furthermore, in a model biodiesel synthesis system, we demonstrate high reaction rates for the enzymatic transesterification of Miglyol® oil 812 with methanol, catalyzed by Novozym® 435 in choline acetate/glycerol (1 : 1.5 molar ratio). The high conversion (97%) of the triglyceride obtained within 3 h, under optimal conditions, suggests that these novel eutectic solvents warrant further exploration as potential media in the enzymatic production of biodiesel. PMID:21283901

  1. Decreased enzymatic activity of 5,10-methylene tetrahydrofolate reductase affects the development of several diseases

    Directory of Open Access Journals (Sweden)

    Maša Vidmar

    2016-07-01

    Full Text Available The importance of folates in human physiology is well known, as are various pathologies associated with low folate status. Folate deficiency can occur due to low dietary intake, genetic predisposition or treatment with medicines affecting the folate status. The aim of this paper is to explore the importance of determining genetic polymorphisms which influence the levels of biologically active folate. MTHFR is involved in the transformation of 5,10-methylene-THF to 5-methyl-THF. Polymorphisms of the MTHRF gene are associated with decreased enzymatic activity.Only 9.3 % of the population in Slovenia displays full activity of the MTHFR enzyme; these subjects are non-mutated homozygotes (wild-type alleles. In contrast, the average enzymatic activity in subjects with mutated alleles is between 50 and 60 %. MTHFR polymorphism is associated with an increased risk of hyperhomocysteinemia and cardiovascular diseases, neurological disorders and various types of cancer. There is also an increased risk for congenital malformations. Folic acid food fortification was introduced in some countries in order to assure an adequate folate status in the population. However, this approach does not address the decreased activity of MTHFR.Polymorphism in the key enzymes of the folate cycle is common. Determination of the genetic predisposition is therefore plausible in the most vulnerable groups of the population, such as pregnant women and patients receiving medicines influencing the folate cycle in various ways, e.g. 5-fluorouracil, methotrexate and 6-mercaptopurine. Genotyping would allow the identification of patients at high risk for suboptimal folate status.

  2. Real-time ESI-MS of enzymatic conversion: impact of organic solvents and multiplexing.

    Science.gov (United States)

    Scheerle, Romy K; Grassmann, Johanna; Letzel, Thomas

    2012-01-01

    Different enzymatic assays were characterized systematically by real-time electrospray ionization mass spectrometry (ESI-MS) in the presence of organic solvents as well as in multiplex approaches and in a combination of both. Typically, biological enzymatic reactions are studied in aqueous solutions, since most enzymes show their full activity solely in aqueous solutions. However, in recent years, the use of organic solvents in combination with enzymatic reactions has gained increasing interest due to biotechnological advantages in chemical synthesis, development of online coupled setups screening for enzyme regulatory compounds, advantages regarding mass spectrometric detection and others. In the current study, the influence of several common organic solvents (methanol, ethanol, isopropanol, acetone, acetonitrile) on enzymatic activity (hen egg white lysozyme, chitinase, α-chymotrypsin, elastase from human neutrophils and porcine pancreas, acetylcholinesterase) was tested. Moreover, multiplexing is a promising approach enabling fast and cost-efficient screening methods, e.g. for determination of inhibitors in complex mixtures or in the field of biomedical research. Although in multiplexed setups the enzymatic activity may be affected by the presence of other substrates and/or enzymes, the expected advantages possibly will predominate. To investigate those effects, we measured multiple enzymatic assays simultaneously. For all conducted measurements, the conversion rate of the substrate(s) was calculated, which reflects the enzymatic activity. The results provide an overview about the susceptibility of the selected enzymes towards diverse factors and a reference point for many applications in analytical chemistry and biotechnology.

  3. Photoelectrochemical enzymatic biosensors.

    Science.gov (United States)

    Zhao, Wei-Wei; Xu, Jing-Juan; Chen, Hong-Yuan

    2017-06-15

    Enzymatic biosensors have been valuable bioanalytical devices for analysis of diverse targets in disease diagnosis, biological and biomedical research, etc. Photoelectrochemical (PEC) bioanalysis is a recently emerged method that promptly becoming a subject of new research interests due to its attractive potential for future bioanalysis with high sensitivity and specificity. PEC enzymatic biosensors integrate the inherent sensitivities of PEC bioanalysis and the selectivity of enzymes and thus share their both advantages. Currently, PEC enzymatic biosensors have become a hot topic of significant research and the recent impetus has grown rapidly as demonstrated by increased research papers. Given the pace of advances in this area, this review will make a thorough discussion and survey on the fundamentals, sensing strategies, applications and the state of the art in PEC enzymatic biosensors, followed by future prospects based on our own opinions. We hope this work could provide an accessible introduction to PEC enzymatic biosensors for any scientist. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Mechano-Enzymatic Deconstruction with a New Enzymatic Cocktail to Enhance Enzymatic Hydrolysis and Bioethanol Fermentation of Two Macroalgae Species

    Directory of Open Access Journals (Sweden)

    Sameh Amamou

    2018-01-01

    Full Text Available The aim of this study was to explore the efficiency of a mechano-enzymatic deconstruction of two macroalgae species for sugars and bioethanol production, by using a new enzymatic cocktail (Haliatase and two types of milling modes (vibro-ball: VBM and centrifugal milling: CM. By increasing the enzymatic concentration from 3.4 to 30 g/L, the total sugars released after 72 h of hydrolysis increased (from 6.7 to 13.1 g/100 g TS and from 7.95 to 10.8 g/100 g TS for the green algae U. lactuca and the red algae G. sesquipedale, respectively. Conversely, total sugars released from G. sesquipedale increased (up to 126% and 129% after VBM and CM, respectively. The best bioethanol yield (6 geth/100 g TS was reached after 72 h of fermentation of U. lactuca and no increase was obtained after centrifugal milling. The latter led to an enhancement of the ethanol yield of G. sesquipedale (from 2 to 4 g/100 g TS.

  5. Enzymatic- and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs

    Directory of Open Access Journals (Sweden)

    Ortega Ryan A

    2011-02-01

    Full Text Available Abstract Background Drug and contrast agent delivery systems that achieve controlled release in the presence of enzymatic activity are becoming increasingly important, as enzymatic activity is a hallmark of a wide array of diseases, including cancer and atherosclerosis. Here, we have synthesized clusters of ultrasmall superparamagnetic iron oxides (USPIOs that sense enzymatic activity for applications in magnetic resonance imaging (MRI. To achieve this goal, we utilize amphiphilic poly(propylene sulfide-bl-poly(ethylene glycol (PPS-b-PEG copolymers, which are known to have excellent properties for smart delivery of drug and siRNA. Results Monodisperse PPS polymers were synthesized by anionic ring opening polymerization of propylene sulfide, and were sequentially reacted with commercially available heterobifunctional PEG reagents and then ssDNA sequences to fashion biofunctional PPS-bl-PEG copolymers. They were then combined with hydrophobic 12 nm USPIO cores in the thin-film hydration method to produce ssDNA-displaying USPIO micelles. Micelle populations displaying complementary ssDNA sequences were mixed to induce crosslinking of the USPIO micelles. By design, these crosslinking sequences contained an EcoRV cleavage site. Treatment of the clusters with EcoRV results in a loss of R2 negative contrast in the system. Further, the USPIO clusters demonstrate temperature sensitivity as evidenced by their reversible dispersion at ~75°C and re-clustering following return to room temperature. Conclusions This work demonstrates proof of concept of an enzymatically-actuatable and thermoresponsive system for dynamic biosensing applications. The platform exhibits controlled release of nanoparticles leading to changes in magnetic relaxation, enabling detection of enzymatic activity. Further, the presented functionalization scheme extends the scope of potential applications for PPS-b-PEG. Combined with previous findings using this polymer platform that

  6. Enzymatic- and temperature-sensitive controlled release of ultrasmall superparamagnetic iron oxides (USPIOs).

    Science.gov (United States)

    Yu, Shann S; Scherer, Randy L; Ortega, Ryan A; Bell, Charleson S; O'Neil, Conlin P; Hubbell, Jeffrey A; Giorgio, Todd D

    2011-02-27

    Drug and contrast agent delivery systems that achieve controlled release in the presence of enzymatic activity are becoming increasingly important, as enzymatic activity is a hallmark of a wide array of diseases, including cancer and atherosclerosis. Here, we have synthesized clusters of ultrasmall superparamagnetic iron oxides (USPIOs) that sense enzymatic activity for applications in magnetic resonance imaging (MRI). To achieve this goal, we utilize amphiphilic poly(propylene sulfide)-bl-poly(ethylene glycol) (PPS-b-PEG) copolymers, which are known to have excellent properties for smart delivery of drug and siRNA. Monodisperse PPS polymers were synthesized by anionic ring opening polymerization of propylene sulfide, and were sequentially reacted with commercially available heterobifunctional PEG reagents and then ssDNA sequences to fashion biofunctional PPS-bl-PEG copolymers. They were then combined with hydrophobic 12 nm USPIO cores in the thin-film hydration method to produce ssDNA-displaying USPIO micelles. Micelle populations displaying complementary ssDNA sequences were mixed to induce crosslinking of the USPIO micelles. By design, these crosslinking sequences contained an EcoRV cleavage site. Treatment of the clusters with EcoRV results in a loss of R2 negative contrast in the system. Further, the USPIO clusters demonstrate temperature sensitivity as evidenced by their reversible dispersion at ~75°C and re-clustering following return to room temperature. This work demonstrates proof of concept of an enzymatically-actuatable and thermoresponsive system for dynamic biosensing applications. The platform exhibits controlled release of nanoparticles leading to changes in magnetic relaxation, enabling detection of enzymatic activity. Further, the presented functionalization scheme extends the scope of potential applications for PPS-b-PEG. Combined with previous findings using this polymer platform that demonstrate controlled drug release in oxidative

  7. Biocolloids with ordered urease multilayer shells as enzymatic reactors.

    Science.gov (United States)

    Lvov, Y; Caruso, F

    2001-09-01

    The preparation of biocolloids with organized enzyme-containing multilayer shells for exploitation as colloidal enzymatic nanoreactors is described. Urease multilayers were assembled onto submicrometer-sized polystyrene spheres by the sequential adsorption of urease and polyelectrolyte, in a predetermined order, utilizing electrostatic interactions for layer growth. The catalytic activity of the biocolloids increased proportionally with the number of urease layers deposited on the particles, demonstrating that biocolloid particles with tailored enzymatic activities can be produced. It was further found that precoating the latex spheres with nanoparticles (40-nm silica or 12-nm magnetite) enhanced both the stability (with respect to adsorption) and enzymatic activity of the urease multilayers. The presence of the magnetite nanoparticle coating also provided a magnetic function that allowed the biocolloids to be easily and rapidly separated with a permanent magnet. The fabrication of such colloids opens new avenues for the application of bioparticles and represents a promising route for the creation of complex catalytic particles.

  8. [ACTIVITY OF Ca2+,Mg(2+)-ATPase OF SARCOPLASMIC RETICULUM AND CONTRACTION STRENGTH OF THE FROG SKELETAL MUSCLES UNDER THE EFFECT OF ORGANOPHOSPHORUS INSECTICIDES].

    Science.gov (United States)

    Nozdrenko, D M; Korchinska, L V; Soroca, V M

    2015-01-01

    The results of an experimental study of organophosphorus insecticides, including pirimiphosmethyl, diazinon and chlorpyrifos caused a decline of the contraction properties in m. tibialis anterior fiber bundles of Rana temporaria, as well as sarcoplasmic reticulum Ca2+, Mg(2+)-ATPase enzymatic activity reduction are outlined in this paper. Concentration-dependent strengths response diminishing in isolated skeletal muscle fiber bundles as a result of non-cholinergic influence of organophosphorus insecticides were found. A decrease of Ca2+, Mg(2+)-ATPase enzymatic activity in sarcoplasmic reticulum was observed after administration of each insecticide. The most significant inhibition of this enzyme was observed when using chlorpyrifos.

  9. Recent advances in the elucidation of enzymatic function in natural product biosynthesis [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Tan Gao-Yi

    2015-12-01

    Full Text Available With the successful production of artemisinic acid in yeast, the promising potential of synthetic biology for natural product biosynthesis is now being realized. The recent total biosynthesis of opioids in microbes is considered to be another landmark in this field. The importance and significance of enzymes in natural product biosynthetic pathways have been re-emphasized by these advancements. Therefore, the characterization and elucidation of enzymatic function in natural product biosynthesis are undoubtedly fundamental for the development of new drugs and the heterologous biosynthesis of active natural products. Here, discoveries regarding enzymatic function in natural product biosynthesis over the past year are briefly reviewed.

  10. The effect of chlorsulfurone and MCPB-Na on the enzymatic activity of microorganisms

    Directory of Open Access Journals (Sweden)

    Filimon Marioara Nicoleta

    2014-01-01

    Full Text Available herbicides, have a broad spectrum effect on weeds, in relatively low doses and with a much reduced toxicity on livestock. In this study were used two herbicides: dacsulfuron with the active substance chlorsulfuron (0.005 - 0.035 μg/g soil and butoxone with the active substance MCPB-Na (0.005 - 0.035 mg/L/g soil. The samples were collected from a depth of 0-20 cm from chernozem soil. The effect of herbicide was estimated by measuring the activity of catalase, actual and potential dehydrogenase, urease and cellulase activities. All samples being incubated for 10 days at 27°C using Sapp medium for isolation and study of cellulosolytic bacteria. The inhibitory effect of the tested herbicides was the most intense for the urease and dehydrogenase enzymatic activities. The most resistant cellulosolytic bacteria to the effects of dacsulfuron were Cellfalcicula fusca, Cellfalcicula viridis, Cellvibrio fulvus and Fuseaux veris and for butoxone Cellfalcicula mucosa, C. viridis and C. fulvus.

  11. Vancomycin analogues containing monosaccharides exhibit improved antibiotic activity: a combined one-pot enzymatic glycosylation and chemical diversification strategy.

    Science.gov (United States)

    Thayer, Desiree A; Wong, Chi-Huey

    2006-09-18

    Many natural products contain carbohydrate moieties that contribute to their biological activity. Manipulation of the carbohydrate domain of natural products through multiple glycosylations to identify new derivatives with novel biological activities has been a difficult and impractical process. We report a practical one-pot enzymatic approach with regeneration of cosubstrates to synthesize analogues of vancomycin that contain an N-alkyl glucosamine, which exhibited marked improvement in antibiotic activity against a vancomycin-resistant strain of Enterococcus.

  12. Cryptococcus gattii urease as a virulence factor and the relevance of enzymatic activity in cryptococcosis pathogenesis.

    Science.gov (United States)

    Feder, Vanessa; Kmetzsch, Lívia; Staats, Charley Christian; Vidal-Figueiredo, Natalia; Ligabue-Braun, Rodrigo; Carlini, Célia Regina; Vainstein, Marilene Henning

    2015-04-01

    Ureases (EC 3.5.1.5) are Ni(2+) -dependent metalloenzymes produced by plants, fungi and bacteria that hydrolyze urea to produce ammonia and CO2 . The insertion of nickel atoms into the apo-urease is better characterized in bacteria, and requires at least three accessory proteins: UreD, UreF, and UreG. Our group has demonstrated that ureases possess ureolytic activity-independent biological properties that could contribute to the pathogenicity of urease-producing microorganisms. The presence of urease in pathogenic bacteria strongly correlates with pathogenesis in some human diseases. Some medically important fungi also produce urease, including Cryptococcus neoformans and Cryptococcus gattii. C. gattii is an etiological agent of cryptococcosis, most often affecting immunocompetent individuals. The cryptococcal urease might play an important role in pathogenesis. It has been proposed that ammonia produced via urease action might damage the host endothelium, which would enable yeast transmigration towards the central nervous system. To analyze the role of urease as a virulence factor in C. gattii, we constructed knockout mutants for the structural urease-coding gene URE1 and for genes that code the accessory proteins Ure4 and Ure6. All knockout mutants showed reduced multiplication within macrophages. In intranasally infected mice, the ure1Δ (lacking urease protein) and ure4Δ (enzymatically inactive apo-urease) mutants caused reduced blood burdens and a delayed time of death, whereas the ure6Δ (enzymatically inactive apo-urease) mutant showed time and dose dependency with regard to fungal burden. Our results suggest that C. gattii urease plays an important role in virulence, in part possibly through enzyme activity-independent mechanism(s). © 2015 FEBS.

  13. Multi-Scale Computational Enzymology: Enhancing Our Understanding of Enzymatic Catalysis

    Science.gov (United States)

    Gherib, Rami; Dokainish, Hisham M.; Gauld, James W.

    2014-01-01

    Elucidating the origin of enzymatic catalysis stands as one the great challenges of contemporary biochemistry and biophysics. The recent emergence of computational enzymology has enhanced our atomistic-level description of biocatalysis as well the kinetic and thermodynamic properties of their mechanisms. There exists a diversity of computational methods allowing the investigation of specific enzymatic properties. Small or large density functional theory models allow the comparison of a plethora of mechanistic reactive species and divergent catalytic pathways. Molecular docking can model different substrate conformations embedded within enzyme active sites and determine those with optimal binding affinities. Molecular dynamics simulations provide insights into the dynamics and roles of active site components as well as the interactions between substrate and enzymes. Hybrid quantum mechanical/molecular mechanical (QM/MM) can model reactions in active sites while considering steric and electrostatic contributions provided by the surrounding environment. Using previous studies done within our group, on OvoA, EgtB, ThrRS, LuxS and MsrA enzymatic systems, we will review how these methods can be used either independently or cooperatively to get insights into enzymatic catalysis. PMID:24384841

  14. Multi-Scale Computational Enzymology: Enhancing Our Understanding of Enzymatic Catalysis

    Directory of Open Access Journals (Sweden)

    Rami Gherib

    2013-12-01

    Full Text Available Elucidating the origin of enzymatic catalysis stands as one the great challenges of contemporary biochemistry and biophysics. The recent emergence of computational enzymology has enhanced our atomistic-level description of biocatalysis as well the kinetic and thermodynamic properties of their mechanisms. There exists a diversity of computational methods allowing the investigation of specific enzymatic properties. Small or large density functional theory models allow the comparison of a plethora of mechanistic reactive species and divergent catalytic pathways. Molecular docking can model different substrate conformations embedded within enzyme active sites and determine those with optimal binding affinities. Molecular dynamics simulations provide insights into the dynamics and roles of active site components as well as the interactions between substrate and enzymes. Hybrid quantum mechanical/molecular mechanical (QM/MM can model reactions in active sites while considering steric and electrostatic contributions provided by the surrounding environment. Using previous studies done within our group, on OvoA, EgtB, ThrRS, LuxS and MsrA enzymatic systems, we will review how these methods can be used either independently or cooperatively to get insights into enzymatic catalysis.

  15. Effect of Enzymatic Hydrolysis on the Antioxidant Properties of Alcoholic Extracts of Oilseed Cakes

    Directory of Open Access Journals (Sweden)

    Jacek Arct

    2013-01-01

    Full Text Available The aim of the present study is to compare changes in the total phenolic, flavonoid and reducing sugar content and antioxidant activity of alcoholic extracts of Oenothera biennis, Borago officinalis and Nigella sativa oilseed cakes before and after enzymatic hydrolysis. Extraction with ethanol and hydrolysis with different commercially available glycosidases: α-amylase, β-glucosidase, β-glucanase and their combinations in a ratio of 1:1:1 were investigated. Total phenolic, flavonoid and reducing sugar content, iron-chelating activity and antioxidant activity according to DPPH and ABTS tests were measured in non-hydrolysed extracts and compared with the results obtained for the extracts after the application of immobilised enzymes. As a result, the hydrolysed extracts had a higher phenolic and reducing sugar content as well as higher iron-chelating and antioxidant activities. Total phenolic content of Oenothera biennis, Borago officinalis and Nigella sativa oilseed cake extracts after enzymatic hydrolysis was higher in comparison with non-hydrolysed extracts, i.e. 2 times (for the enzyme combination, and 1.5 and 2 times (for β-glucanase (p<0.05, respectively. The best results in increasing the flavonoid and sugar content as well as in iron-chelating activity were obtained after enzymatic hydrolysis of oilseed cake extracts by β-glucanase. Oilseed cake extracts after hydrolysis with an enzyme combination in a ratio of 1:1:1 had the highest increase in antioxidant activity.

  16. The mechanism of action of lymphokines. IX. The enzymatic basis of hydrogen peroxide production by lymphokine-activated macrophages.

    Science.gov (United States)

    Freund, M; Pick, E

    1986-08-15

    The purpose of this study was to elucidate the biochemical basis of the enhanced hydrogen peroxide (H2O2) production by guinea pig peritoneal macrophages (MP) cultured in lymphokine (LK)-containing medium. The markedly augmented H2O2 generation by these cells, demonstrable by the horseradish peroxidase (HRP)-catalyzed oxidation of phenol red, is distinguished by its lack of dependence on a second stimulus. We demonstrate that H2O2 production is truly spontaneous and is not caused by a stimulant present among the H2O2 assay reagents. The principal candidate for such a role was HRP type II (a mixture of five isoenzymes) that was reported to be capable of eliciting an oxidative burst in MP. Four distinct HRP isoenzymes that were found incapable of provoking an oxidative response were nevertheless adequate for demonstrating H2O2 production by LK-activated MP. Blocking the MP receptor for mannose by the addition of mannan to the assay system resulted in enhanced detection of H2O2 by low concentrations of HRP type II and by three out of four HRP isoenzymes. Treatment of MP with LK-containing medium for 72 hr did not result in a significant change in the activity of cellular superoxide dismutase (SOD) compared with MP cultured for the same length of time in control medium. By using the specific inhibitor of copper, zinc-containing SOD, sodium diethyldithiocarbamate (DDC), and the universal SOD inhibitor, sodium nitroprusside, we found that the predominant enzyme in guinea pig peritoneal MP is probably manganese-containing SOD. Incubation of LK-activated MP with nitroprusside resulted in almost total inhibition of H2O2 production and a simultaneous switch to superoxide (O2-) liberation. Similar exposure to DDC had no effect. These data indicate that H2O2 produced by LK-activated MP is derived exclusively by enzymatic dismutation of O2- mediated by a manganese-containing SOD. The increase in spontaneous H2O2 production induced by LK is therefore secondary to augmented O2

  17. Chromophoric dissolved organic matter and microbial enzymatic activity. A biophysical approach to understand the marine carbon cycle.

    Science.gov (United States)

    Gonnelli, Margherita; Vestri, Stefano; Santinelli, Chiara

    2013-12-01

    This study reports the first information on extracellular enzymatic activity (EEA) combined with a study of DOM dynamics at the Arno River mouth. DOM dynamics was investigated from both a quantitative (dissolved organic carbon, DOC) and a qualitative (absorption and fluorescence of chromophoric DOM, CDOM) perspective. The data here reported highlight that the Arno River was an important source of both DOC and CDOM for this coastal area. CDOM optical properties suggested that terrestrial DOM did not undergo simple dilution at the river mouth but, other physical-chemical and biological processes were probably at work to change its molecular characteristics. This observation was further supported by the "potential" enzymatic activity of β-glucosidase (BG) and leucine aminopeptidase (LAP). Their Vmax values were markedly higher in the river water than in the seawater and their ratio suggested that most of the DOM used by microbes in the Arno River was polysaccharide-like, while in the seawater it was mainly protein-like. © 2013. Published by Elsevier B.V. All rights reserved.

  18. Activity of Ca(2+,Mg(2+-ATPase of sarcoplasmic reticulum and contraction strength of the frog skeletal muscles under the effect of organophosphorus insecticides

    Directory of Open Access Journals (Sweden)

    D. M. Nozdrenko

    2015-08-01

    Full Text Available The results of an experimental study of organo­phosphorus insecticides, including pirimiphosmethyl, diazinon and chlorpyrifos caused a decline of the contraction properties in m. tibialis anterior fiber bundles of Rana temporaria, as well as sarcoplasmic reticulum Ca2+,Mg2+-ATPase enzymatic activity reduction are outlined in this paper. Concentration-dependent strengths response diminishing in isolated skeletal muscle fiber bundles as a result of non-cholinergic influence of organophosphorus insecticides were found. A decrease of Ca2+,Mg2+-ATPase enzymatic activity in sarcoplasmic reticulum was observed after administration of each insecticide. The most significant inhibition of this enzyme was observed when using chlorpyrifos.

  19. Urease immobilized polymer hydrogel: Long-term stability and enhancement of enzymatic activity.

    Science.gov (United States)

    Kutcherlapati, S N Raju; Yeole, Niranjan; Jana, Tushar

    2016-02-01

    A method has been developed in which an enzyme namely urease was immobilized inside hydrogel matrix to study the stability and enzymatic activity in room temperature (∼27-30°C). This urease coupled hydrogel (UCG) was obtained by amine-acid coupling reaction and this procedure is such that it ensured the wider opening of mobile flap of enzyme active site. A systematic comparison of urea-urease assay and the detailed kinetic data clearly revealed that the urease shows activity for more than a month when stored at ∼27-30°C in case of UCG whereas it becomes inactive in case of free urease (enzyme in buffer solution). The aqueous microenvironment inside the hydrogel, unusual morphological features and thermal behaviour were believed to be the reasons for unexpected behaviour. UCG displayed enzyme activity at basic pH and up to 60°C. UCG showed significant enhancement in activity against thermal degradation compared to free urease. In summary, this method is a suitable process to stabilize the biomacromolecules in standard room temperature for many practical uses. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Enzymatic activity of anthropogenic proto-organic soils in soilless farming

    Science.gov (United States)

    Bireescu, Geanina; Dazzi, Carmelo; Laudicina, Vito Armando; Lo Papa, Giuseppe

    2017-04-01

    In soilless agriculture and horticulture coir is the more used substratum to grow plants because it is widely available and more environmentally friendly than sphagnum or peat. In Italy, soilless agriculture concerns an area of about 1,000 hectares, particularly concentrated in Sicily. The southern coastal belt of this region is the area interested by the most significant experiences in the application of techniques of soilless cultivation that, recently, has been used also for growing table grapes. Starting from the above consideration we suppose that the features of the coconut fiber underlay an evident transformation and that even after few years of table grape cultivation, such organic material undergone to a transformation that allows for the formation of a proto-organic soil (a proto-Histosol, we supposed). If this is true, we believe that, in this case, to speak about soilless cultivation is for sure misleading for the common people, as we should define this cultivation "on anthropogenic soils" instead. To fit the aims of this survey we used a big greenhouse devoted to soilless cultivation of table grape in a farm in the Southern Sicily We have considered the enzymatic activity that characterized the coconut fiber after 3 cycles of cultivation of table grapes. We used as a control the coconut fiber that the farmer used to prepare pots for soilless cultivation and coconut fiber of: 6 pots at the end of the first productive cycle 6 pots at the end of the second cycle and 3 pots at the end of the third cycle. On these organic samples we investigated three enzymes, belonging to oxydoreductase (catalase and dehydrogenase) and hydrolase (urease) classes. Statistical analysis of the investigated enzymes was developed using IBM Statistic SPSS v20 by ANOVA, Tukey test HSD for p ≤ 0.01 and Multivariate Statistical Analysis. Results have shown significant differences in enzymes content and quality among coir tests. The use of the coco fiber, as nutritive substratum

  1. Bioelectrocatalytic NAD+/NADH inter-conversion: transformation of an enzymatic fuel cell into an enzymatic redox flow battery.

    Science.gov (United States)

    Quah, Timothy; Milton, Ross D; Abdellaoui, Sofiene; Minteer, Shelley D

    2017-07-25

    Diaphorase and a benzylpropylviologen redox polymer were combined to create a bioelectrode that can both oxidize NADH and reduce NAD + . We demonstrate how bioelectrocatalytic NAD + /NADH inter-conversion can transform a glucose/O 2 enzymatic fuel cell (EFC) with an open circuit potential (OCP) of 1.1 V into an enzymatic redox flow battery (ERFB), which can be rapidly recharged by operation as an EFC.

  2. Enzymatic Processes in Marine Biotechnology.

    Science.gov (United States)

    Trincone, Antonio

    2017-03-25

    In previous review articles the attention of the biocatalytically oriented scientific community towards the marine environment as a source of biocatalysts focused on the habitat-related properties of marine enzymes. Updates have already appeared in the literature, including marine examples of oxidoreductases, hydrolases, transferases, isomerases, ligases, and lyases ready for food and pharmaceutical applications. Here a new approach for searching the literature and presenting a more refined analysis is adopted with respect to previous surveys, centering the attention on the enzymatic process rather than on a single novel activity. Fields of applications are easily individuated: (i) the biorefinery value-chain, where the provision of biomass is one of the most important aspects, with aquaculture as the prominent sector; (ii) the food industry, where the interest in the marine domain is similarly developed to deal with the enzymatic procedures adopted in food manipulation; (iii) the selective and easy extraction/modification of structurally complex marine molecules, where enzymatic treatments are a recognized tool to improve efficiency and selectivity; and (iv) marine biomarkers and derived applications (bioremediation) in pollution monitoring are also included in that these studies could be of high significance for the appreciation of marine bioprocesses.

  3. The enzymatic activity of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC) is enhanced by NPM-ALK

    DEFF Research Database (Denmark)

    Boccalatte, Francesco E; Voena, Claudia; Riganti, Chiara

    2009-01-01

    . A well-defined set of ALK-associated tyrosine phospho-peptides, including metabolic enzymes, kinases, ribosomal and cytoskeletal proteins was identified. Validation studies confirmed that VASP and ATIC associated with NPM-ALK and their phosphorylation required ALK activity. ATIC phosphorylation was also...... documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampering the methotrexate-mediated transformylase activity inhibition...

  4. Development of a heavy metals enzymatic-based assay using papain

    International Nuclear Information System (INIS)

    Shukor, Yunus; Baharom, Nor Azlan; Rahman, Fadhil Abd.; Abdullah, Mohd. Puad; Shamaan, Nor Aripin; Syed, Mohd. Arif

    2006-01-01

    A heavy metals enzymatic-based assay using papain was developed. Papain was assayed using the Casein-coomassie-dye-binding assay. The assay is sensitive to several heavy metals. The IC 50 (concentration of toxicant giving 50% inhibition) of Hg 2+ , Ag 2+ , Pb 2 , Zn 2+ is 0.39, 0.40, 2.16, 2.11 mg l -1 , respectively. For Cu 2+ and Cd 2+ the LOQ (limits of quantitation) is 0.004 and 0.1 mg l -1 , respectively. The IC 50 and LOQ values were found to be generally comparable to several other enzymatic and bioassays tests such as: immobilized urease, 15-min Microtox TM , 48 h Daphnia magna, and 96 h Rainbow trout. The papain assay is xenobiotics tolerant, has a wide pH for optimum activity, is temperature stable, and has a relatively quick assay time. The papain assay was used to identify polluted water samples from industrial sources in Penang, Malaysia. We found one site where the assay gave a positive toxic response. The toxicity of the site was confirmed using Atomic Emission Spectrometry analysis

  5. Improved antimelanogenesis and antioxidant effects of polysaccharide from Cuscuta chinensis Lam seeds after enzymatic hydrolysis.

    Science.gov (United States)

    Liu, Zi-Jun; Wang, Ya-Lan; Li, Qi-Ling; Yang, Liu

    2018-01-01

    Cuscuta chinensis polysaccharide (CPS) was extracted using hot water and enzymatically hydrolyzed C. chinensis polysaccharide (ECPS) was produced by the mannase enzymatic hydrolysis process. The purpose of this research was to investigate the antimelanogenic activity of ECPS and CPS in B16F10 melanoma cells. The in vitro antioxidant activity was assessed by their ferric iron reducing power and DPPH free radical scavenging activities. The molecular mass distribution of polysaccharides was determined using SEC-MALLS-RI. CPS was successfully enzymatically degraded using mannase and the weighted average molecular weights of CPS and ECPS were 434.6 kDa and 211.7 kDa. The results of biological activity assays suggested that the enzymatically hydrolyzed polysaccharide had superior antimelanogenic activity and antioxidant effect than the original polysaccharide. ECPS exhibited antimelanogenic activity by down-regulating the expression of tyrosinase, MITF, and TRP-1 without cytotoxic effects in B16F10 melanoma cells. In conclusion, ECPS have the potential to become a skin whitening product.

  6. Identification of Key Residues for Enzymatic Carboxylate Reduction

    Directory of Open Access Journals (Sweden)

    Holly Stolterfoht

    2018-02-01

    Full Text Available Carboxylate reductases (CARs, E.C. 1.2.1.30 generate aldehydes from their corresponding carboxylic acid with high selectivity. Little is known about the structure of CARs and their catalytically important amino acid residues. The identification of key residues for carboxylate reduction provides a starting point to gain deeper understanding of enzymatic carboxylate reduction. A multiple sequence alignment of CARs with confirmed activity recently identified in our lab and from the literature revealed a fingerprint of conserved amino acids. We studied the function of conserved residues by multiple sequence alignments and mutational replacements of these residues. In this study, single-site alanine variants of Neurospora crassa CAR were investigated to determine the contribution of conserved residues to the function, expressability or stability of the enzyme. The effect of amino acid replacements was investigated by analyzing enzymatic activity of the variants in vivo and in vitro. Supported by molecular modeling, we interpreted that five of these residues are essential for catalytic activity, or substrate and co-substrate binding. We identified amino acid residues having significant impact on CAR activity. Replacement of His 237, Glu 433, Ser 595, Tyr 844, and Lys 848 by Ala abolish CAR activity, indicating their key role in acid reduction. These results may assist in the functional annotation of CAR coding genes in genomic databases. While some other conserved residues decreased activity or had no significant impact, four residues increased the specific activity of NcCAR variants when replaced by alanine. Finally, we showed that NcCAR wild-type and mutants efficiently reduce aliphatic acids.

  7. Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

    International Nuclear Information System (INIS)

    Marcondes, Marcelo F.; Torquato, Ricardo J.S.; Assis, Diego M.; Juliano, Maria A.; Hayashi, Mirian A.F.; Oliveira, Vitor

    2010-01-01

    In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, and enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.

  8. Thioredoxin is involved in endothelial cell extracellular transglutaminase 2 activation mediated by celiac disease patient IgA.

    Directory of Open Access Journals (Sweden)

    Cristina Antonella Nadalutti

    Full Text Available PURPOSE: To investigate the role of thioredoxin (TRX, a novel regulator of extracellular transglutaminase 2 (TG2, in celiac patients IgA (CD IgA mediated TG2 enzymatic activation. METHODS: TG2 enzymatic activity was evaluated in endothelial cells (HUVECs under different experimental conditions by ELISA and Western blotting. Extracellular TG2 expression was studied by ELISA and immunofluorescence. TRX was analysed by Western blotting and ELISA. Serum immunoglobulins class A from healthy subjects (H IgA were used as controls. Extracellular TG2 enzymatic activity was inhibited by R281. PX12, a TRX inhibitor, was also employed in the present study. RESULTS: We have found that in HUVECs CD IgA is able to induce the activation of extracellular TG2 in a dose-dependent manner. Particularly, we noted that the extracellular modulation of TG2 activity mediated by CD IgA occurred only under reducing conditions, also needed to maintain antibody binding. Furthermore, CD IgA-treated HUVECs were characterized by a slightly augmented TG2 surface expression which was independent from extracellular TG2 activation. We also observed that HUVECs cultured in the presence of CD IgA evinced decreased TRX surface expression, coupled with increased secretion of the protein into the culture medium. Intriguingly, inhibition of TRX after CD IgA treatment was able to overcome most of the CD IgA-mediated effects including the TG2 extracellular transamidase activity. CONCLUSIONS: Altogether our findings suggest that in endothelial cells CD IgA mediate the constitutive activation of extracellular TG2 by a mechanism involving the redox sensor protein TRX.

  9. Enzymatic fuel cells with an oxygen resistant variant of pyranose-2-oxidase as anode biocatalyst.

    Science.gov (United States)

    Şahin, Samet; Wongnate, Thanyaporn; Chuaboon, Litavadee; Chaiyen, Pimchai; Yu, Eileen Hao

    2018-06-01

    In enzymatic fuel cells (EnFCs), hydrogen peroxide formation is one of the main problems when enzymes, such as, glucose oxidase (GOx) is used due to the conversion of oxygen to hydrogen peroxide in the catalytic reaction. To address this problem, we here report the first demonstration of an EnFC using a variant of pyranose-2-oxidase (P2O-T169G) which has been shown to have low activity towards oxygen. A simple and biocompatible immobilisation approach incorporating multi-walled-carbon nanotubes within ferrocene (Fc)-Nafion film was implemented to construct EnFCs. Successful immobilisation of the enzymes was demonstrated showing 3.2 and 1.7-fold higher current than when P2O-T169G and GOx were used in solution, respectively. P2O-T169G showed 25% higher power output (maximum power density value of 8.45 ± 1.6 μW cm -2 ) and better stability than GOx in aerated glucose solutions. P2O-T169G maintained > 70% of its initial current whereas GOx lost activity > 90% during the first hour of 12 h operation at 0.15 V (vs Ag/Ag + ). A different fuel cell configuration using gas-diffusion cathode and carbon paper electrodes were used to improve the power output of the fuel cell to 29.8 ± 6.1 µW cm -2 . This study suggests that P2O-T169G with low oxygen activity could be a promising anode biocatalyst for EnFC applications. Copyright © 2018. Published by Elsevier B.V.

  10. Application of enzymatic methods for chia (Salvia hispanica L oil extraction

    Directory of Open Access Journals (Sweden)

    Norma Ciau-Solís

    2016-07-01

    Full Text Available Aim. The aim was to evaluate the use of different enzymatic treatments on the oil extraction yield from Chia (Salvia hispanica L. seeds Methods. Enzymatic extraction was performed by treating of whole and degummed chia flours with different conditions of enzyme concentration, pH and temperature. Commercial enzymes were employed: Viscozyme LTM (endo-1,3 (4-betaglucanase derived from Aspergillus aculeatus, with 100 FBG g (Beta Glucanase-unit Fungal and Neutrase0.8LTM, neutral protease with 0.8 AU-NH/g of activity, derived from Bacillus amyloliquefaciens. Results. All treatments of enzymatic oil extraction were different (P <0.05 and the maximum oil yield obtained was 9.35%. Conclusion. Oil extraction using enzymatic methods is not a viable for chia seed

  11. Enzymatic studies on the metabolism of the tetrahydrofurfuryl mercaptan moiety of thiamine tetrahydrofurfuryl disulfide, 2

    International Nuclear Information System (INIS)

    Fujita, Takeshi; Suzuoki, Ziro; Kozuka, Seizi; Oae, Shigeru.

    1973-01-01

    The second step in the enzymatic process responsible for the novel metabolic pathway of foreign mercaptans leading to methylsulfonyl metabolites was shown to be sulfoxidation, subsequent to S-methylation. By using [ 35 S] methyl tetrahydrofurfuryl sulfide (MTFS) and [ 35 S] methyl tetrahydrofurfuryl sulfoxide (MTFSO) as substrates, the occurrence and involvement of both sulfide and sulfoxide oxygenases were demonstrated in rat liver microsomes. Both activities required reduced nicotinamide adenine dinucleotide phosphate (NADPH) and O 2 . The reaction products were isolated and identified as MTFSO and its sulfone, respectively. The apparent Michaelis constants were 6.7x10 -4 M for MTFS and 9.1x10 -15 M for NADPH with sulfide oxygenase and 5.6x10 -3 M for MTFSO and 5.0x10 -5 M for NADPH with sulfoxide oxygenase, respectively. P-chloromercuribenzoate, P-chloromercuribenzenesulfonate, HgCl 2 , and menadione strongly inhibited both oxygenases. Polyanions, such as inorganic phosphate, pyrophosphate, sulfate, and ATP stimulated both enzyme activities, especially that of sulfoxide oxygenase. One atom of 18 O 2 was incorporated into the products in both enzyme reactions. No appreciable incorporation was observed from H 2 18 O. These results indicate that both enzyme systems are typical monooxygenases. (auth.)

  12. LysoPC and PAF Trigger Arachidonic Acid Release by Divergent Signaling Mechanisms in Monocytes

    Directory of Open Access Journals (Sweden)

    Janne Oestvang

    2011-01-01

    Full Text Available Oxidized low-density lipoproteins (LDLs play an important role during the development of atherosclerosis characterized by intimal inflammation and macrophage accumulation. A key component of LDL is lysophosphatidylcholine (lysoPC. LysoPC is a strong proinflammatory mediator, and its mechanism is uncertain, but it has been suggested to be mediated via the platelet activating factor (PAF receptor. Here, we report that PAF triggers a pertussis toxin- (PTX- sensitive intracellular signaling pathway leading to sequential activation of sPLA2, PLD, cPLA2, and AA release in human-derived monocytes. In contrast, lysoPC initiates two signaling pathways, one sequentially activating PLD and cPLA2, and a second parallel PTX-sensitive pathway activating cPLA2 with concomitant activation of sPLA2, all leading to AA release. In conclusion, lysoPC and PAF stimulate AA release by divergent pathways suggesting involvement of independent receptors. Elucidation of monocyte lysoPC-specific signaling mechanisms will aid in the development of novel strategies for atherosclerosis prevention, diagnosis, and therapy.

  13. Enzymatic desulfurization of coal

    Energy Technology Data Exchange (ETDEWEB)

    Boyer, Y.N.; Crooker, S.C.; Kitchell, J.P.; Nochur, S.V.

    1991-05-16

    The overall objective of this program was to investigate the feasibility of an enzymatic desulfurization process specifically intended for organic sulfur removal from coal. Toward that end, a series of specific objectives were defined: (1) establish the feasibility of (bio)oxidative pretreatment followed by biochemical sulfate cleavage for representative sulfur-containing model compounds and coals using commercially-available enzymes; (2) investigate the potential for the isolation and selective use of enzyme preparations from coal-utilizing microbial systems for desulfurization of sulfur-containing model compounds and coals; and (3) develop a conceptual design and economic analysis of a process for enzymatic removal of organic sulfur from coal. Within the scope of this program, it was proposed to carry out a portion of each of these efforts concurrently. (VC)

  14. Role of low density lipoprotein in the activation of plasma lysolecithin acyltransferase activity. Effect of chemical and enzymatic modifications of the lipoprotein on enzyme activity.

    Science.gov (United States)

    Subbaiah, P V; Chen, C H; Bagdade, J D; Albers, J J

    1985-01-01

    The effect of various chemical and enzymatic modifications of low density lipoprotein (LDL) on its ability to activate the isolated human plasma lysolecithin acyltransferase (LAT) was studied. Removal of all lipids from LDL resulted in the complete loss of LAT activation. Removal of only neutral lipids by extraction with heptane retained up to 50% of the original activity, which was not increased further by reconstitution of the LDL with the extracted lipids. Hydrolysis of the diacylphosphoglycerides of the LDL with phospholipases resulted in complete loss of LAT activation which was partially restored by the addition of egg lecithin. Hydrolysis of more than 4% of LDL protein by trypsin led to a linear decrease in activity with complete loss of activity occurring when about 25% of the LDL protein is hydrolyzed. Modification of the arginine groups of LDL reversibly inhibited the activation of LAT. Modification of lysine residues of LDL by acetylation, acetoacetylation or succinylation also abolished its ability to activate lysolecithin acylation.

  15. Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays.

    Science.gov (United States)

    Kojima, Takaaki; Mizoguchi, Takuro; Ota, Eri; Hata, Jumpei; Homma, Keisuke; Zhu, Bo; Hitomi, Kiyotaka; Nakano, Hideo

    2016-02-01

    A novel DNA-binding protein tag, scCro-tag, which is a single-chain derivative of the bacteriophage lambda Cro repressor, has been developed to immobilize proteins of interest (POI) on a solid support through binding OR consensus DNA (ORC) that is tightly bound by the scCro protein. The scCro-tag successfully bound a transglutaminase 2 (TGase 2) substrate and manganese peroxidase (MnP) to microbeads via scaffolding DNA. The resulting protein-coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  16. Enzymatic hydrolysis of plant extracts containing inulin

    Energy Technology Data Exchange (ETDEWEB)

    Guiraud, J.P.; Galzy, P.

    1981-10-01

    Inulin-rich extracts of chicory and Jerusalem artichoke are a good potential source of fructose. Total enzymatic hydrolysis of these extracts can be effected by yeast inulinases (EC 3.2.1.7). Chemical prehydrolysis is unfavourable. Enzymatic hydrolysis has advantages over chemical hydrolysis: it does not produce a dark-coloured fraction or secondary substances. It is possible to envisage the preparation of high fructose syrups using this process. (Refs. 42).

  17. Scale-up and integration of alkaline hydrogen peroxide pretreatment, enzymatic hydrolysis, and ethanolic fermentation.

