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Sample records for epimerase enzymatic function

  1. Crystal structure of methylmalonyl-coenzyme A epimerase from P. shermanii: a novel enzymatic function on an ancient metal binding scaffold.

    Science.gov (United States)

    McCarthy, A A; Baker, H M; Shewry, S C; Patchett, M L; Baker, E N

    2001-07-03

    Methylmalonyl-CoA epimerase (MMCE) is an essential enzyme in the breakdown of odd-numbered fatty acids and of the amino acids valine, isoleucine, and methionine. Present in many bacteria and in animals, it catalyzes the conversion of (2R)-methylmalonyl-CoA to (2S)-methylmalonyl-CoA, the substrate for the B12-dependent enzyme, methylmalonyl-CoA mutase. Defects in this pathway can result in severe acidosis and cause damage to the central nervous system in humans. The crystal structure of MMCE from Propionibacterium shermanii has been determined at 2.0 A resolution. The MMCE monomer is folded into two tandem betaalphabetabetabeta modules that pack edge-to-edge to generate an 8-stranded beta sheet. Two monomers then pack back-to-back to create a tightly associated dimer. In each monomer, the beta sheet curves around to create a deep cleft, in the floor of which His12, Gln65, His91, and Glu141 provide a binding site for a divalent metal ion, as shown by the binding of Co2+. Modeling 2-methylmalonate into the active site identifies two glutamate residues as the likely essential bases for the epimerization reaction. The betaalphabetabetabeta modules of MMCE correspond with those found in several other proteins, including bleomycin resistance protein, glyoxalase I, and a family of extradiol dioxygenases. Differences in connectivity are consistent with the evolution of these very different proteins from a common precursor by mechanisms of gene duplication and domain swapping. The metal binding residues also align precisely, and striking structural similarities between MMCE and glyoxalase I suggest common mechanisms in their respective epimerization and isomerization reactions.

  2. 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.

  3. Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets.

    Directory of Open Access Journals (Sweden)

    Paul A Mann

    2016-05-01

    Full Text Available Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro β-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA and S. epidermidis (MRSE. Likewise, the β-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore β-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation.

  4. Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets

    Energy Technology Data Exchange (ETDEWEB)

    Mann, Paul A.; Müller, Anna; Wolff, Kerstin A.; Fischmann, Thierry; Wang, Hao; Reed, Patricia; Hou, Yan; Li, Wenjin; Müller, Christa E.; Xiao, Jianying; Murgolo, Nicholas; Sher, Xinwei; Mayhood, Todd; Sheth, Payal R.; Mirza, Asra; Labroli, Marc; Xiao, Li; McCoy, Mark; Gill, Charles J.; Pinho, Mariana G.; Schneider, Tanja; Roemer, Terry (Merck); (Bonn); (FCT/UNL)

    2016-05-04

    Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro β-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the β-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore β-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation.

  5. Functional analysis of mutations in UDP-galactose-4-epimerase (GALE) associated with galactosemia in Korean patients using mammalian GALE-null cells.

    Science.gov (United States)

    Bang, You-Lim; Nguyen, Trang T T; Trinh, Tram T B; Kim, Yun J; Song, Junghan; Song, Young-Han

    2009-04-01

    Galactosemia is caused by defects in the galactose metabolic pathway, which consists of three enzymes, including UDP-galactose-4-epimerase (GALE). We previously reported nine mutations in Korean patients with epimerase-deficiency galactosemia. In order to determine the functional consequences of these mutations, we expressed wild-type and mutant GALE proteins in 293T cells. GALE(E165K) and GALE(W336X) proteins were unstable, had reduced half-life, formed aggregates and were partly degraded by the proteasome complex. When expressed in GALE-null ldlD cells GALE(E165K), GALE(R239W), GALE(G302D) and GALE(W336X) had no detectable enzyme activity, although substantial amounts of protein were detected in western blots. The relative activities of other mutants were lower than that of wild-type. In addition, unlike wild-type, GALE(R239W) and GALE(G302D) were not able to rescue galactose-sensitive cell proliferation when stably expressed in ldlD cells. The four inactive mutant proteins did not show defects in dimerization or affect the activity of other mutant alleles identified in patients. Our observations show that altered protein stability is due to misfolding and that loss or reduction of enzyme activity is responsible for the molecular defects underlying GALE-deficiency galactosemia.