    Science.gov (United States)

    Banerjee, Goutami; Car, Suzana; Liu, Tongjun; Williams, Daniel L; Meza, Sarynna López; Walton, Jonathan D; Hodge, David B

    2012-04-01

    Alkaline hydrogen peroxide (AHP) has several attractive features as a pretreatment in the lignocellulosic biomass-to-ethanol pipeline. Here, the feasibility of scaling-up the AHP process and integrating it with enzymatic hydrolysis and fermentation was studied. Corn stover (1 kg) was subjected to AHP pretreatment, hydrolyzed enzymatically, and the resulting sugars fermented to ethanol. The AHP pretreatment was performed at 0.125 g H(2) O(2) /g biomass, 22°C, and atmospheric pressure for 48 h with periodic pH readjustment. The enzymatic hydrolysis was performed in the same reactor following pH neutralization of the biomass slurry and without washing. After 48 h, glucose and xylose yields were 75% and 71% of the theoretical maximum. Sterility was maintained during pretreatment and enzymatic hydrolysis without the use of antibiotics. During fermentation using a glucose- and xylose-utilizing strain of Saccharomyces cerevisiae, all of the Glc and 67% of the Xyl were consumed in 120 h. The final ethanol titer was 13.7 g/L. Treatment of the enzymatic hydrolysate with activated carbon prior to fermentation had little effect on Glc fermentation but markedly improved utilization of Xyl, presumably due to the removal of soluble aromatic inhibitors. The results indicate that AHP is readily scalable and can be integrated with enzyme hydrolysis and fermentation. Compared to other leading pretreatments for lignocellulosic biomass, AHP has potential advantages with regard to capital costs, process simplicity, feedstock handling, and compatibility with enzymatic deconstruction and fermentation. Biotechnol. Bioeng. 2012; 109:922-931. © 2011 Wiley Periodicals, Inc. Copyright © 2011 Wiley Periodicals, Inc.

  18. Feeding indices and enzymatic activities of carob moth Ectomyelois ceratoniae (Zeller (Lepidoptera: pyrallidae on two commercial pistachio cultivars and an artificial diet

    Directory of Open Access Journals (Sweden)

    Naeimeh Teimouri

    2015-01-01

    Full Text Available Feeding indices and enzymatic activities of Ectomyelois ceratoniae (Zeller were studied in a growth chamber under controlled conditions (29 ± 2 °C, relative humidity of 70 ± 5% and a photoperiod of 16:8 (L:D hours on two commercial Pistachio cultivars (Akbari and Kalequchi and an artificial diet. Feeding indices of E. ceratoniae larvae differed significantly on three hosts (P < 0.05. The relative consumption rate was calculated to be 5.36 ± 0.009, 11.10 ± 1.49 and 10.631 ± 0.599 (mg/mg/day on artificial diet, Akbari and Kalequchi cultivars, respectively. Carob moth larvae reared on Akbari cultivar showed the highest efficiency of conversion of digested food (ECD (5.64 ± 0.43. The highest amount of efficiency of conversion of ingested food (ECI was obtained on artificial diet but approximate digestibility (AD was the lowest on this diet. The highest enzymatic activities of alpha-amylase, general proteases and lipase were observed in the midgut of larvae reared on artificial diet. Total protein and lipid value were highest in larvae that were reared on artificial diet.

  19. Ultrasound-assisted enzymatic extraction and antioxidant activity of polysaccharides from pumpkin (Cucurbita moschata).

    Science.gov (United States)

    Wu, Hao; Zhu, Junxiang; Diao, Wenchao; Wang, Chengrong

    2014-11-26

    An efficient ultrasound-assisted enzymatic extraction (UAEE) of Cucurbita moschata polysaccharides (CMCP) was established and the CMCP antioxidant activities were studied. The UAEE operating parameters (extraction temperature, ultrasonic power, pH, and liquid-to-material ratio) were optimized using the central composite design (CCD) and the mass transfer kinetic study in UAEE procedure was used to select the optimal extraction time. Enzymolysis and ultrasonication that were simultaneously conducted was selected as the UAEE synergistic model and the optimum extraction conditions with a maximum polysaccharide yield of 4.33 ± 0.15% were as follows: extraction temperature, 51.5 °C; ultrasonic power, 440 W; pH, 5.0; liquid-to-material ratio, 5.70:1 mL/g; and extraction time, 20 min. Evaluation of the antioxidant activity in vitro suggested that CMCP has good potential as a natural antioxidant used in the food or medicine industry because of their high reducing power and positive radical scavenging activity for DPPH radical. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Beneficial effect of mixture of additives amendment on enzymatic activities, organic matter degradation and humification during biosolids co-composting.

    Science.gov (United States)

    Awasthi, Mukesh Kumar; Wang, Quan; Chen, Hongyu; Awasthi, Sanjeev Kumar; Wang, Meijing; Ren, Xiuna; Zhao, Junchao; Zhang, Zengqiang

    2018-01-01

    The objective of this study was to identify the effect of mixture of additives to improve the enzymatic activities, organic matter humification and diminished the bioavailability of heavy metals (HMs) during biosolids co-composting. In this study, zeolite (Z) (10%, 15% and 30%) with 1%lime (L) (dry weight basis of biosolids) was blended into the mixture of biosolids and wheat straw, respectively. The without any amendment and 1%lime applied treatments were run for comparison (Control). The Z+L addition resulted rapid organic matter degradation and humification with maximum enzymatic activities. In addition, higher dosage of Z+1%L amendment reduced the bioavailability of HMs (Cu and Zn) and improved the end product quality as compared to control and 1%L applied treatments. However, the 30%Z+1%L applied treatment showed maximum humification and low bioavailability of HMs but considering the economic feasibility and compost quality results, the treatment with 10%Z+1%L is recommended for biosolids co-composting. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Therapeutic effectiveness of a new enzymatic bleaching dentifrice.

    Science.gov (United States)

    Forner, Leopaldo; Amengual, José; Liena, Carmen; Riutord, Pere

    2012-01-01

    Research into bleaching focuses on new products in order to minimize undesirable effects. This study evaluated the bleaching effectiveness of a new enzymatic-activated dentifrice. A total of 20 volunteers were bleached with a dentifrice containing 5% lactoperoxidase and 3% carbamide peroxide applied three times a day for two minutes over 21 days. Color was recorded before and after the treatment using a spectrophotometer. CIELAB differences were calculated before and after treatment using the paired t test (P whitening teeth. Enzymatic dental bleaching is able to increase the efficiency of low concentration peroxides, reducing the potential risk of peroxides on oral tissues.

  2. Enzymatic processing of pigmented and non pigmented rice bran on changes in oryzanol, polyphenols and antioxidant activity.

    Science.gov (United States)

    Prabhu, Ashish A; Jayadeep, A

    2015-10-01

    Bran from different rice varieties is a treasure of nutrients and nutraceuticals, and its use is limited due to the poor sensory and functional properties. Application of enzymes can alter the functional and phytochemical properties. So the effect of endo-xylanase, cellulase and their combination on microstructural, nutraceutical and antioxidant properties of pigmented (Jyothi) and non-pigmented (IR64) rice bran were investigated. Scanning electron micrograph revealed micro structural changes in fibre structures on processing. All the enzymatic processing methods resulted in an increase in the content of oryzanol, soluble, bound and total polyphenols, flavonoid and tannin. It also showed an increase in the bioactivity with respect to free radical scavenging activity and total antioxidant activity. However, extent of the increase in bio-actives varied with the type of bran and enzyme application method. Endo-xylanase showed higher percentage difference compared to controls of Jyothi and IR64 bran extracts respectively in the content of the bound (10 & 19 %) and total (20 & 14 %) polyphenols. Combination of both the enzymes resulted in higher percentage increase of bioactive components and properties. It resulted in greater percentage difference compared to controls of Jyothi and IR64 extracts respectively in the content of soluble (58 & 17 %) and total (21 & 14 %) polyphenols, flavonoids (12 & 38 %), γ-oryzanol (10 & 12 %), free radical scavenging activity (64 & 30 %) and total antioxidant activity (82 & 136 %). It may be concluded that enzymatic bio-processing of bran with cellulose and hemicellulose degrading enzymes can improve its nutraceutical properties, and it may be used for development of functional foods.

  3. Proximity-activated nanoparticles: in vitro performance of specific structural modification by enzymatic cleavage

    Science.gov (United States)

    Adam Smith, R; Sewell, Sarah L; Giorgio, Todd D

    2008-01-01

    The development and in vitro performance of a modular nanoscale system capable of specific structural modification by enzymatic activity is described in this work. Due to its small physical size and adaptable characteristics, this system has the potential for utilization in targeted delivery systems and biosensing. Nanoparticle probes were synthesized containing two distinct fluorescent species including a quantum dot base particle and fluorescently labeled cleavable peptide substrate. Activity of these probes was monitored by gel electrophoresis with quantitative cleavage measurements made by fluorometric analysis. The model proximity-activated nanoparticles studied here exhibit significant susceptibility to cleavage by matrix metalloprotease-7 (MMP-7) at physiologically relevant concentrations, with nearly complete cleavage of available substrate molecules after 24 hours. This response is specific to MMP-7 enzyme activity, as cleavage is completely inhibited with the addition of EDTA. Utilization of enzyme-specific modification is a sensitive approach with broad applications for targeted therapeutics and biosensing. The versatility of this nanoparticle system is highlighted in its modular design, as it has the capability to integrate characteristics for detection, biosensing, targeting, and payload delivery into a single, multifunctional nanoparticle structure. PMID:18488420

  4. Screening for Genes Coding for Putative Antitumor Compounds, Antimicrobial and Enzymatic Activities from Haloalkalitolerant and Haloalkaliphilic Bacteria Strains of Algerian Sahara Soils

    Directory of Open Access Journals (Sweden)

    Okba Selama

    2014-01-01

    Full Text Available Extreme environments may often contain unusual bacterial groups whose physiology is distinct from those of normal environments. To satisfy the need for new bioactive pharmaceuticals compounds and enzymes, we report here the isolation of novel bacteria from an extreme environment. Thirteen selected haloalkalitolerant and haloalkaliphilic bacteria were isolated from Algerian Sahara Desert soils. These isolates were screened for the presence of genes coding for putative antitumor compounds using PCR based methods. Enzymatic, antibacterial, and antifungal activities were determined by using cultural dependant methods. Several of these isolates are typical of desert and alkaline saline soils, but, in addition, we report for the first time the presence of a potential new member of the genus Nocardia with particular activity against the yeast Saccharomyces cerevisiae. In addition to their haloalkali character, the presence of genes coding for putative antitumor compounds, combined with the antimicrobial activity against a broad range of indicator strains and their enzymatic potential, makes them suitable for biotechnology applications.

  5. Enzymatic hydrolysis of pretreated barley and wheat straw

    DEFF Research Database (Denmark)

    Rosgaard, Lisa

    2007-01-01

    . The work involved evaluation of 1) possible ways to increase the glucose release from the commercial cellulase product Celluclast by boosting with other enzyme activities to increase the enzymatic hydrolysis, 2) comparing differently pretreated feedstock substrates and 3) evaluating a fed-batch substrate...... mixture resulted in a glucose release corresponding to ~84 % of the glucose release from Celluclast. It was therefore suggested that other enzyme activities than the 4 four main cellulase activities in Celluclast are necessary for optimal hydrolysis of lignocellulose. Even though Celluclast...... is a multicomponent cellulase mixture, there are still possibilities for further improvement in terms of providing the most efficient cellulase mixture for lignocellulose hydrolysis. It was shown that substrates evaluated all had some residual hemicellulose in the solid cellulose fraction after pretreatment...

  6. Quinolone resistance-associated amino acid substitutions affect enzymatic activity of Mycobacterium leprae DNA gyrase.

    Science.gov (United States)

    Yamaguchi, Tomoyuki; Yokoyama, Kazumasa; Nakajima, Chie; Suzuki, Yasuhiko

    2017-07-01

    Quinolones are important antimicrobials for treatment of leprosy, a chronic infectious disease caused by Mycobacterium leprae. Although it is well known that mutations in DNA gyrase are responsible for quinolone resistance, the effect of those mutations on the enzymatic activity is yet to be studied in depth. Hence, we conducted in vitro assays to observe supercoiling reactions of wild type and mutated M. leprae DNA gyrases. DNA gyrase with amino acid substitution Ala91Val possessed the highest activity among the mutants. DNA gyrase with Gly89Cys showed the lowest level of activity despite being found in clinical strains, but it supercoiled DNA like the wild type does if applied at a sufficient concentration. In addition, patterns of time-dependent conversion from relaxed circular DNA into supercoiled DNA by DNA gyrases with clinically unreported Asp95Gly and Asp95Asn were observed to be distinct from those by the other DNA gyrases.

  7. Enzymatic browning and antioxidant activities in harvested litchi fruit as influenced by apple polyphenols.

    Science.gov (United States)

    Zhang, Zhengke; Huber, Donald J; Qu, Hongxia; Yun, Ze; Wang, Hui; Huang, Zihui; Huang, Hua; Jiang, Yueming

    2015-03-15

    'Guiwei' litchi fruit were treated with 5 ga.i. L(-1) apple polyphenols (APP) and then stored at 25°C to investigate the effects on pericarp browning. APP treatment effectively reduced pericarp browning and retarded the loss of red colour. APP-treated fruit exhibited higher levels of anthocyanins and cyanidin-3-rutinoside, which correlated with suppressed anthocyanase activity. APP treatment also maintained membrane integrity and reduced oxidative damage, as indicated by a lower relative leakage rate, malondialdehyde content, and reactive oxygen species (ROS) generation. The data suggest that decompartmentalisation of peroxidase and polyphenoloxidase and respective browning substrates was reduced. In addition, APP treatment enhanced the activities of antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase), as well as non-enzymatic antioxidant capacity (DPPH radical-scavenging activity and reducing power), which might be beneficial in scavenging ROS. We propose that APP treatment is a promising safe strategy for controlling postharvest browning of litchi fruit. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Non-Enzymatic Glucose Biosensor Based on CuO-Decorated CeO2 Nanoparticles

    Directory of Open Access Journals (Sweden)

    Panpan Guan

    2016-08-01

    Full Text Available Copper oxide (CuO-decorated cerium oxide (CeO2 nanoparticles were synthesized and used to detect glucose non-enzymatically. The morphological characteristics and structure of the nanoparticles were characterized through transmission electron microscopy, X-ray photoelectron spectroscopy, and X-ray diffraction. The sensor responses of electrodes to glucose were investigated via an electrochemical method. The CuO/CeO2 nanocomposite exhibited a reasonably good sensitivity of 2.77 μA mM−1cm−2, an estimated detection limit of 10 μA, and a good anti-interference ability. The sensor was also fairly stable under ambient conditions.

  9. Sequence homolog-based molecular engineering for shifting the enzymatic pH optimum

    Directory of Open Access Journals (Sweden)

    Fuqiang Ma

    2016-09-01

    Full Text Available Cell-free synthetic biology system organizes multiple enzymes (parts from different sources to implement unnatural catalytic functions. Highly adaption between the catalytic parts is crucial for building up efficient artificial biosynthetic systems. Protein engineering is a powerful technology to tailor various enzymatic properties including catalytic efficiency, substrate specificity, temperature adaptation and even achieve new catalytic functions. However, altering enzymatic pH optimum still remains a challenging task. In this study, we proposed a novel sequence homolog-based protein engineering strategy for shifting the enzymatic pH optimum based on statistical analyses of sequence-function relationship data of enzyme family. By two statistical procedures, artificial neural networks (ANNs and least absolute shrinkage and selection operator (Lasso, five amino acids in GH11 xylanase family were identified to be related to the evolution of enzymatic pH optimum. Site-directed mutagenesis of a thermophilic xylanase from Caldicellulosiruptor bescii revealed that four out of five mutations could alter the enzymatic pH optima toward acidic condition without compromising the catalytic activity and thermostability. Combination of the positive mutants resulted in the best mutant M31 that decreased its pH optimum for 1.5 units and showed increased catalytic activity at pH < 5.0 compared to the wild-type enzyme. Structure analysis revealed that all the mutations are distant from the active center, which may be difficult to be identified by conventional rational design strategy. Interestingly, the four mutation sites are clustered at a certain region of the enzyme, suggesting a potential “hot zone” for regulating the pH optima of xylanases. This study provides an efficient method of modulating enzymatic pH optima based on statistical sequence analyses, which can facilitate the design and optimization of suitable catalytic parts for the construction

  10. Use of [1,2-3 h] testosterone in 5 α- reductase enzymatic activity dosing in dermal fibroblast cultures from polycystic ovarian patients

    International Nuclear Information System (INIS)

    Matei, Lidia; Postolache, Cristian; Condac, Eduard

    2003-01-01

    Polycystic ovarian syndrome is an endocrine malady very frequent in women characterized by the presence of ovarian cysts, visible or not by ultrasonography, menstrual cycle deregulation and sometimes by high plasmatic concentrations of androgen hormones. Many cases of polycystic syndrome could not be easily diagnosed or had an erroneous diagnostic. Therefore, is useful to know the plasmatic androgen hormone profile. This profile could indicate the cause for observed clinical manifestations; this cause may be observed in ovarian, suprarenal glands or hypothalamo-hypophysis level. In vitro studies on dermal fibroblasts permit the detail determination of steroid hormones metabolism in target organs and offer important information regarding action mechanism. This study follows the identification of testosterone metabolites in fibroblasts and enzymatic activities of 5α-reductase using testosterone radioactively labeled with tritium. (authors)

  11. Contrasting effects of untreated textile wastewater onto the soil available nitrogen-phosphorus and enzymatic activities in aridisol.

    Science.gov (United States)

    Arif, Muhammad Saleem; Riaz, Muhammad; Shahzad, Sher Muhammad; Yasmeen, Tahira; Buttler, Alexandre; Garcıa-Gil, Juan Carlos; Roohi, Mahnaz; Rasool, Akhtar

    2016-02-01

    Water shortage and soil qualitative degradation are significant environmental problems in arid and semi-arid regions of the world. The increasing demand for water in agriculture and industry has resulted in the emergence of wastewater use as an alternative in these areas. Textile wastewater is produced in surplus amounts which poses threat to the environment as well as associated flora and fauna. A 60-day incubation study was performed to assess the effects of untreated textile wastewater at 0, 25, 50, 75, and 100% dilution levels on the physico-chemical and some microbial and enzymatic properties of an aridisol soil. The addition of textile wastewater provoked a significant change in soil pH and electrical conductivity and soil dehydrogenase and urease activities compared to the distilled-water treated control soil. Moreover, compared to the control treatment, soil phosphomonoesterase activity was significantly increased from 25 to 75% application rates, but decreased at 100% textile wastewater application rate. Total and available soil N contents increased significantly in response to application of textile wastewater. Despite significant increases in the soil total P contents after the addition of textile wastewater, soil available P content decreased with increasing concentration of wastewater. Changes in soil nutrient contents and related enzymatic activities suggested a dynamic match between substrate availability and soil N and P contents. Aridisols have high fixation and low P availability, application of textile wastewater to such soils should be considered only after careful assessment.

  12. Visualization of red-ox proteins on the gold surface using enzymatic polypyrrole formation

    International Nuclear Information System (INIS)

    Ramanaviciene, A.; Kausaite-Minkstimiene, A.; Voronovic, J.; Ramanavicius, A.; Oztekin, Y.; Carac, G.; German, N.

    2011-01-01

    We describe a new method for the visualization of the activity of red-ox proteins on a gold interface. Glucose oxidase was selected as a model system. Surfaces were modified by adhesion of glucose oxidase on (a) electrochemically cleaned gold; (b) gold films modified with gold nanoparticles, (c) a gold surface modified with self-assembled monolayer, and (d) covalent immobilization of protein on the gold surface modified with a self-assembled monolayer. The simple optical method for the visualization of enzyme on the surfaces is based on the enzymatic formation of polypyrrole. The activity of the enzyme was quantified via enzymatic formation of polypyrrole, which was detected and investigated by quartz microbalance and amperometric techniques. The experimental data suggest that the enzymatic formation of the polymer may serve as a method to indicate the adhesion of active redox enzyme on such surfaces. (author)

  13. The influence of Fe2+ on growth and development of cells enzymatically isolated from Porphyra yezoensis blades

    Science.gov (United States)

    Songdong, Shen; Jixun, Dai

    2005-03-01

    Fe2+ acted as an accessorial factor for many cellular enzymatic reactions is very important for seaweed growth and development, but the Fe2+ requirement in nori had not been seen, Porphyra yezoensis cells were separated enzymatically and cultured in a series of sterilized seawater media containing various concentrations of Fe2+. The growth development and cell were investigated in this work. Through this experiment, two biologically-meant concentration scales were found, one is low concentrations, 12.1-102.1 μg/L, 10-100 times than that in seawater, favoring the development of isolated cells of Porphyra and the other was high concentrations, more than 10mg/L inhibiting the cell growth, leading to the deformity and shrinkage of the cells. At the concentration of 50 mg/L, the cells stopped growing and died eventually.

  14. The Membrane-anchored Serine Protease Prostasin (CAP1/PRSS8) Supports Epidermal Development and Postnatal Homeostasis Independent of Its Enzymatic Activity

    DEFF Research Database (Denmark)

    Peters, Diane E; Szabo, Roman; Friis, Stine

    2014-01-01

    The membrane-anchored serine protease prostasin (CAP1/PRSS8) is part of a cell surface proteolytic cascade that is essential for epithelial barrier formation and homeostasis. Here, we report the surprising finding that prostasin executes these functions independent of its own enzymatic activity. ...

  15. Kinetic modelling of enzymatic starch hydrolysis

    NARCIS (Netherlands)

    Bednarska, K.A.

    2015-01-01

    Kinetic modelling of enzymatic starch hydrolysis – a summary

    K.A. Bednarska

    The dissertation entitled ‘Kinetic modelling of enzymatic starch hydrolysis’ describes the enzymatic hydrolysis and kinetic modelling of liquefaction and saccharification of wheat starch.

  16. New Biocatalyst with Multiple Enzymatic Activities for Treatment of Complex Food Wastewaters

    Directory of Open Access Journals (Sweden)

    Olga Senko

    2008-01-01

    Full Text Available The cells of filamentous fungus R. oryzae entrapped in the polyvinyl alcohol cryogelare capable of producing various extracellular hydrolytic enzymes (proteases, amylases, lipases and are used for the treatment of complex wastewaters of food industry. Five types of media simulating the wastewater of various food enterprises were treated under batch conditions for 600 h. Fats containing mostly residues of unsaturated fatty acids, as well as casein, glucose, sucrose, starch, soybean flour and various salts were the main components of the treated wastewaters. The immobilized cells concurrently possessed lipolytic, amylolytic and proteolytic activities. The level of each enzymatic activity depended on the wastewater content. The physiological state of immobilized cells was monitored by bioluminescent method. The intracellular adenosine triphosphate (ATP concentration determined in the granules with immobilized cells was high enough and almost constant for all the period of biocatalyst application confirming thereby the active metabolic state of the cells. The study of mechanical strength of biocatalyst granules allowed revealing the differences in the values of modulus of biocatalyst elasticity at the beginning and at the end of its use for the wastewater treatment. The decrease in chemical oxygen demand of the tested media after their processing by immobilized biocatalyst was 68–79 % for one working cycle.

  17. Mutation of Asn28 Disrupts the Dimerization and Enzymatic Activity of SARS 3CL

    Energy Technology Data Exchange (ETDEWEB)

    Barrila, J.; Gabelli, S; Bacha, U; Amzel, M; Freire, E

    2010-01-01

    Coronaviruses are responsible for a significant proportion of annual respiratory and enteric infections in humans and other mammals. The most prominent of these viruses is the severe acute respiratory syndrome coronavirus (SARS-CoV) which causes acute respiratory and gastrointestinal infection in humans. The coronavirus main protease, 3CL{sup pro}, is a key target for broad-spectrum antiviral development because of its critical role in viral maturation and high degree of structural conservation among coronaviruses. Dimerization is an indispensable requirement for the function of SARS 3CL{sup pro} and is regulated through mechanisms involving both direct and long-range interactions in the enzyme. While many of the binding interactions at the dimerization interface have been extensively studied, those that are important for long-range control are not well-understood. Characterization of these dimerization mechanisms is important for the structure-based design of new treatments targeting coronavirus-based infections. Here we report that Asn28, a residue 11 {angstrom} from the closest residue in the opposing monomer, is essential for the enzymatic activity and dimerization of SARS 3CLpro. Mutation of this residue to alanine almost completely inactivates the enzyme and results in a 19.2-fold decrease in the dimerization K{sub d}. The crystallographic structure of the N28A mutant determined at 2.35 {angstrom} resolution reveals the critical role of Asn28 in maintaining the structural integrity of the active site and in orienting key residues involved in binding at the dimer interface and substrate catalysis. These findings provide deeper insight into complex mechanisms regulating the activity and dimerization of SARS 3CL{sup pro}.

  18. Enzymatic saccharification of biologically pre-treated wheat straw with white-rot fungi.

    Science.gov (United States)

    Dias, Albino A; Freitas, Gil S; Marques, Guilhermina S M; Sampaio, Ana; Fraga, Irene S; Rodrigues, Miguel A M; Evtuguin, Dmitry V; Bezerra, Rui M F

    2010-08-01

    Wheat straw was submitted to a pre-treatment by the basidiomycetous fungi Euc-1 and Irpex lacteus, aiming to improve the accessibility of cellulose towards enzymatic hydrolysis via previous selective bio-delignification. This allowed the increase of substrate saccharification nearly four and three times while applying the basidiomycetes Euc-1 and I. lacteus, respectively. The cellulose/lignin ratio increased from 2.7 in the untreated wheat straw to 5.9 and 4.6 after the bio-treatment by the basidiomycetes Euc-1 and I. lacteus, respectively, thus evidencing the highly selective lignin biodegradation. The enzymatic profile of both fungi upon bio-treatment of wheat straw have been assessed including laccase, manganese-dependent peroxidase, lignin peroxidase, carboxymethylcellulase, xylanase, avicelase and feruloyl esterase activities. The difference in efficiency and selectivity of delignification within the two fungi treatments was interpreted in terms of specific lignolytic enzyme profiles and moderate xylanase and cellulolytic activities. (c) 2010 Elsevier Ltd. All rights reserved.

  19. The influence of conformational fluctuations on enzymatic activity: modelling the functional motion of β-secretase

    International Nuclear Information System (INIS)

    Neri, M; Cascella, M; Micheletti, C

    2005-01-01

    Considerable insight into the functional activity of proteins and enzymes can be obtained by studying the low energy conformational distortions that the biopolymer can sustain. We carry out the characterization of these large scale structural changes for a protein of considerable pharmaceutical interest, the human β-secretase. Starting from the crystallographic structure of the protein, we use the recently introduced β-Gaussian model to identify, with negligible computational expenditure, the most significant distortions occurring in thermal equilibrium and the associated timescales. The application of this strategy helps us to gain considerable insight into the putative functional movements and, furthermore, allows us to identify a handful of key regions in the protein which have an important mechanical influence on the enzymatic activity despite being spatially distant from the active site. The results obtained within the Gaussian model are validated through an extensive comparison against an all-atom molecular dynamics simulation

  20. Enzymatic Kinetic Resolution of 2-Piperidineethanol for the Enantioselective Targeted and Diversity Oriented Synthesis

    Directory of Open Access Journals (Sweden)

    Dario Perdicchia

    2015-12-01

    Full Text Available 2-Piperidineethanol (1 and its corresponding N-protected aldehyde (2 were used for the synthesis of several natural and synthetic compounds. The existence of a stereocenter at position 2 of the piperidine skeleton and the presence of an easily-functionalized group, such as the alcohol, set 1 as a valuable starting material for enantioselective synthesis. Herein, are presented both synthetic and enzymatic methods for the resolution of the racemic 1, as well as an overview of synthesized natural products starting from the enantiopure 1.

  1. Ectomycorrhizal Fungal Communities and Enzymatic Activities Vary across an Ecotone between a Forest and Field.

    Science.gov (United States)

    Rúa, Megan A; Moore, Becky; Hergott, Nicole; Van, Lily; Jackson, Colin R; Hoeksema, Jason D

    2015-08-28

    Extracellular enzymes degrade macromolecules into soluble substrates and are important for nutrient cycling in soils, where microorganisms, such as ectomycorrhizal (ECM) fungi, produce these enzymes to obtain nutrients. Ecotones between forests and fields represent intriguing arenas for examining the effect of the environment on ECM community structure and enzyme activity because tree maturity, ECM composition, and environmental variables may all be changing simultaneously. We studied the composition and enzymatic activity of ECM associated with loblolly pine (Pinus taeda) across an ecotone between a forest where P. taeda is established and an old field where P. taeda saplings had been growing for <5 years. ECM community and environmental characteristics influenced enzyme activity in the field, indicating that controls on enzyme activity may be intricately linked to the ECM community, but this was not true in the forest. Members of the Russulaceae were associated with increased phenol oxidase activity and decreased peroxidase activity in the field. Members of the Atheliaceae were particularly susceptible to changes in their abiotic environment, but this did not mediate differences in enzyme activity. These results emphasize the complex nature of factors that dictate the distribution of ECM and activity of their enzymes across a habitat boundary.

  2. Catabolite regulation of enzymatic activities in a white pox pathogen and commensal bacteria during growth on mucus polymers from the coral Acropora palmata.

    Science.gov (United States)

    Krediet, Cory J; Ritchie, Kim B; Teplitski, Max

    2009-11-16

    Colonization of host mucus surfaces is one of the first steps in the establishment of coral-associated microbial communities. Coral mucus contains a sulfated glycoprotein (in which oligosaccharide decorations are connected to the polypeptide backbone by a mannose residue) and molecules that result from its degradation. Mucus is utilized as a growth substrate by commensal and pathogenic organisms. Two representative coral commensals, Photobacterium mandapamensis and Halomonas meridiana, differed from a white pox pathogen Serratia marcescens PDL100 in the pattern with which they utilized mucus polymers of Acropora palmata. Incubation with the mucus polymer increased mannopyranosidase activity in S. marcescens, suggestive of its ability to cleave off oligosaccharide side chains. With the exception of glucosidase and N-acetyl galactosaminidase, glycosidases in S. marcescens were subject to catabolite regulation by galactose, glucose, arabinose, mannose and N-acetyl-glucosamine. In commensal P. mandapamensis, at least 10 glycosidases were modestly induced during incubation on coral mucus. Galactose, arabinose, mannose, but not glucose or N-acetyl-glucosamine had a repressive effect on glycosidases in P. mandapamensis. Incubation with the mucus polymers upregulated 3 enzymatic activities in H. meridiana; glucose and galactose appear to be the preferred carbon source in this bacterium. Although all these bacteria were capable of producing the same glycosidases, the differences in the preferred carbon sources and patterns of enzymatic activities induced during growth on the mucus polymer in the presence of these carbon sources suggest that to establish themselves within the coral mucus surface layer commensals and pathogens rely on different enzymatic activities.

  3. Enzymatic determination of cadmium, zinc, and lead in plant materials

    International Nuclear Information System (INIS)

    Muginova, S.V.; Veselova, I.A.; Parova, L.M.; Shekhovtseva, T.N.

    2008-01-01

    Prospects are outlined for using the following enzymes (native and immobilized on polyurethane foam) in the rapid and highly sensitive determination of cadmium, zinc, and lead ions in plant materials (wild grass, fresh pea, and grape): horseradish peroxidase and alkaline phosphatases isolated from chicken intestine and Greenland seal small intestine. The analytical ranges of the above metals are 1x10 -3 -25; 7x10 -3 -250, and 3x10 -2 -67 mg/kg dry matter, respectively. The enzymatic determination procedures developed are based on the inhibiting effect of metal ions on the catalytic activity of peroxidase in the oxidation of o-dianisidine with hydrogen peroxide and alkaline phosphatases in the hydrolysis of p-nitrophenyl phosphate. The rates of enzymatic reactions were monitored spectrophotometrically or visually. In the analysis of plant extracts, their high acidity was diminished by choosing optimum dilution factors and pH values for test samples and the nature and concentration of a buffer solution. The interference of iron(III) was removed by introducing a 0.1 M tartaric acid solution into the indicator reaction. The accuracy of the results of the enzymatic determination of cadmium, zinc, and lead in plant materials was supported by atomic absorption spectrometry and anodic stripping voltammetry [ru

  4. Improving enzymatic activities and thermostability of a tri-functional enzyme with SOD, catalase and cell-permeable activities.

    Science.gov (United States)

    Luangwattananun, Piriya; Eiamphungporn, Warawan; Songtawee, Napat; Bülow, Leif; Isarankura Na Ayudhya, Chartchalerm; Prachayasittikul, Virapong; Yainoy, Sakda

    2017-04-10

    Synergistic action of major antioxidant enzymes, e.g., superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) is known to be more effective than the action of any single enzyme. Recently, we have engineered a tri-functional enzyme, 6His-MnSOD-TAT/CAT-MnSOD (M-TAT/CM), with SOD, CAT and cell-permeable activities. The protein actively internalized into the cells and showed superior protection against oxidative stress-induced cell death over native enzymes fused with TAT. To improve its molecular size, enzymatic activity and stability, in this study, MnSOD portions of the engineered protein were replaced by CuZnSOD, which is the smallest and the most heat resistant SOD isoform. The newly engineered protein, CAT-CuZnSOD/6His-CuZnSOD-TAT (CS/S-TAT), had a 42% reduction in molecular size and an increase in SOD and CAT activities by 22% and 99%, respectively. After incubation at 70°C for 10min, the CS/S-TAT retained residual SOD activity up to 54% while SOD activity of the M-TAT/CM was completely abolished. Moreover, the protein exhibited a 5-fold improvement in half-life at 70°C. Thus, this work provides insights into the design and synthesis of a smaller but much more stable multifunctional antioxidant enzyme with ability to enter mammalian cells for further application as protective/therapeutic agent against oxidative stress-related conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Coated tube for immunochemical and enzymatic assays

    International Nuclear Information System (INIS)

    Brown, J.L.; Lin, W.H.-T.; Woods, J.W.

    1979-01-01

    Containers such as test tubes suitable for use in solid phase immunochemical, enzymatical and particularly radioimmunoassay procedures are described. The lower part of the tube is a polymer, coated with an inert protein to which a biologically active substance eg an antibody to triiodothyronine, thyroxine or digoxin, is attached. (U.K.)

  6. PLD2 has both enzymatic and cell proliferation-inducing capabilities, that are differentially regulated by phosphorylation and dephosphorylation

    International Nuclear Information System (INIS)

    Henkels, Karen M.; Short, Stephen; Peng, Hong-Juan; Fulvio, Mauricio Di; Gomez-Cambronero, Julian

    2009-01-01

    Phospholipase D2 (PLD2) overexpression in mammalian cells results in cell transformation. We have hypothesized that this is due to an increase of de novo DNA synthesis. We show here that overexpression of PLD2-WT leads to an increased DNA synthesis, as measured by the expression levels of the proliferation markers PCNA, p27 KIP1 and phospho-histone-3. The enhancing effect was even higher with phosphorylation-deficient PLD2-Y179F and PLD2-Y511F mutants. The mechanism for this did not involve the enzymatic activity of the lipase, but, rather, the presence of the protein tyrosine phosphatase CD45, as silencing with siRNA for CD45 abrogated the effect. The two Y→F mutants had in common a YxN consensus site that, in the phosphorylated counterparts, could be recognized by SH2-bearing proteins, such as Grb2. Even though Y179F and Y511F cannot bind Grb2, they could still find other protein partners, one of which, we have reasoned, could be CD45 itself. Affinity purified PLD2 is indeed activated by Grb2 and deactivated by CD45 in vitro. We concluded that phosphorylated PLD2, aided by Grb2, mediates lipase activity, whereas dephosphorylated PLD2 mediates an induction of cell proliferation, and the specific residues involved in this newly discovered regulation of PLD2 are Y 179 and Y 511 .

  7. Recent Advances in Enzymatic Fuel Cells: Experiments and Modeling

    Directory of Open Access Journals (Sweden)

    Ivan Ivanov

    2010-04-01

    Full Text Available Enzymatic fuel cells convert the chemical energy of biofuels into electrical energy. Unlike traditional fuel cell types, which are mainly based on metal catalysts, the enzymatic fuel cells employ enzymes as catalysts. This fuel cell type can be used as an implantable power source for a variety of medical devices used in modern medicine to administer drugs, treat ailments and monitor bodily functions. Some advantages in comparison to conventional fuel cells include a simple fuel cell design and lower cost of the main fuel cell components, however they suffer from severe kinetic limitations mainly due to inefficiency in electron transfer between the enzyme and the electrode surface. In this review article, the major research activities concerned with the enzymatic fuel cells (anode and cathode development, system design, modeling by highlighting the current problems (low cell voltage, low current density, stability will be presented.

  8. Enzymatic biomarkers can portray nanoCuO-induced oxidative and neuronal stress in freshwater shredders.

    Science.gov (United States)

    Pradhan, Arunava; Silva, Carla O; Silva, Carlos; Pascoal, Cláudia; Cássio, Fernanda

    2016-11-01

    Commercial applications of nanometal oxides have increased concern about their release into natural waters and consequent risks to aquatic biota and the processes they drive. In forest streams, the invertebrate shredder Allogamus ligonifer plays a key role in detritus food webs by transferring carbon and energy from plant litter to higher trophic levels. We assessed the response profiles of oxidative and neuronal stress enzymatic biomarkers in A. ligonifer after 96h exposure to nanoCuO at concentration ranges stress, Cu 2+ released from nanoCuO was quantified and the enzymatic responses to Cu 2+ exposure at similar effective concentrations were compared. The highest activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) were observed at concentrations stress at low concentrations (released ionic copper on enzyme activities were concentration-dependent, and led to oxidative stress and even to animal death. The activity of acetylcholinesterase (AChE) was strongly inhibited even at concentrations stress in A. ligonifer. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Wild Roman chamomile extracts and phenolic compounds: enzymatic assays and molecular modelling studies with VEGFR-2 tyrosine kinase.

    Science.gov (United States)

    Guimarães, Rafaela; Calhelha, Ricardo C; Froufe, Hugo J C; Abreu, Rui M V; Carvalho, Ana Maria; Queiroz, Maria João R P; Ferreira, Isabel C F R

    2016-01-01

    Angiogenesis is a process by which new blood vessels are formed from the pre-existing vasculature, and it is a key process that leads to tumour development. Some studies have recognized phenolic compounds as chemopreventive agents; flavonoids, in particular, seem to suppress the growth of tumor cells modifying the cell cycle. Herein, the antiangiogenic activity of Roman chamomile (Chamaemelum nobile L.) extracts (methanolic extract and infusion) and the main phenolic compounds present (apigenin, apigenin-7-O-glucoside, caffeic acid, chlorogenic acid, luteolin, and luteolin-7-O-glucoside) was evaluated through enzymatic assays using the tyrosine kinase intracellular domain of the Vascular Endothelium Growth Factor Receptor-2 (VEGFR-2), which is a transmembrane receptor expressed fundamentally in endothelial cells involved in angiogenesis, and molecular modelling studies. The methanolic extract showed a lower IC50 value (concentration that provided 50% of VEGFR-2 inhibition) than the infusion, 269 and 301 μg mL(-1), respectively. Regarding phenolic compounds, luteolin and apigenin showed the highest capacity to inhibit the phosphorylation of VEGFR-2, leading us to believe that these compounds are involved in the activity revealed by the methanolic extract.

  10. Interrogating the activities of conformational deformed enzyme by single-molecule fluorescence-magnetic tweezers microscopy

    Science.gov (United States)

    Guo, Qing; He, Yufan; Lu, H. Peter

    2015-01-01

    Characterizing the impact of fluctuating enzyme conformation on enzymatic activity is critical in understanding the structure–function relationship and enzymatic reaction dynamics. Different from studying enzyme conformations under a denaturing condition, it is highly informative to manipulate the conformation of an enzyme under an enzymatic reaction condition while monitoring the real-time enzymatic activity changes simultaneously. By perturbing conformation of horseradish peroxidase (HRP) molecules using our home-developed single-molecule total internal reflection magnetic tweezers, we successfully manipulated the enzymatic conformation and probed the enzymatic activity changes of HRP in a catalyzed H2O2–amplex red reaction. We also observed a significant tolerance of the enzyme activity to the enzyme conformational perturbation. Our results provide a further understanding of the relation between enzyme behavior and enzymatic conformational fluctuation, enzyme–substrate interactions, enzyme–substrate active complex formation, and protein folding–binding interactions. PMID:26512103

  11. An investigation of nitrile transforming enzymes in the chemo-enzymatic synthesis of the taxol sidechain.

    Science.gov (United States)

    Wilding, Birgit; Veselá, Alicja B; Perry, Justin J B; Black, Gary W; Zhang, Meng; Martínková, Ludmila; Klempier, Norbert

    2015-07-28

    Paclitaxel (taxol) is an antimicrotubule agent widely used in the treatment of cancer. Taxol is prepared in a semisynthetic route by coupling the N-benzoyl-(2R,3S)-3-phenylisoserine sidechain to the baccatin III core structure. Precursors of the taxol sidechain have previously been prepared in chemoenzymatic approaches using acylases, lipases, and reductases, mostly featuring the enantioselective, enzymatic step early in the reaction pathway. Here, nitrile hydrolysing enzymes, namely nitrile hydratases and nitrilases, are investigated for the enzymatic hydrolysis of two different sidechain precursors. Both sidechain precursors, an openchain α-hydroxy-β-amino nitrile and a cyanodihydrooxazole, are suitable for coupling to baccatin III directly after the enzymatic step. An extensive set of nitrilases and nitrile hydratases was screened towards their activity and selectivity in the hydrolysis of two taxol sidechain precursors and their epimers. A number of nitrilases and nitrile hydratases converted both sidechain precursors and their epimers.

  12. Mussel-inspired co-deposition to enhance bisphenol A removal in a bifacial enzymatic membrane reactor

    DEFF Research Database (Denmark)

    Cao, Xiaotong; Luo, Jianquan; Woodley, John M.

    2018-01-01

    were used as the matrix to further exploit the potential of the biocatalytic membranes. such prepared biocatalytic membranes were enzymatically active on both sides, making it possible to construct a bifacial enzymatic membrane reactor (EMR) for highly efficient micro-pollutants removal (taking...

  13. ETHANOL ORGANOSOLV PRETREATMENT OF BAMBOO FOR EFFICIENT ENZYMATIC SACCHARIFICATION

    Directory of Open Access Journals (Sweden)

    Zhiqiang Li,

    2012-06-01

    Full Text Available Bamboo is a potential lignocellulosic biomass for the production of bioethanol because of its high cellulose and hemicelluloses content. In this research, ethanol organosolv pretreatment with dilute sulfuric acid as the catalyst was studied in order to enhance enzymatic saccharification of moso bamboo. The addition of 2% (w/w bamboo dilute sulfuric acid in 75% ethanol had a particularly strong effect on fractionation of bamboo. It yielded a solids fraction containing 83.4% cellulose in the treated substrate. The cellulose conversion to glucose yield reached 77.1 to 83.4% after enzymatic hydrolysis of the solids fraction for 48 h at an enzyme loading of 15 FPU cellulase/g cellulose and 30 IU β-glucosidase/g cellulose. The enzymatic hydrolysis rate was significantly accelerated as the ethanol organosolv pretreatment time increased, reaching the highest enzymatic glucose yield of 83.4% after 48 h at 50 °C. The concentrations of fermentation inhibitors such as HMF (5-hydroxy-2-methyl furfural and furfural were 0.96 g/L and 4.38 g/L in the spent liquor after the ethanol organosolv pretreatment, which were slightly lower than the concentrations quantified during H2SO4-water treatment. Spent liquor was diluted with water, and more than 87.2% of lignin in raw bamboo was recovered as ethanol organosolv lignin through the filtration process.

  14. pH catalyzed pretreatment of corn bran for enhanced enzymatic arabinoxylan degradation

    DEFF Research Database (Denmark)

    Agger, Jane; Johansen, Katja Salomon; Meyer, Anne S.

    2011-01-01

    Corn bran is mainly made up of the pericarp of corn kernels and is a byproduct stream resulting from the wet milling step in corn starch processing. Through statistic modeling this study examined the optimization of pretreatment of corn bran for enzymatic hydrolysis. A low pH pretreatment (pH 2......, 150°C, 65min) boosted the enzymatic release of xylose and glucose and maximized biomass solubilization. With more acidic pretreatment followed by enzymatic hydrolysis the total xylose release was maximized (at pH 1.3) reaching ∼50% by weight of the original amount present in destarched corn bran......, but the enzyme catalyzed xylose release was maximal after pretreatment at approx. pH 2. The total glucose release peaked after pretreatment of approx. pH 1.5 with an enzymatic release of approx. 68% by weight of the original amounts present in destarched corn bran. For arabinose the enzymatic release...

  15. Using temperature-responsive zwitterionic surfactant to enhance the enzymatic hydrolysis of lignocelluloses and recover cellulase by cooling.

    Science.gov (United States)

    Cai, Cheng; Pang, Yuxia; Zhan, Xuejuan; Zeng, Meijun; Lou, Hongming; Qian, Yong; Yang, Dongjie; Qiu, Xueqing

    2017-11-01

    Some zwitterionic surfactants exhibit upper critical solution temperature (UCST) in aqueous solutions. For the zwitterionic surfactant solution mixed with cellulase, when its temperature is below UCST, the cellulase can be recovered by coprecipitation with zwitterionic surfactant. In this work, 3-(Hexadecyldimethylammonio) propanesulfonate (SB3-16) was selected to enhance the enzymatic hydrolysis of lignocelluloses and recover the cellulase. After adding 2mmol/L of SB3-16, the enzymatic digestibility of eucalyptus pretreated by dilute acid (Eu-DA) and by sulfite (Eu-SPORL) increased from 27.9% and 35.1% to 72.6% and 89.7%, respectively. The results showed that SB3-16 could reduce the non-productive adsorption of cellulase on hydrophobic interface, while it did not significantly inhibit the activity of cellulase. For the solution contained 1wt% SB3-16 and 200mg protein/L CTec2 cellulase, 55.2% of protein could be recovered by cooling. The filter paper activity of the recovered cellulase was 1.93FPU/mg protein, which was 95.8% of its initial activity. Copyright © 2017. Published by Elsevier Ltd.