  6. Immobilization on graphene oxide improves the thermal stability and bioconversion efficiency of D-psicose 3-epimerase for rare sugar production.

    Science.gov (United States)

    Dedania, Samir R; Patel, Manisha J; Patel, Dijit M; Akhani, Rekha C; Patel, Darshan H

    2017-12-01

    D-Psicose (D-ribo-2-hexulose or D-allulose), an epimer of D-fructose is considered as a rare low-calorie sugar displaying important physiological functions. Enzymatic production using ketose 3-epimerases is the feasible process for the production of D-Psicose. However, major drawbacks in application of ketose 3-epimerases are bioconversion efficiency and reusability of the enzyme. We have attempted immobilization of ketose 3-epimerases from Agrobacterium tumefaciens (agtu) D-psicose 3-epimerase (DPEase) on graphene oxide. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and Thermo gravimetric analysis (TGA) showed that the enzyme was successfully immobilized on the graphene oxide. Graphene oxide immobilized agtu-DPEase (GO-agtu-DPEase) shows pH optima at 7.5 and 60°C as higher working temperature. Significant improvement in thermal stability was observed which showed half-life of 720min at 60°C whereas Agrobacterium tumefaciens (agtu) DPEase displayed 3.99min. At equilibrium, 40:60 (D-psicose: D-fructose) the bioconversion efficiency was accounted for Graphene oxide immobilized DPEase which is higher than the agtu-DPEase. Graphene oxide immobilized DPEase showed bioconversion efficiency up to 10 cycles of reusability. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Hits, Fhits and Nits: beyond enzymatic function.

    Science.gov (United States)

    Huebner, Kay; Saldivar, Joshua C; Sun, Jin; Shibata, Hidetaka; Druck, Teresa

    2011-01-01

    We have briefly summarized what is known about these proteins, but in closing wish to feature the outstanding questions. Hint1 was discovered mistakenly as an inhibitor of Protein Kinase C and designated Pkci, a designation that still confuses the literature. The other Hint family members were discovered by homology to Hint1. Aprataxin was discovered as a result of the hunt for a gene responsible for AOA1. Fhit was discovered through cloning of a familial chromosome translocation breakpoint on chromosome 3 that interrupts the large FHIT gene within an intron, in the FRA3B chromosome region (Ohta et al., 1996), now known to be the region of the human genome most susceptible to DNA damage due to replication stress (Durkin et al., 2008). The NitFhit fusion genewas discovered during searches for Fhit homologs in flies and worms because the fly/worm Nit polypeptide is fused to the 5'-end of the Fhit gene; the mammalian Nit gene family was discovered because of the NitFhit fusion gene, in searches for homologs to the Nit polypeptide of the NitFhit gene. Each of the Hit family member proteins is reported to have enzymatic activities toward putative substrates involving nucleosides or dinucleosides. Most surprisingly, each of the Hit family proteins discussed has been implicated in important DNA damage response pathways and/or tumor suppression pathways. And for each of them it has been difficult to assign definite substrates, to know if the substrates and catalytic products have biological functions, to know if that function is related to the DNA damage response and suppressor functions, and to precisely define the pathways through which tumor suppression occurs. When the fly Nit sequence was found at the 5'-end of the fly Fhit gene, this gene was hailed as a Rosetta stone gene/protein that would help in discovery of the function of Fhit, because the Nit protein should be in the same signal pathway (Pace et al., 2000). However, the mammalian Nit family proteins have

  8. Structural and functional characterization of the R-modules in alginate C-5 epimerases AlgE4 and AlgE6 from Azotobacter vinelandii.

    Science.gov (United States)

    Buchinger, Edith; Knudsen, Daniel H; Behrens, Manja A; Pedersen, Jan Skov; Aarstad, Olav A; Tøndervik, Anne; Valla, Svein; Skjåk-Bræk, Gudmund; Wimmer, Reinhard; Aachmann, Finn L

    2014-11-07

    The bacterium Azotobacter vinelandii produces a family of seven secreted and calcium-dependent mannuronan C-5 epimerases (AlgE1-7). These epimerases are responsible for the epimerization of β-D-mannuronic acid (M) to α-L-guluronic acid (G) in alginate polymers. The epimerases display a modular structure composed of one or two catalytic A-modules and from one to seven R-modules having an activating effect on the A-module. In this study, we have determined the NMR structure of the three individual R-modules from AlgE6 (AR1R2R3) and the overall structure of both AlgE4 (AR) and AlgE6 using small angle x-ray scattering. Furthermore, the alginate binding ability of the R-modules of AlgE4 and AlgE6 has been studied with NMR and isothermal titration calorimetry. The AlgE6 R-modules fold into an elongated parallel β-roll with a shallow, positively charged groove across the module. Small angle x-ray scattering analyses of AlgE4 and AlgE6 show an overall elongated shape with some degree of flexibility between the modules for both enzymes. Titration of the R-modules with defined alginate oligomers shows strong interaction between AlgE4R and both oligo-M and MG, whereas no interaction was detected between these oligomers and the individual R-modules from AlgE6. A combination of all three R-modules from AlgE6 shows weak interaction with long M-oligomers. Exchanging the R-modules between AlgE4 and AlgE6 resulted in a novel epimerase called AlgE64 with increased G-block forming ability compared with AlgE6. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Functional characterization and transcriptional analysis of galE gene encoding a UDP-galactose 4-epimerase in Xanthomonas campestris pv. campestris.

    Science.gov (United States)

    Li, Chien-Te; Liao, Chao-Tsai; Du, Shin-Chiao; Hsiao, Yu-Ping; Lo, Hsueh-Hsia; Hsiao, Yi-Min

    2014-01-01

    The Gram-negative plant pathogen Xanthomonas campestris pv. campestris (Xcc) is the causative agent of black rot in crucifers, a disease that causes tremendous agricultural loss. In this study, the Xcc galE gene was characterized. Sequence and mutational analysis demonstrated that the Xcc galE encodes a UDP-galactose 4-epimerase (EC 5.1.3.2), which catalyzes the interconversion of UDP-galactose and UDP-glucose. Alanine substitution of the putative catalytic residues (Ser124, Tyr147, and Lys151) of GalE caused loss of epimerase activity. Further study showed that the Xcc galE mutant had reduced biofilm formation ability. Furthermore, reporter assays revealed that galE transcription exhibits a distinct expression profile under different culture conditions, is subject to catabolite repression, and is positively regulated by Clp and RpfF. In addition, the galE transcription initiation site was mapped. This is the first time that UDP-galactose 4-epimerase has been characterized in the crucifer pathogen Xcc. Copyright © 2013 Elsevier GmbH. All rights reserved.

  10. Galactose Epimerase Deficiency: Expanding the Phenotype

    NARCIS (Netherlands)

    Dias Costa, Filipa; Ferdinandusse, Sacha; Pinto, Carla; Dias, Andrea; Keldermans, Liesbeth; Quelhas, Dulce; Matthijs, Gert; Mooijer, Petra A.; Diogo, Luísa; Jaeken, Jaak; Garcia, Paula

    2017-01-01

    Galactose epimerase deficiency is an inborn error of metabolism due to uridine diphosphate-galactose-4'-epimerase (GALE) deficiency. We report the clinical presentation, genetic and biochemical studies in two siblings with generalized GALE deficiency.Patient 1: The first child was born with a

  11. Enzymatic and non-enzymatic functions of the lysyl oxidase family in bone.

    Science.gov (United States)

    Trackman, Philip C

    2016-01-01

    Advances in the understanding of the biological roles of the lysyl oxidase family of enzyme proteins in bone structure and function are reviewed. This family of proteins is well-known as catalyzing the final reaction required for cross-linking of collagens and elastin. Novel emerging roles for these proteins in the phenotypic development of progenitor cells and in angiogenesis are highlighted and which point to enzymatic and non-enzymatic roles for this family in bone development and homeostasis and in disease. The explosion of interest in the lysyl oxidase family in the cancer field highlights the need to have a better understanding of the functions of this protein family in normal and abnormal connective tissue homeostasis at fundamental molecular and cellular levels including in mineralized tissues. Copyright © 2015 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

  12. Functional bio-based polyesters by enzymatic polymerization

    DEFF Research Database (Denmark)