  16. Oxidant and enzymatic antioxidant status (gene expression and activity) in the brain of chickens with cold-induced pulmonary hypertension

    Science.gov (United States)

    Hassanpour, Hossein; Khalaji-Pirbalouty, Valiallah; Nasiri, Leila; Mohebbi, Abdonnaser; Bahadoran, Shahab

    2015-11-01

    To evaluate oxidant and antioxidant status of the brain (hindbrain, midbrain, and forebrain) in chickens with cold-induced pulmonary hypertension, the measurements of lipid peroxidation, protein oxidation, antioxidant capacity, enzymatic activity, and gene expression (for catalase, glutathione peroxidase, and superoxide dismutases) were done. There were high lipid peroxidation/protein oxidation and low antioxidant capacity in the hindbrain of cold-induced pulmonary hypertensive chickens compared to control ( P pulmonary hypertension.

  17. Enzymatic Synthesis and Anti-Allergic Activities of Curcumin Oligosaccharides

    Directory of Open Access Journals (Sweden)

    Kei Shimoda

    2010-01-01

    Full Text Available Curcumin 4‘- O -glucooligosaccharides were synthesized by a two step-enzymatic method using almond β-glucosidase and cyclodextrin glucanotransferase (CGTase. Curcumin was glucosylated to curcumin 4‘- O -β-D-glucopyranoside by almond β-glucosidase in 19% yield. Curcumin 4‘- O -β-D-glucopyranoside was converted into curcumin 4‘- O -β-glucooligosaccharides, i.e. 4‘- O -β-maltoside (51% and 4‘- O -β-maltotrioside (25%, by further CGTase-catalyzed glycosylation. Curcumin 4‘- O -β-glycosides showed suppressive action on IgE antibody formation and inhibitory effects on histamine release from rat peritoneal mast cells.

  18. Secretory phospholipase A2 responsive liposomes exhibit a potent anti-neoplastic effect in vitro, but induce unforeseen severe toxicity in vivo

    DEFF Research Database (Denmark)

    Østrem, Ragnhild Garborg; Parhamifar, Ladan; Pourhassan, Houman

    2017-01-01

    The clinical use of liposomal drug delivery vehicles is often hindered by insufficient drug release. Here we present the rational design of liposomes optimized for secretory phospholipase A2 (sPLA2) triggered drug release, and test their utility in vitro and in vivo. We hypothesized...

  19. Imbalanced nutrient recycling in a warmer ocean driven by differential response of extracellular enzymatic activities

    KAUST Repository

    Ayo, Begoñ a; Abad, Naiara; Artolozaga, Itxaso; Azua, Iñ igo; Bañ a, Zuriñ e; Unanue, Marian; Gasol, Josep M.; Duarte, Carlos M.; Iriberri, Juan

    2017-01-01

    Ocean oligotrophication concurrent with warming weakens the capacity of marine primary producers to support marine food webs and act as a CO2 sink, and is believed to result from reduced nutrient inputs associated to the stabilization of the thermocline. However, nutrient supply in the oligotrophic ocean is largely dependent on the recycling of organic matter. This involves hydrolytic processes catalyzed by extracellular enzymes released by bacteria, which temperature-dependence has not yet been evaluated. Here we report a global assessment of the temperature-sensitivity, as represented by the activation energies (Ea ), of extracellular β-glucosidase (βG), leucine aminopeptidase (LAP) and alkaline phosphatase (AP) enzymatic activities, which enable the uptake by bacteria of substrates rich in carbon, nitrogen and phosphorus, respectively. These Ea were calculated from two different approaches, temperature experimental manipulations and a space-for-time substitution approach, which generated congruent results. The three activities showed contrasting Ea in the subtropical and tropical ocean, with βG increasing the fastest with warming, followed by LAP, while AP showed the smallest increase. The estimated activation energies predict that the hydrolysis products under projected warming scenarios will have higher C:N, C:P and N:P molar ratios than those currently generated, and suggest that the warming of oceanic surface waters leads to a decline in the nutrient supply to the microbial heterotrophic community relative to that of carbon, particularly so for phosphorus, slowing down nutrient recycling and contributing to further ocean oligotrophication. This article is protected by copyright. All rights reserved.

  20. Imbalanced nutrient recycling in a warmer ocean driven by differential response of extracellular enzymatic activities

    KAUST Repository

    Ayo, Begoña

    2017-06-08

    Ocean oligotrophication concurrent with warming weakens the capacity of marine primary producers to support marine food webs and act as a CO2 sink, and is believed to result from reduced nutrient inputs associated to the stabilization of the thermocline. However, nutrient supply in the oligotrophic ocean is largely dependent on the recycling of organic matter. This involves hydrolytic processes catalyzed by extracellular enzymes released by bacteria, which temperature-dependence has not yet been evaluated. Here we report a global assessment of the temperature-sensitivity, as represented by the activation energies (Ea ), of extracellular β-glucosidase (βG), leucine aminopeptidase (LAP) and alkaline phosphatase (AP) enzymatic activities, which enable the uptake by bacteria of substrates rich in carbon, nitrogen and phosphorus, respectively. These Ea were calculated from two different approaches, temperature experimental manipulations and a space-for-time substitution approach, which generated congruent results. The three activities showed contrasting Ea in the subtropical and tropical ocean, with βG increasing the fastest with warming, followed by LAP, while AP showed the smallest increase. The estimated activation energies predict that the hydrolysis products under projected warming scenarios will have higher C:N, C:P and N:P molar ratios than those currently generated, and suggest that the warming of oceanic surface waters leads to a decline in the nutrient supply to the microbial heterotrophic community relative to that of carbon, particularly so for phosphorus, slowing down nutrient recycling and contributing to further ocean oligotrophication. This article is protected by copyright. All rights reserved.

  1. Imbalanced nutrient recycling in a warmer ocean driven by differential response of extracellular enzymatic activities.

    Science.gov (United States)

    Ayo, Begoña; Abad, Naiara; Artolozaga, Itxaso; Azua, Iñigo; Baña, Zuriñe; Unanue, Marian; Gasol, Josep M; Duarte, Carlos M; Iriberri, Juan

    2017-10-01

    Ocean oligotrophication concurrent with warming weakens the capacity of marine primary producers to support marine food webs and act as a CO 2 sink, and is believed to result from reduced nutrient inputs associated to the stabilization of the thermocline. However, nutrient supply in the oligotrophic ocean is largely dependent on the recycling of organic matter. This involves hydrolytic processes catalyzed by extracellular enzymes released by bacteria, which temperature dependence has not yet been evaluated. Here, we report a global assessment of the temperature-sensitivity, as represented by the activation energies (E a ), of extracellular β-glucosidase (βG), leucine aminopeptidase (LAP) and alkaline phosphatase (AP) enzymatic activities, which enable the uptake by bacteria of substrates rich in carbon, nitrogen, and phosphorus, respectively. These E a were calculated from two different approaches, temperature experimental manipulations and a space-for-time substitution approach, which generated congruent results. The three activities showed contrasting E a in the subtropical and tropical ocean, with βG increasing the fastest with warming, followed by LAP, while AP showed the smallest increase. The estimated activation energies predict that the hydrolysis products under projected warming scenarios will have higher C:N, C:P and N:P molar ratios than those currently generated, and suggest that the warming of oceanic surface waters leads to a decline in the nutrient supply to the microbial heterotrophic community relative to that of carbon, particularly so for phosphorus, slowing down nutrient recycling and contributing to further ocean oligotrophication. © 2017 John Wiley & Sons Ltd.

  2. Reduced ceramide synthase 2 activity causes progressive myoclonic epilepsy

    DEFF Research Database (Denmark)

    Mosbech, Mai-Britt; Olsen, Anne S B; Neess, Ditte

    2014-01-01

    between genes involved in SL metabolism and epilepsy. METHODS: We used quantitative real-time PCR, Western blotting, and enzymatic assays to determine the mRNA, protein, and activity levels of ceramide synthase 2 (CERS2) in fiibroblasts isolated from parental control subjects and from a patient diagnosed...... with progressive myoclonic epilepsy (PME). Mass spectrometry and fluorescence microscopy were used to examine the effects of reduced CERS2 activity on cellular lipid composition and plasma membrane functions. RESULTS: We identify a novel 27 kb heterozygous deletion including the CERS2 gene in a proband diagnosed...... with PME. Compared to parental controls, levels of CERS2 mRNA, protein, and activity were reduced by ˜50% in fibroblasts isolated from this proband, resulting in significantly reduced levels of ceramides and sphingomyelins containing the very long-chain fatty acids C24:0 and C26:0. The change in SL...

  3. Continuous enzymatic hydrolysis of lignocellulosic biomass with simultaneous detoxification and enzyme recovery.

    Science.gov (United States)

    Gurram, Raghu N; Menkhaus, Todd J

    2014-07-01

    Recovering hydrolysis enzymes and/or alternative enzyme addition strategies are two potential mechanisms for reducing the cost during the biochemical conversion of lignocellulosic materials into renewable biofuels and biochemicals. Here, we show that enzymatic hydrolysis of acid-pretreated pine wood with continuous and/or fed-batch enzyme addition improved sugar conversion efficiencies by over sixfold. In addition, specific activity of the hydrolysis enzymes (cellulases, hemicellulases, etc.) increased as a result of continuously washing the residual solids with removal of glucose (avoiding the end product inhibition) and other enzymatic inhibitory compounds (e.g., furfural, hydroxymethyl furfural, organic acids, and phenolics). As part of the continuous hydrolysis, anion exchange resin was tested for its dual application of simultaneous enzyme recovery and removal of potential enzymatic and fermentation inhibitors. Amberlite IRA-96 showed favorable adsorption profiles of inhibitors, especially furfural, hydroxymethyl furfural, and acetic acid with low affinity toward sugars. Affinity of hydrolysis enzymes to adsorb onto the resin allowed for up to 92 % of the enzymatic activity to be recovered using a relatively low-molar NaCl wash solution. Integration of an ion exchange column with enzyme recovery into the proposed fed-batch hydrolysis process can improve the overall biorefinery efficiency and can greatly reduce the production costs of lignocellulosic biorenewable products.

  4. In Situ Enzymatically Generated Photoswitchable Oxidase Mimetics and Their Application for Colorimetric Detection of Glucose Oxidase.

    Science.gov (United States)

    Cao, Gen-Xia; Wu, Xiu-Ming; Dong, Yu-Ming; Li, Zai-Jun; Wang, Guang-Li

    2016-07-09

    In this study, a simple and amplified colorimetric assay is developed for the detection of the enzymatic activity of glucose oxidase (GOx) based on in situ formation of a photoswitchable oxidase mimetic of PO₄(3-)-capped CdS quantum dots (QDs). GOx catalyzes the oxidation of 1-thio-β-d-glucose to give 1-thio-β-d-gluconic acid which spontaneously hydrolyzes to β-d-gluconic acid and H₂S; the generated H₂S instantly reacts with Cd(2+) in the presence of Na₃PO₄ to give PO₄(3-)-stabilized CdS QDs in situ. Under visible-light (λ ≥ 400 nm) stimulation, the PO₄(3-)-capped CdS QDs are a new style of oxidase mimic derived by producing some active species, such as h⁺, (•)OH, O₂(•-) and a little H₂O₂, which can oxidize the typical substrate (3,3,5,5-tetramethylbenzydine (TMB)) with a color change. Based on the GOx-triggered growth of the oxidase mimetics of PO₄(3-)-capped CdS QDs in situ, we developed a simple and amplified colorimetric assay to probe the enzymatic activity of GOx. The proposed method allowed the detection of the enzymatic activity of GOx over the range from 25 μg/L to 50 mg/L with a low detection limit of 6.6 μg/L. We believe the PO₄(3-)-capped CdS QDs generated in situ with photo-stimulated enzyme-mimicking activity may find wide potential applications in biosensors.

  5. Automatic single- and multi-label enzymatic function prediction by machine learning

    Directory of Open Access Journals (Sweden)

    Shervine Amidi

    2017-03-01

    Full Text Available The number of protein structures in the PDB database has been increasing more than 15-fold since 1999. The creation of computational models predicting enzymatic function is of major importance since such models provide the means to better understand the behavior of newly discovered enzymes when catalyzing chemical reactions. Until now, single-label classification has been widely performed for predicting enzymatic function limiting the application to enzymes performing unique reactions and introducing errors when multi-functional enzymes are examined. Indeed, some enzymes may be performing different reactions and can hence be directly associated with multiple enzymatic functions. In the present work, we propose a multi-label enzymatic function classification scheme that combines structural and amino acid sequence information. We investigate two fusion approaches (in the feature level and decision level and assess the methodology for general enzymatic function prediction indicated by the first digit of the enzyme commission (EC code (six main classes on 40,034 enzymes from the PDB database. The proposed single-label and multi-label models predict correctly the actual functional activities in 97.8% and 95.5% (based on Hamming-loss of the cases, respectively. Also the multi-label model predicts all possible enzymatic reactions in 85.4% of the multi-labeled enzymes when the number of reactions is unknown. Code and datasets are available at https://figshare.com/s/a63e0bafa9b71fc7cbd7.

  6. Degradation of di(2-ethyl hexyl) phthalate by Fusarium culmorum: Kinetics, enzymatic activities and biodegradation pathway based on quantum chemical modelingpathway based on quantum chemical modeling

    International Nuclear Information System (INIS)

    Ahuactzin-Pérez, Miriam; Tlecuitl-Beristain, Saúl; García-Dávila, Jorge; González-Pérez, Manuel; Gutiérrez-Ruíz, María Concepción; Sánchez, Carmen

    2016-01-01

    Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer widely used in the manufacture of plastics, and it is an environmental contaminant. The specific growth rate (μ), maximum biomass (X_m_a_x), biodegradation constant of DEHP (k), half-life (t_1_/_2) of DEHP biodegradation and removal efficiency of DEHP, esterase and laccase specific activities, and enzymatic yield parameters were evaluated for Fusarium culmorum grown on media containing glucose and different concentrations of DEHP (0, 500 and 1000 mg/L). The greatest μ and the largest X_m_a_x occurred in media supplemented with 1000 mg of DEHP/L. F. culmorum degraded 95% of the highest amount of DEHP tested (1000 mg/L) within 60 h of growth. The k and t_1_/_2 were 0.024 h"−"1 and 28 h, respectively, for both DEHP concentrations. The removal efficiency of DEHP was 99.8% and 99.9% for 1000 and 500 mg/L, respectively. Much higher specific esterase activity than specific laccase activity was observed in all media tested. The compounds of biodegradation of DEHP were identified by GC–MS. A DEHP biodegradation pathway by F. culmorum was proposed on the basis of the intermolecular flow of electrons of the identified intermediate compounds using quantum chemical modeling. DEHP was fully metabolized by F. culmorum with butanediol as the final product. This fungus offers great potential in bioremediation of environments polluted with DEHP. - Highlights: • F. culmorum degraded 95% of DEHP (1000 mg/L) within 60 h. • Removal efficiency of DEHP was 99.8% and 99.9% for 1000 and 500 mg/L, respectively. • DEHP was fully metabolized by F. culmorum, with butanediol as the final product. • A DEHP biodegradation pathway was proposed using on quantum chemical modeling.

  7. Degradation of di(2-ethyl hexyl) phthalate by Fusarium culmorum: Kinetics, enzymatic activities and biodegradation pathway based on quantum chemical modelingpathway based on quantum chemical modeling

    Energy Technology Data Exchange (ETDEWEB)

    Ahuactzin-Pérez, Miriam [Doctorado en Biología Experimental, Universidad Autónoma Metropolitana-Iztapalapa (UAM-I) (Mexico); Facultad de Agrobiología, Universidad Autónoma de Tlaxcala, Ixtacuixtla, Tlaxcala (Mexico); Tlecuitl-Beristain, Saúl; García-Dávila, Jorge [Universidad Politécnica de Tlaxcala, San Pedro Xalcatzinco, Tepeyanco, Tlaxcala CP 90180 (Mexico); González-Pérez, Manuel [Universidad Popular Autónoma del Estado de Puebla, Puebla CP 72410 (Mexico); Gutiérrez-Ruíz, María Concepción [Departamento de Ciencias de la Salud, Universidad Autónoma Metropolitana-Iztapalapa, D.F (Mexico); Sánchez, Carmen, E-mail: sanher6@hotmail.com [Laboratory of Biotechnology, Research Centre for Biological Sciences, Universidad Autónoma de Tlaxcala, Ixtacuixtla, Tlaxcala CP. 90062 (Mexico)

    2016-10-01

    Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer widely used in the manufacture of plastics, and it is an environmental contaminant. The specific growth rate (μ), maximum biomass (X{sub max}), biodegradation constant of DEHP (k), half-life (t{sub 1/2}) of DEHP biodegradation and removal efficiency of DEHP, esterase and laccase specific activities, and enzymatic yield parameters were evaluated for Fusarium culmorum grown on media containing glucose and different concentrations of DEHP (0, 500 and 1000 mg/L). The greatest μ and the largest X{sub max} occurred in media supplemented with 1000 mg of DEHP/L. F. culmorum degraded 95% of the highest amount of DEHP tested (1000 mg/L) within 60 h of growth. The k and t{sub 1/2} were 0.024 h{sup −1} and 28 h, respectively, for both DEHP concentrations. The removal efficiency of DEHP was 99.8% and 99.9% for 1000 and 500 mg/L, respectively. Much higher specific esterase activity than specific laccase activity was observed in all media tested. The compounds of biodegradation of DEHP were identified by GC–MS. A DEHP biodegradation pathway by F. culmorum was proposed on the basis of the intermolecular flow of electrons of the identified intermediate compounds using quantum chemical modeling. DEHP was fully metabolized by F. culmorum with butanediol as the final product. This fungus offers great potential in bioremediation of environments polluted with DEHP. - Highlights: • F. culmorum degraded 95% of DEHP (1000 mg/L) within 60 h. • Removal efficiency of DEHP was 99.8% and 99.9% for 1000 and 500 mg/L, respectively. • DEHP was fully metabolized by F. culmorum, with butanediol as the final product. • A DEHP biodegradation pathway was proposed using on quantum chemical modeling.

  8. Identification and enzymatic characterization of the yeasts isolated from Erzincan tulum cheese

    Directory of Open Access Journals (Sweden)

    S. Karasu-Yalcin

    2012-03-01

    Full Text Available In this study, 146 yeast isolates were obtained from 45 Erzincan tulum cheese samples. By using API ID 32C test system and some complementary morphological, physiological and biochemica tests, 121 of the isolates could be identified at species level, while 12 of them were identified at genus level. The identified yeast isolates belonged to six different genera which were Candida, Geotrichum, Kluyveromyces, Pichia, Saccharomyces and Zygosaccharomyces. The most aboundant species was C. lambica, followed by C. zeylanoides, C. famata var. famata, G. candidum and C. kefyr. Enzymatic characterization of the strains was determined by using API-ZYM test system. All of the isolates had leucin arylamidase activity. Eight strains belonging to S. cerevisiae, Z. mellis, G. candidum and P. fermentans were found to have high leucin arylamidase activities. Most of the isolates had β-galactosidase, acid phosphatase and esterase lipase (C8 activities. Eight investigated C. lambica strains had high acid phosphatase activities. Such enzymatic properties of investigated yeast isolates could be fundamental factor for their application as starter culture candidates in production of Erzincan tulum cheese. It was demonstrated that the strain C. lambica T103 had superior enzymatic characteristics with the potential to be used in further technological investigations as an adjunct starter.

  9. Enzymatic reduction of U(VI) in groundwaters

    International Nuclear Information System (INIS)

    Addelouas, A.; Gong, W.; Lutze, W.; Nuttall, E.; Fritz, B.; Crovisier, J.L.

    1999-01-01

    The use of enzymatic reduction of U(VI) in remediation of groundwater contaminated with U(VI) is receiving considerable attention. Certain strains of bacteria can combine the oxidation of an organic compound to the reduction of U(VI) to U(IV), which precipitates as uraninite. In the present study, we tested the reduction of U(VI) in groundwaters with various origins and compositions. In all groundwaters u(VI) was reduced by sulfate reducing bacteria that had been activated by ethanol and tri-metaphosphate. The reduction rate of U(VI) depends on sulfate concentration in water and the abundance of bacteria in the system. This work shows that bacteria capable of U(VI) reduction are ubiquitous in nature, and suggests the possibility of a large application of the enzymatic reduction of U(VI) for in situ clean up of groundwaters contaminated with uranium. (authors)

  10. Tandem and sequential multi-enzymatic syntheses

    NARCIS (Netherlands)

    Kim, B.G.; Ahn, J.H.; Sello, G.; Di Gennaro, P.; van Herk, T.; Hartog, A.F.; Wever, R.; Oroz-Guinea, I.; Sánchez-Moreno, I.; García-Junceda, E.; Wu, B.; Szymanski, W.; Feringa, B.L.; Janssen, D.B.; Villo, L.; Kreen, M.; Kudryashova, M.; Metsala, A.; Tamp, S.; Lille, ü.; Pehk, T.; Parve, O.; McClean, K.; Eddowes, P.; Whittall, J.; Sutton, P.W.

    2012-01-01

    This chapter contains sections titled: Production of Isorhamnetin 3-O-Glucoside in Escherichia coli Using Engineered Glycosyltransferase Multienzymatic Preparation of (−)-3-(Oxiran-2-yl)Benzoic Acid Enzymatic Synthesis of Carbohydrates from Dihydroxyacetone and Aldehydes by a One Pot Enzyme Cascade

  11. Bacterial Production and Enzymatic Activities in Deep-Sea Sediments of the Pacific Ocean: Biogeochemical Implications of Different Temperature Constraints

    Science.gov (United States)

    Danovaro, R.; Corinaldesi, C.; dell'Anno, A.

    2002-12-01

    The deep-sea bed, acting as the ultimate sink for organic material derived from the upper oceans primary production, is now assumed to play a key role in biogeochemical cycling of organic matter on global scale. Early diagenesis of organic matter in marine sediments is dependent upon biological processes (largely mediated by bacterial activity) and by molecular diffusion. Organic matter reaching the sea floor by sedimentation is subjected to complex biogeochemical transformations that make organic matter largely unsuitable for direct utilization by benthic heterotrophs. Extracellular enzymatic activities in the sediment is generally recognized as the key step in the degradation and utilization of organic polymers by bacteria and a key role in biopolymeric carbon mobilization is played by aminopeptidase, alkaline phosphatase and glucosidase activities. In the present study we investigated bacterial density, bacterial C production and exo-enzymatic activities (aminopeptidase, glucosidase and phosphatase activity) in deep-sea sediments of the Pacific Ocean in relation with the biochemical composition of sediment organic matter (proteins, carbohydrates and lipids), in order to gather information on organic matter cycling and diagenesis. Benthic viral abundance was also measured to investigate the potential role of viruses on microbial loop functioning. Sediment samples were collected at eight stations (depth ranging from 2070-3100 m) along two transects located at the opposite side (north and south) of ocean seismic ridge Juan Fernandez (along latitudes 33° 20' - 33° 40'), constituted by the submerged vulcanoes, which connects the Chilean coasts to Rapa Nui Island. Since the northern and southern sides of this ridge apparently displayed small but significant differences in deep-sea temperature (related to the general ocean circulation), this sampling strategy allowed also investigating the role of different temperature constraints on bacterial activity and

  12. Application of photocatalytic cadmium sulfide nanoparticles to detection of enzymatic activities of glucose oxidase and glutathione reductase using oxidation of 3,3′,5,5′-tetramethylbenzidine

    Energy Technology Data Exchange (ETDEWEB)

    Grinyte, Ruta; Garai-Ibabe, Gaizka; Saa, Laura; Pavlov, Valeri, E-mail: vpavlov@cicbiomagune.es

    2015-06-30

    Highlights: • The light-powered nanosensor fabricated by enzymatic reactions was reported. • The sensor use energy of photons for oxidation of chromogenic enzymatic substrates. • Enzymatic assays for glucose oxidase and glutathione reductase were developed. - Abstract: It was found out that semiconductor CdS nanoparticles (NPs) are able to catalyze photooxidation of the well known chromogenic enzymatic substrate 3,3′,5,5′-tetramethylbenzidine (TMB) by oxygen. The photocatalytical oxidation of TMB does not require hydrogen peroxide and its rate is directly proportional to the quantity of CdS NPs produced in situ through the interaction of Cd{sup 2+} and S{sup 2−} ions in an aqueous medium. This phenomenon was applied to development of colorimetric sensitive assays for glucose oxidase and glutathione reductase based on enzymatic generation of CdS NPs acting as light-powered catalysts. Sensitivity of the developed chromogenic assays was of the same order of magnitude or even better than that of relevant fluorogenic assays. The present approach opens the possibility for the design of simple and sensitive colorimetric assays for a number of enzymes using inexpensive and available TMB as a universal chromogenic compound.

  13. Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid.

    Directory of Open Access Journals (Sweden)

    Yan Huang

    Full Text Available Glutamate decarboxylase (GAD catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement γ-aminobutyric acid (GABA. In this study, GAD from Escherichia coli K12, a pyridoxal phosphate (PLP-dependent enzyme, was overexpressed in E. coli. The GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analog pyridoxine hydrochloride (GAD-V activity was 154.8 U mL-1, 1.8-fold higher than that of GAD obtained without supplementation (GAD-C. Purified GAD-V exhibited increased activity (193.4 U mg-1, 1.5-fold higher than that of GAD-C, superior thermostability (2.8-fold greater than that of GAD-C, and higher kcat/Km (1.6-fold higher than that of GAD-C. Under optimal conditions in reactions mixtures lacking added PLP, crude GAD-V converted 500 g L-1 monosodium glutamate (MSG to GABA with a yield of 100%, and 750 g L-1 MSG with a yield of 88.7%. These results establish the utility of pyridoxine supplementation and lay the foundation for large-scale enzymatic production of GABA.

  14. ALUMINUM CHLORIDE EFFECT ON Ca2+,Mg(2+)-ATPase ACTIVITY AND DYNAMIC PARAMETERS OF SKELETAL MUSCLE CONTRACTION.

    Science.gov (United States)

    Nozdrenko, D M; Abramchuk, O M; Soroca, V M; Miroshnichenko, N S

    2015-01-01

    We studied enzymatic activity and measured strain-gauge contraction properties of the frog Rana temporaria m. tibialis anterior muscle fascicles during the action of aluminum chloride solution. It was shown that AlCl3 solutions did not affect the dynamic properties of skeletal muscle preparation in concentrations less than 10(-4) M Increasing the concentration of AlCl3 to 10(-2) M induce complete inhibition of muscle contraction. A linear correlation between decrease in Ca2+,Mg(2+)-ATPase activity of sarcoplasmic reticulum and the investigated concentrations range of aluminum chloride was observed. The reduction in the dynamic contraction performance and the decrease Ca2+,Mg(2+)-ATPase activity of the sarcoplasmic reticulum under the effect of the investigated AlCl3 solution were minimal in pre-tetanus period of contraction.

  15. Aluminum chloride effect on Ca(2+,Mg(2+-ATPase activity and dynamic parameters of skeletal muscle contraction

    Directory of Open Access Journals (Sweden)

    D. M. Nozdrenko

    2015-10-01

    Full Text Available We studied enzymatic activity and measured strain-gauge contraction properties of the frog Rana temporaria m. tibialis anterior muscle fascicles during the action of aluminum chloride solution. It was shown that AlCl3 solutions did not affect the dynamic properties of skeletal muscle preparation in concentrations less than 10-4 M. Increasing the concentration of AlCl3 to 10-2 M induce complete inhibition of muscle contraction. A linear correlation between decrease in Ca2+,Mg2+-ATPase activity of sarcoplasmic reticulum and the investigated concentrations range of aluminum chloride was observed. The reduction in the dynamic contraction performance and the decrease Ca2+,Mg2+-ATPase activity of the sarcoplasmic reticulum under the effect of the investigated AlCl3 solution were minimal in pre-tetanus period of contraction.

  16. Inhibition of Secretory Phospholipase A(2) in Patients with Acute Coronary Syndromes: Rationale and Design of the Vascular Inflammation Suppression to Treat Acute Coronary Syndrome for 16 Weeks (VISTA-16) Trial

    NARCIS (Netherlands)

    Nicholls, Stephen J.; Cavender, Matthew A.; Kastelein, John J. P.; Schwartz, Gregory; Waters, David D.; Rosenson, Robert S.; Bash, Dianna; Hislop, Colin

    2012-01-01

    Background The action of secretory phospholipase A(2) (sPLA(2)) on lipoproteins may render them more susceptible to oxidation, thereby promoting vascular inflammation and increasing cardiovascular risk. Patients with acute coronary syndrome face a high risk of early, recurrent cardiovascular events

  17. Platelike WO3 sensitized with CdS quantum dots heterostructures for photoelectrochemical dynamic sensing of H2O2 based on enzymatic etching.

    Science.gov (United States)

    Wang, Yanhu; Gao, Chaomin; Ge, Shenguang; Yu, Jinghua; Yan, Mei

    2016-11-15

    A platelike tungsten trioxide (WO3) sensitized with CdS quantum dots (QDs) heterojunction is developed for solar-driven, real-time, and selective photoelectrochemical (PEC) sensing of H2O2 in the living cells. The structure is synthesized by hydrothermally growing platelike WO3 on fluorine doped tin oxide (FTO) and subsequently sensitized with CdS QDs. The as-prepared WO3-CdS QDs heterojunction achieve significant photocurrent enhancement, which is remarkably beneficial for light absorption and charge carrier separation. Based on the enzymatic etching of CdS QDs enables the activation of quenching the charge transfer efficiency, thus leading to sensitive PEC recording of H2O2 level in buffer and cellular environments. The results indicated that the proposed method will pave the way for the development of excellent PEC sensing platform with the quantum dot sensitization. This study could also provide a new train of thought on designing of self-operating photoanode in PEC sensing, promoting the application of semiconductor nanomaterials in photoelectrochemistry. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Effect of the exposure to suspended solids on the enzymatic activity in the bivalve Sinonovacula constricta

    Directory of Open Access Journals (Sweden)

    Guojun Yang

    2017-01-01

    Full Text Available Aquatic animals are susceptible to sudden changes of their living environment but they adopt strategies to cope with adverse environmental challenges. Contamination by suspended solids, often associated with a dramatic change in the concentrations of important water-quality variables is a frequent occurrence in China's coastal waters and estuaries. Here we studied the impact of suspended solids on the activities of the antioxidant enzymes superoxide dismutase (SOD and catalase (CAT, as well as adenosine triphosphates (including Na+ K+-ATPase, Mg+ +-ATPase, Ca+ +-ATPase and H+ K+-ATPase in the gills and visceral mass tissues of the molluscan bivalve Sinonovacula constricta exposed (4, 8, 12, 16, 20, and 24 days to various concentrations of suspended solids. Our results showed that the antioxidant enzymes cooperated closely to effectively scavenge superoxide anion free radicals and H2O2 (which can ultimately inhibit gill activity through the modification of SOD and/or CAT enzymatic activities. ATPases activity (considered to be a sensitive indicator of toxicity could play an effective role in the maintenance of functional integrity of the plasma membranes as well as some other intracellular functions. After the exposure, a decrease in the Na+ K+-ATPase, Mg+ +-ATPase, and Ca+ +-ATPase activity of the gills was observed suggesting that they were inhibited by the treatments. These results also indicated that, from day 4 to day 16, exposure to high concentrations of suspended solids had an inhibitory effect on the activity of H+-K+-ATPase in the visceral mass of S. constricta. However, after a period of adaptation the H+-K+-ATPase activity was restored to original levels. Our results suggest that long-term exposure to high levels of suspended solids disturb osmoregulation, gastric acid secretion and digestion, cause oxidative damage, as a consequence of antioxidant enzymes inactivation which eventually damages the gills, affect the food intake

  19. Fabrication of Nickel/nanodiamond/boron-doped diamond electrode for non-enzymatic glucose biosensor

    International Nuclear Information System (INIS)

    Dai, Wei; Li, Mingji; Gao, Sumei; Li, Hongji; Li, Cuiping; Xu, Sheng; Wu, Xiaoguo; Yang, Baohe

    2016-01-01

    Highlights: • Nanodiamonds (NDs) were electrophoretically deposited on the BDD film. • The NDs significantly extended the potential window. • Ni/NDs/BDD electrode was prepared by electrodeposition. • The electrode shows good catalytic activity for glucose oxidation. - Abstract: A stable and sensitive non-enzymatic glucose sensor was prepared by modifying a boron-doped diamond (BDD) electrode with nickel (Ni) nanosheets and nanodiamonds (NDs). The NDs were electrophoretically deposited on the BDD surface, and acted as nucleation sites for the subsequent electrodeposition of Ni. The morphology and composition of the modified BDD electrodes were characterized by field-emission scanning electron microscopy and energy-dispersive X-ray spectroscopy, respectively. The Ni nanosheet-ND modified BDD electrode exhibited good current response towards the non-enzymatic oxidation of glucose in alkaline media. The NDs significantly extended the potential window. The response to glucose was linear over the 0.2–1055.4-μM range. The limit of detection was 0.05 μM, at a signal-to-noise ratio of 3. The Ni nanosheet-ND/BDD electrode exhibited good selectivity, reproducibility and stability. Its electrochemical performance, low cost and simple preparation make it a promising non-enzymatic glucose sensor.

  20. ENCAPSULATION OF HORSERADISH PEROXIDASE-GLUCOSE OXIDASE (HRP-GOx IN SILICA AQUAGEL SYNTHESIZED FROM RICE HULL ASH FOR ENZYMATIC REACTION OF GLUCOSE

    Directory of Open Access Journals (Sweden)

    Nuryono Nuryono

    2010-06-01

    Full Text Available In recent years, the sol-gel technique has attracted increasing interest as a unique approach to immobilize biomolecules for bioanalytical applications as well as biochemical and biophysical studies. In this research, encapsulation of Horseradish peroxidase-Glucose oxidase (HRP-GOx enzymes in silica aquagel from rice hull ash by sol-gel process has been carried out. In addition, the effect of several parameters (weight ratio of HRP to GOx, pH, temperature, sodium ion concentration on enzyme activity was studied, as well. Rice hull ash, which was produced by ashing at 700 °C, was extracted it's silika by NaOH solution 1 M at 100 °C for two hours to produce sodium silikate (Na2SiO3 solution. The Na2SiO3 solution with pH of 13 was added with a strong cation exchanger resin, to produce sol solution with the pH of 4. Encapsulation was emphasized by mixing sol solution and phosphate buffer pH 7 containing HRP-GOx solution at volume ratio of buffer to sol solution 1:5. The mixture was transferred into 96-microwell plate and was aged for 24 hours. Enzymatic reaction was carried out by adding chromogenic solution of phenol and 4-aminoantipyrine (4-AAP and b-D-glucose solution (as substrate into the microwell. Enzymatic activity was examined by measuring absorbance of product solution at 490 nm with ELISA reader. Result of enzymatic activity for encapsulated enzymes (SGE was compared to that for free enzymes (EB. Results showed that at the investigated condition, HRP-GOx enzymes gave high activity at weight ratio of HRP to GOx 10:1 and pH 7 for both SGE and EB. Encapsulation caused the enzymes activity decrease to 53.0±0.2 %. However, SGE was observed to be more stable on pH and temperature changes than EB. Study on the effect of sodium concentration showed that the increase of sodium concentration from 0.10 to 0.37 M decreased the enzymatic activity to 56±0.2%. Reusability test showed that the synthesized SGE was reusable with activity decrease of 60

  1. Aldehyde PEGylation of laccase from Trametes versicolor in route to increase its stability: effect on enzymatic activity.

    Science.gov (United States)

    Mayolo-Deloisa, Karla; González-González, Mirna; Simental-Martínez, Jesús; Rito-Palomares, Marco

    2015-03-01

    Laccase is a multicopper oxidase that catalyzes the oxidation of phenolic compounds. Laccase can be used in bioremediation, beverage (wine, fruit juice, and beer) processing, ascorbic acid determination, sugar beet pectin gelation baking, and as a biosensor. Recently, the antiproliferative activity of laccase toward tumor cells has been reported. Because of the potential applications of this enzyme, the efforts for enhancing and stabilizing its activity have increased. Thus, the PEGylation of laccase can be an alternative. PEGylation is the covalent attachment of one or more molecules of methoxy poly(ethylene glycol) (mPEG) to a protein. Normally, during the PEGylation reaction, the activity is reduced but the stability increases; thus, it is important to minimize the loss of activity. In this work, the effects of molar ratio (1:4, 1:8, and 1:12), concentration of laccase (6 and 12 mg/ml), reaction time (4 and 17 h), molecular weight, and type of mPEG (20, 30, 40 kDa and 40 kDa-branched) were analyzed. The activity was measured using three substrates: ABTS, 2,6-dimethoxyphenol, and syringaldazine. The best conditions for laccase PEGylation were 12 mg/ml of laccase, molar ratio 1:4, and 4 h reaction time. Under these conditions, the enzyme was able to maintain nearly 100% of its enzymatic activity with ABTS. The PEGylation of laccase has not been extensively explored, so it is important to analyze the effects of this bioconjugation in route to produce a robust modified enzyme. Copyright © 2015 John Wiley & Sons, Ltd.

  2. Effect of enzymatic treatment of extracted sunflower proteins on solubility, amino acid composition, and surface activity.

    Science.gov (United States)

    Conde, José Miñones; Escobar, María del Mar Yust; Pedroche Jiménez, Justo J; Rodríguez, Francisco Millán; Rodríguez Patino, Juan M

    2005-10-05

    Industrial proteins from agriculture of either animal or vegetable origin, including their peptide derivatives, are of great importance, from the qualitative and quantitative point of view, in food formulations (emulsions and foams). A fundamental understanding of the physical, chemical, and functional properties of these proteins is essential if the performance of proteins in foods is to be improved and if underutilized proteins, such as plant proteins (and their hydrolysates and peptides derivatives), are to be increasingly used in traditional and new processed food products (safe, high-quality, health foods with good nutritional value). In this contribution we have determined the main physicochemical characteristics (solubility, composition, and analysis of amino acids) of a sunflower protein isolate (SPI) and its hydrolysates with low (5.62%), medium (23.5%), and high (46.3%) degrees of hydrolysis. The hydrolysates were obtained by enzymatic treatment with Alcalase 2.4 L for DH 5.62 and 23.5% and with Alcalase 2.4 L and Flavorzyme 1000 MG sequentially for DH 46.3%. The protein concentration dependence on surface pressure (surface pressure isotherm), a measure of the surface activity of the products (SPI and its hydrolysates), was obtained by tensiometry. We have observed that the degree of hydrolysis has an effect on solubility, composition, and content of the amino acids of the SPI and its hydrolysates. The superficial activity and the adsorption efficiency were also affected by the degree of hydrolysis.

  3. Phenylpropanoid Glycoside Analogues: Enzymatic Synthesis, Antioxidant Activity and Theoretical Study of Their Free Radical Scavenger Mechanism

    Science.gov (United States)

    López-Munguía, Agustín; Hernández-Romero, Yanet; Pedraza-Chaverri, José; Miranda-Molina, Alfonso; Regla, Ignacio; Martínez, Ana; Castillo, Edmundo

    2011-01-01

    Phenylpropanoid glycosides (PPGs) are natural compounds present in several medicinal plants that have high antioxidant power and diverse biological activities. Because of their low content in plants (less than 5% w/w), several chemical synthetic routes to produce PPGs have been developed, but their synthesis is a time consuming process and the achieved yields are often low. In this study, an alternative and efficient two-step biosynthetic route to obtain natural PPG analogues is reported for the first time. Two galactosides were initially synthesized from vanillyl alcohol and homovanillyl alcohol by a transgalactosylation reaction catalyzed by Kluyveromyces lactis β-galactosidase in saturated lactose solutions with a 30%–35% yield. To synthesize PPGs, the galactoconjugates were esterified with saturated and unsaturated hydroxycinnamic acid derivatives using Candida antarctica Lipase B (CaL-B) as a biocatalyst with 40%–60% yields. The scavenging ability of the phenolic raw materials, intermediates and PPGs was evaluated by the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) method. It was found that the biosynthesized PPGs had higher scavenging abilities when compared to ascorbic acid, the reference compound, while their antioxidant activities were found similar to that of natural PPGs. Moreover, density functional theory (DFT) calculations were used to determine that the PPGs antioxidant mechanism proceeds through a sequential proton loss single electron transfer (SPLET). The enzymatic process reported in this study is an efficient and versatile route to obtain PPGs from different phenylpropanoid acids, sugars and phenolic alcohols. PMID:21674039

  4. Superpower proton linear accelerators for neutron generators and electronuclear facilities

    International Nuclear Information System (INIS)

    Lazarev, N.V.; Kozodaev, A.M.

    2000-01-01

    The report is a review of projects on the superpower proton linear accelerators (SPLA) for neutron generators (NG) and electronuclear facilities, proposed in the recent years. The beam average output capacity in these projects reaches 100 MW. The basic parameters of certain operating NGs, as well as some projected NGs will the SPLA drivers are presented. The problems on application of superconducting resonators in the SPLA as well as the issues of the SPLA reliability and costs are discussed [ru

  5. Cleaved CD147 shed from the surface of malignant melanoma cells activates MMP2 produced by fibroblasts.

    Science.gov (United States)

    Hatanaka, Miho; Higashi, Yuko; Fukushige, Tomoko; Baba, Naoko; Kawai, Kazuhiro; Hashiguchi, Teruto; Su, Juan; Zeng, Weiqi; Chen, Xiang; Kanekura, Takuro

    2014-12-01

    Cluster of differentiation 147 (CD147)/basigin on the malignant tumor cell surface is critical for tumor proliferation, invasiveness, metastasis, and angiogenesis. CD147 expressed on malignant melanoma cells can induce tumor cell invasion by stimulating the production of matrix metalloproteinases (MMPs) by surrounding fibroblasts. Membrane vesicles, microvesicles and exosomes have attracted attention, as vehicles of functional molecules and their association with CD147 has been reported. Cleaved CD147 fragments released from tumor cells were reported to interact with fibroblasts. We investigated the intercellular mechanisms by which CD147 stimulates fibroblasts to induce MMP2 activity. CD147 was knocked-down using short hairpin RNA (shRNA). The stimulatory effect of CD147 in cell culture supernatants, microvesicles, and exosomes on the enzymatic activity of MMP2 was examined by gelatin zymography. Supernatants from A375 control cells induced increased enzymatic activity of fibroblasts; such activity was significantly lower in CD147 knock-down cells. Cleaved CD147 plays a pivotal role in stimulating fibroblasts to induce MMP2 activity. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  6. Bioethanol production: Pretreatment and enzymatic hydrolysis of softwood

    Energy Technology Data Exchange (ETDEWEB)

    Tengborg, Charlotte

    2000-05-01

    The enzymatic hydrolysis process can be used to produce bioethanol from softwood, which are the dominating raw material in the Northern hemisphere. This thesis deals with the development of the process focusing on the pretreatment and the enzymatic hydrolysis stages. The influence of pretreatment conditions on sugar yield, and the effect of inhibitors on the ethanol yield, were investigated for spruce and pine. The maximum yields of hemicellulose sugars and glucose were obtained under different pretreatment conditions. This indicates that two-stage pretreatment may be preferable. The added catalysts, H{sub 2}SO{sub 4} and SO{sub 2}, resulted in similar total sugar yields about 40 g/100 g dry raw material. However, the fermentability of SO{sub 2}-impregnated material was better. This pretreatment resulted in the formation of inhibitors to the subsequent process steps, e.g. sugar and lignin degradation products. The glucose yield in the enzymatic hydrolysis stage was affected by various parameters such as enzyme loading, temperature, pH, residence time, substrate concentration, and agitation. To decrease the amount of fresh water used and thereby waste water produced, the sugar-rich prehydrolysate from the pretreatment step was included in the enzymatic hydrolysis of the solid fraction, resulting in a reduction in the cellulose conversion of up to 36%. Different prehydrolysate detoxification methods, such as treatment with Ca(OH){sub 2}, laccase, and fermentation using yeast, were investigated. The latter was shown to be very efficient. The amount of fresh water used can be further reduced by recycling various process streams. This was simulated experimentally in a bench-scale process. A reduction in fresh water demand of 50% was obtained without any further negative effects on either hydrolysis or fermentation.

  7. Isoprene Production on Enzymatic Hydrolysate of Peanut Hull Using Different Pretreatment Methods

    Directory of Open Access Journals (Sweden)

    Sumeng Wang

    2016-01-01

    Full Text Available The present study is about the use of peanut hull for isoprene production. In this study, two pretreatment methods, hydrogen peroxide-acetic acid (HPAC and popping, were employed prior to enzymatic hydrolysis, which could destroy the lignocellulosic structure and accordingly improve the efficiency of enzymatic hydrolysis. It is proven that the isoprene production on enzymatic hydrolysate with HPAC pretreatment is about 1.9-fold higher than that of popping pretreatment. Moreover, through High Performance Liquid Chromatography (HPLC analysis, the amount and category of inhibitors such as formic acid, acetic acid, and HMF were assayed and were varied in different enzymatic hydrolysates, which may be the reason leading to a decrease in isoprene production during fermentation. To further increase the isoprene yield, the enzymatic hydrolysate of HPAC was detoxified by activated carbon. As a result, using the detoxified enzymatic hydrolysate as the carbon source, the engineered strain YJM21 could accumulate 297.5 mg/L isoprene, which accounted for about 90% of isoprene production by YJM21 fermented on pure glucose (338.6 mg/L. This work is thought to be the first attempt on isoprene production by E. coli using peanut hull as the feedstock. More importantly, it also shows the prospect of peanut hull to be considered as an alternative feedstock for bio-based chemicals or biofuels production due to its easy access and high polysaccharide content.

  8. Conversion of 1-alkyl-2-acetyl-sn-glycerols to platelet activating factor and related phospholipids by rabbit platelets

    International Nuclear Information System (INIS)

    Blank, M.L.; Lee, T.; Cress, E.A.; Malone, B.; Fitzgerald, V.; Snyder, F.

    1984-01-01

    The metabolic pathway for 1-alkyl-2-acetyl-sn-glycerols, a recently discovered biologically active neutral lipid class, was elucidated in experiments conducted with rabbit platelets. The total lipid extract obtained from platelets incubated with 1-[1-,2- 3 H]alkyl-2-acetyl-sn-glycerols or 1-alkyl-2-[ 3 H]acetyl-sn-glycerols contained at least six metabolic products. The six metabolites, identified on the basis of chemical and enzymatic reactions combined with thin-layer or high-performance liquid chromatographic analyses, corresponded to 1-alkyl-sn-glycerols, 1-alkyl-2-acetyl-sn-glycero-3-phosphates, 1-alkyl-2-acyl(long-chain)-sn-glycero-3-phosphoethanolamines, 1-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamines, 1-alkyl-2-acyl(long-chain)-sn-glycero-3-phosphocholines, and 1-alkyl-2-actyl-sn-glycero-3-phosphocholines (platelet activating factor). These results indicate that the metabolic pathway for alkylacetylglycerols involves reaction steps catalyzed by the following enzymatic activities: choline- and ethanolamine- phosphotransferases, acetyl-hydrolase, an acyltransferase, and a phosphotransferase. The step responsible for the biosynthesis of platelet activating factor would appear to be the most important reaction in this pathway and this product could explain the hypotensive activities previously described for alkylacetyl-(or propionyl)-glycerols. Of particular interest was the preference exhibited for the utilization of the 1-hexadecyl-2-acetyl-sn-glycerol species in the formation of platelet activating factor

  9. Enzymatic labelling of. gamma. -globulin and insulin with iodine-125

    Energy Technology Data Exchange (ETDEWEB)

    Lucka, B; Russin, K [Institute of Nuclear Physics, Krakow (Poland)

    1979-01-01

    The parameters of enzymatic labelling of proteins with iodine 125 were examined. The manner and sequence of reagent addition, the effects of reagent concentration, reaction time and total Na/sup 125/I activity on the labelling yield were determined.

  10. Dissipation of S-metolachlor in plant and soil and effect on enzymatic activities.

    Science.gov (United States)

    Wołejko, Elżbieta; Kaczyński, Piotr; Łozowicka, Bożena; Wydro, Urszula; Borusiewicz, Andrzej; Hrynko, Izabela; Konecki, Rafał; Snarska, Krystyna; Dec, Dorota; Malinowski, Paweł

    2017-07-01

    The present study aimed at evaluating the dissipation of S-metolachlor (S-MET) at three doses in maize growing on diverse physico-chemical properties of soil. The effect of herbicide on dehydrogenase (DHA) and acid phosphatase (ACP) activity was estimated. A modified QuEChERS method using LC-MS/MS has been developed. The limit of quantification (0.001 mg kg -1 ) and detection (0.0005 mg kg -1 ) were very low for soil and maize samples. The mean recoveries and RSDs for the six spiked levels (0.001-0.5 mg kg -1 ) were 91.3 and 5.8%. The biggest differences in concentration of S-MET in maize were observed between the 28th and 63rd days. The dissipation of S-MET in the alkaline soil was the slowest between the 2nd and 7th days, and in the acidic soil between the 5th and 11th days. DT 50 of S-MET calculated according to the first-order kinetics model was 11.1-14.7 days (soil) and 9.6-13.9 days (maize). The enzymatic activity of soil was higher in the acidic environment. One observed the significant positive correlation of ACP with pH of soil and contents of potassium and magnesium and negative with contents of phosphorus and organic carbon. The results indicated that at harvest time, the residues of S-MET in maize were well below the safety limit for maize. The findings of this study will foster the research on main parameters influencing the dissipation in maize ecosystems.

  11. Recycle of enzymes and substrate following enzymatic hydrolysis of steam-pretreated aspenwood

    Energy Technology Data Exchange (ETDEWEB)

    Mes-Hartree, M.; Hogan, C.M.; Saddler, J.N.

    1987-09-01

    The commercial production of chemicals and fuels from lignocellulosic residues by enzymatic means still requires considerable research on both the technical and economic aspects. Two technical problems that have been identified as requiring further research are the recycle of the enzymes used in hydrolysis and the reuse of the recalcitrant cellulose remaining after incomplete hydrolysis. Enzyme recycle is required to lower the cost of the enzymes, while the reuse of the spent cellulose will lower the feedstock cost. The conversion process studied was a combined enzymatic hydrolysis and fermentation (CHF) procedure that utilized the cellulolytic enzymes derived from the fungus Trichoderma harzianum E58 and the yeast Saccharomyces cerevisiae. The rate and extent of hydrolysis and ethanol production was monitored as was the activity and hydrolytic potential of the enzymes remaining in the filtrate after the hydrolysis period. When a commercial cellulose was used as the substrate for a routine 2-day CHF process, 60% of the original filter paper activity could be recovered. When steam-treated, water-extracted aspenwood was used as the substrate, only 13% of the original filter paper activity was detected after a similar procedure. The combination of 60% spent enzymes with 40% fresh enzymes resulted in the production of 30% less reducing sugars than the original enzyme mixture. Since 100% hydrolysis of the cellulose portion is seldom accomplished in an enzymatic hydrolysis process, the residual cellulose was used as a substrate for the growth of T. harzianum E58 and production of cellulolytic enzymes. The residue remaining after the CHF process was used as a substrate for the production of the cellulolytic enzymes. The production of enzymes from the residue of the Solka Floc hydrolysis was greater than the production of enzymes from the original Solka Floc. (Refs. 14).

  12. Characterization of two MODY2 mutations with different susceptibility to activation

    Energy Technology Data Exchange (ETDEWEB)

    Langer, Sara; Platz, Christian; Waterstradt, Rica; Baltrusch, Simone, E-mail: simone.baltrusch@med.uni-rostock.de

    2015-09-04

    Glucokinase plays a key role in glucose sensing in pancreatic beta cells and in liver metabolism. Heterozygous inactivating glucokinase mutations cause the autosomal dominantly inherited MODY2 subtype of maturity-onset diabetes of the young. The goal of this study was to elucidate the pathogenicity of the recently described glucokinase mutants L304P and L315H, located in an alpha-helix and connecting region, respectively, at the outer region of the large domain of glucokinase. Both mutants showed wild-type-like cytosolic localization, but faster protein degradation in insulin-secreting MIN6 cells. However, strongly reduced nuclear/cytoplasmic localization of the mutants was observed in primary hepatocytes suggesting reduced interaction with the liver specific glucokinase regulatory protein. Both mutants displayed a significantly lowered glucokinase activity compared to the wild-type protein. Even though the L315H protein showed the lowest enzymatic activity, this mutant was very sensitive to allosteric activation. The endogenous activator fructose-2,6-bisphosphatase evoked an increase in glucokinase activity for both mutants, but much stronger for L315H compared to L304P. The synthetic activator RO281675 was ineffective against the L304P mutant. Expression of the mutant proteins evoked loss of glucose-induced insulin secretion in MIN6 cells. Administration of RO281675 increased insulin secretion, however, only for the L315H mutant. Thus, a glucokinase activator drug therapy may help MODY2 patients not in general, but seems to be a useful strategy for carriers of the L315H glucokinase mutation. - Highlights: • The GK mutants L304P and L315H display a highly reduced enzymatic activity. • In hepatocytes both mutations lower the nuclear/cytoplasmic localization ratio of GK. • Both mutants inhibit stimulus-secretion coupling in insulin-producing cells. • Activation by fructose-2,6-bisphosphatase and by RO281675 is stronger for L315H. • RO281675 stimulates

  13. Enzymatic Detoxication, Conformational Selection, and the Role of Molten Globule Active Sites*

    Science.gov (United States)

    Honaker, Matthew T.; Acchione, Mauro; Zhang, Wei; Mannervik, Bengt; Atkins, William M.

    2013-01-01

    The role of conformational ensembles in enzymatic reactions remains unclear. Discussion concerning “induced fit” versus “conformational selection” has, however, ignored detoxication enzymes, which exhibit catalytic promiscuity. These enzymes dominate drug metabolism and determine drug-drug interactions. The detoxication enzyme glutathione transferase A1–1 (GSTA1–1), exploits a molten globule-like active site to achieve remarkable catalytic promiscuity wherein the substrate-free conformational ensemble is broad with barrierless transitions between states. A quantitative index of catalytic promiscuity is used to compare engineered variants of GSTA1–1 and the catalytic promiscuity correlates strongly with characteristics of the thermodynamic partition function, for the substrate-free enzymes. Access to chemically disparate transition states is encoded by the substrate-free conformational ensemble. Pre-steady state catalytic data confirm an extension of the conformational selection model, wherein different substrates select different starting conformations. The kinetic liability of the conformational breadth is minimized by a smooth landscape. We propose that “local” molten globule behavior optimizes detoxication enzymes. PMID:23649628

  14. Experimental primers containing synthetic and natural compounds reduce enzymatic activity at the dentin–adhesive interface under cyclic loading

    Science.gov (United States)

    Sousa, Ana Beatriz Silva; de Mattos Pimenta Vidal, Cristina; Leme-Kraus, Ariene Arcas; de Carvalho Panzeri Pires-de-Souza, Fernanda; Bedran-Russo, Ana K.

    2016-01-01

    Objective To evaluate the effect of experimental primers (chlorhexidine, enriched mixture of proanthocyanidins and doxycycline) on the adhesive properties and gelatinolytic activity at dentin-resin interfaces of occlusal Class I restorations. Methods The inactivation of enzymes by the experimental primers was assessed by fluorescence assay and gelatin zymography. To assess the adhesive properties, occlusal Class I cavities were prepared in sound human molars, etched with phosphoric acid and restored with one of the primers and an etch-and-rinse adhesive system (Adper Single Bond Plus - 3M ESPE). After the restorative procedures, the specimens were divided into two subgroups (n = 6) consisting of storage in incubation buffer or axial cyclic loading at 50 N and 1,000,000 cycles. Then, the sectioned and sliced specimens were assigned to in situ zymography assay and microtensile bond strength (TBS) test. Results Fluorescence assay and gelatin zymography revealed that the experimental primers inactivated rMMPs. In situ zymography (2-way ANOVA, Tukey, p 0.05). Significance The use of experimental primers impaired the enzymatic activity at the dentin-adhesive interface after cyclic loading and the activity of rMMPs. Cyclic loading did not have a significant effect on the bond strength. PMID:27524231

  15. Characterizing Enzymatic Deposition for Microelectrode Neurotransmitter Detection

    Energy Technology Data Exchange (ETDEWEB)

    Hosein, W. K. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Yorita, A. M. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Tolosa, V. M. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2016-08-12

    The enzyme immobilization process, one step in creating an enzymatic biosensor, was characterized and analyzed as a function of its physical properties. The neural glutamic biosensor is a flexible device, effectively minimizing trauma to the area of implantation. The Multielectrode Array (MEA) is composed primarily of a proprietary polymer which has been successfully implanted into human subjects in recent years. This polymer allows the device the pliability that other devices normally lack, though this poses some challenges to implantation. The electrodes are made of Platinum (Pt), and can range in number from eight to thirty two electrodes per device. These electrodes are electroplated with a semipermeable polymer layer to improve selectivity of the electrode to the neurotransmitter of interest, in this case glutamate. A signal is created from the interaction of glutamate in the brain with the glutamate oxidase (GluOx) which is immobilized on the surface of the electrode by using crosslinking chemistry in conjunction with glutaraldehyde and Bovine Serum Albumin (BSA). The glutamate is oxidized by glutamate oxidase, producing α-ketoglutarate and hydrogen peroxide (H2O2) as a by-product. The production of H2O2 is crucial for detection of the presence of the glutamate within the enzymatic coating, as it diffuses through the enzyme layer and oxidizes at the surface of the electrode. This oxidation is detectable by measurable change in the current using amperometry. Hence, the MEA allows for in vivo monitoring of neurotransmitter activity in real time. The sensitivity of the sensor to these neurotransmitters is dependent on the thickness of the layer, which is investigated in these experiments in order to optimize the efficacy of the device to detecting the substrate, once implanted.

  16. Cerebral ischemia is exacerbated by extracellular nicotinamide phosphoribosyltransferase via a non-enzymatic mechanism.

    Directory of Open Access Journals (Sweden)

    Bing Zhao

    Full Text Available Intracellular nicotinamide phosphoribosyltransferase (iNAMPT in neuron has been known as a protective factor against cerebral ischemia through its enzymatic activity, but the role of central extracellular NAMPT (eNAMPT is not clear. Here we show that eNAMPT protein level was elevated in the ischemic rat brain after middle-cerebral-artery occlusion (MCAO and reperfusion, which can be traced to at least in part from blood circulation. Administration of recombinant NAMPT protein exacerbated MCAO-induced neuronal injury in rat brain, while exacerbated oxygen-glucose-deprivation (OGD induced neuronal injury only in neuron-glial mixed culture, but not in neuron culture. In the mixed culture, NAMPT protein promoted TNF-α release in a time- and concentration-dependent fashion, while TNF-α neutralizing antibody protected OGD-induced, NAMPT-enhanced neuronal injury. Importantly, H247A mutant of NAMPT with essentially no enzymatic activity exerted similar effects on ischemic neuronal injury and TNF-α release as the wild type protein. Thus, eNAMPT is an injurious and inflammatory factor in cerebral ischemia and aggravates ischemic neuronal injury by triggering TNF-α release from glia cells, via a mechanism not related to NAMPT enzymatic activity.

  17. Enzymatic Synthesis of Esculin Ester in Ionic Liquids Buffered with Organic Solvents

    DEFF Research Database (Denmark)

    Hu, Yifan; Guo, Zheng; Lue, Bena-Marie

    2009-01-01

    The enzymatic esterification of esculin catalyzed by Candida antarctica lipase B (Novozym 435) was carried out in ionic liquid (IL)-organic solvent mixed systems in comparison with individual systems. The reaction behaviors in IL-organic solvents were systemically evaluated using acetone as a model...... in IL-acetone mixtures made it possible to improve the solubility of esculin while the effects of ILs on lipase activity were minimized. Following the benignity of ILs to lipase activity, the anions of ILs were ranked in the order as [Tf2N](-) > [PF6](-) > [BF4](-) > [CF3SO3](-) > [C4F9SO3](-) > [TAF...

  18. IMPACT OF ALTITUDES ON SOIL CHARACTERISTICS AND ENZYMATIC ACTIVITIES IN FOREST AND FALLOW LANDS OF ALMORA DISTRICT OF CENTRAL HIMALAYA

    OpenAIRE

    B. R. Maurya; Vimal Singh; P. P. Dhyani

    2014-01-01

    Abstract: Altitude is one of the major topographical factors which influence the fertility status of soil. Population explosion has rooted deforestation at different altitudes to bring more area under cultivation leading to fallow lands. Objective of this study was to assess the impact of altitude on electro-chemical properties and enzymatic activities of forest and fallow land soils of Almora district of Central Himalaya. Seventy soil samples were collected from different altitudes of forest...

  19. D-Amino acid oxidase bio-functionalized platforms: Toward an enhanced enzymatic bio-activity

    Science.gov (United States)

    Herrera, Elisa; Valdez Taubas, Javier; Giacomelli, Carla E.

    2015-11-01

    The purpose of this work is to study the adsorption process and surface bio-activity of His-tagged D-amino acid oxidase (DAAO) from Rhodotorula gracilis (His6-RgDAAO) as the first step for the development of an electrochemical bio-functionalized platform. With such a purpose this work comprises: (a) the His6-RgDAAO bio-activity in solution determined by amperometry, (b) the adsorption mechanism of His6-RgDAAO on bare gold and carboxylated modified substrates in the absence (substrate/COO-) and presence of Ni(II) (substrate/COO- + Ni(II)) determined by reflectometry, and (c) the bio-activity of the His6-RgDAAO bio-functionalized platforms determined by amperometry. Comparing the adsorption behavior and bio-activity of His6-RgDAAO on these different solid substrates allows understanding the contribution of the diverse interactions responsible for the platform performance. His6-RgDAAO enzymatic performance in solution is highly improved when compared to the previously used pig kidney (pk) DAAO. His6-RgDAAO exhibits an amperometrically detectable bio-activity at concentrations as low as those expected on a bio-functional platform; hence, it is a viable bio-recognition element of D-amino acids to be coupled to electrochemical platforms. Moreover, His6-RgDAAO bio-functionalized platforms exhibit a higher surface activity than pkDAAO physically adsorbed on gold. The platform built on Ni(II) modified substrates present enhanced bio-activity because the surface complexes histidine-Ni(II) provide with site-oriented, native-like enzymes. The adsorption mechanism responsible of the excellent performance of the bio-functionalized platform takes place in two steps involving electrostatic and bio-affinity interactions whose prevalence depends on the degree of surface coverage.

  20. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity.

    Science.gov (United States)

    Petzold, Christine; Marceau, Aimee H; Miller, Katherine H; Marqusee, Susan; Keck, James L

    2015-06-05

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity*

    Science.gov (United States)

    Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L.

    2015-01-01

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. PMID:25903123

  2. STUDY ON QUALITY PARAMETERS AND ENZYMATIC ACTIVITY OF GRAIN MILL PRODUCTS REGION IN TRANSYLVANIA

    Directory of Open Access Journals (Sweden)

    Glevitzky Mirel

    2011-07-01

    Full Text Available This paper aims at determining the main quality parameters of grain mill products in the Transylvania region, also studying and emphasizing the enzymatic activity of flour. Determination of quality characteristics of grain mill products entails establishing physical, chemical and sensory parameters and assessing them against the limits imposed by law. Analysis was performed on samples formed by mixing basic medium extracted from different batches. Incremental size, sampling tools, how to extract them, the training sample and laboratory environments, packaging and labeling of samples were performed according to STAS 1068 69. Determination of the fall (Falling Number, an empirical test that relies on the ability of endogenous ?-amylase to reduce viscosity of the treated warm flour suspension is used, large scale milling and bakery industry to predict and assess the Baking quality of flour. In sprouted wheat, characterised by a low Falling number, dextrin produced by the action of ?-amylase leads to a sticky bread core. Experiments suggest that the values fall turnover (FN does not shrink in direct proportion to the percentage of germinating seeds. Amylolytic activity depends on the stage of sprouting of grains. Lack of ?-amylase activity can be corrected by adding malt grain ?-amylase or fungal ?-amylase.

  3. Anti-oxidant, anti-inflammatory and immunomodulating properties of an enzymatic protein hydrolysate from yellow field pea seeds.

    Science.gov (United States)

    Ndiaye, Fatou; Vuong, Tri; Duarte, Jairo; Aluko, Rotimi E; Matar, Chantal

    2012-02-01

    Enzymatic protein hydrolysates of yellow pea seed have been shown to possess high anti-oxidant and anti-bacterial activities. The aim of this work was to confirm the anti-oxidant, anti-inflammatory and immunomodulating activities of an enzymatic protein hydrolysate of yellow field pea seeds. The anti-oxidant and anti-inflammatory properties of peptides from yellow field pea proteins (Pisum sativum L.) were investigated in LPS/IFN-γ-activated RAW 264.7 NO⁻ macrophages. The immunomodulating potential of pea protein hydrolysate (PPH) was then studied in a murine model. Pea protein hydrolysate, after a 12 h pre-treatment, showed significant inhibition of NO production by activated macrophages up to 20%. Moreover, PPH significantly inhibited their secretion of pro-inflammatory cytokines, TNF-α- and IL-6, up to 35 and 80%, respectively. Oral administration of PPH in mice enhanced the phagocytic activity of their peritoneal macrophages and stimulated the gut mucosa immune response. The number of IgA+ cells was elevated in the small intestine lamina propria, accompanied by an increase in the number of IL-4+, IL-10+ and IFN-γ+ cells. This was correlated to up-regulation of IL-6 secretion by small intestine epithelial cells (IEC), probably responsible for B-cell terminal differentiation to IgA-secreting cells. Moreover, PPH might have increased IL-6 production in IECs via the stimulation of toll-like receptors (TLRs) family, especially TLR2 and TLR4 since either anti-TLR2 or anti-TLR4 was able to completely abolish PPH-induced IL-6 secretion. Enzymatic protein degradation confers anti-oxidant, anti-inflammatory and immunomodulating potentials to pea proteins, and the resulted peptides could be used as an alternative therapy for the prevention of inflammatory-related diseases.

  4. Process technology for multi-enzymatic reaction systems

    DEFF Research Database (Denmark)

    Xue, Rui; Woodley, John M.

    2012-01-01

    In recent years, biocatalysis has started to provide an important green tool in synthetic organic chemistry. Currently, the idea of using multi-enzymatic systems for industrial production of chemical compounds becomes increasingly attractive. Recent examples demonstrate the potential of enzymatic...... synthesis and fermentation as an alternative to chemical-catalysis for the production of pharmaceuticals and fine chemicals. In particular, the use of multiple enzymes is of special interest. However, many challenges remain in the scale-up of a multi-enzymatic system. This review summarizes and discusses...... the technology options and strategies that are available for the development of multi-enzymatic processes. Some engineering tools, including kinetic models and operating windows, for developing and evaluating such processes are also introduced....

  5. Enzymatic U(VI) reduction by Desulfosporosinus species

    International Nuclear Information System (INIS)

    Suzuki, Y.; Kelly, S.D.; Kemner, K.M.; Banfield, J.F.

    2004-01-01

    Here we tested U(VI) reduction by a Desulfosporosinus species (sp.) isolate and type strain (DSM 765) in cell suspensions (pH 7) containing 1 mM U(VI) and lactate, under an atmosphere containing N 2 -CO 2 -H 2 (90: 5: 5). Although neither Desulfosporosinus species (spp.) reduced U(VI) in cell suspensions with 0.25% Na-bicarbonate or 0.85% NaCl, U(VI) was reduced in these solutions by a control strain, desulfovibrio desulfuricans (ATCC 642). However, both Desulfosporosinus strains reduced U(VI) in cell suspensions depleted in bicarbonate and NaCl. No U(VI) reduction was observed without lactate and H 2 electron donors or with heat-killed cells, indicating enzymatic U(VI) reduction. Uranium(VI) reduction by both strains was inhibited when 1 mM CuCl 2 was added to the cell suspensions. Because the Desulfosporosinus DSM 765 does not contain cytochrome c 3 used by Desulfovibrio spp. to reduce U(VI), Desulfosporosinus species reduce uranium via a different enzymatic pathway. (orig.)

  6. Antioxidant Enzyme Activities of some Brassica Species

    Directory of Open Access Journals (Sweden)

    Rodica SOARE

    2017-11-01

    Full Text Available This paper set out to comparatively study five species: white cabbage (Brassica oleracea L. var. capitata alba Alef., red cabbage (Brassica oleracea L. var. capitata f. rubra Alef., Kale (Brassica oleracea L. var. Acephala, cauliflower (Brassica oleracea var. botrytis and broccoli (Brassica oleracea var. cymosa in order to identify those with high enzymatic and antioxidant activities. The enzymatic activity of superoxide dismutase (SOD, catalase (CAT and soluble peroxidase (POX as well as the antioxidant activity against 2.2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS radical cation were determined. Total superoxide dismutase activity was measured spectrophotometrically based on inhibition in the photochemical reduction of nitroblue tetrazolium. Total soluble peroxidase was assayed by measuring the increase in A436 due to the guaiacol oxidation and the catalase activity was assayed through the colorimetric method. The capacity of extracts to scavenge the ABTS radical cation was assessed colorimetric using Trolox as a standard. The obtained results show that studied enzymatic activities and the antioxidant activity against ABTS vary depending on the analyzed species. So, among the studied Brassicaceae species, it emphasize red cabbage with the highest enzymatic activity (CAT 22.54 mM H2O2/min/g and POX 187.2 mM ΔA/1min/1g f.w. and kale with highest antioxidant activity, of 767 μmol TE/100g f.w. The results of this study recommendintroducing the studied varieties in diet due to the rich antioxidant properties.

  7. The effect of TGF-beta2 on MMP-2 production and activity in highly metastatic human bladder carcinoma cell line 5637.

    Science.gov (United States)

    Dehnavi, Ehsan; Soheili, Zahra-Soheila; Samiei, Shahram; Ataei, Zahra; Aryan, Hajar

    2009-06-01

    Transforming growth factor-beta (TGF-beta) superfamily regulates matrix metalloproteinases (MMP), which intrinsically regulate various cell behaviors leading to metastasis. We investigated the effect of TGF-beta(2) on MMP-2 regulation in human bladder carcinoma cell line 5637. Zymography, ELISA, and real-time polymerase chain reaction revealed that TGF-beta(2) stimulated MMP-2 production, but the transcription of its gene remained unchanged. Wortmannin could not inhibit MMP-2 secretion and activity and conversely the amount of the protein and its enzymatic activity were increased. These data suggest that TGF-beta(2) increased MMP-2 at the posttranscriptional level and this upregulation was independent of phosphatidylinositol 3-kinase signaling pathway.

  8. Role of Ca-bentonite to improve the humification, enzymatic activities, nutrient transformation and end product quality during sewage sludge composting.

    Science.gov (United States)

    Awasthi, Mukesh Kumar; Awasthi, Sanjeev Kumar; Wang, Quan; Awasthi, Mrigendra Kumar; Zhao, Junchao; Chen, Hongyu; Ren, Xiuna; Wang, Meijing; Zhang, Zengqiang

    2018-04-10

    This study was aimed to examine the response of Ca-bentonite (CB) amendment to improve the sewage sludge (SS) composting along with wheat straw (WS) as bulking agent. Five treatments (SS + WS) were mixed with or without blending of discrepant concentration of CB (2%, 4%, 6%, and 10%), respectively, and without CB added treatment applied as the control. The results showed that compared to control and 2%CB blended treatments, while the 6-10%CB -amended treatment indicated maximum enzymatic activities with the composting progress and highest organic matter degradation and loss. The amendment of 6-10%CB increased the humic acid, HA/FA ratio, DON, NH 4 + -N, NO 3 and DOC but reduced the fulvic acids content and the maturity period by 2 weeks as compared to control. In addition, maturity parameters also confirmed that the highest seed germination was observed with the 10%CB applied compost followed by 6%CB, 4%CB and 2%CB applied treatments, respectively. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. Novel synthesis and characterization of Ag@TiO2 core shell nanostructure for non-enzymatic glucose sensor

    Science.gov (United States)

    T, Dayakar; Venkateswara Rao, K.; Vinodkumar, M.; Bikshalu, K.; Chakradhar, B.; Ramachandra Rao, K.

    2018-03-01

    Ag@TiO2 core-shell nano composite (ATCSNC) were synthesized by using Ocimum tenuiflorum leaves extract through a simple one-step hydrothermal route for Non-enzymatic glucose sensing material. The prepared NCs were characterized and found high crystallinity, red shift absorbance, interface-bonding parameters, rough surface and network like microstructure through XRD, Uv-vis, FTIR, SEM, and TEM. The prepared ATCSNC have been used for fabrication of glassy carbon electrode (GCE) and the same was applied to test its electro catalytic activity of glucose in 0.1 M NaOH. The promising results were recorded for ATCSNC/GCE with a high sensitivity (1968.72 μAm M-1cm-2), wide linear range (1 μM-8.1 mM), good response time (3 s), and excellent low detection limit (0.19 μM, S/N = 3). Furthermore, the designed sensor exhibits admirable stability and reproducibility, as well as attractive achievability for real sample analysis. As such, the proposed ATCSNC could be highly beneficial in the development of sustainable and eco-friendly glucose sensing devices.

  10. Activation and inactivation of the volume-sensitive taurine leak pathway in NIH3T3 fibroblasts and Ehrlich Lettre ascites cells

    DEFF Research Database (Denmark)

    Lambert, Ian Henry

    2007-01-01

    Hypotonic exposure provokes the mobilization of arachidonic acid, production of ROS, and a transient increase in taurine release in Ehrlich Lettre cells. The taurine release is potentiated by H(2)O(2) and the tyrosine phosphatase inhibitor vanadate and reduced by the phospholipase A(2) (PLA(2......)) inhibitors bromoenol lactone (BEL) and manoalide, the 5-lipoxygenase (5-LO) inhibitor ETH-615139, the NADPH oxidase inhibitor diphenyl iodonium (DPI), and antioxidants. Thus, swelling-induced taurine efflux in Ehrlich Lettre cells involves Ca(2+)-independent (iPLA(2))/secretory PLA(2) (sPLA(2)) plus 5-LO...... activity and modulation by ROS. Vanadate and H(2)O(2) stimulate arachidonic acid mobilization and vanadate potentiates ROS production in Ehrlich Lettre cells and NIH3T3 fibroblasts under hypotonic conditions. However, vanadate-induced potentiation of the volume-sensitive taurine efflux is, in both cell...

  11. Protective effect of enzymatic hydrolysates from highbush blueberry (Vaccinium corymbosum L.) against hydrogen peroxide-induced oxidative damage in Chinese hamster lung fibroblast cell line.

    Science.gov (United States)

    Senevirathne, Mahinda; Kim, Soo-Hyun; Jeon, You-Jin

    2010-06-01

    Blueberry was enzymatically hydrolyzed using selected commercial food grade carbohydrases (AMG, Celluclast, Termamyl, Ultraflo and Viscozyme) and proteases (Alcalase, Flavourzyme, Kojizyme, Neutrase and Protamex) to obtain water soluble compounds, and their protective effect was investigated against H(2)O(2)-induced damage in Chinese hamster lung fibroblast cell line (V79-4) via various published methods. Both AMG and Alcalase hydrolysates showed higher total phenolic content as well as higher cell viability and ROS scavenging activities, and hence, selected for further antioxidant assays. Both AMG and Alcalase hydrolysates also showed higher protective effects against lipid peroxidation, DNA damage and apoptotic body formation in a dose-dependent fashion. Thus, the results indicated that water soluble compounds obtained by enzymatic hydrolysis of blueberry possess good antioxidant activity against H(2)O(2)-induced cell damage in vitro.

  12. Effect of elevated CO2 on chlorpyriphos degradation and soil microbial activities in tropical rice soil.

    Science.gov (United States)

    Adak, Totan; Munda, Sushmita; Kumar, Upendra; Berliner, J; Pokhare, Somnath S; Jambhulkar, N N; Jena, M

    2016-02-01

    Impact of elevated CO2 on chlorpyriphos degradation, microbial biomass carbon, and enzymatic activities in rice soil was investigated. Rice (variety Naveen, Indica type) was grown under four conditions, namely, chambered control, elevated CO2 (550 ppm), elevated CO2 (700 ppm) in open-top chambers and open field. Chlorpyriphos was sprayed at 500 g a.i. ha(-1) at maximum tillering stage. Chlorpyriphos degraded rapidly from rice soils, and 88.4% of initially applied chlorpyriphos was lost from the rice soil maintained under elevated CO2 (700 ppm) by day 5 of spray, whereas the loss was 80.7% from open field rice soil. Half-life values of chlorpyriphos under different conditions ranged from 2.4 to 1.7 days with minimum half-life recorded with two elevated CO2 treatments. Increased CO2 concentration led to increase in temperature (1.2 to 1.8 °C) that played a critical role in chlorpyriphos persistence. Microbial biomass carbon and soil enzymatic activities specifically, dehydrogenase, fluorescien diacetate hydrolase, urease, acid phosphatase, and alkaline phosphatase responded positively to elevated CO2 concentrations. Generally, the enzyme activities were highly correlated with each other. Irrespective of the level of CO2, short-term negative influence of chlorpyriphos was observed on soil enzymes till day 7 of spray. Knowledge obtained from this study highlights that the elevated CO2 may negatively influence persistence of pesticide but will have positive effects on soil enzyme activities.

  13. Pretreatment of corn stover using wet oxidation to enhance enzymatic digestibility.

    Science.gov (United States)

    Varga, Eniko; Schmidt, Anette S; Réczey, Kati; Thomsen, Anne Belinda

    2003-01-01

    Corn stover is an abundant, promising raw material for fuel ethanol production. Although it has a high cellulose content, without pretreatment it resists enzymatic hydrolysis, like most lignocellulosic materials. Wet oxidation (water, oxygen, mild alkali or acid, elevated temperature and pressure) was investigated to enhance the enzymatic digestibility of corn stover. Six different combinations of reaction temperature, time, and pH were applied. The best conditions (60 g/L of corn stover, 195 degrees C, 15 min, 12 bar O2, 2 g/L of Na2CO3) increased the enzymatic conversion of corn stover four times, compared to untreated material. Under these conditions 60% of hemicellulose and 30% of lignin were solubilized, whereas 90% of cellulose remained in the solid fraction. After 24-h hydrolysis at 50 degrees C using 25 filter paper units (FPU)/g of drymatter (DM) biomass, the achieved conversion of cellulose to glucose was about 85%. Decreasing the hydrolysis temperature to 40 degrees C increased hydrolysis time from 24 to 72 h. Decreasing the enzyme loading to 5 FPU/g of DM biomass slightly decreased the enzymatic conversion from 83.4 to 71%. Thus, enzyme loading can be reduced without significantly affecting the efficiency of hydrolysis, an important economical aspect.

  14. Metabolic activation of pyrrolizidine alkaloids: insights into the structural and enzymatic basis.

    Science.gov (United States)

    Ruan, Jianqing; Yang, Mengbi; Fu, Peter; Ye, Yang; Lin, Ge

    2014-06-16

    Pyrrolizidine alkaloids (PAs) are natural toxins widely distributed in plants. The toxic potencies of different PAs vary significantly. PAs are mono- or diesters of necine acids with a necine base. On the basis of the necine bases, PAs are classified into three types: retronecine-type, otonecine-type, and platynecine-type. Hepatotoxic PAs contain an unsaturated necine base. PAs exert hepatotoxicity through metabolic activation by hepatic cytochromes P450s (CYPs) to generate reactive intermediates which form pyrrole-protein adducts. These adducts provide a mechanism-based biomarker to assess PA toxicity. In the present study, metabolic activation of 12 PAs from three structural types was investigated first in mice to demonstrate significant variations in hepatic metabolic activation of different PAs. Subsequently, the structural and enzymatic factors affecting metabolic activation of these PAs were further investigated by using human liver microsomes and recombinant human CYPs. Pyrrole-protein adducts were detected in the liver and blood of mice and the in vitro systems treated with toxic retronecine-type and otonecine-type PAs having unsaturated necine bases but not with a platynecine-type PA containing a saturated necine base. Retronecine-type PAs produced more pyrrole-protein adducts than otonecine-type PAs with similar necine acids, demonstrating that the structure of necine base affected PA toxic potency. Among retronecine-type PAs, open-ring diesters generated the highest amount of pyrrole-protein adducts, followed by macrocyclic diesters, while monoesters produced the least. Only CYP3A4 and CYP3A5 activated otonecine-type PAs, while all 10 CYPs studied showed the ability to activate retronecine-type PAs. Moreover, the contribution of major CYPs involved also varied significantly among retronecine-type PAs. In conclusion, our findings provide a scientific basis for predicting the toxicities of individual PAs in biological systems based on PA structural

  15. Highly selective apo-arginase based method for sensitive enzymatic assay of manganese (II) and cobalt (II) ions

    Science.gov (United States)

    Stasyuk, Nataliya; Gayda, Galina; Zakalskiy, Andriy; Zakalska, Oksana; Errachid, Abdelhamid; Gonchar, Mykhailo

    2018-03-01

    A novel enzymatic method of manganese (II) and cobalt (II) ions assay, based on using apo-enzyme of Mn2 +-dependent recombinant arginase I (arginase) and 2,3-butanedione monoxime (DMO) as a chemical reagent is proposed. The principle of the method is the evaluation of the activity of L-arginine-hydrolyzing of arginase holoenzyme after the specific binding of Mn2 + or Co2 + with apo-arginase. Urea, which is the product of enzymatic hydrolysis of L-arginine (Arg), reacts with DMO and the resulted compound is detected by both fluorometry and visual spectrophotometry. Thus, the content of metal ions in the tested samples can be determined by measuring the level of urea generated after enzymatic hydrolysis of Arg by reconstructed arginase holoenzyme in the presence of tested metal ions. The linearity range of the fluorometric apo-arginase-DMO method in the case of Mn2 + assay is from 4 pM to 1.10 nM with a limit of detection of 1 pM Mn2 +, whereas the linearity range of the present method in the case of Co2 + assay is from 8 pM to 45 nM with a limit of detection of 2.5 pM Co2 +. The proposed method being highly sensitive, selective, valid and low-cost, may be useful to monitor Mn2 + and Co2 + content in clinical laboratories, food industry and environmental control service.

  16. Enzymatic assays for the diagnosis of bradykinin-dependent angioedema.

    Directory of Open Access Journals (Sweden)

    Federica Defendi

    Full Text Available BACKGROUND: The kinins (primarily bradykinin, BK represent the mediators responsible for local increase of vascular permeability in hereditary angioedema (HAE, HAE I-II associated with alterations of the SERPING1 gene and HAE with normal C1-Inhibitor function (HAE-nC1INH. Besides C1-Inhibitor function and concentration, no biological assay of kinin metabolism is actually available to help physicians for the diagnosis of angioedema (AE. We describe enzymatic tests on the plasma for diagnosis of BK-dependent AE. METHODS: The plasma amidase assays are performed using the Pro-Phe-Arg-p-nitroanilide peptide substrate to evaluate the spontaneous amidase activity and the proenzyme activation. We analyzed data of 872 patients presenting with BK-dependent AE or BK-unrelated diseases, compared to 303 controls. Anti-high MW kininogen (HK immunoblot was achieved to confirm HK cleavage in exemplary samples. Reproducibility, repeatability, limit of blank, limit of detection, precision, linearity and receiver operating characteristics (ROC were used to calculate the diagnostic performance of the assays. RESULTS: Spontaneous amidase activity was significantly increased in all BK-dependent AE, associated with the acute phase of disease in HAE-nC1INH, but preserved in BK-unrelated disorders. The increase of the amidase activity was associated to HK proteolysis, indicating its relevance to identify kininogenase activity. The oestrogens, known for precipitating AE episodes, were found as triggers of enzymatic activity. Calculations from ROC curves gave the optimum diagnostic cut-off for women (9.3 nmol⋅min(-1⋅mL(-1, area under curve [AUC] 92.1%, sensitivity 80.0%, and specificity 90.1% and for men (6.6 nmol·min(-1⋅mL(-1, AUC 91.0%, sensitivity 87.0% and specificity 81.2%. CONCLUSION: The amidase assay represents a diagnostic tool to help physicians in the decision to distinguish between BK-related and -unrelated AE.

  17. Inhibition of Lipid Peroxidation by Enzymatic Hydrolysates from Wheat Bran

    Directory of Open Access Journals (Sweden)

    Yanping Cao

    2011-01-01

    Full Text Available Wheat bran, an important by-product of the cereal industry, is rich in potentially health-promoting phenolic compounds. The phenolics are mainly esterified to the cell wall polysaccharides. In our previous paper, wheat bran was destarched and deproteinated by α-amylase, protease and amyloglucosidase successively and further hydrolyzed using Bacillus subtilis xylanases, and the enzymatic hydrolysates from wheat bran (EHWB showed good scavenging activity in vitro. The aim of this study is to further characterize the antioxidant potential of EHWB against various systems, both ex vivo and in vivo, namely, rat liver microsomal lipid peroxidation systems induced by Fe2+/H2O2 and Fe3+-adenosine diphosphate (ADP/dihydronicotinamide adenine dinucleotide phosphate (NADPH, copper- and 2,2’-azo-bis(2-amidinopropane dihydrochloride (AAPH-induced human low-density lipoprotein (LDL oxidation systems, and alloxan-induced in vivo lipid peroxidation in mice. EHWB inhibited lipid peroxidation in rat liver microsomes induced by Fe2+/H2O2 and Fe3+-ADP/NADPH in a concentration-dependent manner with 90.3 and 87 % inhibition of lipid peroxidation at 50 mg/L, respectively, which were similar to that of butylated hydroxytoluene (BHT at 20 mg/L. The antioxidant potential of EHWB at a concentration ranging from 10 to 20 mg/L in the nonenzymatic system was more effective than in the enzymatic system. EHWB strongly inhibited in vitro copper- and AAPH-mediated oxidation of LDL in a concentration- and time-dependent manner with 52.41 and 63.03 % inhibition at 20 mg/L, respectively, which were similar to that of ascorbate at 10 mg/L. EHWB significantly decreased the level of thiobarbituric acid reactive substances (TBARS and increased the activities of glutathione peroxidase (GSH-Px, catalase (CAT and superoxide dismutase (SOD in serum and liver of alloxan-treated mice compared with the control. These results demonstrated that EHWB might be efficient in the protection of

  18. Modification of chemical properties, Cu fractionation and enzymatic activities in an acid vineyard soil amended with winery wastes: A field study.

    Science.gov (United States)

    Rodríguez-Salgado, Isabel; Pérez-Rodríguez, Paula; Gómez-Armesto, Antía; Díaz-Raviña, Montserrat; Nóvoa-Muñoz, Juan Carlos; Arias-Estévez, Manuel; Fernández-Calviño, David

    2017-11-01

    The effects of adding two winery wastes, perlite waste (PW) and bentonite waste (BW), to an acid vineyard soil were assessed using some chemical and biological soil properties in a field study that lasted 18 months. The addition of PW (up to 81 Mg ha -1 ) had neither significant nor permanent effects on soil characteristics such as the pH, organic matter content or nutrient concentrations, the amounts of copper or zinc, or the electrical conductivity. Moreover, no persistent negative effects were found on the enzymatic activities after PW application. In contrast, soil that was amended with up to 71 Mg BW ha -1 showed increases in its soil pH values, exchangeable potassium and water soluble potassium and phosphorus contents. In addition, it caused significant increases in the electrical conductivity and water-soluble Cu. In addition, the phosphomonoesterase enzymatic activity decreased significantly (up to 28%) in response to the amendment with 71 Mg BW ha -1 . These results showed that adding BW and PW to the soil may be a good agronomic practice for recycling these types of wastes. However, in the case of PW, its use as a soil amendment must be performed with caution to control its possible harmful effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Longitudinal changes in PON1 enzymatic activities in Mexican-American mothers and children with different genotypes and haplotypes

    International Nuclear Information System (INIS)

    Huen, Karen; Harley, Kim; Bradman, Asa; Eskenazi, Brenda; Holland, Nina

    2010-01-01

    The paraoxonase 1 (PON1) enzyme prevents low-density lipoprotein oxidation and also detoxifies the oxon derivatives of certain neurotoxic organophosphate (OP) pesticides. PON1 activity in infants is low compared to adults, rendering them with lower metabolic and antioxidant capacities. We made a longitudinal comparison of the role of genetic variability on control of PON1 phenotypes in Mexican-American mothers and their children at the time of delivery (n = 388 and 338, respectively) and again 7 years later (n = 280 and 281, respectively) using generalized estimating equations models. At age 7, children's mean PON1 activities were still lower than those of mothers. This difference was larger in children with genotypes associated with low PON1 activities (PON1 -108TT , PON1 192QQ , and PON1 -909CC ). In mothers, PON1 activities were elevated at delivery and during pregnancy compared to 7 years later when they were not pregnant (p < 0.001). In non-pregnant mothers, PON1 polymorphisms and haplotypes accounted for almost 2-fold more variation of arylesterase (AREase) and chlorpyrifos-oxonase (CPOase) activity than in mothers at delivery. In both mothers and children, the five PON1 polymorphisms (192, 55, -108, -909, -162) explained a noticeably larger proportion of variance of paraoxonase activity (62-78%) than AREase activity (12.3-26.6%). Genetic control of PON1 enzymatic activity varies in children compared to adults and is also affected by pregnancy status. In addition to known PON1 polymorphisms, unidentified environmental, genetic, or epigenetic factors may also influence variability of PON1 expression and therefore susceptibility to OPs and oxidative stress.

  20. Role of enzymatic and non enzymatic antioxidant in ameliorating salinity induced damage in nostoc muscorum

    International Nuclear Information System (INIS)

    Hend, A.; Abeer, A.; Allah, A.

    2015-01-01

    Presence of high salt concentration in the growth medium adversely affected the plant growth and productivity by altering its metabolic activities. Experiments were conducted on cyanobacteriaum Nostoc muscorum grown in nitrogen free medium supplemented with 250 mM NaCl to evaluate the salt stress induced changes in growth, antioxidants and lipid composition. Salt stress significantly reduced the growth and physio-biochemical attributes. Salt stress increased malonaldehyde content thereby causing alterations in the lipid fraction. Significant reduction in polyunsaturated fatty acids including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI) and phosphatidylserine (PS) was observed. Where as diacylglycerol, sterol ester and non-esterified fatty acids were increased. Activities of antioxidant enzymes and contents of non-enzymatic antioxidants including glutathione enhanced due to salt stress. An increase in accumulation of proline was also observed. Hence increased activity of antioxidants and altered fatty acid composition was observed in salt stressed Nostoc muscorum. (author)

  1. Enzymatic Synthesis of Biobased Polyesters and Polyamides

    Directory of Open Access Journals (Sweden)

    Yi Jiang

    2016-06-01

    Full Text Available Nowadays, “green” is a hot topic almost everywhere, from retailers to universities to industries; and achieving a green status has become a universal aim. However, polymers are commonly considered not to be “green”, being associated with massive energy consumption and severe pollution problems (for example, the “Plastic Soup” as a public stereotype. To achieve green polymers, three elements should be entailed: (1 green raw materials, catalysts and solvents; (2 eco-friendly synthesis processes; and (3 sustainable polymers with a low carbon footprint, for example, (biodegradable polymers or polymers which can be recycled or disposed with a gentle environmental impact. By utilizing biobased monomers in enzymatic polymerizations, many advantageous green aspects can be fulfilled. For example, biobased monomers and enzyme catalysts are renewable materials that are derived from biomass feedstocks; enzymatic polymerizations are clean and energy saving processes; and no toxic residuals contaminate the final products. Therefore, synthesis of renewable polymers via enzymatic polymerizations of biobased monomers provides an opportunity for achieving green polymers and a future sustainable polymer industry, which will eventually play an essential role for realizing and maintaining a biobased and sustainable society.

  2. Dual role of the carboxyl-terminal region of pig liver L-kynurenine 3-monooxygenase: mitochondrial-targeting signal and enzymatic activity.

    Science.gov (United States)

    Hirai, Kumiko; Kuroyanagi, Hidehito; Tatebayashi, Yoshitaka; Hayashi, Yoshitaka; Hirabayashi-Takahashi, Kanako; Saito, Kuniaki; Haga, Seiich; Uemura, Tomihiko; Izumi, Susumu

    2010-12-01

    l-kynurenine 3-monooxygenase (KMO) is an NAD(P)H-dependent flavin monooxygenase that catalyses the hydroxylation of l-kynurenine to 3-hydroxykynurenine, and is localized as an oligomer in the mitochondrial outer membrane. In the human brain, KMO may play an important role in the formation of two neurotoxins, 3-hydroxykynurenine and quinolinic acid, both of which provoke severe neurodegenerative diseases. In mosquitos, it plays a role in the formation both of eye pigment and of an exflagellation-inducing factor (xanthurenic acid). Here, we present evidence that the C-terminal region of pig liver KMO plays a dual role. First, it is required for the enzymatic activity. Second, it functions as a mitochondrial targeting signal as seen in monoamine oxidase B (MAO B) or outer membrane cytochrome b(5). The first role was shown by the comparison of the enzymatic activity of two mutants (C-terminally FLAG-tagged KMO and carboxyl-terminal truncation form, KMOΔC50) with that of the wild-type enzyme expressed in COS-7 cells. The second role was demonstrated with fluorescence microscopy by the comparison of the intracellular localization of the wild-type, three carboxyl-terminal truncated forms (ΔC20, ΔC30 and ΔC50), C-terminally FLAG-tagged wild-type and a mutant KMO, where two arginine residues, Arg461-Arg462, were replaced with Ser residues.

  3. Pretreatment of corn stover using wet oxidation to enhance enzymatic digestibility

    DEFF Research Database (Denmark)

    Varga, E.; Schmidt, A.S.; Reczey, K.

    2003-01-01

    was about 85%. Decreasing the hydrolysis temperature to 40degreesC increased hydrolysis time from 24 to 72 h. Decreasing the enzyme loading to 5 FPU/g of DM biomass slightly decreased the enzymatic conversion from 83.4 to 71%. Thus, enzyme loading can be reduced without significantly affecting......) was investigated to enhance the enzymatic digestibility of corn stover. Six different combinations of reaction temperature, time, and pH were applied. The best conditions (60 g/L of corn stover, 195degreesC, 15 min, 12 bar O-2, 2 g/L of Na2CO) increased the enzymatic conversion of corn stover four times, compared...... to untreated material. Under these conditions 60% of hemicellulose and 30% of lignin were solubilized, whereas 90% of cellulose remained in the solid fraction. After 24-h hydrolysis at 50degreesC using 25 filter paper units (FPU)/g of drymatter (DM) biomass, the achieved conversion of cellulose to glucose...

  4. Kinetic study of enzymatic hydrolysis of acid-pretreated coconut coir

    Science.gov (United States)

    Fatmawati, Akbarningrum; Agustriyanto, Rudy

    2015-12-01

    Biomass waste utilization for biofuel production such as bioethanol, has become more prominent currently. Coconut coir is one of lignocellulosic food wastes, which is abundant in Indonesia. Bioethanol production from such materials consists of more than one step. Pretreatment and enzymatic hydrolysis is crucial steps to produce sugar which can then be fermented into bioethanol. In this research, ground coconut coir was pretreated using dilute sulfuric acid at 121°C. This pretreatment had increased the cellulose content and decreased the lignin content of coconut coir. The pretreated coconut coir was hydrolyzed using a mix of two commercial cellulase enzymes at pH of 4.8 and temperature of 50°C. The enzymatic hydrolysis was conducted at several initial coconut coir slurry concentrations (0.1-2 g/100 mL) and reaction times (2-72 hours). The reducing sugar concentration profiles had been produced and can be used to obtain reaction rates. The highest reducing sugar concentration obtained was 1,152.567 mg/L, which was produced at initial slurry concentration of 2 g/100 mL and 72 hours reaction time. In this paper, the reducing sugar concentrations were empirically modeled as a function of reaction time using power equations. Michaelis-Menten kinetic model for enzymatic hydrolysis reaction is adopted. The kinetic parameters of that model for sulfuric acid-pretreated coconut coir enzymatic hydrolysis had been obtained which are Vm of 3.587×104 mg/L.h, and KM of 130.6 mg/L.

  5. Enzymatic saccharification and fermentation of paper and pulp industry effluent for biohydrogen production

    Energy Technology Data Exchange (ETDEWEB)

    Lakshmidevi, Rajendran; Muthukumar, Karuppan [Department of Chemical Engineering, Alagappa College of Technology Campus, Anna University Chennai, Chennai 600 025 (India)

    2010-04-15

    Paper and pulp industry effluent was enzymatically hydrolysed using crude cellulase enzyme (0.8-2.2FPU/ml) obtained from Trichoderma reesei and from the hydrolysate biohydrogen was produced using Enterobacter aerogenes. The influence of temperature and incubation time on enzyme production was studied. The optimum temperature for the growth of T. reesei was found to be around 29 C. The enzyme activity of 2.5 FPU/ml was found to produce about 22 g/l of total sugars consisting mainly of glucose, xylose and arabinose. Relevant kinetic parameters with respect to sugars production were estimated using two fraction model. The enzymatic hydrolysate was used for the biohydrogen production using E. aerogenes. The growth data obtained for E. aerogenes were fitted well with Monod and Logistic equations. The maximum hydrogen yield of 2.03 mol H{sub 2}/mol sugar and specific hydrogen production rate of 225 mmol of H{sub 2}/g cell/h were obtained with an initial concentration of 22 g/l of total sugars. The colour and COD of effluent was also decreased significantly during the production of hydrogen. The results showed that the paper and pulp industry effluent can be used as a substrate for biohydrogen production. (author)

  6. Enzymatic determination of rare earth elements using pyrophosphatases

    International Nuclear Information System (INIS)

    Shekhovtsova, T.N.; Pirogova, S.V.; Fedorova, O.M.; Dolmanova, I.F.; Bajkov, A.A.

    1993-01-01

    A highly sensitive(determination limit 8x10 -6 -4x10 -4 μ g/m) and selective enzymatic method for determination of rare earth elements has been developed. The method is based on inhibition action of rare earths on the catalytic activity of pyrophosphates isolated from bakery geast and E.Coli. The mechanism of the rare earth element action, corresponding to competitive inhibition, has been established

  7. Highly Atom Economic Synthesis of d?2?Aminobutyric Acid through an In?Vitro Tri?enzymatic Catalytic System

    OpenAIRE

    Chen, Xi; Cui, Yunfeng; Cheng, Xinkuan; Feng, Jinhui; Wu, Qiaqing; Zhu, Dunming

    2017-01-01

    Abstract d?2?Aminobutyric acid is an unnatural amino acid serving as an important intermediate in pharmaceutical production. Developing a synthetic method that uses cheaper starting materials and produces less by?product is a pressing demand. A tri?enzymatic catalytic system, which is composed of l?threonine ammonia lyase (l?TAL), d?amino acid dehydrogenase (d?AADH), and formate dehydrogenase (FDH), has thus been developed for the synthesis of d?2?aminobutyric acid with high optical purity. I...

  8. Enzymatic transesterification of used frying oils

    Energy Technology Data Exchange (ETDEWEB)

    Kovacs, S.; Hancsok, J. (Univ. of Pannonia, Veszprem (HU)), Email: hancsokj@almos.uni-pannon.hu

    2009-07-01

    The research of converting used frying oils to less harmful products with much higher value was forced by environmental, human biological and economical reasons. One possible pathway of the transformation is the enzymatic transesterification. Through the research work used frying oils (UFO) and sunflower oils (SO) from different origins were first properly pre-treated. Then the previously mentioned feeds and different mixtures of them were transesterified in the presence of Novozym 435 enzyme catalyst under different process conditions. Characteristics of the produced methyl esters were evaluated according to the requirements of EN 14214:2009 standard. We determined that the transesterification of used frying oils is not expediential in the presence of enzyme catalyst because the significant decreasing of catalyst activity. We have found proper UFO and SO mixtures and combination of process conditions (pressure: atmospheric, temperature: 54 +-1 deg C; methanol to triglyceride molar ratio: 4:1; reaction time: 16 hours) resulting in high (>90 %) yield of monoesters. We clearly established that the best results through the enzymatic transesterification were obtained with the improved sunflower oils containing the highest amount (>88 %) of oleic acid and the used frying oils originated from this source. (orig.)

  9. Characterization of Volatile Flavor Compounds in Chinese Rice Wine Fermented from Enzymatic Extruded Rice.

    Science.gov (United States)

    Xu, Enbo; Long, Jie; Wu, Zhengzong; Li, Hongyan; Wang, Fang; Xu, Xueming; Jin, Zhengyu; Jiao, Aiquan

    2015-07-01

    Enzymatic extrusion, instead of traditional steam cooking, to treat rice is an efficient and alternative pretreatment for Chinese rice wine fermentation. In order to determine the formation of volatiles in enzymatic extrusion-processed rice wine (EE), and to confirm its characteristic flavor compounds, headspace solid-phase micro-extraction followed by GC-MS was used. A total of 66 volatile compounds were identified in EE. During fermentation, most volatiles generated from enzymatic extruded rice had the similar trends with those from steam-cooked rice, but the differences in the concentration of volatiles indicated a changed balance of flavors release caused by enzymatic extrusion. Besides, the concentrations and sorts of volatiles in EEs fermented from different rice particle sizes, were not dramatically different. By principal component analysis, EE could be distinctly separated from other traditional Chinese rice wines according to its characteristic volatiles, namely, 2-heptanol, 1-octen-3-ol, ethyl 4-hydroxybenzoate, methylpentyl 2-propenoate, γ-hexalactone, and 4-vinylguaiacol. Enzymatic extrusion liquefaction has been a popular thermal treatment for cereals, and gradually being applied in fermentation and liquor-making industry all over the world. The characterization of volatile flavor compounds in Chinese rice wine processed by enzymatic extrusion liquefaction pretreatment, might be made use not only for a better understanding of this new-type rice wine, but for the further utilization of enzymatic extrusion in other wine or alcohol production as well. © 2015 Institute of Food Technologists®

  10. Enzymatic hydrolysis of pretreated soybean straw

    International Nuclear Information System (INIS)

    Xu Zhong; Wang Qunhui; Jiang Zhaohua; Yang Xuexin; Ji Yongzhen

    2007-01-01

    In order to produce lactic acid, from agricultural residues such as soybean straw, which is a raw material for biodegradable plastic production, it is necessary to decompose the soybean straw into soluble sugars. Enzymatic hydrolysis is one of the methods in common use, while pretreatment is the effective way to increase the hydrolysis rate. The optimal conditions of pretreatment using ammonia and enzymatic hydrolysis of soybean straw were determined. Compared with the untreated straw, cellulose in straw pretreated by ammonia liquor (10%) soaking for 24 h at room temperature increased 70.27%, whereas hemicellulose and lignin in pretreated straw decreased to 41.45% and 30.16%, respectively. The results of infrared spectra (IR), scanning electron microscope (SEM) and X-ray diffraction (XRD) analysis also showed that the structure and the surface of the straw were changed through pretreatment that is in favor of the following enzymatic hydrolysis. maximum enzymatic hydrolysis rate of 51.22% was achieved at a substrate concentration of 5% (w/v) at 50 deg. C and pH 4.8 using cellulase (50 fpu/g of substrate) for 36 h

  11. Radiation pretreatments of cellulose materials for the enhancement of enzymatic hydrolysis

    International Nuclear Information System (INIS)

    Ardica, S.; Calderaro, E.; Cappadona, C.

    1985-01-01

    The effect of γ-ray pre-irradiation of cellulose materials such as wood chips, paper, grain straw, hay and kapok on glucose production on enzymatic hydrolysis by cellulase has been investigated. These materials have been irradiated in air, water and acetate buffer solution over the dose range 10 3 to 4 x 10 6 Gy. In the relatively low dose range, up to about 5 x 10 5 Gy, the glucose yields after enzymatic hydrolysis are practically insensitive to radiation. At higher dose levels, up to 1.7 to 2 x 10 6 Gy, the pre-irradiation becomes very effective on enzymatic cellulose conversion. It has been found that the radiation-induced degradation of cellulose into low molecular weight polysaccharides is dependent on the nature and chemical composition of the cellulose materials and on the radiation environmental conditions. Further increases of dose causes radiation-induced structural modifications in polysaccharides previously produced, which can lead to a decrease in glucose production by enzymatic hydrolysis. (author)

  12. Enzymatic approaches to rare sugar production.

    Science.gov (United States)

    Zhang, Wenli; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

    Rare sugars have recently attracted much attention because of their potential applications in the food, nutraceutical, and pharmaceutical industries. A systematic strategy for enzymatic production of rare sugars, named Izumoring, was developed >10years ago. The strategy consists of aldose-ketose isomerization, ketose C-3 epimerization, and monosaccharide oxidation-reduction. Recent development of the Izumoring strategy is reviewed herein, especially the genetic approaches to the improvement of rare sugar-producing enzymes and the applications of target-oriented bioconversion. In addition, novel non-Izumoring enzymatic approaches are also summarized, including enzymatic condensation, phosphorylation-dephosphorylation cascade reaction, aldose epimerization, ulosonic acid decarboxylation, and biosynthesis of rare disaccharides. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Radiolytic and enzymatic dimerization of tyrosyl residues in insulin, ribonuclease, papain and collagen

    Energy Technology Data Exchange (ETDEWEB)

    Boguta, G; Dancewicz, A M [Institute of Nuclear Research, Warsaw (Poland)

    1983-03-01

    Insulin ribonuclease, papain and collagen solutions saturated with nitrogen, N/sub 2/O or air were irradiated with doses of 10 to 640 Gy of gamma rays. Protein solutions were also oxidized enzymatically in a system of horse-radish peroxidase: hydrogen peroxide. Column chromatography (Sephadex G-75 or Sephacryl S-200) of treated protein solutions revealed that they contain protein molecular aggregates. Nitrogen saturation of solution before irradiation was most favourable for radiation-induced aggregation of proteins. Fluorescence analysis of protein solutions resulted in detection of dityrosyl structures in irradiated as well as in enzymatically oxidized proteins. Concentration of dityrosine in proteins studied was determined fluorimetrically in their hydrolysates separated on BioGel P-2 column. In irradiated proteins, dityrosine was present almost exclusively in their aggregated forms. In proteins oxidized enzymatically, dityrosine was also present in fractions containing apparently unchanged protein. Mechanisms which could account for differences in the yield of dityrosine formation in radiolysis and in enzymatic oxidation of proteins are suggested.

  14. Radiolytic and enzymatic dimerization of tyrosyl residues in insulin, ribonuclease, papain and collagen

    International Nuclear Information System (INIS)

    Boguta, G.; Dancewicz, A.M.

    1983-01-01

    Insulin ribonuclease, papain and collagen solutions saturated with nitrogen, N 2 O or air were irradiated with doses of 10 to 640 Gy of gamma rays. Protein solutions were also oxidized enzymatically in a system of horse-radish peroxidase: hydrogen peroxide. Column chromatography (Sephadex G-75 or Sephacryl S-200) of treated protein solutions revealed that they contain protein molecular aggregates. Nitrogen saturation of solution before irradiation was most favourable for radiation-induced aggregation of proteins. Fluorescence analysis of protein solutions resulted in detection of dityrosyl structures in irradiated as well as in enzymatically oxidized proteins. Concentration of dityrosine in proteins studied was determined fluorimetrically in their hydrolysates separated on BioGel P-2 column. In irradiated proteins, dityrosine was present almost exclusively in their aggregated forms. In proteins oxidized enzymatically, dityrosine was also present in fractions containing apparently unchanged protein. Mechanisms which could account for differences in the yield of dityrosine formation in radiolysis and in enzymatic oxidation of proteins are suggested. (author)

  15. Inhibition of Enzymatic Browning of Chlorogenic Acid by Sulfur-Containing Compounds

    NARCIS (Netherlands)

    Kuijpers, T.F.M.; Narvaez Cuenca, C.E.; Vincken, J.P.; Verloop, J.W.; Berkel, van W.J.H.; Gruppen, H.

    2012-01-01

    The antibrowning activity of sodium hydrogen sulfite (NaHSO3) was compared to that of other sulfur-containing compounds. Inhibition of enzymatic browning was investigated using a model browning system consisting of mushroom tyrosinase and chlorogenic acid (5-CQA). Development of brown color

  16. Reinterpreting the best biomarker of oxidative stress: The 8-iso-PGF(2α)/PGF(2α) ratio distinguishes chemical from enzymatic lipid peroxidation.

    Science.gov (United States)

    van 't Erve, Thomas J; Lih, Fred B; Kadiiska, Maria B; Deterding, Leesa J; Eling, Thomas E; Mason, Ronald P

    2015-06-01

    The biomarker 8-iso-prostaglandin F2α (8-iso-PGF2α) is regarded as the gold standard for detection of excessive chemical lipid peroxidation in humans. However, biosynthesis of 8-iso-PGF2α via enzymatic lipid peroxidation by prostaglandin-endoperoxide synthases (PGHSs), which are significantly induced in inflammation, could lead to incorrect biomarker interpretation. To resolve the ambiguity with this biomarker, the ratio of 8-iso-PGF2α to prostaglandin F2α (PGF2α) is established as a quantitative measure to distinguish enzymatic from chemical lipid peroxidation in vitro, in animal models, and in humans. Using this method, we find that chemical lipid peroxidation contributes only 3% to the total 8-iso-PGF2α in the plasma of rats. In contrast, the 8-iso-PGF2α levels in plasma of human males are generated >99% by chemical lipid peroxidation. This establishes the potential for an alternate pathway of biomarker synthesis, and draws into question the source of increases in 8-iso-PGF2α seen in many human diseases. In conclusion, increases in 8-iso-PGF2α do not necessarily reflect increases in oxidative stress; therefore, past studies using 8-iso-PGF2α as a marker of oxidative stress may have been misinterpreted. The 8-iso-PGF2α/PGF2α ratio can be used to distinguish biomarker synthesis pathways and thus confirm the potential change in oxidative stress in the myriad of disease and chemical exposures known to induce 8-iso-PGF2α. Published by Elsevier Inc.

  17. Bioconversion and enzymatic activities of neurospora sitophila grown under solid state and submerged fermentation on Sago Hamps

    International Nuclear Information System (INIS)

    Shojaosadati, S. A.; Vikineswary, S.; Looi, C. C.

    2000-01-01

    N.Sitophila was grown under controlled conditions of solid state and submerged fermentation on Sago Hampas. The optimum conditions of protein enrichment previously established for sugar beet pulp was used for this study. Under this condition the protein content of Sago Hampas under solid state increased from 1.4 to 14.45% (W/W) whereas for Sago Hampas and Sago starch, the protein content under submerged condition increased from 1.4% (W/W) and 0.7% (W/W) to 18.56% (W/W) and 43/16% (W/W) based on dry weight of product respectively. The cellulase, a-amylase and glucoamylase activities of N.Sitophila under solid state condition on Sago Hampas were, 9.0, 0.6 and 11.8 U/g of wet fermented solid respectively. the enzymatic activities were also measured under submerged fermentation using both Sago Hampas and Sago starch as substrate

  18. Evaluation of wet oxidation pretreatment for enzymatic hydrolysis of softwood

    DEFF Research Database (Denmark)

    Palonen, H.; Thomsen, A.B.; Tenkanen, M.

    2004-01-01

    The wet oxidation pretreatment (water, oxygen, elevated temperature, and pressure) of softwood (Picea abies) was investigated for enhancing enzymatic hydrolysis. The pretreatment was preliminarily optimized. Six different combinations of reaction time, temperature, and pH were applied......, and the compositions of solid and liquid fractions were analyzed. The solid fraction after wet oxidation contained 58-64% cellulose, 2-16% hemicellulose, and 24-30% lignin. The pretreatment series gave information about the roles of lignin and hemicellulose in the enzymatic hydrolysis. The temperature...

  19. [Response surface method optimize of nano-silica solid dispersion technology assistant enzymatic hydrolysis preparation genistein].

    Science.gov (United States)

    Jin, Xin; Zhang, Zhen-Hai; Zhu, Jing; Sun, E; Yu, Dan-Hong; Chen, Xiao-Yun; Liu, Qi-Yuan; Ning, Qing; Jia, Xiao-Bin

    2012-04-01

    This article reports that nano-silica solid dispersion technology was used to raise genistein efficiency through increasing the enzymatic hydrolysis rate. Firstly, genistin-nano-silica solid dispersion was prepared by solvent method. And differential scanning calorimetry (DSC) and transmission electron microscopy (TEM) were used to verify the formation of solid dispersion, then enzymatic hydrolysis of solid dispersion was done by snailase to get genistein. With the conversion of genistein as criteria, single factor experiments were used to study the different factors affecting enzymatic hydrolysis of genistin and its solid dispersion. And then, response surface method was used to optimize of nano-silica solid dispersion technology assistant enzymatic hydrolysis. The optimum condition to get genistein through enzymatic hydrolysis of genistin-nano-silica solid dispersion was pH 7.1, temperature 52.2 degrees C, enzyme concentration 5.0 mg x mL(-1) and reaction time 7 h. Under this condition, the conversion of genistein was (93.47 +/- 2.40)%. Comparing with that without forming the genistin-nano-silica solid dispersion, the conversion increased 2.62 fold. At the same time, the product of hydrolysis was purified to get pure genistein. The method of enzymatic hydrolysis of genistin-nano-silica solid dispersion by snailase to obtain genistein is simple, efficiency and suitable for the modern scale production.

  20. Topical formulations with superoxide dismutase: influence of formulation composition on physical stability and enzymatic activity.

    Science.gov (United States)

    Di Mambro, Valéria M; Borin, Maria F; Fonseca, Maria J V

    2003-04-24

    Three different topical formulations were supplemented with superoxide dismutase (SOD) and evaluated concerning physical and chemical stabilities in order to determine the most stable formulation that would maintain SOD activity. Physical stability was evaluated by storing the formulation at room temperature, and at 37 and 45 degrees C for 28 days. Samples were collected at 7-day intervals for assessment of rheological behavior. Chemical stability was evaluated by the measurement of enzymatic activity in formulations stored at room temperature and at 45 degrees C for 75 days. The formulations showed a pseudoplastic behavior, with a flow index of less than 1. There was no significant difference in the initial values of flow index, hysteresis loop or minimum apparent viscosity. The simple emulsion and the one stabilized with hydroxyethylcellulose showed decreased viscosity by the 21st day and with higher temperature, but no significant changes concerning the presence of SOD. Although there were no significant changes concerning storage time or temperature, the formulation stabilized with hydroxyethylcellulose showed a marked loss of SOD activity. The addition of SOD to the formulations studied did not affect their physical stability. Simple emulsions or emulsions stabilized with carboxypolymethylene seem to be better bases for enzyme addition than emulsion stabilized with hydroxyethylcellulose.

  1. Enzymatic hydrolysis of biomimetic bacterial cellulose-hemicellulose composites.

    Science.gov (United States)

    Penttilä, Paavo A; Imai, Tomoya; Hemming, Jarl; Willför, Stefan; Sugiyama, Junji

    2018-06-15

    The production of biofuels and other chemicals from lignocellulosic biomass is limited by the inefficiency of enzymatic hydrolysis. Here a biomimetic composite material consisting of bacterial cellulose and wood-based hemicelluloses was used to study the effects of hemicelluloses on the enzymatic hydrolysis with a commercial cellulase mixture. Bacterial cellulose synthesized in the presence of hemicelluloses, especially xylan, was found to be more susceptible to enzymatic hydrolysis than hemicellulose-free bacterial cellulose. The reason for the easier hydrolysis could be related to the nanoscale structure of the substrate, particularly the packing of cellulose microfibrils into ribbons or bundles. In addition, small-angle X-ray scattering was used to show that the average nanoscale morphology of bacterial cellulose remained unchanged during the enzymatic hydrolysis. The reported easier enzymatic hydrolysis of bacterial cellulose produced in the presence of wood-based xylan offers new insights to overcome biomass recalcitrance through genetic engineering. Copyright © 2018 Elsevier Ltd. All rights reserved.

  2. Plant oligoadenylates: enzymatic synthesis, isolation, and biological activities

    International Nuclear Information System (INIS)

    Devash, Y.; Reichman, M.; Sela, I.; Reichenbach, N.L.; Suhadolnik, R.J.

    1985-01-01

    An enzyme that converts [ 3 H, 32 P]ATP, with a 3 H: 32 P ratio of 1:1, to oligoadenylates with the same 3 H: 32 P ratio was increased in plants following treatment with human leukocyte interferon or plant antiviral factor or inoculation with tobacco mosaic virus. The enzyme was extracted from tobacco leaves, callus tissue cultures, or cell suspension cultures. The enzyme, a putative plant oligoadenylate synthetase, was immobilized on poly(rI) . poly(rC)-agarose columns and converted ATP into plant oligoadenylates. These oligoadenylates were displaced from DEAE-cellulose columns with 350 mM KCl buffer, dialyzed, and further purified by high-performance liquid chromatography (HPLC) and DEAE-cellulose gradient chromatography. In all steps of purification, the ratio of 3 H: 32 P in the oligoadenylates remained 1:1. The plant oligoadenylates isolated by displacement with 350 mM KCl had a molecular weight greater than 1000. The plant oligoadenylates had charges of 5- and 6-. HPLC resolved five peaks, three of which inhibited protein synthesis in reticulocyte and wheat germ systems. Partial structural elucidation of the plant oligoadenylates has been determined by enzymatic and chemical treatments. An adenylate with a 3',5'-phosphodiester and/or a pyrophosphoryl linkage with either 3'- or 5'-terminal phosphates is postulated on the basis of treatment of the oligoadenylates with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase and acid and alkaline hydrolyses. The plant oligoadenylates at 8 X 10(-7) M inhibit protein synthesis by 75% in lysates from rabbit reticulocytes and 45% in wheat germ cell-free systems

  3. A role for non-covalent SUMO interaction motifs in Pc2/CBX4 E3 activity.

    Directory of Open Access Journals (Sweden)

    Jacqueline C Merrill

    2010-01-01

    Full Text Available Modification of proteins by the small ubiquitin like modifier (SUMO is an essential process in mammalian cells. SUMO is covalently attached to lysines in target proteins via an enzymatic cascade which consists of E1 and E2, SUMO activating and conjugating enzymes. There is also a variable requirement for non-enzymatic E3 adapter like proteins, which can increase the efficiency and specificity of the sumoylation process. In addition to covalent attachment of SUMO to target proteins, specific non-covalent SUMO interaction motifs (SIMs that are generally short hydrophobic peptide motifs have been identified.Intriguingly, consensus SIMs are present in most SUMO E3s, including the polycomb protein, Pc2/Cbx4. However, a role for SIMs in SUMO E3 activity remains to be shown. We show that Pc2 contains two functional SIMs, both of which contribute to full E3 activity in mammalian cells, and are also required for sumoylation of Pc2 itself. Pc2 forms distinct sub-nuclear foci, termed polycomb bodies, and can recruit partner proteins, such as the corepressor CtBP. We demonstrate that mutation of the SIMs in Pc2 prevents Pc2-dependent CtBP sumoylation, and decreases enrichment of SUMO1 and SUMO2 at polycomb foci. Furthermore, mutational analysis of both SUMO1 and SUMO2 reveals that the SIM-interacting residues of both SUMO isoforms are required for Pc2-mediated sumoylation and localization to polycomb foci.This work provides the first clear evidence for a role for SIMs in SUMO E3 activity.

  4. High volumetric power density, non-enzymatic, glucose fuel cells.

    Science.gov (United States)

    Oncescu, Vlad; Erickson, David

    2013-01-01

    The development of new implantable medical devices has been limited in the past by slow advances in lithium battery technology. Non-enzymatic glucose fuel cells are promising replacement candidates for lithium batteries because of good long-term stability and adequate power density. The devices developed to date however use an "oxygen depletion design" whereby the electrodes are stacked on top of each other leading to low volumetric power density and complicated fabrication protocols. Here we have developed a novel single-layer fuel cell with good performance (2 μW cm⁻²) and stability that can be integrated directly as a coating layer on large implantable devices, or stacked to obtain a high volumetric power density (over 16 μW cm⁻³). This represents the first demonstration of a low volume non-enzymatic fuel cell stack with high power density, greatly increasing the range of applications for non-enzymatic glucose fuel cells.

  5. Enzymatic production of biodiesel from microalgal oil using ethyl acetate as an acyl acceptor.

    Science.gov (United States)

    Alavijeh, Razieh Shafiee; Tabandeh, Fatemeh; Tavakoli, Omid; Karkhane, Aliasghar; Shariati, Parvin

    2015-01-01

    Microalgae have become an important source of biomass for biodiesel production. In enzymatic transesterification reaction, the enzyme activity is decreased in presence of alcohols. The use of different acyl acceptors such as methyl/ethyl acetate is suggested as an alternative and effective way to overcome this problem. In this study, ethyl acetate was used for the first time in the enzymatic production of biodiesel by using microalga, Chlorella vulgaris, as a triglyceride source. Enzymatic conversion of such fatty acids to biodiesel was catalyzed by Novozym 435 as an efficient immobilized lipase which is extensively used in biodiesel production. The best conversion yield of 66.71% was obtained at the ethyl acetate to oil molar ratio of 13:1 and Novozym 435 concentration of 40%, based on the amount of oil, and a time period of 72 h at 40℃. The results showed that ethyl acetate have no adverse effect on lipase activity and the biodiesel amount was not decreased even after seven transesterification cycles, so ethyl acetate has a great potential to be substituted for short-chain alcohols in transesterification reaction.

  6. Computational study on a puzzle in the biosynthetic pathway of anthocyanin: Why is an enzymatic oxidation/ reduction process required for a simple tautomerization?

    Science.gov (United States)

    Sato, Hajime; Wang, Chao; Yamazaki, Mami; Saito, Kazuki; Uchiyama, Masanobu

    2018-01-01

    In the late stage of anthocyanin biosynthesis, dihydroflavonol reductase (DFR) and anthocyanidin synthase (ANS) mediate a formal tautomerization. However, such oxidation/reduction process requires high energy and appears to be unnecessary, as the oxidation state does not change during the transformation. Thus, a non-enzymatic pathway of tautomerization has also been proposed. To resolve the long-standing issue of whether this non-enzymatic pathway is the main contributor for the biosynthesis, we carried out density functional theory (DFT) calculations to examine this non-enzymatic pathway from dihydroflavonol to anthocyanidin. We show here that the activation barriers for the proposed non-enzymatic tautomerization are too high to enable the reaction to proceed under normal aqueous conditions in plants. The calculations also explain the experimentally observed requirement for acidic conditions during the final step of conversion of 2-flaven-3,4-diol to anthocyanidin; a thermodynamically and kinetically favorable concerted pathway can operate under these conditions.

  7. Enzymatic extraction of star gooseberry (Phyllanthus acidus) juice with high antioxidant level

    Science.gov (United States)

    Loan, Do Thi Thanh; Tra, Tran Thi Thu; Nguyet, Ton Nu Minh; Man, Le Van Viet

    2017-09-01

    Ascorbic acid and phenolic compounds are main antioxidants in star gooseberry (Phyllanthus acidus) fruit. In this study, Pectinex Ultra SP-L preparation with pectinase activity was used in the extraction of star gooseberry juice. The effects of pectinase concentration and biocatalytic time on the content of ascorbic acid, phenolic compounds and antioxidant activity of the fruit juice were firstly investigated. Response surface methodology was then used to optimize the conditions of enzymatic extraction for maximizing the antioxidant activity of the star gooseberry juice. The optimal pectinase concentration and biocatalytic time were 19 polygalacturonase units per 100g pulp dry weight and 67 min, respectively under which the maximal antioxidant activity achieved 5595±6 µmol Trolox equivalent per 100g juice dry weight. On the basis of kinetic model of second-order extraction, the extraction rate constant of ascorbic acid and phenolic compounds in the enzymatic extraction increased approximately 21% and 157%, respectively in comparison with that in the conventional extraction. Application of pectinase preparation to the fruit juice extraction was therefore potential for improvement in antioxidant level of the product.

  8. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity

    Energy Technology Data Exchange (ETDEWEB)

    Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L. (UW-MED); (UCB)

    2015-04-22

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.

  9. Protein-Rich Fraction of Cnidoscolus urens (L. Arthur Leaves: Enzymatic Characterization and Procoagulant and Fibrinogenolytic Activities

    Directory of Open Access Journals (Sweden)

    Yamara A. S. de Menezes

    2014-03-01

    Full Text Available Proteolytic enzymes are important macromolecules in the regulation of biochemical processes in living organisms. Additionally, these versatile biomolecules have numerous applications in the industrial segment. In this study we have characterized a protein-rich fraction of Cnidoscolus urens (L. Arthur leaves, rich in proteolytic enzymes, and evaluated its effects on the coagulation cascade. Three protein-rich fractions were obtained from the crude extract of C. urens leaves by precipitation with acetone. Fraction F1.0 showed higher proteolytic activity upon azocasein, and thus, was chosen for subsequent tests. The proteolytic activity of F1.0 on fibrinogen was dose-dependent and time-dependent. The extract demonstrated procoagulant activity on citrated plasma and reduced the APTT, not exerting effects on PT. Despite the fibrin(ogenolytic activity, F1.0 showed no defibrinogenating activity in vivo. The fraction F1.0 did not express hemorrhagic nor hemolytic activities. The proteolytic activity was inhibited by E-64, EDTA and in the presence of metal ions, and increased when pretreated with reducing agents, suggesting that the observed activity was mostly due to cysteine proteases. Several bands with proteolytic activity were detected by zymography with gelatin, albumin and fibrinogen. The optimal enzymatic activity was observed in temperature of 60 °C and pH 5.0, demonstrating the presence of acidic proteases. In conclusion, these results could provide basis for the pharmacological application of C. urens proteases as a new source of bioactive molecules to treat bleeding and thrombotic disorders.

  10. Extracellular enzyme activities of aquatic bacteria in polluted environment: 2. Amylolytic activity

    International Nuclear Information System (INIS)

    Arbaciauskiene, V.

    2003-01-01

    Water samples were taken from Lake Drukshiai tributaries (Ricanka); Gulbinele Stream affected by urban rain sewerage from Visaginas; Gulbinele Stream into which municipal sewage from Visaginas (MS) and industrial rain sewerage from the Ignalina NPP and their mouth, and Lake Dringis. Lake Dringis, in Aukstaitija National Park, was selected as an ecosystem pattern of a weak anthropogenic influence, while Lake Drukshiai was chosen as a regularly polluted water body. Lake Drukshiai, the cooling basin of the Ignalina NPP (IRS-1.2), is being polluted with industrial and municipal sewage through its tributaries. The amylolytic activity (AA) of heterotrophic aquatic bacteria was tested. The highest total mean AA of aquatic bacteria was calculated in Lake Dringis. Here, the results were significantly higher than in Lake Drukshiai tributaries and their mouths, excepting the mouths of the Ricanka and MS. The lowest mean of AA in Lake Drukshiai was characteristic of the IRS-1.2 tributary. A comparison of the mean AA of active isolates showed that certain bacterial strains from the sites of varying degrees of pollution could be noted for a relatively high level of enzymatic activity. Thus, anthropogenic pollution exerts a negative effect on the total mean AA, although certain strains of bacteria are able to adapt to the stressful environment and remain active. (author)

  11. Apixaban Enhances Vasodilatation Mediated by Protease-Activated Receptor 2 in Isolated Rat Arteries

    Directory of Open Access Journals (Sweden)

    Ambra Villari

    2017-07-01

    Full Text Available Apixaban (APX is a direct inhibitor of factor X (FXa approved for prophylaxis and treatment of deep venous thrombosis and atrial fibrillation. Because FXa activates protease-activated receptor 2 (PAR-2 in endothelium and vascular smooth muscle, inhibition of FXa by APX may affect vasomotor function. The effect of APX was assessed in vitro, by wire myography, in rat mesenteric resistance arteries (MRAs and basilar arteries challenged with vasoconstrictors [phenylephrine (PE; 5-hydroxytryptamine (5-HT], vasodilators [acetylcholine (ACh; sodium nitroprusside (SNP] or with the PAR-2 peptide agonist SLIGRL. APX (10 μM reduced the vasoconstriction to PE and 5-HT while did not change the vasodilatation to ACh or SNP. SLIGRL induced concentration-dependent vasodilation in pre-constricted arteries, that was reduced by incubation with the NO inhibitor NG-nitro-L-arginine (L-NNA and abolished by endothelium removal. APX enhanced vasodilation to SLIGRL either in the presence or in the absence of L-NNA, but was ineffective in endothelium-denuded vessels. In preparations from heparin-treated rats (to inhibit FXa APX did not change the vasodilation to SLIGRL. FXa enzymatic activity, detected in mesentery homogenates from controls, was inhibited by APX, whereas APX-sensitive enzymatic activity was undetectable in homogenates from heparin-treated rats. Immunoblot analysis showed that incubation of MRA or aorta with APX increased the abundance of PAR-2, an effect not seen in MRA from heparin-treated rats or in endothelium-denuded aortas. In conclusion, inhibition of FXa by APX increases vasodilatation mediated by PAR-2. APX may act by inhibiting PAR-2 desensitization induced by endogenous FXa. This effect could be useful in the context of endothelial dysfunction associated to cardiovascular diseases.

  12. A δ38 deletion variant of human transketolase as a model of transketolase-like protein 1 exhibits no enzymatic activity.

    Directory of Open Access Journals (Sweden)

    Stefan Schneider

    Full Text Available Besides transketolase (TKT, a thiamin-dependent enzyme of the pentose phosphate pathway, the human genome encodes for two closely related transketolase-like proteins, which share a high sequence identity with TKT. Transketolase-like protein 1 (TKTL1 has been implicated in cancerogenesis as its cellular expression levels were reported to directly correlate with invasion efficiency of cancer cells and patient mortality. It has been proposed that TKTL1 exerts its function by catalyzing an unusual enzymatic reaction, a hypothesis that has been the subject of recent controversy. The most striking difference between TKTL1 and TKT is a deletion of 38 consecutive amino acids in the N-terminal domain of the former, which constitute part of the active site in authentic TKT. Our structural and sequence analysis suggested that TKTL1 might not possess transketolase activity. In order to test this hypothesis in the absence of a recombinant expression system for TKTL1 and resilient data on its biochemical properties, we have engineered and biochemically characterized a "pseudo-TKTL1" Δ38 deletion variant of human TKT (TKTΔ38 as a viable model of TKTL1. Although the isolated protein is properly folded under in vitro conditions, both thermal stability as well as stability of the TKT-specific homodimeric assembly are markedly reduced. Circular dichroism and NMR spectroscopic analysis further indicates that TKTΔ38 is unable to bind the thiamin cofactor in a specific manner, even at superphysiological concentrations. No transketolase activity of TKTΔ38 can be detected for conversion of physiological sugar substrates thus arguing against an intrinsically encoded enzymatic function of TKTL1 in tumor cell metabolism.

  13. Enzymatic synthesis of bioactive compounds with high potential for cosmeceutical application

    OpenAIRE

    Antonopoulou, Io; Varriale, Simona; Topakas, Evangelos; Rova, Ulrika; Christakopoulos, Paul; Faraco, Vincenza

    2016-01-01

    Cosmeceuticals are cosmetic products containing biologically active ingredients purporting to offer a pharmaceutical therapeutic benefit. The active ingredients can be extracted and purified from natural sources (botanicals, herbal extracts, or animals) but can also be obtained biotechnologically by fermentation and cell cultures or by enzymatic synthesis and modification of natural compounds. A cosmeceutical ingredient should possess an attractive property such as anti-oxidant, anti-inflamma...

  14. Conferring specificity in redox pathways by enzymatic thiol/disulfide exchange reactions.

    Science.gov (United States)

    Netto, Luis Eduardo S; de Oliveira, Marcos Antonio; Tairum, Carlos A; da Silva Neto, José Freire

    2016-01-01

    Thiol-disulfide exchange reactions are highly reversible, displaying nucleophilic substitutions mechanism (S(N)2 type). For aliphatic, low molecular thiols, these reactions are slow, but can attain million times faster rates in enzymatic processes. Thioredoxin (Trx) proteins were the first enzymes described to accelerate thiol-disulfide exchange reactions and their high reactivity is related to the high nucleophilicity of the attacking thiol. Substrate specificity in Trx is achieved by several factors, including polar, hydrophobic, and topological interactions through a groove in the active site. Glutaredoxin (Grx) enzymes also contain the Trx fold, but they do not share amino acid sequence similarity with Trx. A conserved glutathione binding site is a typical feature of Grx that can reduce substrates by two mechanisms (mono and dithiol). The high reactivity of Grx enzymes is related to the very acid pK(a) values of reactive Cys that plays roles as good leaving groups. Therefore, although distinct oxidoreductases catalyze similar thiol–disulfide exchange reactions, their enzymatic mechanisms vary. PDI and DsbA are two other oxidoreductases, but they are involved in disulfide bond formation, instead of disulfide reduction, which is related to the oxidative environment where they are found. PDI enzymes and DsbC are endowed with disulfide isomerase activity, which is related with their tetra-domain architecture. As illustrative description of specificity in thiol-disulfide exchange, redox aspects of transcription activation in bacteria, yeast, and mammals are presented in an evolutionary perspective. Therefore, thiol-disulfide exchange reactions play important roles in conferring specificity to pathways, a required feature for signaling.

  15. Archetypal sandwich-structured CuO for high performance non-enzymatic sensing of glucose

    Science.gov (United States)

    Meher, Sumanta Kumar; Rao, G. Ranga

    2013-02-01

    In the quest to enhance the selectivity and sensitivity of novel structured metal oxides for electrochemical non-enzymatic sensing of glucose, we report here a green synthesis of unique sandwich-structured CuO on a large scale under microwave mediated homogeneous precipitation conditions. The physicochemical studies carried out by XRD and BET methods show that the monoclinic CuO formed via thermal decomposition of Cu2(OH)2CO3 possesses monomodal channel-type pores with largely improved surface area (~43 m2 g-1) and pore volume (0.163 cm3 g-1). The fascinating surface morphology and pore structure of CuO is formulated due to homogeneous crystallization and microwave induced self assembly during synthesis. The cyclic voltammetry and chronoamperometry studies show diffusion controlled glucose oxidation at ~0.6 V (vs. Ag/AgCl) with extremely high sensitivity of 5342.8 μA mM-1 cm-2 and respective detection limit and response time of ~1 μM and ~0.7 s, under a wide dynamic concentration range of glucose. The chronoamperometry measurements demonstrate that the sensitivity of CuO to glucose is unaffected by the absence of dissolved oxygen and presence of poisoning chloride ions in the reaction medium, which essentially implies high poison resistance activity of the sandwich-structured CuO. The sandwich-structured CuO also shows insignificant interference/significant selectivity to glucose, even in the presence of high concentrations of other sugars as well as reducing species. In addition, the sandwich-structured CuO shows excellent reproducibility (relative standard deviation of ~2.4% over ten identically fabricated electrodes) and outstanding long term stability (only ~1.3% loss in sensitivity over a period of one month) during non-enzymatic electrochemical sensing of glucose. The unique microstructure and suitable channel-type pore architecture provide structural stability and maximum accessible electroactive surface for unimpeded mobility of glucose as well as the

  16. Enzymatic production of pectic oligosaccharides from onion skins.

    Science.gov (United States)

    Babbar, Neha; Baldassarre, Stefania; Maesen, Miranda; Prandi, Barbara; Dejonghe, Winnie; Sforza, Stefano; Elst, Kathy

    2016-08-01

    Onion skins are evaluated as a new raw material for the enzymatic production of pectic oligosaccharides (POS) with a targeted degree of polymerization (DP). The process is based on a two-stage process consisting of a chelator-based crude pectin extraction followed by a controlled enzymatic hydrolysis. Treatment of the extracted crude onion skin's pectin with various enzymes (EPG-M2, Viscozyme and Pectinase) shows that EPG-M2 is the most appropriate enzyme for tailored POS production. The experiments reveal that the highest amount of DP2 and DP3 is obtained at a time scale of 75-90min with an EPG-M2 concentration of 26IU/mL. At these conditions the production amounts 2.5-3.0% (w/w) d.m for DP2 and 5.5-5.6% (w/w) d.m for DP3 respectively. In contrast, maximum DP4 production of 5.2-5.5% (w/w) d.m. is obtained with 5.2IU/mL at a time scale of 15-30min. Detailed LC-MS analysis reveals the presence of more methylated oligomers compared to acetylated forms in the digests. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Enzymatic Dissolution of Biocomposite Solids Consisting of Phosphopeptides to Form Supramolecular Hydrogels

    KAUST Repository

    Shi, Junfeng; Yuan, Dan; Haburcak, Richard; Zhang, Qiang; Zhao, Chao; Zhang, Xixiang; Xu, Bing

    2015-01-01

    Enzyme-catalyzed dephosphorylation is essential for biomineralization and bone metabolism. Here we report the exploration of using enzymatic reaction to transform biocomposites of phosphopeptides and calcium (or strontium) ions to supramolecular hydrogels as a mimic of enzymatic dissolution of biominerals. 31P NMR shows that strong affinity between the phosphopeptides and alkaline metal ions (e.g., Ca2+ or Sr2+) induces the formation of biocomposites as precipitates. Electron microscopy reveals that the enzymatic reaction regulates the morphological transition from particles to nanofibers. Rheology confirms the formation of a rigid hydrogel. As the first example of enzyme-instructed dissolution of a solid to form supramolecular nanofibers/hydrogels, this work provides an approach to generate soft materials with desired properties, expands the application of supramolecular hydrogelators, and offers insights to control the demineralization of calcified soft tissues.

  18. Enzymatic Dissolution of Biocomposite Solids Consisting of Phosphopeptides to Form Supramolecular Hydrogels

    KAUST Repository

    Shi, Junfeng

    2015-10-14

    Enzyme-catalyzed dephosphorylation is essential for biomineralization and bone metabolism. Here we report the exploration of using enzymatic reaction to transform biocomposites of phosphopeptides and calcium (or strontium) ions to supramolecular hydrogels as a mimic of enzymatic dissolution of biominerals. 31P NMR shows that strong affinity between the phosphopeptides and alkaline metal ions (e.g., Ca2+ or Sr2+) induces the formation of biocomposites as precipitates. Electron microscopy reveals that the enzymatic reaction regulates the morphological transition from particles to nanofibers. Rheology confirms the formation of a rigid hydrogel. As the first example of enzyme-instructed dissolution of a solid to form supramolecular nanofibers/hydrogels, this work provides an approach to generate soft materials with desired properties, expands the application of supramolecular hydrogelators, and offers insights to control the demineralization of calcified soft tissues.

  19. Postprandial incretin and islet hormone responses and dipeptidyl-peptidase 4 enzymatic activity in patients with maturity onset diabetes of the young

    DEFF Research Database (Denmark)

    Østoft, Signe Harring; Bagger, Jonatan Ising; Hansen, Torben

    2015-01-01

    Objective: The role of the incretin hormones in the pathophysiology of maturity onset diabetes of the young (MODY) is unclear. Design: We studied the postprandial plasma responses of glucagon, incretin hormones (glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP......)), and dipeptidyl-peptidase 4 (DPP-4) enzymatic activity in patients with glucokinase (GCK)-diabetes (MODY2), hepatocyte nuclear factor 1α (HNF1A)-diabetes (MODY3), and in matched healthy individuals (CTRLs). Subjects and methods: Ten patients with GCK-diabetes (age: 43±5 years; BMI: 24±2 kg/m2; FPG: 7.1±0.3 mmol....../l: HbA1c: 6.6±0.2%), 10 patients with HNF1A-diabetes (age: 31±3 years (mean ± SEM); body mass index (BMI): 24±1 kg/m2; fasting plasma glucose (FPG): 8.9±0.8 mmol/l; haemoglobin A1c (HbA1c): 7.0±0.3%), and 10 CTRLs (age: 40±5 years; BMI: 24±1 kg/m2; FPG: 5.1±0.1 mmol/l; HbA1c: 5.3±0.1%) were examined...

  20. Direct organogenesis of Mandevilla illustris (Vell) Woodson and effects of its aqueous extract on the enzymatic and toxic activities of Crotalus durissus terrificus snake venom.

    Science.gov (United States)

    Biondo, R; Soares, A M; Bertoni, B W; França, S C; Pereira, A M S

    2004-03-01

    In order to produce explants of Mandevilla illustris (Vell) Woodson for the "Cerrado in vitro", the Germplasm Bank of UNAERP, we carried out a micropropagation protocol using MS or MS/3 medium supplemented with different concentrations of 6-benzyladeninepurine (BA), Zeatin or 2-isopentenyladenine for nodal segment growth, and alpha-naphthaleneacetic acid, indole-3-butyric acid (IBA) or 1,4 dithiothreitol for rooting. For nodal segments, all the cytokinins tested yielded similar results. However, 2.22 micro M BA is more economical to use. MS/3 medium supplemented with 0.49 micro M IBA was the most appropriate medium for rooting, resulting in 29% rooted explants. The crude aqueous extract from the subterranean system (SS) of M. illustris was assayed for its inhibitory action on the enzymatic activity of Crotalus durissus terrificus snake venom, isolated basic phospholipase A2 (CB) and crotoxin. It totally inhibited the phospholipase activity of crude Cdt venom and CB toxin and inhibited the phospholipase activity of crotoxin by 49%. The toxic action of both the crude venom and crotoxin was partially inhibited-there was a prolonged survival time and a 40.0% decrease in lethality.

  1. Drug Delivery by an Enzyme-Mediated Cyclization of a Lipid Prodrug with Unique Bilayer-Formation Properties

    DEFF Research Database (Denmark)

    Linderoth, Lars; Peters, Günther H.j.; Madsen, Robert

    2009-01-01

    Special delivery: Liposomal drug-delivery systems in which prodrugs are activated specifically by disease-associated enzymes have great potential for the treatment of severe diseases, such as cancer. A new type of phospholipid-based prodrug has the ability to form stable small unilamellar vesicle...... (see picture). Activation of the prodrug vesicles by the enzyme sPLA2 initiates a cyclization reaction, which leads to the release of the drug....

  2. The effects of fermentation and enzymatic treatment of pea on nutrient digestibility and growth performance of broilers.

    Science.gov (United States)

    Goodarzi Boroojeni, F; Senz, M; Kozłowski, K; Boros, D; Wisniewska, M; Rose, D; Männer, K; Zentek, J

    2017-10-01

    The present study examined the impacts of native, fermented or enzymatically treated peas (Pisum sativum L.) inclusion in broiler diets, on growth performance and nutrient digestibility. For the fermentation process, Madonna pea was mixed with water (1/1) containing 2.57×108 Bacillus subtilis (GalliPro®) spores/kg pea and then, incubated for 48 h at 30 °C. For the enzymatic treatment process, the used water for dough production contained three enzymes, AlphaGalTM (α-galactosidase), RONOZYME® ProAct and VP (protease and pectinases respectively - DSM, Switzerland) and the pea dough incubated for 24 h at 30°C. Nine corn-wheat-soybean diets were formulated by supplying 10%, 20% and 30% of the required CP with either native, fermented or enzymatically treated peas. Performance was recorded weekly and at the end of the experiment (day 35), apparent ileal digestibility (AID) of CP, amino acids (AA), crude fat, starch, Ca, P and K were determined. Data were subjected to ANOVA using GLM procedure with a 3×3 factorial arrangement of treatments. Both processes reduced α-galactosides, phytate, trypsin inhibitor activity and resistant starch in peas. Increasing levels of pea products up to 300 g/kg diet, reduced BW gain and feed intake (P⩽0.05). Broilers fed diets containing enzymatically treated pea had the best feed conversion ratio at day 35. Different types of pea product and their inclusion levels had no effect on AID of all nutrients. The interaction between type of the pea products and inclusion levels was significant for AID of starch. For native pea diets, 10% group showed similar AID of starch to 20% native pea but it had higher AID than 30% native pea. For fermented and enzymatically treated groups, all three levels displayed similar AID of starch. In conclusion, enzymatic treatment and fermentation could improve the nutritional quality of pea. Inclusion of enzymatically treated pea in broiler diets could improve broiler performance compared with other pea

  3. Inhibition of tyrosinase-mediated enzymatic browning by sulfite and natural alternatives

    NARCIS (Netherlands)

    Kuijpers, T.F.M.; Vincken, J.P.

    2013-01-01

    Although sulfite is widely used to counteract enzymatic browning, its mechanism has remained largely unknown. We describe a double inhibitory mechanism of sulfite on enzymatic browning, affecting both the enzymatic oxidation of phenols into o‑quinones, as well as the non‑enzymatic

  4. Conformational changes in a hyperthermostable glycoside hydrolase: enzymatic activity is a consequence of the loop dynamics and protonation balance.

    Directory of Open Access Journals (Sweden)

    Leandro C de Oliveira

    Full Text Available Endo-β-1, 4-mannanase from Thermotoga petrophila (TpMan is a modular hyperthermostable enzyme involved in the degradation of mannan-containing polysaccharides. The degradation of these polysaccharides represents a key step for several industrial applications. Here, as part of a continuing investigation of TpMan, the region corresponding to the GH5 domain (TpManGH5 was characterized as a function of pH and temperature. The results indicated that the enzymatic activity of the TpManGH5 is pH-dependent, with its optimum activity occurring at pH 6. At pH 8, the studies demonstrated that TpManGH5 is a molecule with a nearly spherical tightly packed core displaying negligible flexibility in solution, and with size and shape very similar to crystal structure. However, TpManGH5 experiences an increase in radius of gyration in acidic conditions suggesting expansion of the molecule. Furthermore, at acidic pH values, TpManGH5 showed a less globular shape, probably due to a loop region slightly more expanded and flexible in solution (residues Y88 to A105. In addition, molecular dynamics simulations indicated that conformational changes caused by pH variation did not change the core of the TpManGH5, which means that only the above mentioned loop region presents high degree of fluctuations. The results also suggested that conformational changes of the loop region may facilitate polysaccharide and enzyme interaction. Finally, at pH 6 the results indicated that TpManGH5 is slightly more flexible at 65°C when compared to the same enzyme at 20°C. The biophysical characterization presented here is well correlated with the enzymatic activity and provide new insight into the structural basis for the temperature and pH-dependent activity of the TpManGH5. Also, the data suggest a loop region that provides a starting point for a rational design of biotechnological desired features.

  5. Dual functional rhodium oxide nanocorals enabled sensor for both non-enzymatic glucose and solid-state pH sensing.

    Science.gov (United States)

    Dong, Qiuchen; Huang, Yikun; Song, Donghui; Wu, Huixiang; Cao, Fei; Lei, Yu

    2018-07-30

    Both pH-sensitive and glucose-responsive rhodium oxide nanocorals (Rh 2 O 3 NCs) were synthesized through electrospinning followed by high-temperature calcination. The as-prepared Rh 2 O 3 NCs were systematically characterized using various advanced techniques including scanning electron microscopy, X-ray powder diffraction and Raman spectroscopy, and then employed as a dual functional nanomaterial to fabricate a dual sensor for both non-enzymatic glucose sensing and solid-state pH monitoring. The sensing performance of the Rh 2 O 3 NCs based dual sensor toward pH and glucose was evaluated using open circuit potential, cyclic voltammetry and amperometric techniques, respectively. The results show that the as-prepared Rh 2 O 3 NCs not only maintain accurate and reversible pH sensitivity of Rh 2 O 3 , but also demonstrate a good electrocatalytic activity toward glucose oxidation in alkaline medium with a sensitivity of 11.46 μA mM -1 cm -2 , a limit of detection of 3.1 μM (S/N = 3), and a reasonable selectivity against various interferents in non-enzymatic glucose detection. Its accuracy in determining glucose in human serum samples was further demonstrated. These features indicate that the as-prepared Rh 2 O 3 NCs hold great promise as a dual-functional sensing material in the development of a high-performance sensor forManjakkal both solid-state pH and non-enzymatic glucose sensing. Copyright © 2018 Elsevier B.V. All rights reserved.

  6. Production of Fish Hydrolysates Protein From Waste of Fish Carp (Cyprinus Carpio) by Enzymatic Hydrolysis

    OpenAIRE

    Saputra, Dede; Nurhayati, Tati

    2016-01-01

    Fish Protein Hydrolysates (FPH) is the mixed products of polypeptide, dipeptides, and amino acid. It can be produced from materials that contained of protein by acid reaction, base reaction or enzymatic hydrolysis. The objectives of this study were to study the production of FPH from fish carp meat at post rigor phase and viscera by enzymatic hydrolysis, to determine the specific activity of papain enzyme, and to determine the solubility of FPH. Capacity of fish hydrolyzing can be identified ...

  7. Physiological and enzymatic analyses of pineapple subjected to ionizing radiation

    International Nuclear Information System (INIS)

    Silva, Josenilda Maria da; Silva, Juliana Pizarro; Spoto, Marta Helena Fillet

    2007-01-01

    The physiological and enzymatic post-harvest characteristics of the pineapple cultivar Smooth Cayenne were evaluated after the fruits were gamma-irradiated with doses of 100 and 150 Gy and the fruits were stored for 10, 20 and 30 days at 12 deg C (±1) and relative humidity of 85% (±5). Physiological and enzymatic analyses were made for each storage period to evaluate the alterations resulting from the application of ionizing radiation. Control specimens showed higher values of soluble pectins, total pectins, reducing sugars, sucrose and total sugars and lower values of polyphenyloxidase and polygalacturonase enzyme activities. All the analyses indicated that storage time is a significantly influencing factor. The 100 Gy dosage and 20-day storage period presented the best results from the standpoint of maturation and conservation of the fruits quality. (author)

  8. Enzymatic Modification of Sphingomyelin

    DEFF Research Database (Denmark)

    Due to its major role in maintaining the water-retaining properties of the epidermis, ceramide is of great commercial potential in cosmetic and pharmaceuticals such as hair and skin care products. Currently, chemical synthesis of ceramide is a costly process, and developments of alternative cost......-efficient, high yield production methods are of great interest. In the present study, the potential of producing ceramide through the enzymatic hydrolysis of sphingomyelin have been studied. sphingomyelin is a ubiquitous membrane-lipid and rich in dairy products or by-products. It has been verified...... that sphingomyelin modification gives a feasible approach to the potential production of ceramide. The reaction system has been improved through system evaluation and the optimization of several important factors, and phospholipase C from Clostridium perfringens shows higher activity towards the hydrolysis reaction...

  9. Enzymatic modification of starch

    DEFF Research Database (Denmark)

    Jensen, Susanne Langgård

    In the food industry approaches for using bioengineering are investigated as alternatives to conventional chemical and physical starch modification techniques in development of starches with specific properties. Enzyme-assisted post-harvest modification is an interesting approach to this, since...... it is considered a clean and energy saving technology. This thesis aimed to investigate the effect of using reaction conditions, simulating an industrial process, for enzymatic treatment of starch with branching enzyme (BE) from Rhodothermus obamensis. Thus treatements were conducted at 70°C using very high...... substrate concentration (30-40% dry matter (DM)) and high enzyme activity (750-2250 BE units (BEU)/g sample). Starches from various botanical sources, representing a broad range of properties, were used as substrates. The effects of the used conditions on the BE-reaction were evaluated by characterization...

  10. Enzymatic epoxidation of biodiesel optimized by response surface ...

    African Journals Online (AJOL)

    During the enzymatic epoxidation of biodiesel, stearic acid was selected as oxygen carrier. Enzyme screening and the load of stearic acid were investigated. The effects of four main reaction conditions including reaction time, temperature, enzyme load, and mole ratio of H2O2/C=C-bonds on the epoxy oxygen group content ...

  11. Haematology, genotoxicity, enzymatic activity and histopathology as biomarkers of metal pollution in the shrew Crocidura russula

    International Nuclear Information System (INIS)

    Sanchez-Chardi, A.; Marques, C.C.; Gabriel, S.I.; Capela-Silva, F.; Cabrita, A.S.; Lopez-Fuster, M.J.; Nadal, J.; Mathias, M.L.

    2008-01-01

    Haematological (WBC, RBC, Hgb and Hct) and genotoxicity (MNT) parameters, hepatic enzymatic activities (GST, GPx and GR), and a histopathological evaluation of liver, kidneys and gonads were assessed as general biomarkers of metal pollution in the shrew Crocidura russula inhabiting a pyrite mining area. Specimens exposed to metals presented a few significant alterations when compared with reference animals: GST activity decreased; micronuclei increased; and evident liver alterations related to metal exposure were observed. On the basis of all the parameters studied, age was an important factor that partly explained the observed variation, whereas sex was the least important factor. Significant correlations were also found between heavy metal concentrations and biomarkers evaluated, demonstrating the great influence of these metals in the metabolic alterations. To the best of our knowledge, these data constitute the first measurements of a battery of biomarkers in shrews from a mine site and are among the few available for insectivorous mammals. - Metals from an abandoned pyrite mine produce alterations in haematological parameters, GST, MNT, and histopathology in shrews

  12. The properties of main oxidases associated with enzymatic ...

    African Journals Online (AJOL)

    Lenovo User

    2012-07-03

    Jul 3, 2012 ... 1College of Food Science and Engineering, Shandong Agricultural University, Taian, Shandong, 271018,. People's Republic of China. 2The Central Hospital of Taian, Taian, Shandong ... pear vinegar, preserved pear and canned pear. However, enzymatic browning during processing impairs sensory.

  13. Development of a Novel NMR-based Rheb GTPase Assay and Molecular Characterization of TSC2 GAP Activity

    Science.gov (United States)

    2010-05-01

    GTPase) that belongs to the Ras superfamily and has homologs in yeast, fungi , slime mold, fruit fly, zebra fish, and mammals (1–3). Ge- netic and...characterization of TSC2 disease mutations affecting its GAP activity (months 9-12) While the final aspects of this task are yet to be completed, we have...domain mutants of TSC2 that we examined affected its enzymatic activ- ity. This method can now be applied to study the function and regulation of other

  14. Encapsulation and covalent binding of molecular payload in enzymatically activated micellar nanocarriers.

    Science.gov (United States)

    Rosenbaum, Ido; Harnoy, Assaf J; Tirosh, Einat; Buzhor, Marina; Segal, Merav; Frid, Liat; Shaharabani, Rona; Avinery, Ram; Beck, Roy; Amir, Roey J

    2015-02-18

    The high selectivity and often-observed overexpression of specific disease-associated enzymes make them extremely attractive for triggering the release of hydrophobic drug or probe molecules from stimuli-responsive micellar nanocarriers. Here we utilized highly modular amphiphilic polymeric hybrids, composed of a linear hydrophilic polyethylene glycol (PEG) and an esterase-responsive hydrophobic dendron, to prepare and study two diverse strategies for loading of enzyme-responsive micelles. In the first type of micelles, hydrophobic coumarin-derived dyes were encapsulated noncovalently inside the hydrophobic core of the micelle, which was composed of lipophilic enzyme-responsive dendrons. In the second type of micellar nanocarrier the hydrophobic molecular cargo was covalently linked to the end-groups of the dendron through enzyme-cleavable bonds. These amphiphilic hybrids self-assembled into micellar nanocarriers with their cargo covalently encapsulated within the hydrophobic core. Both types of micelles were highly responsive toward the activating enzyme and released their molecular cargo upon enzymatic stimulus. Importantly, while faster release was observed with noncovalent encapsulation, higher loading capacity and slower release rate were achieved with covalent encapsulation. Our results clearly indicate the great potential of enzyme-responsive micellar delivery platforms due to the ability to tune their payload capacities and release rates by adjusting the loading strategy.

  15. Chondroitin sulphate extracted from antler cartilage using high hydrostatic pressure and enzymatic hydrolysis

    Directory of Open Access Journals (Sweden)

    Chong-Tai Kim

    2014-12-01

    Full Text Available Chondroitin sulphate (CS, a major glycosaminoglycan, is an essential component of the extracellular matrix in cartilaginous tissues. Wapiti velvet antlers are a rich source of these molecules. The purpose of the present study was to develop an effective isolation procedure of CS from fresh velvet antlers using a combination of high hydrostatic pressure (100 MPa and enzymatic hydrolysis (papain. High CS extractability (95.1 ± 2.5% of total uronic acid was obtained following incubation (4 h at 50 °C with papain at pH 6.0 in 100 MPa compared to low extractability (19 ± 1.1% in ambient pressure (0.1 MPa. Antler CS fractions were isolated by Sephacryl S-300 chromatography and identified by western blot using an anti-CS monoclonal antibody. The antler CS fraction did not aggregate with hyaluronic acid in CL-2B chromatography and possessed DPPH radical scavenging activity at 78.3 ± 1.5%. The results indicated that high hydrostatic pressure and enzymatic hydrolysis procedure may be a useful tool for the isolation of CS from antler cartilaginous tissues.

  16. Production of spray-dried honey jackfruit (Artocarpus heterophyllus) powder from enzymatic liquefied puree.

    Science.gov (United States)

    Wong, Chen Wai; Tan, Hong Hock

    2017-02-01

    This paper presents the enzymatic liquefaction process for honey jackfruit optimized with Pectinex ® Ultra SP-L and Celluclast ® 1.5 L individually or in combinations at different concentrations (0-2.5% v/w) and incubation time (0-2.5 h). Treatment with combinations of enzymes showed a greater effect in the reduction of viscosity (83.9-98.8%) as compared to single enzyme treatment (64.8-87.3%). The best parameter for enzymatic liquefaction was obtained with 1.0% (v/w) Pectinex ® Ultra SP-L and 0.5% (v/w) Celluclast ® 1.5 L for 1.5 h. Spray drying process was carried out using different inlet temperatures (140-180 °C) and maltodextrin concentrations (10-30% w/w). Results indicated that the spray-dried honey jackfruit powder produced at 160 °C with 30% w/w maltodextrin gave the highest product yield (66.90%) with good powder qualities in terms of water activity, solubility, moisture content, hygroscopicity, color and bulk density. The spray-dried honey jackfruit powder could potentially be incorporated into various food products.

  17. Susceptibility to antifungal agents and enzymatic activity of Candida haemulonii and Cutaneotrichosporon dermatis isolated from soft corals on the Brazilian reefs.

    Science.gov (United States)

    Pagani, Danielle M; Heidrich, Daiane; Paulino, Gustavo V B; de Oliveira Alves, Karine; Dalbem, Paula T; de Oliveira, Caroline F; Andrade, Zélia M M; Silva, Carolini; Correia, Monica D; Scroferneker, Maria Lúcia; Valente, Patricia; Landell, Melissa Fontes

    2016-12-01

    Candida is a common fungus with the capacity to cause infections in humans. However, most studies have concentrated on clinical isolates and little is known about the identity, ecology and drug resistance of free living species/strains. Here, we isolate eight strains of Candida haemulonii and four strains of Cutaneotrichosporon dermatis from three marine cnidarian zoanthids species (Palythoa caribaeorum, Palythoa variabilis and Zoanthus sociatus) collected from Brazilian coral reefs. Strains were identified by sequencing of the D1/D2 domain LSU rDNA and ITS region. We tested these environmental isolates for their capacity to grow in media with increasing concentration of NaCl, capacity to grow in different temperatures, enzymatic activity and antifungal susceptibility. For C. haemulonii, all strains strongly produced gelatinase, esterase and albuminase and were either able to express lipase, phospholipase and keratinase, but not express urease and DNase. The strains were able to grow at 37 °C, but not at 39 °C, and except for LMS 40, all of them could grow in a 10 % NaCl medium. All isolates were resistant to all antifungals tested, with exception for ketoconazole and tioconazole (MIC = 2 µg/mL). For C. dermatis, all strains could grow at 39 °C and could not express phospholipase, keratinase or gelatinase. However, all were capable of expressing urease, lipase and esterase. Three out of four strains could grow in a 10 % NaCl medium, but none grew in a 30 % NaCl medium. The strains showed high values of minimal inhibitory concentration. LMPV 90 was resistant to tioconazole, terbinafine, fluconazole and posaconazole, and LMS 38 was resistant to all antifungal agents tested. We discuss the characterization of C. haemulonii and C. dermatis as a possible emerging pathogen due to its animal-related enzymatic arsenal and antifungal resistance.

  18. Label-free and sensitive detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplification.

    Science.gov (United States)

    Cheng, Rui; Tao, Mangjuan; Shi, Zhilu; Zhang, Xiafei; Jin, Yan; Li, Baoxin

    2015-11-15

    Several fluorescence signal amplification strategies have been developed for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity, but they need fluorescence dye labeled DNA probe. We have addressed the limitation and report here a label-free strategy for sensitive detection of PNK activity by coupling DNA strand displacement reaction with enzymatic-aided amplification. A hairpin oligonucleotide (hpDNA) with blunt ends was used as the substrate for T4 PNK phosphorylation. In the presence of T4 PNK, the stem of hpDNA was phosphorylated and further degraded by lambda exonuclease (λ exo) from 5' to 3' direction to release a single-stranded DNA as a trigger of DNA strand displacement reaction (SDR). The trigger DNA can continuously displace DNA P2 from P1/P2 hybrid with the help of specific cleavage of nicking endonuclease (Nt.BbvCI). Then, DNA P2 can form G-quadruplex in the presence of potassium ions and quadruplex-selective fluorphore, N-methyl mesoporphyrin IX (NMM), resulting in a significant increase in fluorescence intensity of NMM. Thus, the accumulative release of DNA P2 led to fluorescence signal amplification for determining T4 PNK activity with a detection limit of 6.6×10(-4) U/mL, which is superior or comparative with established approaches. By ingeniously utilizing T4 PNK-triggered DNA SDR, T4 PNK activity can be specifically and facilely studied in homogeneous solution containing complex matrix without any external fluorescence labeling. Moreover, the influence of different inhibitors on the T4 PNK activity revealed that it also can be explored to screen T4 PNK inhibitors. Therefore, this label-free amplification strategy presents a facile and cost-effective approach for nucleic acid phosphorylation related research. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Rational design of functional and tunable oscillating enzymatic networks

    Science.gov (United States)

    Semenov, Sergey N.; Wong, Albert S. Y.; van der Made, R. Martijn; Postma, Sjoerd G. J.; Groen, Joost; van Roekel, Hendrik W. H.; de Greef, Tom F. A.; Huck, Wilhelm T. S.

    2015-02-01

    Life is sustained by complex systems operating far from equilibrium and consisting of a multitude of enzymatic reaction networks. The operating principles of biology's regulatory networks are known, but the in vitro assembly of out-of-equilibrium enzymatic reaction networks has proved challenging, limiting the development of synthetic systems showing autonomous behaviour. Here, we present a strategy for the rational design of programmable functional reaction networks that exhibit dynamic behaviour. We demonstrate that a network built around autoactivation and delayed negative feedback of the enzyme trypsin is capable of producing sustained oscillating concentrations of active trypsin for over 65 h. Other functions, such as amplification, analog-to-digital conversion and periodic control over equilibrium systems, are obtained by linking multiple network modules in microfluidic flow reactors. The methodology developed here provides a general framework to construct dissipative, tunable and robust (bio)chemical reaction networks.

  20. Positive effects of temperature and growth conditions on enzymatic and antioxidant status in lettuce plants.

    Science.gov (United States)

    Boo, Hee-Ock; Heo, Buk-Gu; Gorinstein, Shela; Chon, Sang-Uk

    2011-10-01

    The contents of two bioactive compounds (polyphenols and flavonoids) and their antioxidant and enzyme activities were determined in the leaves of six lettuce (Latuca sativa L.) cultivars subjected to 4 different day/night temperatures for 6 weeks. The total polyphenol and anthocyanin contents and the corresponding antioxidant activities were the highest at 13/10°C and 20/13°C, followed by 25/20°C and 30/25°C. The enzymatic activities of polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) were also the highest at low day/night temperatures, but the peroxidase (POD) activity was decreased at low day/night temperatures and increased at high day/night temperatures. The most significant positive correlation existed between anthocyanin content and PPO activity, total polyphenols and their antioxidant activities. The results showed that at relatively low temperatures, lettuce plants have a high antioxidant and enzymatic status. These results provide additional information for the lettuce growers. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  1. A high performance non-enzymatic glucose sensor based on nickel hydroxide modified nitrogen-incorporated nanodiamonds.

    Science.gov (United States)

    Ko, Chih-Yu; Huang, Jin-Hua; Raina, Supil; Kang, Weng P

    2013-06-07

    A highly selective, sensitive, and stable non-enzymatic glucose sensor based on Ni hydroxide modified nitrogen-incorporated nanodiamonds (Ni(OH)2-NND) was developed. The sensor was fabricated by e-beam evaporation of a thin Ni film on NND followed by the growth of Ni(OH)2 using an electrochemical process. It was found that the Ni film thickness greatly affects the morphology and electro-catalytic activity of the as-synthesized electrode for non-enzymatic glucose oxidation. Owing to its nanostructure characteristics, the best sensor fabricated by 150 nm Ni deposition showed two wide response ranges, namely, 0.02-1 mM and 1-9 mM, with sensitivities of 3.20 and 1.41 mA mM(-1) cm(-2), respectively, and a detection limit of 1.2 μM (S/N = 3). The sensor also showed good long-term stability as well as high selectivity in the presence of interferences such as ascorbic acid, acetaminophen, and uric acid. This finding reveals the possibility of exploiting the NND as an electrochemical biosensor platform where high performance addressable sensor arrays could be built.

  2. Enzymatic conversion of all-trans-β-carotene to retinal by a cytosolic enzyme from rabbit and rat intestinal mucosa

    International Nuclear Information System (INIS)

    Lakshman, M.R.; Mychkovsky, I.; Attlesey, M.

    1989-01-01

    Enzymatic conversion of all-trans-β-carotene to retinal by a partially purified enzyme from rabbit and rat intestinal mucosa was demonstrated. The enzymatic product was characterized based on the following evidence: (i) the product gave rise to its O-ethyloxime by treatment with O-ethylhydroxylamine with an absorption maximum at 363 nm in ethanol characteristics of authentic retinal O-ethyloxime. High-pressure liquid chromatography (HPLC) of this derivative yielded a sharp peak with a retention time of 7.99 min corresponding to the authentic compound; (ii) the mass spectrum of the O-ethyloxime of the enzymatic product was identical to that of authentic retinal O-ethyloxime; (iii) the specific activity of the enzymatically formed [ 14 C]retinal O-ethyloxime remained constant even after repeated crystallization; (iv) the enzymatic product exhibited an absorption maximum at 370 nm in light petroleum characteristic of authentic retinal. This retinol was enzymatically esterified to retinyl palmitate by rat pancreatic esterase with a retention time of 10 min on HPLC corresponding to authentic retinyl palmitate. Thus, the enzymatic product of β-carotene cleavage by the partially purified intestinal enzyme was unequivocally confirmed to be retinal

  3. THE KINETICS OF THE REACTIONS CATALYZED BY AN ENZYMATIC PREPARATION PRODUCED BY A BACILLUS LICHENIFORMIS STRAIN

    Directory of Open Access Journals (Sweden)

    MONICA DRAGOMIRESCU

    2007-05-01

    Full Text Available Robust immobilization techniques that preserve the activity of biomolecules have manypotential applications. In recent years, a number of new bioimobilisation methods in solgel-derived materials were reported. The interactions between the biomolecule and theinorganic material determine the degree to which the biomolecule retains its nativeproperties. The newer technological developments in the field of immobilizedbiocatalysts can offer the possibility of a wider and more economical exploitation ofbiocatalysts in biological applications, food and feed industry, medicine, and in thedevelopment of bioprocess monitoring devices, like the biosensors.The aim of this study was to obtain immobilized enzymatic preparations by methodswhich affect enzyme conformations and kinetic parameters as less as possible. Weimmobilized the enzymatic preparation with protease activity produced by a Bacilluslicheniformis B 40 local strain by physical bonding on ceramics and entrapment into solgel-derived glasses obtained from tetraethyl orthosilicate (TEOS, deposited in thin layeron a ceramic support (entrapment/deposition. Both physically adsorbed andentrapped/deposited enzymes follow Michaelis-Menten kinetics, similar with the solubleenzyme. In the case of immobilized enzymes, the apparent Michaelis constant, Km, wasgreater than that of the native one, as it was expected. The kinetic parameters indicatethat the enzymatic preparations adsorbed on ceramic support and entrapped/depositedshow less affinity for the substrate, Km being 1.3 and 2.1 times higher than that of thenative enzyme, respectively. The maximum velocity increased also by 3.5 and 7.9 timesrespectively, compared with the free counterpart (according to Lineweaver-Burklinearization.

  4. Enhanced enzymatic cellulose degradation by cellobiohydrolases via product removal

    DEFF Research Database (Denmark)

    Ahmadi Gavlighi, Hassan; Meyer, Anne S.; Mikkelsen, Jørn Dalgaard

    2013-01-01

    Product inhibition by cellobiose decreases the rate of enzymatic cellulose degradation. The optimal reaction conditions for two Emericella (Aspergillus) nidulans-derived cellobiohydrolases I and II produced in Pichia pastoris were identified as CBHI: 52 °C, pH 4.5–6.5, and CBHII: 46 °C, pH 4.......8. The optimum in a mixture of the two was 50 °C, pH 4.9. An almost fourfold increase in enzymatic hydrolysis yield was achieved with intermittent product removal of cellobiose with membrane filtration (2 kDa cut-off): The conversion of cotton cellulose after 72 h was ~19 % by weight, whereas the conversion...

  5. From Fed-batch to Continuous Enzymatic Biodiesel Production

    DEFF Research Database (Denmark)

    Price, Jason Anthony; Nordblad, Mathias; Woodley, John M.

    2015-01-01

    In this this paper, we use mechanistic modelling to guide the development of acontinuous enzymatic process that is performed as a fed-batch operation. In this workwe use the enzymatic biodiesel process as a case study. A mechanistic model developedin our previous work was used to determine...... measured components (triglycerides, diglycerides, monoglycerides, free fatty acid and fatty acid methyl esters(biodiesel)) much better than using fed-batch data alone given the smaller residuals. We also observe a reduction in the correlation between the parameters.The model was then used to predict that 5...... reactors are required (with a combined residence time of 30 hours) to reach a final biodiesel concentration within 2 % of the95.6 mass % achieved in a fed-batch operation, for 24 hours....

  6. Modification of chemical reactivity of enzymatic hydrolysis lignin by ultrasound treatment in dilute alkaline solutions.

    Science.gov (United States)

    Ma, Zhuoming; Li, Shujun; Fang, Guizhen; Patil, Nikhil; Yan, Ning

    2016-12-01

    In this study, we have explored various ultrasound treatment conditions for structural modification of enzymatic hydrolysis lignin (EHL) for enhanced chemical reactivity. The key structural modifications were characterized by using a combination of analytical methods, including, Fourier Transform-Infrared spectroscopy (FTIR), Proton Nuclear Magnetic Resonance ( 1 H NMR), Gel permeation chromatography (GPC), X-ray photoelectron spectroscopy (XPS), and Folin-Ciocalteu (F-C) method. Chemical reactivity of the modified EHL samples was determined by both 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity and their reactivity towards formaldehyde. It was observed that the modified EHL had a higher phenolic hydroxyl group content, a lower molecular weight, a higher reactivity towards formaldehyde, and a greater antioxidant property. The higher reactivity demonstrated by the samples after treatment suggesting that ultrasound is a promising method for modifying enzymatic hydrolysis lignin for value-added applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Microstructural study of pre-treated and enzymatic hydrolyzed bamboo

    Directory of Open Access Journals (Sweden)

    Funsho O. KOLAWOLE

    2016-07-01

    Full Text Available Bamboo was used as biomass feedstock which was pre-treated using dilute acid hydrolysis followed by enzymatic hydrolysis. The bamboo was mechanical ground to particle sizes 212–500µm, followed by pre-treatment with dilute sulfuric acid at a concentration of 0.5 and 1.0 (%v/v at temperatures of 25, 110, 120, 150 and 200°C with time intervals of 2 and 4 hours. Pre-hydrolyzate was later analyzed for reducing sugar using UV-Vis spectrophotometry. Under the above conditions, a maximum glucose yield of 153.1 mg/g was obtained at 200°C and acid concentrations of 1% for 4 hours. Water insoluble solids obtained were subsequently hydrolyzed with Celluclast (Trichoderma reesi and β-glucosidase (Novozyme 188 for 72 hours. Optical Microscope and ESEM images of bamboo samples were obtained at various stages of pre-treatment and enzymatic hydrolysis. Result reveals a breakdown in the ligno-cellulosic structure of the bamboo during exposure to dilute acid and enzymatic hydrolysis.

  8. Graphene paper based bioelectrodes for enzymatic biofuel cells

    DEFF Research Database (Denmark)

    Werchmeister, Rebecka Maria Larsen; Shen, Fei; Zhang, Jingdong

    We aim at developing bioelectrodes for enzymatic biofuel cells, where sustainable and renewable enzymes are used for catalyzing the oxidation and reduction of fuel molecules. Here glucose is chosen as fuel molecule and glucose oxidase (GOx) is target enzyme which catalyzes the oxidation of glucose...... of glucose. This indicates that the enzyme has been successfully immobilized and is actively consuming glucose while transferring electrons to the graphene paper-GOx bioanode. Stability and efficiency of the bioelectrodes are under investigation....

  9. Recombinant EXLX1 from Bacillus subtilis for enhancing enzymatic ...

    African Journals Online (AJOL)

    AJL

    2012-06-21

    Jun 21, 2012 ... enhancing enzymatic hydrolysis of corn stover with low cellulase loadings. Zhang Yan1, He Ming-Xiong2,3*, Wu Bo1, ... University, Chengdu 610064, China. 2Biogas Institute of Ministry of Agriculture, Biomass Energy Technology Research Centre, Section 4-13, Renming Nanlu,. Chengdu 610041, China.

  10. Enzymatic biodiesel production: Technical and economical considerations

    DEFF Research Database (Denmark)

    Munk Nielsen, Per; Brask, Jesper; Fjerbæk, Lene

    2008-01-01

    It is well documented in the literature that enzymatic processing of oils and fats for biodiesel is technically feasible. However, with very few exceptions, enzyme technology is not currently used in commercial-scale biodiesel production. This is mainly due to non-optimized process design...... and a lack of available costeffective enzymes. The technology to re-use enzymes has typically proven insufficient for the processes to be competitive. However, literature data documenting the productivity of enzymatic biodiesel together with the development of new immobilization technology indicates...... that enzyme catalysts can become cost effective compared to chemical processing. This work reviews the enzymatic processing of oils and fats into biodiesel with focus on process design and economy....

  11. Radiation pretreatments of cellulose materials for the enhancement of enzymatic hydrolysis

    International Nuclear Information System (INIS)

    Ait Si Mamar, S.; Hadjadj, A.

    1990-01-01

    The conversion of wheat straw agricultural cellulosic wastes to reducing sugars and glucose has been studied by pretreatments by acid hydrolysis and gamma radiolysis over the dose 0-2 MGy. The pretreatment of cellulosic wastes by gamma radiolysis in the presence of sulfuric acid solution shows that the reducing sugars yield increases with the irradiation dose. The effect of radiation degradation on cellulosic wastes between 0.1 MGy and 2 MGy shows the glucose and reducing sugars yields after enzymatic hydrolysis by cellulase vary with the dose. In the relatively low dose range, up to about 0.5 MGy, the reducing sugars yields vary slightly. For an acid hydrolysis followed by radiation at dose range below 0.5 MGy the reducing sugars yields are practically insensitive to radiation. On the other hand, the pretreatment by radiation in higher dose range from 0.5 to 2 MGy followed by enzymatic hydrolysis is effective for the conversion of cellulosic wastes into glucose. The radiation induced degradation of cellulose into glucose depends on the type of acid hydrolysis and on the enzymatic hydrolysis time by cellulase. Pre-irradiation in air is more effective than in acid solution. (author)

  12. Radiation pretreatments of cellulose materials for the enhancement of enzymatic hydrolysis

    Science.gov (United States)

    Mamar, S. Ait Si; Hadjadj, A.

    The conversion of wheat straw agricultural cellulosic wastes to reduning sugars and glucose has been studied by pretreatments by acid hydrolysis and gamma radiolysis over the dose 0-2 MGy. The pretreatment of cellulosic wastes by gamma radiolysis in the presence of sulfuric acid solution shows that the reducing sugars yield increases with the irradiation dose. The effect of radiation degradation on cellulosic wastes between 0.1 MGy and 2 MGy shows the glucose and reducing sugars yields after enzymatic hydrolysis by cellulase vary with the dose. In the relatively low dose range, up to about 0.5 MGy, the reducing sugars yields vary slightly. For an acid hydrolysis followed by radiation at dose range below 0.5 MGy the reducing sugars yields are practically insensitive to radiation. On the other hand, the pretreatment by radiation in higher dose range from 0.5 to 2 MGy followed by enzymatic hydrolysis is effective for the conversion of cellulosic wastes into glucose. The radiation induced degradation of cellulose into glucose depends on the type of acid hydrolysis and on the enzymatic hydrolysis time by cellulase. Pre-irradiation in air is more effective than in acid solution.

  13. BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates.

    Science.gov (United States)

    Yang, Bin; Wyman, Charles E

    2006-07-05

    Cellulase and bovine serum albumin (BSA) were added to Avicel cellulose and solids containing 56% cellulose and 28% lignin from dilute sulfuric acid pretreatment of corn stover. Little BSA was adsorbed on Avicel cellulose, while pretreated corn stover solids adsorbed considerable amounts of this protein. On the other hand, cellulase was highly adsorbed on both substrates. Adding a 1% concentration of BSA to dilute acid pretreated corn stover prior to enzyme addition at 15 FPU/g cellulose enhanced filter paper activity in solution by about a factor of 2 and beta-glucosidase activity in solution by about a factor of 14. Overall, these results suggested that BSA treatment reduced adsorption of cellulase and particularly beta-glucosidase on lignin. Of particular note, BSA treatment of pretreated corn stover solids prior to enzymatic hydrolysis increased 72 h glucose yields from about 82% to about 92% at a cellulase loading of 15 FPU/g cellulose or achieved about the same yield at a loading of 7.5 FPU/g cellulose. Similar improvements were also observed for enzymatic hydrolysis of ammonia fiber explosion (AFEX) pretreated corn stover and Douglas fir treated by SO(2) steam explosion and for simultaneous saccharification and fermentation (SSF) of BSA pretreated corn stover. In addition, BSA treatment prior to hydrolysis reduced the need for beta-glucosidase supplementation of SSF. The results are consistent with non-specific competitive, irreversible adsorption of BSA on lignin and identify promising strategies to reduce enzyme requirements for cellulose hydrolysis. (c) 2006 Wiley Periodicals, Inc.

  14. Enzymatic biodiesel synthesis. Key factors affecting efficiency of the process

    Energy Technology Data Exchange (ETDEWEB)

    Szczesna Antczak, Miroslawa; Kubiak, Aneta; Antczak, Tadeusz; Bielecki, Stanislaw [Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Stefanowskiego 4/10, 90-924 Lodz (Poland)

    2009-05-15

    Chemical processes of biodiesel production are energy-consuming and generate undesirable by-products such as soaps and polymeric pigments that retard separation of pure methyl or ethyl esters of fatty acids from glycerol and di- and monoacylglycerols. Enzymatic, lipase-catalyzed biodiesel synthesis has no such drawbacks. Comprehension of the latter process and an appreciable progress in production of robust preparations of lipases may soon result in the replacement of chemical catalysts with enzymes in biodiesel synthesis. Engineering of enzymatic biodiesel synthesis processes requires optimization of such factors as: molar ratio of substrates (triacylglycerols: alcohol), temperature, type of organic solvent (if any) and water activity. All of them are correlated with properties of lipase preparation. This paper reports on the interplay between the crucial parameters of the lipase-catalyzed reactions carried out in non-aqueous systems and the yield of biodiesel synthesis. (author)

  15. Comparison of enzymatic activities in different Candida species isolated from women with vulvovaginitis.

    Science.gov (United States)

    Fatahinia, M; Halvaeezadeh, M; Rezaei-Matehkolaei, A

    2017-06-01

    Comparing the activities of secreted enzymes in different fungal species can improve our understanding of their pathogenic role. Secretion of various enzymes by Candida species has been considered for determination of their virulence in different Candida infections including vulvovaginitis. The aim of this study was to determine and compare the activity of secreted enzymes in Candidia strains isolated from women suspected to vulvovaginal candidiasis (VVC) and referred to some health centers in Khuzestan, Southwestern Iran. The vaginal secretion samples were taken by swap from 250 suspected women with symptoms of vulvovaginal candidiasis and cultured on CHROMagar Candida medium. Identification of the isolated Candida from culture positive samples performed by the color of colonies and some standard mycological procedures. Activities of phospholipase, hemolysin-α, hemolysin-β, esterase and proteinase were measured in vitro by standard laboratory protocols. The enzymatic activity index (EAI) was calculated for each enzyme in accordance to relevant protocols. Totally in eighty cases (32%), a Candida strain was isolated which found to be as 52 (65%) Candida albicans; 12 (15%) C. glabrata; 10 (12.5%) C. dubliniensis; 4 (5%) C. krusei, C. tropicalis and C. parapsilosis species (each=1; 1.3%). Among C. albicans strains, 89.1% produced all studied enzymes, while 86% of C. glabrata strains failed to produce proteinase and phospholipase. The EAIs in decreasing order were as hemolysin-β=0.2895, hemolysin-α=0.5420, esterase=0.5753, proteinase=0.7413, and phospholipase=0.7446, respectively. Activity of phospholipase, esterase and proteinase secreted by C. albicans and C. dubliniensis were significantly more than those released by C. glabrata and C. krusei, while 86% of C. glabrata strains did not show esterase activity. On the other hand, the activity rates of hemolysin α and β among all studied isolates were almost similar. In the present study, the prevalence

  16. Preclinical Evidence of Anti-Tumor Activity Induced by EZH2 Inhibition in Human Models of Synovial Sarcoma.

    Directory of Open Access Journals (Sweden)

    Satoshi Kawano

    Full Text Available The catalytic activities of covalent and ATP-dependent chromatin remodeling are central to regulating the conformational state of chromatin and the resultant transcriptional output. The enzymes that catalyze these activities are often contained within multiprotein complexes in nature. Two such multiprotein complexes, the polycomb repressive complex 2 (PRC2 methyltransferase and the SWItch/Sucrose Non-Fermentable (SWI/SNF chromatin remodeler have been reported to act in opposition to each other during development and homeostasis. An imbalance in their activities induced by mutations/deletions in complex members (e.g. SMARCB1 has been suggested to be a pathogenic mechanism in certain human cancers. Here we show that preclinical models of synovial sarcoma-a cancer characterized by functional SMARCB1 loss via its displacement from the SWI/SNF complex through the pathognomonic SS18-SSX fusion protein-display sensitivity to pharmacologic inhibition of EZH2, the catalytic subunit of PRC2. Treatment with tazemetostat, a clinical-stage, selective and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity reverses a subset of synovial sarcoma gene expression and results in concentration-dependent cell growth inhibition and cell death specifically in SS18-SSX fusion-positive cells in vitro. Treatment of mice bearing either a cell line or two patient-derived xenograft models of synovial sarcoma leads to dose-dependent tumor growth inhibition with correlative inhibition of trimethylation levels of the EZH2-specific substrate, lysine 27 on histone H3. These data demonstrate a dependency of SS18-SSX-positive, SMARCB1-deficient synovial sarcomas on EZH2 enzymatic activity and suggests the potential utility of EZH2-targeted drugs in these genetically defined cancers.

  17. Enzymatic modification of egg lecithin to improve properties.

    Science.gov (United States)

    Asomaning, Justice; Curtis, Jonathan M

    2017-04-01

    This research studied the enzymatic modification of egg yolk phospholipids and its effect on physicochemical properties. Egg yolk lipids were extracted with food grade ethanol and egg phospholipids (ePL) produced by deoiling with acetone. Vegetable oils were used to interesterify ePL utilizing Lipozyme®: sn-1,3 specific lipase. The enzymatic interesterification resulted in a single phase liquid product, whereas simple blending of the ePL and vegetable oil resulted in a product with two phases. In addition solid fat content decreased by 50% at -10°C and 94% at 35°C when compared with egg yolk lipids extract. A decrease in melting temperature resulted from the interesterification process. Interesterification improved emulsion stability index when used as an emulsifier in oil-in-water emulsion and compared to the native and soy lecithin. Enzyme reusability test showed retention of 63% activity after 10 cycles. Overall, the properties of native egg phospholipids were significantly enhanced in a potentially useful manner through interesterification. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Sol-Gel Derived Adsorbents with Enzymatic and Complexonate Functions for Complex Water Remediation

    Directory of Open Access Journals (Sweden)

    Roman P. Pogorilyi

    2017-09-01

    Full Text Available Sol-gel technology is a versatile tool for preparation of complex silica-based materials with targeting functions for use as adsorbents in water purification. Most efficient removal of organic pollutants is achieved by using enzymatic reagents grafted on nano-carriers. However, enzymes are easily deactivated in the presence of heavy metal cations. In this work, we avoided inactivation of immobilized urease by Cu (II and Cd (II ions using magnetic nanoparticles provided with additional complexonate (diethylene triamine pentaacetic acid or DTPA functions. Obtained nanomaterials were characterized by Fourier transform infrared spectroscopy (FTIR, thermogravimetric analysis (TGA, and scanning electron microscopy (SEM. According to TGA, the obtained Fe3O4/SiO2-NH2-DTPA nanoadsorbents contained up to 0.401 mmol/g of DTPA groups. In the concentration range Ceq = 0–50 mmol/L, maximum adsorption capacities towards Cu (II and Cd (II ions were 1.1 mmol/g and 1.7 mmol/g, respectively. Langmuir adsorption model fits experimental data in concentration range Ceq = 0–10 mmol/L. The adsorption mechanisms have been evaluated for both of cations. Crosslinking of 5 wt % of immobilized urease with glutaraldehyde prevented the loss of the enzyme in repeated use of the adsorbent and improved the stability of the enzymatic function leading to unchanged activity in at least 18 cycles. Crosslinking of 10 wt % urease on the surface of the particles allowed a decrease in urea concentration in 20 mmol/L model solutions to 2 mmol/L in up to 10 consequent decomposition cycles. Due to the presence of DTPA groups, Cu2+ ions in concentration 1 µmol/L did not significantly affect the urease activity. Obtained magnetic Fe3O4/SiO2-NH2-DTPA-Urease nanocomposite sorbents revealed a high potential for urease decomposition, even in presence of heavy metal ions.

  19. Enzymatic generation of hydrogen peroxide shows promising antifouling effect

    DEFF Research Database (Denmark)

    Kristensen, J.B.; Olsen, Stefan Møller; Laursen, B.S.

    2010-01-01

    Proteobacteria, tested in microtiter plates. However, enzymatically produced H2O2 released from a coating did not impede biofilm formation by bacteria in natural seawater tested in a biofilm reactor. A field trial revealed a noticeable effect of the enzyme system: after immersion in the North Sea for 97 days...

  20. Warming and organic matter sources impact the proportion of dissolved to total activities in marine extracellular enzymatic rates

    KAUST Repository

    Baltar, Federico

    2017-04-19

    Extracellular enzymatic activities (EEAs) are the rate-limiting step in the degradation of organic matter. Extracellular enzymes can be found associated to cells or dissolved in the surrounding water. The proportion of cell-free EEA constitutes in many marine environments more than half of the total activity. This high proportion causes an uncoupling between hydrolysis rates and the actual bacterial activity. However, we do not know what factors control the proportion of dissolved relative to total EEA, nor how this may change in the future ocean. To resolve this, we performed laboratory experiments with water from the Great Barrier Reef (Australia) to study the effects of temperature and dissolved organic matter sources on EEA and the proportion of dissolved EEA. We found that warming increases the rates of organic matter hydrolysis and reduces the proportion of dissolved relative to total EEA. This suggests a potential increase of the coupling between organic matter hydrolysis and heterotrophic activities with increasing ocean temperatures, although strongly dependent on the organic matter substrates available. Our study suggests that local differences in the organic matter composition in tropical coastal ecosystems will strongly affect the proportion of dissolved EEA in response to ocean warming.

  1. CuO nanoparticles supported on nitrogen and sulfur co-doped graphene nanocomposites for non-enzymatic glucose sensing

    Energy Technology Data Exchange (ETDEWEB)

    Li, Meixia [Hebei University of Engineering, Faculty of Material Science and Engineering (China); Guo, Qingbin, E-mail: guoqingbinhue@163.com [Hebei University of Engineering, Academic Affairs office (China); Xie, Juan; Li, Yongde; Feng, Yapeng [Hebei University of Engineering, Faculty of Material Science and Engineering (China)

    2017-01-15

    Developing highly active catalysts to promote the electrocatalytic glucose oxidation (EGO) is a crucial demand for non-enzymatic glucose sensing. Herein, we reported the use of nitrogen and sulfur co-doped graphene (NSG) as a novel support material for anchoring CuO nanoparticles and obtained CuO/NSG was employed as an efficient EGO catalyst for non-enzymatic glucose sensing. The results showed that the NSG endowed the CuO/NSG with large surface area, increased structural defects, improved conductivity, and strong covalent coupling between NSG and CuO. Owing to the significant contribution of NSG and the synergistic effect of NSG and CuO, the CuO/NSG exhibited a remarkably higher EGO activity than CuO and CuO/reduced graphene oxide. The CuO/NSG-based sensor displayed excellent glucose sensing performances with a considerably low detection limit of 0.07 μM. These findings elucidate that the NSG is a promising support material for non-enzymatic glucose detection.

  2. Effect of alkali lignins with different molecular weights from alkali pretreated rice straw hydrolyzate on enzymatic hydrolysis.

    Science.gov (United States)

    Li, Yun; Qi, Benkun; Luo, Jianquan; Wan, Yinhua

    2016-01-01

    This study investigated the effect of alkali lignins with different molecular weights on enzymatic hydrolysis of lignocellulose. Different alkali lignins fractions, which were obtained from cascade ultrafiltration, were added into the dilute acid pretreated (DAP) and alkali pretreated (AP) rice straws respectively during enzymatic hydrolysis. The results showed that the addition of alkali lignins enhanced the hydrolysis and the enhancement for hydrolysis increased with increasing molecular weights of alkali lignins, with maximum enhancement being 28.69% for DAP and 20.05% for AP, respectively. The enhancement was partly attributed to the improved cellulase activity, and filter paper activity increased by 18.03% when adding lignin with highest molecular weight. It was found that the enhancement of enzymatic hydrolysis was correlated with the adsorption affinity of cellulase on alkali lignins, and the difference in surface charge and hydrophobicity of alkali lignins were responsible for the difference in affinity between cellulase and lignins. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Enzymatic Inverse Opal Hydrogel Particles for Biocatalyst.

    Science.gov (United States)

    Wang, Huan; Gu, Hongcheng; Chen, Zhuoyue; Shang, Luoran; Zhao, Ze; Gu, Zhongze; Zhao, Yuanjin

    2017-04-19

    Enzymatic carriers have a demonstrated value for chemical reactions and industrial applications. Here, we present a novel kind of inverse opal hydrogel particles as the enzymatic carriers. The particles were negatively replicated from spherical colloidal crystal templates by using magnetic nanoparticles tagged acrylamide hydrogel. Thus, they were endowed with the features of monodispersity, small volume, complete penetrating structure, and controllable motion, which are all beneficial for improving the efficiency of biocatalysis. In addition, due to the ordered porous nanostructure, the inverse opal hydrogel particles were imparted with unique photonic band gaps (PBGs) and vivid structural colors for encoding varieties of immobilized enzymes and for constructing a multienzymes biocatalysis system. These features of the inverse opal hydrogel particles indicate that they are ideal enzymatic carriers for biocatalysis.

  4. Chemical and enzymatic stability of amino acid prodrugs containing methoxy, ethoxy and propylene glycol linkers.

    Science.gov (United States)

    Gupta, Deepak; Gupta, Sheeba Varghese; Lee, Kyung-Dall; Amidon, Gordon L

    2009-01-01

    We evaluated the chemical and enzymatic stabilities of prodrugs containing methoxy, ethoxy and propylene glycol linkers in order to find a suitable linker for prodrugs of carboxylic acids with amino acids. l-Valine and l-phenylalanine prodrugs of model compounds (benzoic acid and phenyl acetic acid) containing methoxy, ethoxy and propylene glycol linkers were synthesized. The hydrolysis rate profile of each compound was studied at physiologically relevant pHs (1.2, 4, 6 and 7.4). Enzymatic hydrolysis of propylene glycol containing compounds was studied using Caco-2 homogenate as well as purified enzyme valacyclovirase. It was observed that the stability of the prodrugs increases with the linker length (propyl > ethyl > methyl). The model prodrugs were stable at acidic pH as compared to basic pH. It was observed that the prodrug with the aliphatic amino acid promoiety was more stable compared to its aromatic counterpart. The comparison between benzyl and the phenyl model compounds revealed that the amino acid side chain is significant in determining the stability of the prodrug whereas the benzyl or phenyl carboxylic acid had little or no effect on the stability. The enzymatic activation studies of propylene glycol linker prodrug in the presence of valacyclovirase and cell homogenate showed faster generation of the parent drug at pH 7.4. The half-life of prodrugs at pH 7.4 was more than 12 h, whereas in the presence of cell homogenate the half-lives were less than 1 h. Hydrolysis by Caco-2 homogenate generated the parent compound in two steps, where the prodrug was first converted to the intermediate, propylene glycol benzoate, which was then converted to the parent compound (benzoic acid). Enzymatic hydrolysis of propylene glycol containing prodrugs by valacyclovirase showed hydrolysis of the amino acid ester part to generate the propylene glycol ester of model compound (propylene glycol benzoate) as the major product. The amino acid prodrugs containing methoxy

  5. A green and efficient technology for the degradation of cellulosic materials: structure changes and enhanced enzymatic hydrolysis of natural cellulose pretreated by synergistic interaction of mechanical activation and metal salt.

    Science.gov (United States)

    Zhang, Yanjuan; Li, Qian; Su, Jianmei; Lin, Ye; Huang, Zuqiang; Lu, Yinghua; Sun, Guosong; Yang, Mei; Huang, Aimin; Hu, Huayu; Zhu, Yuanqin

    2015-02-01

    A new technology for the pretreatment of natural cellulose was developed, which combined mechanical activation (MA) and metal salt treatments in a stirring ball mill. Different valent metal nitrates were used to investigate the changes in degree of polymerization (DP) and crystallinity index (CrI) of cellulose after MA+metal salt (MAMS) pretreatment, and Al(NO3)3 showed better pretreatment effect than NaNO3 and Zn(NO3)2. The destruction of morphological structure of cellulose was mainly resulted from intense ball milling, and the comparative studies on the changes of DP and crystal structure of MA and MA+Al(NO3)3 pretreated cellulose samples showed a synergistic interaction of MA and Al(NO3)3 treatments with more effective changes of structural characteristics of MA+Al(NO3)3 pretreated cellulose and substantial increase of reducing sugar yield in enzymatic hydrolysis of cellulose. In addition, the results indicated that the presence of Al(NO3)3 had significant enhancement for the enzymatic hydrolysis of cellulose. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. The inhibition of the mitochondrial F1FO-ATPase activity when activated by Ca2+ opens new regulatory roles for NAD.

    Science.gov (United States)

    Nesci, Salvatore; Trombetti, Fabiana; Ventrella, Vittoria; Pirini, Maurizio; Pagliarani, Alessandra

    2018-01-26

    The mitochondrial F1FO-ATPase is uncompetitively inhibited by NAD+ only when the natural cofactor Mg2+ is replaced by Ca2+, a mode putatively involved in cell death. The Ca2+-dependent F1FO-ATPase is also inhibited when NAD+ concentration in mitochondria is raised by acetoacetate. The enzyme inhibition by NAD+ cannot be ascribed to any de-ac(et)ylation or ADP-ribosylation by sirtuines, as it is not reversed by nicotinamide. Moreover, the addition of acetyl-CoA or palmitate, which would favor the enzyme ac(et)ylation, does not affect the F1FO-ATPase activity. Consistently, NAD+ may play a new role, not associated with redox and non-redox enzymatic reactions, in the Ca2+-dependent regulation of the F1FO-ATPase activity.

  7. Electrodeposition of flower-like platinum on electrophoretically grown nitrogen-doped graphene as a highly sensitive electrochemical non-enzymatic biosensor for hydrogen peroxide detection

    International Nuclear Information System (INIS)

    Tajabadi, M.T.; Sookhakian, M.; Zalnezhad, E.; Yoon, G.H.; Hamouda, A.M.S.; Azarang, Majid; Basirun, W.J.; Alias, Y.

    2016-01-01

    Highlights: • Nitrogen doped graphene with different thickness by electrophoretic deposition. • The conductivity of N-graphene layer depends on the tickness. • Support of platinum shows efficient electrocatalytic performance for biosensor. • CV curves and amperometric responses improved and optimized in the presence of N-graphene. - Abstract: An efficient non-enzymatic biosensor electrode consisting of nitrogen-doped graphene (N-graphene) and platinum nanoflower (Pt NF) with different N-graphene loadings were fabricated on indium tin oxide (ITO) glass using a simple layer-by-layer electrophoretic and electrochemical sequential deposition approach. N-graphene was synthesized by annealing graphene oxide with urea at 900 °C. The structure and morphology of the as-fabricated non-enzymatic biosensor electrodes were determined using X-ray diffraction, field emission electron microscopy, transmission electron microscopy, Raman and X-ray photoelectron spectra. The as-fabricated Pt NF-N-graphene-modified ITO electrodes with different N-graphene loadings were utilized as a non-enzymatic biosensor electrode for the detection of hydrogen peroxide (H_2O_2). The behaviors of the hybrid electrodes towards H_2O_2 reduction were assessed using chronoamperometry, cyclic voltammetry and electrochemical impedance spectroscopy analysis. The Pt NF-N-graphene-modified ITO electrode with a 0.05 mg ml"−"1 N-graphene loading exhibited the lowest detection limit, fastest amperometric sensing, a wide linear response range, excellent stability and reproducibility for the non-enzymatic H_2O_2 detection, due to the synergistic effect between the electrocatalytic activity of the Pt NF and the high conductivity and large surface area of N-graphene.

  8. Active site studies of Escherichia coli 2-keto-4-hydroxyglutarate aldolase

    International Nuclear Information System (INIS)

    Vlahos, C.J.

    1987-01-01

    The data presented delineate the complete amino acid sequence of E. coli KHG aldolase and also identify Lys-133, Glu-45, and Arg-49 as aminoacyl residues required for catalytic activity. Incubation of E. coli KHG aldolase with [ 14 C]pyruvate in the presence of NaCNBH 3 results in the incorporation of one mol of 14 C per mol of enzyme subunit. Digestion of this enzyme-adduct with trypsin, followed by purification of the peptides, allowed for the isolation of a unique radioactive peptide. Its amino acid sequence showed that the pyruvate-binding (i.e., Schiff-base forming) lysine residue is located at position 133 in the intact enzyme. E. coli KHG aldolase activity is lost when the enzyme is reacted with bromopyruvate; saturation kinetics are observed. The substrates, pyruvate and KHG, protect the enzyme from inactivation. Both facts suggest that the reagent is active-site specific. Incubation of the aldolase with [3- 14 C]bromopyruvate is associated with a concomitant loss of enzymatic activity and esterification of Glu-45; if the enzyme is denatured in the presence of excess bromopyruvate, Cys-159 and Cys-180 are also alkylated. Blocking the active-site lysine residue with pyruvate prevents Glu-45 from being esterified but does not eliminate alkylation of these two cysteine residues. Woodward's Reagent K was also found to inactivate the aldolase under conditions that are usually specific for carboxyl group modification. This aldolase is also inactivated by 1,2-cyclohexanedione. Loss of enzymatic activity occurs concomitantly with modification of one arginine residue per enzyme subunit. Treatment of the aldolase with the arginine-specific reagent, 4-(oxyacetyl)phenoxyacetic acid, followed by digestion with trypsin allowed for the isolation of a unique peptide and the identification of Arg-49 as the specific residue involved

  9. Characterization of Amino Acid Profile and Enzymatic Activity in Adult Rat Astrocyte Cultures.

    Science.gov (United States)

    Souza, Débora Guerini; Bellaver, Bruna; Hansel, Gisele; Arús, Bernardo Assein; Bellaver, Gabriela; Longoni, Aline; Kolling, Janaina; Wyse, Angela T S; Souza, Diogo Onofre; Quincozes-Santos, André

    2016-07-01

    Astrocytes are multitasking players in brain complexity, possessing several receptors and mechanisms to detect, participate and modulate neuronal communication. The functionality of astrocytes has been mainly unraveled through the study of primary astrocyte cultures, and recently our research group characterized a model of astrocyte cultures derived from adult Wistar rats. We, herein, aim to characterize other basal functions of these cells to explore the potential of this model for studying the adult brain. To characterize the astrocytic phenotype, we determined the presence of GFAP, GLAST and GLT 1 proteins in cells by immunofluorescence. Next, we determined the concentrations of thirteen amino acids, ATP, ADP, adenosine and calcium in astrocyte cultures, as well as the activities of Na(+)/K(+)-ATPase and acetylcholine esterase. Furthermore, we assessed the presence of the GABA transporter 1 (GAT 1) and cannabinoid receptor 1 (CB 1) in the astrocytes. Cells demonstrated the presence of glutamine, consistent with their role in the glutamate-glutamine cycle, as well as glutamate and D-serine, amino acids classically known to act as gliotransmitters. ATP was produced and released by the cells and ADP was consumed. Calcium levels were in agreement with those reported in the literature, as were the enzymatic activities measured. The presence of GAT 1 was detected, but the presence of CB 1 was not, suggesting a decreased neuroprotective capacity in adult astrocytes under in vitro conditions. Taken together, our results show cellular functionality regarding the astrocytic role in gliotransmission and neurotransmitter management since they are able to produce and release gliotransmitters and to modulate the cholinergic and GABAergic systems.

  10. Enzymatic Synthesis of Psilocybin.

    Science.gov (United States)

    Fricke, Janis; Blei, Felix; Hoffmeister, Dirk

    2017-09-25

    Psilocybin is the psychotropic tryptamine-derived natural product of Psilocybe carpophores, the so-called "magic mushrooms". Although its structure has been known for 60 years, the enzymatic basis of its biosynthesis has remained obscure. We characterized four psilocybin biosynthesis enzymes, namely i) PsiD, which represents a new class of fungal l-tryptophan decarboxylases, ii) PsiK, which catalyzes the phosphotransfer step, iii) the methyltransferase PsiM, catalyzing iterative N-methyl transfer as the terminal biosynthetic step, and iv) PsiH, a monooxygenase. In a combined PsiD/PsiK/PsiM reaction, psilocybin was synthesized enzymatically in a step-economic route from 4-hydroxy-l-tryptophan. Given the renewed pharmaceutical interest in psilocybin, our results may lay the foundation for its biotechnological production. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Enzymatic determination of rare earth elements by use of pyrophosphotases

    International Nuclear Information System (INIS)

    Shekhovtseva, T.N.; Pirogova, S.V.; Fedorova, O.M.; Dolmanova, I.F.; Bajkov, A.A.

    1993-01-01

    A highly sensitive (determination limit 8 x 10 -6 - 4 x 10 -4 μg/ml) and selective enzymatic method for determination of rare earth elements has been developed. The method is based on inhibition action of rare earths on the catalytic activity of pyrophosphates isolated from bakery geast and E. Coli. The mechanism of the rare earth element action, corresponding to competitive inhibition, has been established

  12. Operation and Control of Enzymatic Biodiesel Production

    DEFF Research Database (Denmark)

    Price, Jason Anthony; Huusom, Jakob Kjøbsted; Nordblad, Mathias

    This work explores the control of biodiesel production via an enzymatic catalyst. The process involves the transesterification of oils/fats with an alcohol (usually methanol or ethanol), using enzymatic catalysts to generate mono-alkyl esters (the basis of biodiesel) and glycerol as by......-product. Current literature indicates that enzymatic processing of oils and fats to produce biodiesel is technically feasible and developments in immobilization technology indicate that enzyme catalysts can become cost effective compared to chemical processing. However, with very few exceptions, enzyme technology...... is not currently used in commercial-scale biodiesel production. This is mainly due to non-optimized process designs, which do not use the full potential of the catalysts in a cost-efficient way. Furthermore is it unclear what process variables need to be monitored and controlled to ensure optimal economics...

  13. Colorimetric determination of nitrate plus nitrite in water by enzymatic reduction, automated discrete analyzer methods

    Science.gov (United States)

    Patton, Charles J.; Kryskalla, Jennifer R.

    2011-01-01

    This report documents work at the U.S. Geological Survey (USGS) National Water Quality Laboratory (NWQL) to validate enzymatic reduction, colorimetric determinative methods for nitrate + nitrite in filtered water by automated discrete analysis. In these standard- and low-level methods (USGS I-2547-11 and I-2548-11), nitrate is reduced to nitrite with nontoxic, soluble nitrate reductase rather than toxic, granular, copperized cadmium used in the longstanding USGS automated continuous-flow analyzer methods I-2545-90 (NWQL laboratory code 1975) and I-2546-91 (NWQL laboratory code 1979). Colorimetric reagents used to determine resulting nitrite in aforementioned enzymatic- and cadmium-reduction methods are identical. The enzyme used in these discrete analyzer methods, designated AtNaR2 by its manufacturer, is produced by recombinant expression of the nitrate reductase gene from wall cress (Arabidopsis thaliana) in the yeast Pichia pastoris. Unlike other commercially available nitrate reductases we evaluated, AtNaR2 maintains high activity at 37°C and is not inhibited by high-phenolic-content humic acids at reaction temperatures in the range of 20°C to 37°C. These previously unrecognized AtNaR2 characteristics are essential for successful performance of discrete analyzer nitrate + nitrite assays (henceforth, DA-AtNaR2) described here.

  14. Similar potential ATP-P production and enzymatic activities in the microplankton community off Concepción (Chile) under oxic and suboxic conditions

    Science.gov (United States)

    González, Rodrigo R.; Gutiérrez, Marcelo H.; Quiñones, Renato A.

    2007-11-01

    The effects of the oxygen minimum zone on the metabolism of the heterotrophic microplankton community (0.22-100 μm) in the Humboldt Current System, as well as the factors controlling its biomass production, remain unknown. Here we compare the effect of four sources of dissolved organic carbon (glucose, oxaloacetate, glycine, leucine) on microbial biomass production (such as ATP-P) and the potential enzymatic activities involved in catabolic pathways under oxic and suboxic conditions. Our results show significant differences ( p oxygen minimum zone has the same or greater potential growth than the community inhabiting more oxygenated strata of the water column and that malate dehydrogenase is the activity that best represents the metabolic potential of the community.

  15. Process Evaluation Tools for Enzymatic Cascades Welcome Message

    DEFF Research Database (Denmark)

    Abu, Rohana

    improvement and implementation. Hence, the goal of this thesis is to evaluate the process concepts in enzymatic cascades in a systematic manner, using tools such as thermodynamic and kinetic analysis. Three relevant case studies have been used to exemplify the approach. In the first case study, thermodynamic......Biocatalysis is attracting significant attention from both academic and industrial scientists due to the excellent capability of enzyme to catalyse selective reactions. Recently, much interest has been shown in the application of enzymatic cascades as a useful tool in organic synthesis......, the kinetics can be controlled in a highly efficient way to achieve a sufficiently favourable conversion to a given target product. This is exemplified in the second case study, in the kinetic modelling of the formation of 2-ketoglutarate from glucoronate, the second case study. This cascade consists of 4...

  16. Glucose obtained from rice bran by ultrasound-assisted enzymatic hydrolysis

    Directory of Open Access Journals (Sweden)

    Raquel Cristine Kuhn

    2015-05-01

    Full Text Available In this work ultrasound-assisted solid-state enzymatic hydrolysis of rice bran to obtain fermentable sugars was investigated. For this purpose, process variables such as temperature, enzyme concentration and moisture content were evaluated during the enzymatic hydrolysis with and without ultrasound irradiation. The enzyme used is a blend of amylases derived from genetically modified strains of Trichoderma reesei. Kinetic of the enzymatic hydrolysis of rice bran at the constant-reaction rate period were measured. The best results for the ultrasound-assisted enzymatic hydrolysis was obtained using 3 wt% of enzyme, 60 oC and moisture content of 65 wt%, yielding 0.38 g sugar/g rice bran, whereas for the hydrolysis in the absence of ultrasound the highest yield was 0.20 g sugar/g rice bran using 3 wt% of enzyme, 60 oC and moisture content of 50 wt%. The use of ultrasound-assisted enzymatic hydrolysis of rice bran was intensified, obtaining around 74% more fermentable sugar than in the absence, showing that the use of ultrasound is a promising technology to be used in enzymatic reaction as an alternative of process intensification.

  17. Phospholipase A2 is involved in galactosylsphingosine-induced astrocyte toxicity, neuronal damage and demyelination.

    Directory of Open Access Journals (Sweden)

    Cedric Misslin

    Full Text Available Krabbe disease is a fatal rare inherited lipid storage disorder affecting 1:100,000 births. This illness is caused by mutations in the galc gene encoding for the enzyme galactosylceramidase (GALC. Dysfunction of GALC has been linked to the toxic build-up of the galactolipid, galactosylsphingosine (psychosine, which induces cell death of oligodendrocytes. Previous studies show that phospholipase A2 (PLA2 may play a role in psychosine induce cell death. Here, we demonstrate that non-selective inhibition of cPLA2/sPLA2 and selective inhibition of cPLA2, but not sPLA2, also attenuates psychosine-induced cell death of human astrocytes. This study shows that extracellular calcium is required for psychosine induced cell death, but intracellular calcium release, reactive oxygen species or release of soluble factors are not involved. These findings suggest a cell autonomous effect, at least in human astrocytes. Supporting a role for PLA2 in psychosine-induced cell death of oligodendrocytes and astrocytes, the results show inhibition of PLA2 attenuates psychosine-induced decrease in the expression of astrocyte marker vimentin as well as myelin basic protein (MBP, myelin oligodendrocyte glycoprotein (MOG and the neuronal marker SMI-32 in organotypic slice cultures. These findings provide further mechanistic details of psychosine-induced death of glia and suggest a role for PLA2 in the process. This work also supports the proposal that novel drugs for Krabbe disease may require testing on astrocytes as well as oligodendrocytes for more holistic prediction of pre-clinical and clinical efficacy.

  18. Pregnancy Exercise Increase Enzymatic Antioxidant In Pregnant Women

    Directory of Open Access Journals (Sweden)

    Wagey Freddy Wagey

    2012-01-01

    Full Text Available Objectives: Pregnancy is a vulnerable condition to all kinds of "stress", resulting in changes of physiological and metabolic functions. This research aims to determine effect of exercise during pregnancy in increasing enzymatic antioxidant marked by increase of superoxide dismutase (SOD, gluthation peroxidase (GSHPx, and catalase (CAT levels. Methods: Randomized pre and posttest control group design was employed in this study. A number of 66 pregnant women were recruited in this study and grouped into two groups, i.e 30 of them as control group and the rest as treatment group. Pregnancy exercise was performed to all 36 pregnant women from 20 weeks gestation on treatment group. The exercise was performed in the morning for about 30 minutes, twice a weeks. On the other hand, daily activities was sugested for control group. Student’s t-test was then applied to determine the mean different of treatment and control group with 5 % of significant value. Results: This study reveals that there were significantly higher increase of (superoxide dismutase (SOD, gluthation peroxidase (GSHPx, and catalse (CAT levels of treatment group compare to control group. These enzymatic antioxidant increase among these two group were around 1.36 mg/gHb for SOD; 1.14 IU/gHb for GSHPx; and 0.97 IU/gHb for CAT, (p < 0.05.  Clinical outcomes, such as strengten of pelvic muscle and quality of life of treatment group were significantly better compared to control group (p < 0.05. Conclusions: This means that exercise during pregnancy ages of 20 weeks increase enzymatic antioxidant levels SOD, GSHPx, and CAT higher compare to control group without exercise.  

  19. Pregnancy Exercise Increase Enzymatic Antioxidant In Pregnant Women

    Directory of Open Access Journals (Sweden)

    Wagey Freddy Wagey

    2012-01-01

    Full Text Available Objectives: Pregnancy is a vulnerable condition to all kinds of "stress", resulting in changes of physiological and metabolic functions. This research aims to determine effect of exercise during pregnancy in increasing enzymatic antioxidant marked by increase of superoxide dismutase (SOD, gluthation peroxidase (GSHPx, and catalase (CAT levels. Methods: Randomized pre and posttest control group design was employed in this study. A number of 66 pregnant women were recruited in this study and grouped into two groups, i.e 30 of them as control group and the rest as treatment group. Pregnancy exercise was performed to all 36 pregnant women from 20 weeks gestation on treatment group. The exercise was performed in the morning for about 30 minutes, twice a weeks. On the other hand, daily activities was sugested for control group. Student’s t-test was then applied to determine the mean different of treatment and control group with 5 % of significant value. Results: This study reveals that there were significantly higher increase of (superoxide dismutase (SOD, gluthation peroxidase (GSHPx, and catalse (CAT levels of treatment group compare to control group. These enzymatic antioxidant increase among these two group were around 1.36 mg/gHb for SOD; 1.14 IU/gHb for GSHPx; and 0.97 IU/gHb for CAT, (p < 0.05. Clinical outcomes, such as strengten of pelvic muscle and quality of life of treatment group were significantly better compared to control group (p < 0.05. Conclusions: This means that exercise during pregnancy ages of 20 weeks increase enzymatic antioxidant levels SOD, GSHPx, and CAT higher compare to control group without exercise.

  20. Effect of lignin chemistry on the enzymatic hydrolysis of woody biomass.

    Science.gov (United States)

    Yu, Zhiying; Gwak, Ki-Seob; Treasure, Trevor; Jameel, Hasan; Chang, Hou-min; Park, Sunkyu

    2014-07-01

    The impact of lignin-derived inhibition on enzymatic hydrolysis is investigated by using lignins isolated from untreated woods and pretreated wood pulps. A new method, biomass reconstruction, for which isolated lignins are precipitated onto bleached pulps to mimic lignocellulosic biomass, is introduced, for the first time, to decouple the lignin distribution issue from lignin chemistry. Isolated lignins are physically mixed and reconstructed with bleached pulps. Lignins obtained from pretreated woods adsorb two to six times more cellulase than lignins obtained from untreated woods. The higher adsorption of enzymes on lignin correlates with decreased carbohydrate conversion in enzymatic hydrolysis. In addition, the reconstructed softwood substrate has a lower carbohydrate conversion than the reconstructed hardwood substrate. The degree of condensation of lignin increases significantly after pretreatment, especially with softwood lignins. In this study, the degree of condensation of lignin (0.02 to 0.64) and total OH groups in lignin (1.7 to 1.1) have a critical impact on cellulase adsorption (9 to 70%) and enzymatic hydrolysis (83.2 to 58.2%); this may provide insights into the more recalcitrant nature of softwood substrates. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Enzymatic hydrolysis (pepsin assisted by ultrasound in the functional Properties of hydrolyzates from different collagens

    Directory of Open Access Journals (Sweden)

    Alessandra Roseline Vidal

    2018-03-01

    Full Text Available ABSTRACT: Enzymatic hydrolysis (pepsin assisted with or without ultrasound in the functional properties of hydrolyzates from different collagens were analyzed. Degree of hydrolysis, antioxidant activity (DPPH and antimicrobial activity (MIC were assessed. The treatment that resulted in greater antioxidant activity for the fiber sample was with the use of 4% of enzyme and concomitant ultrasound (40.7%, leading to a degree of hydrolysis of 21.7%. For the powdered fiber sample the hydrolysis treatment with use of 4% of enzyme resulted in lower protein content (6.97mg/mL, higher degree of hydrolysis (19.9% and greater antioxidant activity (38.6%. The hydrolyzates showed inhibitory capacity against gram-negative bacteria Salmonella choleraesuis and gram-positive bacteria Staphylococcus aureus. It can be concluded that enzymatic hydrolysis concomitant or not with the use of ultrasound increased the functionality of the fiber and powdered fiber samples, for the other samples its use as supplementary treatment was not productive, due to the worse results of antioxidant activity (DPPH reported. However, it provided greater hydrolysis degree.

  2. Pretreatment and enzymatic hydrolysis of lignocellulosic biomass

    Science.gov (United States)

    Corredor, Deisy Y.

    The performance of soybean hulls and forage sorghum as feedstocks for ethanol production was studied. The main goal of this research was to increase fermentable sugars' yield through high-efficiency pretreatment technology. Soybean hulls are a potential feedstock for production of bio-ethanol due to their high carbohydrate content (≈50%) of nearly 37% cellulose. Soybean hulls could be the ideal feedstock for fuel ethanol production, because they are abundant and require no special harvesting and additional transportation costs as they are already in the plant. Dilute acid and modified steam-explosion were used as pretreatment technologies to increase fermentable sugars yields. Effects of reaction time, temperature, acid concentration and type of acid on hydrolysis of hemicellulose in soybean hulls and total sugar yields were studied. Optimum pretreatment parameters and enzymatic hydrolysis conditions for converting soybean hulls into fermentable sugars were identified. The combination of acid (H2SO4, 2% w/v) and steam (140°C, 30 min) efficiently solubilized the hemicellulose, giving a pentose yield of 96%. Sorghum is a tropical grass grown primarily in semiarid and dry parts of the world, especially in areas too dry for corn. The production of sorghum results in about 30 million tons of byproducts mainly composed of cellulose, hemicellulose, and lignin. Forage sorghum such as brown midrib (BMR) sorghum for ethanol production has generated much interest since this trait is characterized genetically by lower lignin concentrations in the plant compared with conventional types. Three varieties of forage sorghum and one variety of regular sorghum were characterized and evaluated as feedstock for fermentable sugar production. Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM) and X-Ray diffraction were used to determine changes in structure and chemical composition of forage sorghum before and after pretreatment and enzymatic hydrolysis

  3. Influence of nitrogen additions on litter decomposition, nutrient dynamics, and enzymatic activity of two plant species in a peatland in Northeast China.

    Science.gov (United States)

    Song, Yanyu; Song, Changchun; Ren, Jiusheng; Tan, Wenwen; Jin, Shaofei; Jiang, Lei

    2018-06-01

    Nitrogen (N) availability affects litter decomposition and nutrient dynamics, especially in N-limited ecosystems. We investigated the response of litter decomposition to N additions in Eriophorum vaginatum and Vaccinium uliginosum peatlands. These two species dominate peatlands in Northeast China. In 2012, mesh bags containing senesced leaf litter of Eriophorum vaginatum and Vaccinium uliginosum were placed in N addition plots and sprayed monthly for two years with NH 4 NO 3 solution at dose rates of 0, 6, 12, and 24gNm -2 year -1 (CK, N1, N2 and N3, respectively). Mass loss, N and phosphorus (P) content, and enzymatic activity were measured over time as litter decomposed. In the control plots, V. uliginosum litter decomposed faster than E. vaginatum litter. N1, N2, and N3 treatments increased the mass losses of V. uliginosum litter by 6%, 9%, and 4% respectively, when compared with control. No significant influence of N additions was found on the decomposition of E. vaginatum litter. However, N and P content in E. vaginatum litter and V. uliginosum litter significantly increased with N additions. Moreover, N additions significantly promoted invertase and β-glucosidase activity in E. vaginatum and V. uliginosum litter. However, only in V. uliginosum litter was polyphenol oxidase activity significantly enhanced. Our results showed that initial litter quality and polyphenol oxidase activity influence the response of plant litter to N additions in peatland ecosystems. Increased N availability may change peatland soil N and P cycling by enhancing N and P immobilization during litter decomposition. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Polyphenol Content, Physicochemical Properties, Enzymatic Activity, Anthocyanin Profiles, and Antioxidant Capacity of Cerasus humilis (Bge. Sok. Genotypes

    Directory of Open Access Journals (Sweden)

    Suwen Liu

    2018-01-01

    Full Text Available Seven varieties of Chinese dwarf cherries were evaluated and compared with respect to their weight, diameter, titratable acidity, total soluble solids, color, polyphenol contents, ascorbic acid levels, anthocyanin profiles, enzymatic activity, and antioxidant capacity. The fruits are rich in phenolic content (339.07–770.30 mg/100 g fresh weight. Nine anthocyanins were obtained from fruits after chromatographic separation and their structures analyzed using HPLC-ESI-MS/MS. Cyanidin-3-glucoside was the major anthocyanin with 50.36–78.39% concentration. Three anthocyanins were reported for the first time in these cherries. They exhibit low polyphenol oxidase and peroxidase activities, but their superoxide dismutase activity is high (572.75–800.17 U/g FW. The highest amounts of soluble solid content (15.67 Brix %, total titratable acid (1.90%, ascorbic acid (18.47 mg/100 g FW, and total anthocyanin (152.66 mg/100 g FW were observed. Three methods (DPPH-scavenging ability, oxygen radical absorbance capacity assay, and cellular antioxidant activity assay were employed to evaluate the antioxidant capacity of the phenolic extracts of these cherries. Number 5 has the highest values of ORAC and CAA of 205.68 μmol TE/g DM and 99.67 μmol QE/100 g FW, respectively.

  5. Effect of enzymatic depolymerization on physicochemical and rheological properties of guar gum.

    Science.gov (United States)

    Mudgil, Deepak; Barak, Sheweta; Khatkar, B S

    2012-09-01

    Depolymerization of guar gum using enzymatic hydrolysis was performed to obtain depolymerized guar gum having functional application as soluble dietary fiber. Enzymatic hydrolysis of guar gum significantly affected the physicochemical and rheological characteristics of guar gum. The depolymerized guar gum showed a significant increase in crystallinity index from 3.86% to 13.2% and flow behavior index from 0.31 to 1.7 as compared to native guar gum. Remarkable decrease in intrinsic viscosity and consistency index was also observed from 9 to 0.28 and 4.04 to 0.07, respectively. Results revealed that enzymatic hydrolysis of guar gum resulted in a polysaccharide with low degree of polymerization, viscosity and consistency which could make it useful for incorporation in food products as dietary fiber without affecting the rheology, consistency and texture of the products. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Enzymatic coupling of 2,4-dichlorophenol to stream fulvic acid in the presence of oxidoreductases

    International Nuclear Information System (INIS)

    Sarkar, J.M.; Malcolm, R.L.; Bollag, J.M.

    1988-01-01

    The coupling 14 C-ring-labelled 2,4-dichlorophenol (2,4-DCP) to stream fulvic acid was investigated in the presence of several oxidoreductases including tyrosinase, peroxidase, and laccases of Rhizoctonia praticola and Trametes vesicolor. During 12-h incubation of the oxidoreductases with 14 C-2, 4-DCP and stream fulvic acid, a substantial amount of the radioactivity was incorporated into fulvic acid. Chromatographic analysis indicated that although a large portion of the radioactivity remained in solution, no unbound 14 C-2,4-DCP was present in the supernatant. The effects of pH, temperature, concentration of fulvic acid, and concentration of enzyme on the coupling processes were studied. The results of this research provide evidence that the enzymatic coupling of certain xenobiotic pollutants to humic substances is an important natural process which must be considered in studies of the fate, reactivity, and persistence of these organic compounds in soils and stream waters

  7. Evaluation of Biological and Enzymatic Activity of Soil in a Tropical Dry Forest: Desierto de la Tatacoa (Colombia) with Potential in Mars Terraforming and Other Similar Planets

    Science.gov (United States)

    Moreno Moreno, A. N.

    2009-12-01

    Desierto de la Tatacoa has been determined to be a tropical dry forest bioma, which is located at 3° 13" N 75° 13" W. It has a hot thermal floor with 440 msnm of altitude; it has a daily average of 28° C, and a maximum of 40° C, Its annual rainfall total can be upwards of 1250 mm. Its solar sheen has a daily average of 5.8 hours and its relative humidity is between 60% and 65%. Therefore, the life forms presents are very scant, and in certain places, almost void. It was realized a completely random sampling of soil from its surface down to 6 inches deep, of zones without vegetation and with soils highly loaded by oxides of iron in order to determine the number of microorganisms per gram and its subsequent identification. It was measured the soil basal respiration. Besides, it was determined enzymatic activity (catalase, dehydrogenase, phosphatase and urease). Starting with the obtained results, it is developes an alternative towards the study of soil genesis in Mars in particular, and recommendations for same process in other planets. Although the information found in the experiments already realized in Martian soil they demonstrate that doesnt exist any enzymatic activity, the knowledge of the same topic in the soil is proposed as an alternative to problems like carbonic fixing of the dense Martian atmosphere of CO2, the degradation of inorganic compounds amongst other in order to prepare the substratum for later colonization by some life form.

  8. One-step enzymatic synthesis of nucleosides from low water-soluble purine bases in non-conventional media.

    Science.gov (United States)

    Fernández-Lucas, Jesús; Fresco-Taboada, Alba; de la Mata, Isabel; Arroyo, Miguel

    2012-07-01

    The effect of several water-miscible cosolvents on activity and stability of soluble and immobilized 2'-deoxyribosyltransferase from Lactobacillus reuteri on Sepabeads® has been studied in order to establish optimal conditions for enzymatic synthesis of nucleosides using purine bases with low solubility in aqueous buffer. As a rule of thumb, there was a general reduction of soluble enzyme activity when cosolvent content was gradually increased in reaction medium. In contrast, immobilized enzyme activity was enhanced 1.2-1.4-fold at 20% of methanol, ethanol, 2-propanol, diethylene glycol, and acetone; and at 10% and 30% acetonitrile. Likewise, highest increased activity (1.8-fold) was also obtained in presence of 20% acetonitrile. Immobilized enzyme was successfully used in the synthesis of 2'-deoxyxanthosine and 2'-deoxyguanosine using 2'-deoxyuridine as sugar donor and the corresponding poor water-soluble base in the presence of 30% of methanol, ethanol, 2-propanol, ethylene glycol, acetonitrile, and DMSO, giving high nucleoside yields at 4h. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. Kinetics of enzymatic hydrolysis of methyl ricinoleate

    OpenAIRE

    Neeharika, T. S.V.R.; Lokesh, P.; Prasanna Rani, K. N.; Prathap Kumar, T.; Prasad, R. B.N.

    2015-01-01

    Ricinoleic acid is an unsaturated hydroxy fatty acid that naturally occurs in castor oil in proportions of up to 85–90%. Ricinoleic acid is a potential raw material and finds several applications in coatings, lubricant formulations and pharmaceutical areas. Enzymatic hydrolysis of castor oil is preferred over conventional hydrolysis for the preparation of ricinoleic acid to avoid estolide formation. A kinetics analysis of the enzymatic hydrolysis of Methyl Ricinoleate in the presence of Candi...

  10. Induction of mast cell accumulation by chymase via an enzymatic activity- and intercellular adhesion molecule-1-dependent mechanism.

    Science.gov (United States)

    Zhang, Huiyun; Wang, Junling; Wang, Ling; Zhan, Mengmeng; Li, Shigang; Fang, Zeman; Xu, Ciyan; Zheng, Yanshan; He, Shaoheng

    2018-02-01

    Chymase is a unique, abundant secretory product of mast cells and a potent chemoattractant for eosinophils, monocytes and neutrophils, but little is known of its influence on mast cell accumulation. A mouse peritoneal inflammation model, cell migration assay and flowcytometry analysis, were used to investigate the role of chymase in recruiting mast cells. Chymase increased, by up to 5.4-fold, mast cell numbers in mouse peritoneum. Inhibitors of chymase, heat-inactivation of the enzyme, sodium cromoglycate and terfenadine, and pretreatment of mice with anti-intercellular adhesion molecule 1, anti-L-selectin, anti-CD11a and anti-CD18 antibodies dramatically diminished the chymase-induced increase in mast cell accumulation. These findings indicate that this effect of chymase is dependent on its enzymatic activity and activation of adhesion molecules. In addition, chymase provoked a significant increase in 5-HT and eotaxin release (up to 1.8- and 2.2-fold, respectively) in mouse peritoneum. Since 5-HT, eotaxin and RANTES can induce marked mast cell accumulation, these indirect mechanisms may also contribute to chymase-induced mast cell accumulation. Moreover, chymase increased the trans-endothelium migration of mast cells in vitro indicating it also acts as a chemoattractant. The finding that mast cells accumulate in response to chymase implies further that chymase is a major pro-inflammatory mediator of mast cells. This effect of chymase, a major product of mast cell granules, suggests a novel self-amplification mechanism for mast cell accumulation in allergic inflammation. Mast cell stabilizers and inhibitors of chymase may have potential as a treatment of allergic disorders. © 2017 The British Pharmacological Society.

  11. Expression of an enzymatically active Yb3 glutathione S-transferase in Escherichia coli and identification of its natural form in rat brain.

    Science.gov (United States)

    Abramovitz, M; Ishigaki, S; Felix, A M; Listowsky, I

    1988-11-25

    Glutathione S-transferases containing Yb3 subunits are relatively uncommon forms that are expressed in a tissue-specific manner and have not been identified unequivocally or characterized. A cDNA clone containing the entire coding sequence of Yb3 glutathione S-transferase mRNA was incorporated into a pIN-III expression vector used to transform Escherichia coli. A fusion Yb3-protein containing 14 additional amino acid residues at its N terminus was purified to homogeneity. Recombinant Yb3 was enzymatically active with both 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates but lacked glutathione peroxidase activity. Substrate specificity patterns of recombinant Yb3 were more limited than those of glutathione S-transferase isoenzymes containing Yb1- or Yb2-type subunits. Peptides corresponding to unique amino acid sequences of Yb3 as well as a peptide from a region of homology with Yb1 and Yb2 subunits were synthesized. These synthetic peptides were used to raise antibodies specific to Yb3 and others that cross-reacted with all Yb forms. Immunoblotting was utilized to identify the natural counterpart of recombinant Yb3 among rat glutathione transferases. Brain and testis glutathione S-transferases were rich in Yb3 subunits, but very little was found in liver or kidney. Physical properties, substrate specificities, and binding patterns of the recombinant protein paralleled properties of the natural isoenzyme isolated from brain.

  12. Identification of odor volatile compounds and deodorization of Paphia undulata enzymatic hydrolysate

    Science.gov (United States)

    Chen, Deke; Chen, Xin; Chen, Hua; Cai, Bingna; Wan, Peng; Zhu, Xiaolian; Sun, Han; Sun, Huili; Pan, Jianyu

    2016-12-01

    Unfavorable fishy odour is an inevitable problem in aquatic products. In the present study, headspace solid-phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS) analysis of volatiles from untreated samples and three deodorized samples (under the optimal conditions) of Paphia undulata enzymatic hydrolysate revealed that the compounds contributing to the distinctive odor were 1-octen-3-ol, n-hexanal, n-heptanal, 2,4-heptadienal, and 2,4-decadienal, whereas n-pentanal, n-octanal, n-octanol, benzaldehyde, 2-ethylfuran and 2-pentylfuran were the main contributors to the aromatic flavor. The deodorizing effects of activated carbon (AC) adsorption, yeast extract (YE) masking and tea polyphenol (TP) treatment on a P. undulata enzymatic hydrolysate were investigated using orthogonal experiments with sensory evaluation as the index. The following optimized deodorization conditions were obtained: AC adsorption (35 mg mL-1, 80°C, 40 min), YE masking (7 mg mL-1, 45°C, 30 min) and TP treatment (0.4 mg mL-1, 40°C, 50 min). AC adsorption effectively removed off-flavor volatile aldehydes and ketones. YE masking modified the odor profile by increasing the relative contents of aromatic compounds and decreasing the relative contents of aldehydes and ketones. The TP treatment was not effective in reducing the odor score, but it significantly reduced the relative content of aldehydes while increasing that of alkanes. It is also notable that TP effectively suppressed trimethylamine (TMA) formation in a P. undulate hydrolysate solution for a period of 72 h.

  13. Enzymatic activity and gene expression changes in zebrafish embryos and larvae exposed to pesticides diazinon and diuron.

    Science.gov (United States)

    Velki, Mirna; Meyer-Alert, Henriette; Seiler, Thomas-Benjamin; Hollert, Henner

    2017-12-01

    The zebrafish as a test organism enables the investigation of effects on a wide range of biological levels from molecular level to the whole-organism level. The use of fish embryos represents an attractive model for studies aimed at understanding toxic mechanisms and the environmental risk assessment of chemicals. In the present study, a zebrafish (Danio rerio) in vivo model was employed in order to assess the effects of two commonly used pesticides, the insecticide diazinon and the herbicide diuron, on zebrafish early life stages. Since it was previously established that diazinon and diuron cause effects at the whole-organism level, this study assessed the suborganismic responses to exposure to these pesticides and the enzymatic responses (biochemical level) and the gene expression changes (molecular level) were analyzed. Different exposure scenarios were employed and the following endpoints measured: acetylcholinesterase (AChE), carboxylesterase (CES), ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), catalase (CAT) and glutathione peroxidase (GPx) activities; and gene expressions of the corresponding genes: acetylcholinesterase (ache), carboxylesterase (ces2), cytochrome P450 (cyp1a), glutathione-S-transferase (gstp1), catalase (cat), glutathione peroxidase (gpx1a) and additionally glutathione reductase (gsr). Significant changes at both the biochemical and the molecular level were detected. In addition, different sensitivities of different developmental stages of zebrafish were determined and partial recovery of the enzyme activity 48h after the end of the exposure was observed. The observed disparity between gene expression changes and alterations in enzyme activities points to the necessity of monitoring changes at different levels of biological organization. Different exposure scenarios, together with a comparison of the responses at the biochemical and molecular level, provide valuable data on the effects of diazinon and diuron on low

  14. Seawater operating bio-photovoltaic cells coupling semiconductor photoanodes and enzymatic biocathodes

    DEFF Research Database (Denmark)

    Zhang, Lingling; Alvarez-Martos, Isabel; Vakurov, Alexander

    2017-01-01

    and inexpensive way. Here, we report clean and sustainable conversion of solar energy into electricity by photo-and bio-electrocatalytic recycling of the H2O/O-2 redox couple in a hybrid bio-photovoltaic (BPV) membraneless cell comprising a sunlight-illuminated water-oxidizing semiconductor anode (either Zn......-doped hematite or TiO2) and an oxygen-reducing enzymatic biocathode, in such environmental media as seawater. Upon simulated solar light illumination (AM 1.5G, 100 mW cm(-2)), the maximum power density (P-max) generated by the cell was 236 and 21.4 mu W cm(-2) in 1 M Tris-HCl and seawater, both at pH 8...... thermodynamically feasible coupling of cost-effective photoactive materials such as TiO2 or hematite semiconductors and enzymatic counterparts in seawater media opens a prospective clean and sustainable way of transformation of the most abundant, clean and renewable source of energy - solar light - and the Earth...

  15. [The effect of enzymatic treatment using proteases on properties of persistent sodium current in CA1 pyramidal neurons of rat hippocampus].

    Science.gov (United States)

    Lun'ko, O O; Isaiev, D S; Maxymiuk, O P; Kryshtal', O O; Isaieva, O V

    2014-01-01

    We investigated the effect of proteases, widely used for neuron isolation in electrophysiological studies, on the amplitude and kinetic characteristics of persistent sodium current (I(NaP)) in hippocampal CA1 pyramidal neurons. Properties of I(NaP) were studied on neurons isolated by mechanical treatment (control group) and by mechanical and enzymatic treatment using pronase E (from Streptomyces griseus) or protease type XXIII (from Aspergillus oryzae). We show that in neurons isolated with pronase E kinetic of activation and density of I(NaP) was unaltered. Enzymatic treatment with protease type XXIII did not alter I(NaP) activation but result in significant decrease in I(NaP) density. Our data indicates that enzymatic treatment using pronase E for neuron isolation is preferable for investigation of I(NaP).

  16. Enzymatic synthesis of vanillin

    NARCIS (Netherlands)

    van den Heuvel, RHH; Fraaije, MW; Laane, C; van Berkel, WJH; Heuvel, Robert H.H. van den; Berkel, Willem J.H. van

    Due to increasing interest in natural vanillin, two enzymatic routes for the synthesis of vanillin were developed. The flavoprotein vanillyl alcohol oxidase (VAO) acts on a wide range of phenolic compounds and converts both creosol and vanillylamine to vanillin with high yield. The VAO-mediated

  17. Enzymatic synthesis of vanillin

    NARCIS (Netherlands)

    Heuvel, van den R.H.H.; Fraaije, M.W.; Laane, C.; Berkel, van W.J.H.

    2001-01-01

    Due to increasing interest in natural vanillin, two enzymatic routes for the synthesis of vanillin were developed. The flavoprotein vanillyl alcohol oxidase (VAO) acts on a wide range of phenolic compounds and converts both creosol and vanillylamine to vanillin with high yield. The VAO-mediated

  18. Electrodeposition of flower-like platinum on electrophoretically grown nitrogen-doped graphene as a highly sensitive electrochemical non-enzymatic biosensor for hydrogen peroxide detection

    Energy Technology Data Exchange (ETDEWEB)

    Tajabadi, M.T. [University Malaya Centre for Ionic Liquids, Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603 (Malaysia); Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603 (Malaysia); Sookhakian, M., E-mail: m.sokhakian@gmail.com [University Malaya Centre for Ionic Liquids, Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603 (Malaysia); Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603 (Malaysia); Department of Mechanical Convergence Engineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 133-791, Korea (Korea, Republic of); Zalnezhad, E., E-mail: erfan@hanyang.ac.kr [Department of Mechanical Convergence Engineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 133-791, Korea (Korea, Republic of); Yoon, G.H. [Department of Mechanical Convergence Engineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 133-791, Korea (Korea, Republic of); Hamouda, A.M.S. [Mechanical and Industrial Engineering Department, College of Engineering, Qatar University, 2713, Doha (Qatar); Azarang, Majid [Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603 (Malaysia); Basirun, W.J. [Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603 (Malaysia); Institute of Nanotechnology & Catalysis Research, Institute of Postgraduate Studies, University Malaya, 50603 Kuala Lumpur (Malaysia); Alias, Y., E-mail: yatimah70@um.edu.my [University Malaya Centre for Ionic Liquids, Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603 (Malaysia); Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603 (Malaysia)

    2016-11-15

    Highlights: • Nitrogen doped graphene with different thickness by electrophoretic deposition. • The conductivity of N-graphene layer depends on the tickness. • Support of platinum shows efficient electrocatalytic performance for biosensor. • CV curves and amperometric responses improved and optimized in the presence of N-graphene. - Abstract: An efficient non-enzymatic biosensor electrode consisting of nitrogen-doped graphene (N-graphene) and platinum nanoflower (Pt NF) with different N-graphene loadings were fabricated on indium tin oxide (ITO) glass using a simple layer-by-layer electrophoretic and electrochemical sequential deposition approach. N-graphene was synthesized by annealing graphene oxide with urea at 900 °C. The structure and morphology of the as-fabricated non-enzymatic biosensor electrodes were determined using X-ray diffraction, field emission electron microscopy, transmission electron microscopy, Raman and X-ray photoelectron spectra. The as-fabricated Pt NF-N-graphene-modified ITO electrodes with different N-graphene loadings were utilized as a non-enzymatic biosensor electrode for the detection of hydrogen peroxide (H{sub 2}O{sub 2}). The behaviors of the hybrid electrodes towards H{sub 2}O{sub 2} reduction were assessed using chronoamperometry, cyclic voltammetry and electrochemical impedance spectroscopy analysis. The Pt NF-N-graphene-modified ITO electrode with a 0.05 mg ml{sup −1} N-graphene loading exhibited the lowest detection limit, fastest amperometric sensing, a wide linear response range, excellent stability and reproducibility for the non-enzymatic H{sub 2}O{sub 2} detection, due to the synergistic effect between the electrocatalytic activity of the Pt NF and the high conductivity and large surface area of N-graphene.

  19. Enzymatic Hydrolysis of Alkaline Pretreated Coconut Coir

    Directory of Open Access Journals (Sweden)

    Akbarningrum Fatmawati

    2013-06-01

    Full Text Available The purpose of this research is to study the effect of concentration and temperature on the cellulose and lignin content, and the reducing sugars produced in the enzymatic hydrolysis of coconut coir. In this research, the coconut coir is pretreated using 3%, 7%, and 11% NaOH solution at 60oC, 80oC, and 100oC. The pretreated coir were assayed by measuring the amount of cellulose and lignin and then hydrolysed using Celluclast and Novozyme 188 under various temperature (30oC, 40oC, 50oC and pH (3, 4, 5. The hydrolysis results were assayed for the reducing sugar content. The results showed that the alkaline delignification was effective to reduce lignin and to increase the cellulose content of the coir. The best delignification condition was observed at 11% NaOH solution and 100oC which removed 14,53% of lignin and increased the cellulose content up to 50,23%. The best condition of the enzymatic hydrolysis was obtained at 50oC and pH 4 which produced 7,57 gr/L reducing sugar. © 2013 BCREC UNDIP. All rights reservedReceived: 2nd October 2012; Revised: 31st January 2013; Accepted: 6th February 2013[How to Cite: Fatmawati, A., Agustriyanto, R., Liasari, Y. (2013. Enzymatic Hydrolysis of Alkaline Pre-treated Coconut Coir. Bulletin of Chemical Reaction Engineering & Catalysis, 8 (1: 34-39 (doi:10.9767/bcrec.8.1.4048.34-39[Permalink/DOI: http://dx.doi.org/10.9767/bcrec.8.1.4048.34-39] | View in  |

  20. Dimerization of inositol monophosphatase Mycobacterium tuberculosis SuhB is not constitutive, but induced by binding of the activator Mg2+

    Directory of Open Access Journals (Sweden)

    Nigou Jérôme

    2007-08-01

    Full Text Available Abstract Background The cell wall of Mycobacterium tuberculosis contains a wide range of phosphatidyl inositol-based glycolipids that play critical structural roles and, in part, govern pathogen-host interactions. Synthesis of phosphatidyl inositol is dependent on free myo-inositol, generated through dephosphorylation of myo-inositol-1-phosphate by inositol monophosphatase (IMPase. Human IMPase, the putative target of lithium therapy, has been studied extensively, but the function of four IMPase-like genes in M. tuberculosis is unclear. Results We determined the crystal structure, to 2.6 Å resolution, of the IMPase M. tuberculosis SuhB in the apo form, and analysed self-assembly by analytical ultracentrifugation. Contrary to the paradigm of constitutive dimerization of IMPases, SuhB is predominantly monomeric in the absence of the physiological activator Mg2+, in spite of a conserved fold and apparent dimerization in the crystal. However, Mg2+ concentrations that result in enzymatic activation of SuhB decisively promote dimerization, with the inhibitor Li+ amplifying the effect of Mg2+, but failing to induce dimerization on its own. Conclusion The correlation of Mg2+-driven enzymatic activity with dimerization suggests that catalytic activity is linked to the dimer form. Current models of lithium inhibition of IMPases posit that Li+ competes for one of three catalytic Mg2+ sites in the active site, stabilized by a mobile loop at the dimer interface. Our data suggest that Mg2+/Li+-induced ordering of this loop may promote dimerization by expanding the dimer interface of SuhB. The dynamic nature of the monomer-dimer equilibrium may also explain the extended concentration range over which Mg2+ maintains SuhB activity.

  1. Green-synthesized CdS nano-pesticides: Toxicity on young instars of malaria vectors and impact on enzymatic activities of the non-target mud crab Scylla serrata.

    Science.gov (United States)

    Sujitha, Vasu; Murugan, Kadarkarai; Dinesh, Devakumar; Pandiyan, Amuthvalli; Aruliah, Rajasekar; Hwang, Jiang-Shiou; Kalimuthu, Kandasamy; Panneerselvam, Chellasamy; Higuchi, Akon; Aziz, Al Thabiani; Kumar, Suresh; Alarfaj, Abdullah A; Vaseeharan, Baskaralingam; Canale, Angelo; Benelli, Giovanni

    2017-07-01

    Currently, nano-formulated mosquito larvicides have been widely proposed to control young instars of malaria vector populations. However, the fate of nanoparticles in the aquatic environment is scarcely known, with special reference to the impact of nanoparticles on enzymatic activity of non-target aquatic invertebrates. In this study, we synthesized CdS nanoparticles using a green protocol relying on the cheap extract of Valoniopsis pachynema algae. CdS nanoparticles showed high toxicity on young instars of the malaria vectors Anopheles stephensi and A. sundaicus. The antimalarial activity of the nano-synthesized product against chloroquine-resistant (CQ-r) Plasmodium falciparum parasites was investigated. From a non-target perspective, we focused on the impact of this novel nano-pesticide on antioxidant enzymes acetylcholinesterase (AChE) and glutathione S-transferase (GST) activities of the mud crab Scylla serrata. The characterization of nanomaterials was carried out by UV-vis and FTIR spectroscopy, as well as SEM and XRD analyses. In mosquitocidal assays, LC 50 of V. pachynema-synthesized CdS nanoparticles on A. stephensi ranged from 16.856 (larva I), to 30.301μg/ml (pupa), while for An. sundaicus they ranged from 13.584 to 22.496μg/ml. The antiplasmodial activity of V. pachynema extract and CdS nanoparticles was evaluated against CQ-r and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. IC 50 of V. pachynema extract was 58.1μg/ml (CQ-s) and 71.46μg/ml (CQ-r), while nano-CdS IC 50 was 76.14μg/ml (CQ-s) and 89.21μg/ml (CQ-r). In enzymatic assays, S. serrata crabs were exposed to sub-lethal concentrations, i.e. 4, 6 and 8μg/ml of CdS nanoparticles, assessing changes in GST and AChE activity after 16days. We observed significantly higher activity of GST, if compared to the control, during the whole experiment period. In addition, a single treatment with CdS nanoparticles led to a significant decrease in AChE activity over time. The toxicity of Cd

  2. Enzymatic hydrolsis of pretreated rice straw

    Energy Technology Data Exchange (ETDEWEB)

    Vlasenko, E.Y.; Shoemaker, S.P. [California Inst. of Food and Agricultural Research, Davis, CA (United States); Ding, H. [California Univ., Davis (Canada). Dept. of Food Science and Technology; Labavitch, J.M. [California Univ., Davis, CA (United States). Dept. of Pomology

    1997-02-01

    California rice straw is being evaluated as a feedstock for production of power and fuel. This paper examines the initial steps in the process: pretreatment of rice straw and enzymatic hydrolysis of the polysaccharides in the pretreated material to soluble sugars. Rice straw was subjected to three distinct pretreatment procedures: acid-catalyzed steam explosion (Swan Biomass Company), acid hydrolysis (U.S. DOE National Renewable Energy Laboratory), and ammonia fiber explosion or AFEX (Texas A and M University). Standard conditions for each pretreatment were used, but none was optimized for rice straw specifically. Six commercial cellulases, products of Genencor International (USA), Novo (Denmark), Iogen (Canada) and Fermtech (Russia) were used for hydrolysis. The Swan- and the acid-pretreatments effectively removed hemicellulose from rice straw, providing high yields of fermentable sugars. The AFEX-pretreatment was distinctly different from other pretreatments in that it did not significantly solubilize hemicellulose. All three pretreatment procedures substantially increased enzymatic digestibility of rice straw. Three commercial Trichoderma-reesei-derived enzyme preparations: Cellulase 100L (Iogen), Spezyme CP (Genencor), and Al (Fermtech), were more active on pretreated rice straw compared than others tested. Conditions for hydrolysis of rice straw using Cellulase 100L were evaluated. The supplementation of this enzyme preparation with cellobiase (Novozyme 188) significantly improved the parameters of hydrolysis for the Swan- and the acid-pretreated materials, but did not affect the hydrolysis of the AFEX-pretreated rice straw. (Author)

  3. NMNAT2:HSP90 Complex Mediates Proteostasis in Proteinopathies.

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    Yousuf O Ali

    2016-06-01

    Full Text Available Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2 is neuroprotective in numerous preclinical models of neurodegeneration. Here, we show that brain nmnat2 mRNA levels correlate positively with global cognitive function and negatively with AD pathology. In AD brains, NMNAT2 mRNA and protein levels are reduced. NMNAT2 shifts its solubility and colocalizes with aggregated Tau in AD brains, similar to chaperones, which aid in the clearance or refolding of misfolded proteins. Investigating the mechanism of this observation, we discover a novel chaperone function of NMNAT2, independent from its enzymatic activity. NMNAT2 complexes with heat shock protein 90 (HSP90 to refold aggregated protein substrates. NMNAT2's refoldase activity requires a unique C-terminal ATP site, activated in the presence of HSP90. Furthermore, deleting NMNAT2 function increases the vulnerability of cortical neurons to proteotoxic stress and excitotoxicity. Interestingly, NMNAT2 acts as a chaperone to reduce proteotoxic stress, while its enzymatic activity protects neurons from excitotoxicity. Taken together, our data indicate that NMNAT2 exerts its chaperone or enzymatic function in a context-dependent manner to maintain neuronal health.

  4. Enzymatic activity of proteases and its isoenzymes in fermentation process in cultivars of cocoa (Theobroma cacao L. produced in southern Bahia, Brazil

    Directory of Open Access Journals (Sweden)

    Luciane Santos SOUSA

    Full Text Available Abstract The fermentation of cocoa seeds envolves microbial processes and the action of enzymes. To identify the possible differences in the cocoa fermentation process, with regards to proteolysis, this study has the objective of determining protease activity (under predetermined conditions and its isoenzymes in two cocoa cultivars (PH-16 and HRT-1188 in different cocoa fermentation times, in addition to establishing the microbial load (molds and yeasts and aerobic mesophilic. Protease and its isoenzymes were extracted and partially purified and the enzymatic activities determined by spectrophotometry. The results showed that the proteases activity was higher at 66h of fermentation for both cultivars. When the isoenzymes activity was evaluated, the results demonstrated similar activity behavior for both cultivars, with regards to the isoenzymes aminopeptidase and carboxypeptidase, although the behavior of the endoprotease isoenzyme activity proved to be a little different for TSH-1188 cultivar. Concerning microbiological analyses, the results indicate that the period after molds and yeast counting reduction is consistent with the period of protease activity increase.

  5. Enzymatic network for production of ether amines from alcohols

    NARCIS (Netherlands)

    Palacio, Cyntia M.; Crismaru, Gica Ciprian; Bartsch, Sebastian; Navickas, Vaidotas; Ditrich, Klaus; Breuer, Michael; Abu, Rohana; Woodley, John; Baldenius, Kai-Uwe; Wu, Bian; Janssen, Dick

    We constructed an enzymatic network composed of three different enzymes for the synthesis of valuable ether amines. The enzymatic reactions are interconnected to catalyze the oxidation and subsequent transamination of the substrate and to provide cofactor recycling. This allows production of the

  6. Recent insights in enzymatic synthesis of fructooligosaccharides from inulin.

    Science.gov (United States)

    Singh, Ram Sarup; Singh, Rupinder Pal; Kennedy, John F

    2016-04-01

    In the past few years, people are paying more attention to their dietary habits, and functional foods are playing a key role in maintaining the health of man. Prebiotics are considered as a main component of the functional foods which are usually composed of short chains of carbohydrates. Fructooligosaccharides (FOSs) are considered as one of the main group of prebiotics which have recognisable bifidogenic properties. FOSs are obtained either by extraction from various plant materials or by enzymatic synthesis from different substrates. Enzymatically, these can be obtained either from sucrose using fructosyltransferase or from inulin by endoinulinase. Inulin is a potent substrate for the enzymatic production of FOSs. This review article will provide an overview on the inulin as potent substrate, microbial sources of endoinulinases, enzymatic synthesis of FOSs from inulin, commercial status of FOSs, and their future perspectives. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Method of reduction of nitroaromatics by enzymatic reaction with redox enzymes

    Science.gov (United States)

    Shah, Manish M.

    2000-01-01

    A method for the controlled reduction of nitroaromatic compounds such as nitrobenzene and 2,4,6-trinitrotoluene by enzymatic reaction with redox enzymes, such as Oxyrase (Trademark of Oxyrase, Inc., Mansfield, Ohio).

  8. Analysis of glycosidases activity in Chlamydia trachomatis L2 serotype

    Directory of Open Access Journals (Sweden)

    Dr. Sanjiv Rughooputh

    2006-01-01

    Full Text Available Chlamydiae are prokaryotic, gram negative, intracellular parasites, causing a number of human diseases with serious sequelae. The exact mechanism of chlamydial attachment and entry into the host cell is still unclear. Chlamydia trachomatis, SA2F (L2 was tested for the activity of glycosidases. Glycosidases are involved in the synthesis and breakdown of cell-wall polysaccharides, and have a pivotal role in attachment to the surface of host cells as well as in the invasion process of other infective agents. For these reasons, glycosidases are likely to provide important information regarding the general behaviour of this bacterium. Ten different glycosides bound to p-nitrophenol (pNP substrate were tested. Assays were performed on lysed and unlysed chlamydial extracts.Our results provided strong evidence for the existence of glycosidase activity in C.trachomatis, strain SA2F peaking at pH 5.0 and pH 7.5-8.0 There was a high enzymatic activity against p-NPa mannopyranoside and p-NP b-N acetylglucosamine with a recovery of 88% suggesting that the enzymes were present on the bacterial surface. Activity against these sugars, which form part of either the extracellular filler or in endoplasmic reticulum may suggest of a possible mechanism of cellular invasion.

  9. Coral-like CeO{sub 2}/NiO nanocomposites with efficient enzyme-mimetic activity for biosensing application

    Energy Technology Data Exchange (ETDEWEB)

    Mu, Jianshuai; Zhao, Xin; Li, Jie [College of Chemistry, Key Laboratory of Inorganic-Organic Hybrid Functional Material Chemistry, Ministry of Education, Tianjin Key Laboratory of Structure and Performance for Functional Molecules, Tianjin Normal University, Tianjin 300387 (China); Yang, En-Cui, E-mail: encui_yang@163.com [College of Chemistry, Key Laboratory of Inorganic-Organic Hybrid Functional Material Chemistry, Ministry of Education, Tianjin Key Laboratory of Structure and Performance for Functional Molecules, Tianjin Normal University, Tianjin 300387 (China); Zhao, Xiao-Jun, E-mail: xiaojun_zhao15@163.com [College of Chemistry, Key Laboratory of Inorganic-Organic Hybrid Functional Material Chemistry, Ministry of Education, Tianjin Key Laboratory of Structure and Performance for Functional Molecules, Tianjin Normal University, Tianjin 300387 (China); Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300071 (China)

    2017-05-01

    Development of nanomaterials-based enzymatic mimics has gained considerable attention in recent years, because of their low cost, high stability and efficiently catalytic ability. Here, CeO{sub 2} was successfully incorporated into the coral-like NiO nanostructures assembled by nanoflakes with high surface area, forming the coral-like CeO{sub 2}/NiO nanocomposites. The morphology and composition of CeO{sub 2}/NiO nanocomposites were characterized by XRD, SEM, element mapping and XPS. The results of characterization showed that cerium was highly dispersed in the coral-like NiO nanostructures. The peroxidase-like activity of CeO{sub 2}/NiO nanocomposites was investigated, and they exhibited enhanced peroxidase-like activity in comparison to that of pure NiO or CeO{sub 2}. The catalytic activity was dependent on the cerium content, and the optimal content was 2.5%. The enhanced catalytic activity of CeO{sub 2}/NiO nanocomposites arised from their high ability of electron transfer because of cerium incorporation. The catalytic performance of CeO{sub 2}/NiO nanocomposites was evaluated by steady-state kinetic, which showed that the CeO{sub 2}/NiO nanocomposites exhibited higher affinity for the substrates and similar catalytic efficiency compared with natural peroxidase. Based on the efficient peroxidase-like activity, CeO{sub 2}/NiO was used for H{sub 2}O{sub 2} determination. The constructed colorimetric H{sub 2}O{sub 2} sensor had fast response for only 5 min, a wide linear range from 0.05 to 40 mM and a low detection limit with 0.88 μM. The CeO{sub 2}/NiO nanocomposites were expected to have potential applications in clinical diagnosis and biotechnology as enzymatic mimics. - Highlights: • Coral-like CeO{sub 2}/NiO nanocomposites with different Ce content were synthesized. • CeO{sub 2} was highly dispersed in the NiO matrixes with high surface area. • CeO{sub 2}/NiO nanocomposites exhibited efficient peroxidase-like activity. • A colorimetric H{sub 2}O

  10. Development and Validation of an Enzymatic Method To Determine Stevioside Content from Stevia rebaudiana.

    Science.gov (United States)

    Udompaisarn, Somsiri; Arthan, Dumrongkiet; Somana, Jamorn

    2017-04-19

    An enzymatic method for specific determination of stevioside content was established. Recombinant β-glucosidase BT_3567 (rBT_3567) from Bacteroides thetaiotaomicron HB-13 exhibited selective hydrolysis of stevioside at β-1,2-glycosidic bond to yield rubusoside and glucose. Coupling of this enzyme with glucose oxidase and peroxidase allowed for quantitation of stevioside content in Stevia samples by using a colorimetric-based approach. The series of reactions for stevioside determination can be completed within 1 h at 37 °C. Stevioside determination using the enzymatic assay strongly correlated with results obtained from HPLC quantitation (r 2 = 0.9629, n = 16). The percentages of coefficient variation (CV) of within day (n = 12) and between days (n = 12) assays were lower than 5%, and accuracy ranges were 95-105%. This analysis demonstrates that the enzymatic method developed in this study is specific, easy to perform, accurate, and yields reproducible results.

  11. Enzymatic analysis of Hemiscorpius lepturus scorpion venom using zymography and venom-specific antivenin.

    Science.gov (United States)

    Seyedian, Ramin; Pipelzadeh, Mohammad Hassan; Jalali, Amir; Kim, Euikyung; Lee, Hyunkyoung; Kang, Changkeun; Cha, Mijin; Sohn, Eun-Tae; Jung, Eun-Sun; Rahmani, Ali Hassan; Mirakabady, Abbas Zare

    2010-09-15

    Hemiscorpius lepturus envenomation exhibits various pathological changes in the affected tissues, including skin, blood cells, cardiovascular and central nervous systems. The enzymatic activity and protein component of the venom have not been described previously. In the present study, the electrophoretic profile of H. lepturus venom was determined by SDS-PAGE (12 and 15%), resulting in major protein bands at 3.5-5, 30-35 and 50-60 kDa. The enzymatic activities of the venom was, for the first time, investigated using various zymography techniques, which showed the gelatinolytic, caseinolytic, and hyaluronidase activities mainly at around 50-60 kDa, 30-40 kDa, and 40-50 kDa, respectively. Among these, the proteolytic activities was almost completely disappeared in the presence of a matrix metalloproteinase inhibitor, 1, 10-phenanthroline. Antigen-antibody interactions between the venom and its Iranian antivenin was observed by Western blotting, and it showed several antigenic proteins in the range of 30-160 kDa. This strong antigen-antibody reaction was also demonstrated through an enzyme-linked immunosorbent assay (ELISA). The gelatinase activity of the venom was suppressed by Razi institute polyvalent antivenin, suggesting the inhibitory effect of the antivenin against H. lepturus venom protease activities. Prudently, more extensive clinical studies are necessary for validation of its use in envenomed patients. Copyright 2010 Elsevier Ltd. All rights reserved.

  12. Studies on estradiol-2/4-hydroxylase activity in rat brain and liver

    International Nuclear Information System (INIS)

    Theron, C.N.

    1985-03-01

    A sensitive and specific radio-enzymatic assay was used to study estradiol-2/4-hydroxylase activity in rat liver microsomes and in microsomes obtained from 6 discrete brain areas of the rat. Kinetic parameters were determined for these enzyme activities. The effects of different P-450 inhibitors on estradiol-2/4-hydroxylase activity in brain and liver microsomes were also studied. In both organs these enzyme activities were found to be located mainly in the microsomal fraction and were inhibited by the 3 P-450 inhibitors tested. The hepatic estradiol-2/4-hydroxylase activity in adult male rats was significantly higher than that of females, but the enzyme activity in the brain did not exhibit a similar sex difference. Furthermore, estradiol-2/4-hydroxylase activity in rat liver was strongly induced by phenobarbitone treatment, but not in the brain. The phenobarbitone-induced activity in male and female rats exhibited significant kinetic differences. In female rats sexual maturation was associated with significant changes in the apparent Km of estradiol-2/4-hydroxylases in the liver and hypothalamus. Evidence was found that the in vitro estradiol-2/4-hydroxylase activity in rat brain and liver is due to more than one form of microsomal P-450. Kinetic studies showed important differences between the estradiol-2/4-hydroxylase activities in the hippocampus and hypothalamus. Significant differences in estradiol-2/4-hydroxylase activities were observed in the 6 brain areas studied, with the hippocampus showing the highest, and the hypothalamus the lowest activity at all developmental stages in both male and female rats

  13. Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1

    International Nuclear Information System (INIS)

    Santos, Camila Ramos; Meza, Andreia Navarro; Hoffmam, Zaira Bruna; Silva, Junio Cota; Alvarez, Thabata Maria; Ruller, Roberto; Giesel, Guilherme Menegon; Verli, Hugo; Squina, Fabio Marcio; Prade, Rolf Alexander; Murakami, Mario Tyago

    2010-01-01

    Research highlights: → The hyperthermostable xylanase 10B from Thermotoga petrophila RKU-1 produces exclusively xylobiose at the optimum temperature. → Circular dichroism spectroscopy suggests a coupling effect of temperature-induced structural changes with its enzymatic behavior. → Crystallographic and molecular dynamics studies indicate that conformational changes in the product release area modulate the enzyme action mode. -- Abstract: Endo-xylanases play a key role in the depolymerization of xylan and recently, they have attracted much attention owing to their potential applications on biofuels and paper industries. In this work, we have investigated the molecular basis for the action mode of xylanases 10B at high temperatures using biochemical, biophysical and crystallographic methods. The crystal structure of xylanase 10B from hyperthermophilic bacterium Thermotoga petrophila RKU-1 (TpXyl10B) has been solved in the native state and in complex with xylobiose. The complex crystal structure showed a classical binding mode shared among other xylanases, which encompasses the -1 and -2 subsites. Interestingly, TpXyl10B displayed a temperature-dependent action mode producing xylobiose and xylotriose at 20 o C, and exclusively xylobiose at 90 o C as assessed by capillary zone electrophoresis. Moreover, circular dichroism spectroscopy suggested a coupling effect of temperature-induced structural changes with this particular enzymatic behavior. Molecular dynamics simulations supported the CD analysis suggesting that an open conformational state adopted by the catalytic loop (Trp297-Lys326) provokes significant modifications in the product release area (+1,+2 and +3 subsites), which drives the enzymatic activity to the specific release of xylobiose at high temperatures.

  14. Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Camila Ramos; Meza, Andreia Navarro [Laboratorio Nacional de Biociencias (LNBio), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP (Brazil); Hoffmam, Zaira Bruna; Silva, Junio Cota; Alvarez, Thabata Maria; Ruller, Roberto [Laboratorio Nacional de Ciencia e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP (Brazil); Giesel, Guilherme Menegon; Verli, Hugo [Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Squina, Fabio Marcio [Laboratorio Nacional de Ciencia e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP (Brazil); Prade, Rolf Alexander [Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK (United States); Murakami, Mario Tyago, E-mail: mario.murakami@lnbio.org.br [Laboratorio Nacional de Biociencias (LNBio), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP (Brazil)

    2010-12-10

    Research highlights: {yields} The hyperthermostable xylanase 10B from Thermotoga petrophila RKU-1 produces exclusively xylobiose at the optimum temperature. {yields} Circular dichroism spectroscopy suggests a coupling effect of temperature-induced structural changes with its enzymatic behavior. {yields} Crystallographic and molecular dynamics studies indicate that conformational changes in the product release area modulate the enzyme action mode. -- Abstract: Endo-xylanases play a key role in the depolymerization of xylan and recently, they have attracted much attention owing to their potential applications on biofuels and paper industries. In this work, we have investigated the molecular basis for the action mode of xylanases 10B at high temperatures using biochemical, biophysical and crystallographic methods. The crystal structure of xylanase 10B from hyperthermophilic bacterium Thermotoga petrophila RKU-1 (TpXyl10B) has been solved in the native state and in complex with xylobiose. The complex crystal structure showed a classical binding mode shared among other xylanases, which encompasses the -1 and -2 subsites. Interestingly, TpXyl10B displayed a temperature-dependent action mode producing xylobiose and xylotriose at 20 {sup o}C, and exclusively xylobiose at 90 {sup o}C as assessed by capillary zone electrophoresis. Moreover, circular dichroism spectroscopy suggested a coupling effect of temperature-induced structural changes with this particular enzymatic behavior. Molecular dynamics simulations supported the CD analysis suggesting that an open conformational state adopted by the catalytic loop (Trp297-Lys326) provokes significant modifications in the product release area (+1,+2 and +3 subsites), which drives the enzymatic activity to the specific release of xylobiose at high temperatures.

  15. Enzymatic halogenation and oxidation using an alcohol oxidase-vanadium chloroperoxidase cascade

    NARCIS (Netherlands)

    But, Andrada; Noord, Van Aster; Poletto, Francesca; Sanders, Johan P.M.; Franssen, Maurice C.R.; Scott, Elinor L.

    2017-01-01

    The chemo-enzymatic cascade which combines alcohol oxidase from Hansenula polymorpha (AOXHp) with vanadium chloroperoxidase (VCPO), for the production of biobased nitriles from amino acids was investigated. In the first reaction H2O2 (and acetaldehyde) are generated from ethanol and oxygen by AOXHp.

  16. Cellulase and xylanase activity during the decomposition of three aquatic macrophytes in a tropical oxbow lagoon

    Directory of Open Access Journals (Sweden)

    L Sciessere

    2011-09-01

    Full Text Available Due to the connection between enzymatic activity and degradation of different fractions of organic matter, enzyme assays can be used to estimate degradation rates of particulate and dissolved organic carbon in freshwater systems. The aim of this study was to quantify and model the enzymatic degradation involving the decomposition of macrophytes, describing temporal activity of cellulases (EC 3.2.1.4 and EC 3.2.1.91 and xylanase (EC 3.2.1.8 during in situ decomposition of three aquatic macrophytes (Salvinia sp., Eichhornia azurea and Cyperus giganteus on the surface and water-sediment interface (w-s interface of an oxbow lagoon (Óleo lagoon within a natural Brazilian Savanna Reserve. Overall, the enzymatic degradation of aquatic macrophytes in Óleo lagoon occurred during the whole year and was initiated together with leaching. Xylanase production was ca. 5 times higher than cellulase values due to easy access to this compound by cellulolytic microorganisms. Enzymatic production and detritus mass decay were similar on the surface and w-s interface. Salvinia sp. was the most recalcitrant detritus, with low mass decay and enzymatic activity. E. azurea and C. giganteus decomposition rates and enzymatic production were high and similar. Due to the physicochemical homogeneity observed in the Óleo lagoon, the differences between the decay rates of each species are mostly related with detritus chemical quality.

  17. Enzymatic hydrolysis of lactose of whey permeate

    Directory of Open Access Journals (Sweden)

    Karina Nascimento de Almeida

    2015-09-01

    Full Text Available The whey permeate is the residual of the concentration process of the whey proteins by ultrafiltration method. It contains important nutrients such as lactose, minerals and some proteins and lipids. It is without an ending industrial waste that causes serious damage to the environment. For its full use the lactose must be hydrolyzed to enable its consumption by intolerant people. The enzymatic hydrolysis by lactase (β-galactosidase of Kluyveromyces lactis yeast is a safe method that does not compromise the integrity of other nutrients, enabling further use of the permeate as a raw material. This study aimed to perform tests of enzymatic hydrolysis of lactose in whey permeate formulations in a concentration of 0.2%, 0.7% and 1% at 30, 60 and 90 minutes with pH 6.3 medium and 37 °C. The reactions were monitored by high performance liquid chromatography which showed that the enzyme concentration of 0.7% at time 30 minutes formulations became safe for consumption by lactose intolerant people, according to minimum levels established by law.

  18. Enzymatic lipid oxidation by eosinophils propagates coagulation, hemostasis, and thrombotic disease

    Science.gov (United States)

    Uderhardt, Stefan; Ackermann, Jochen A.; Fillep, Tobias; Hammond, Victoria J.; Willeit, Johann; Stark, Konstantin; Rossaint, Jan; Schubert, Irene; Mielenz, Dirk; Dietel, Barbara; Raaz-Schrauder, Dorette; Ay, Cihan; Thaler, Johannes; Heim, Christian; Collins, Peter W.; Schabbauer, Gernot; Mackman, Nigel; Voehringer, David; Nadler, Jerry L.; Lee, James J.; Massberg, Steffen; Rauh, Manfred; O’Donnell, Valerie B.

    2017-01-01

    Blood coagulation is essential for physiological hemostasis but simultaneously contributes to thrombotic disease. However, molecular and cellular events controlling initiation and propagation of coagulation are still incompletely understood. In this study, we demonstrate an unexpected role of eosinophils during plasmatic coagulation, hemostasis, and thrombosis. Using a large-scale epidemiological approach, we identified eosinophil cationic protein as an independent and predictive risk factor for thrombotic events in humans. Concurrent experiments showed that eosinophils contributed to intravascular thrombosis by exhibiting a strong endogenous thrombin-generation capacity that relied on the enzymatic generation and active provision of a procoagulant phospholipid surface enriched in 12/15-lipoxygenase–derived hydroxyeicosatetraenoic acid–phosphatidylethanolamines. Our findings reveal a previously unrecognized role of eosinophils and enzymatic lipid oxidation as regulatory elements that facilitate both hemostasis and thrombosis in response to vascular injury, thus identifying promising new targets for the treatment of thrombotic disease. PMID:28566277

  19. Increased release of fermentable sugars from elephant grass by enzymatic hydrolysis in the presence of surfactants

    International Nuclear Information System (INIS)

    Menegol, Daiane; Scholl, Angélica Luisi; Fontana, Roselei Claudete; Dillon, Aldo José Pinheiro; Camassola, Marli

    2014-01-01

    Highlights: • Milling is an attractive method to enhance the enzymatic hydrolysis of biomass. • Surfactants improve the efficiency of lignocellulose enzymatic hydrolysis. • Pretreatment with NaOH, smaller particle size and Tween 80® were more efficient. - Abstract: In the search for renewable energy sources, elephant grass is an alternative substrate for ethanol production, but this substrate must be hydrolyzed by cellulases and xylanases to liberate fermentable sugars. During enzymatic hydrolysis, cellulase activity is reduced by the irreversible adsorption of cellulase onto cellulose, decreasing the rate of hydrolysis. Adding surfactants during hydrolysis can improve the process. The effects of Tween® and Triton® surfactants on the enzymatic hydrolysis of elephant grass were evaluated in this context. The data indicate that pretreatment with sodium hydroxide, along with a smaller particle size (0.075–0.152 mm) and the use of Tween 80®, increased the efficiency of releasing reducing sugars from pretreated elephant grass biomass. Thus, it is possible to reduce grinding costs in second-generation ethanol production through the use of surfactants, as they allow efficient hydrolysis of larger biomass particles

  20. L-[4-11C]aspartic acid: enzymatic synthesis, myocardial uptake, and metabolism

    International Nuclear Information System (INIS)

    Barrio, J.R.; Egbert, J.E.; Henze, E.; Schelbert, H.R.; Baumgartner, F.J.

    1982-01-01

    Sterile, pyrogen-free L-[4- 11 C]aspartic acid was prepared from 11 CO 2 using phosphoenolpyruvate carboxylase and glutamic/oxaloacetic acid transaminase immobilized on Sepharose supports to determine if it is a useful indicator for in vivo, noninvasive determination of myocardial metabolism. An intracoronary bolus injection of L-[4- 11 C]aspartic acid into dog myocardium showed a triexponential clearance curve with maximal production of 11 CO 2 100 s after injection. Inactivation of myocardial transaminase activity modified the tracer clearance and inhibited the production of 11 CO 2 . Positron-computed tomography imaging showed that the 11 C activities retained in rhesus monkey myocardium are higher than those observed in dog heart after intravenous injection of L-[4- 11 C]aspartic acid. These findings demonstrated the rapid incorporation of the carbon skeleton of L-aspartic acid into the tricarboxylic acid cycle after enzymatic transamination in myocardium and suggested that L-[4- 11 C]aspartic acid could be of value for in vivo, noninvasive assessment of local myocardial metabolism