    Daugaard, Anders Egede; Hoffmann, Christian; Andersen, Christian

    based on the polyesters were shown to be degradable and could be prepared either from the pure polyester or from prefunctionalized polyesters, though the thiol-ene reactions were found to be less effective. Since then a new monomer, trans-2,5-dihydroxy-3-pentenoic acid methyl ester (DPM) has been...... for grafting. In addition to this, thiol-ene reactions using hexanethiol, mercaptoethanol, mercaptoacetic acid, 2-ethylhexanethiol and thiophenol were conducted on the internal double bond resulting in conversions of 32-100%. Given the lower reactivity of the internal double bond the extent...... when considering monomers from bio-based feedstock that generally contain a high number of functional groups such as a both secondary and primary alcohols. Enzymatic polymerization can be conducted at relatively low temperature and thereby is well suited for sensitive monomers. Recently enzymatic...

  13. Catalytic Mechanism and Mode of Action of the Periplasmic Alginate Epimerase AlgG

    NARCIS (Netherlands)

    Wolfram, Francis; Kitova, Elena N.; Robinson, Howard; Walvoort, Marthe T. C.; Codee, Jeroen D. C.; Klassen, John S.; Howell, P. Lynne

    2014-01-01

    Background: The alginate epimerase AlgG converts mannuronate to its C5 epimer guluronate at the polymer level. Results: The structure of Pseudomonas syringae AlgG has been determined, and the protein has been functionally characterized. Conclusion: His(319) acts as the catalytic base, whereas

  14. Enzymatic regulation of functional vascular networks using gelatin hydrogels

    Science.gov (United States)

    Chuang, Chia-Hui; Lin, Ruei-Zeng; Tien, Han-Wen; Chu, Ya-Chun; Li, Yen-Cheng; Melero-Martin, Juan M.; Chen, Ying-Chieh

    2015-01-01

    To manufacture tissue engineering-based functional tissues, scaffold materials that can be sufficiently vascularized to mimic the functionality and complexity of native tissues are needed. Currently, vascular network bioengineering is largely carried out using natural hydrogels as embedding scaffolds, but most natural hydrogels have poor mechanical stability and durability, factors that critically limit their widespread use. In this study, we examined the suitability of gelatin-phenolic hydroxyl (gelatin-Ph) hydrogels that can be enzymatically crosslinked, allowing tuning of the storage modulus and the proteolytic degradation rate, for use as injectable hydrogels to support the human progenitor cell-based formation of a stable and mature vascular network. Porcine gelatin-Ph hydrogels were found to be cytocompatible with human blood-derived endothelial colony-forming cells and white adipose tissue-derived mesenchymal stem cells, resulting in >87% viability, and cell proliferation and spreading could be modulated by using hydrogels with different proteolytic degradability and stiffness. In addition, gelatin was extracted from mouse dermis and murine gelatin-Ph hydrogels were prepared. Importantly, implantation of human cell-laden porcine or murine gelatin-Ph hydrogels into immunodeficient mice resulted in the rapid formation of functional anastomoses between the bioengineered human vascular network and the mouse vasculature. Furthermore, the degree of enzymatic crosslinking of the gelatin-Ph hydrogels could be used to modulate cell behavior and the extent of vascular network formation in vivo. Our report details a technique for the synthesis of gelatin-Ph hydrogels from allogeneic or xenogeneic dermal skin and suggests that these hydrogels can be used for biomedical applications that require the formation of microvascular networks, including the development of complex engineered tissues. PMID:25749296

  15. UDP-galactose 4' epimerase (GALE) is essential for development of Drosophila melanogaster.

    Science.gov (United States)

    Sanders, Rebecca D; Sefton, Jennifer M I; Moberg, Kenneth H; Fridovich-Keil, Judith L

    2010-01-01

    UDP-galactose 4' epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose in the final step of the Leloir pathway; human GALE (hGALE) also interconverts UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. GALE therefore plays key roles in the metabolism of dietary galactose, in the production of endogenous galactose, and in maintaining the ratios of key substrates for glycoprotein and glycolipid biosynthesis. Partial impairment of hGALE results in the potentially lethal disorder epimerase-deficiency galactosemia. We report here the generation and initial characterization of a first whole-animal model of GALE deficiency using the fruit fly Drosophila melanogaster. Our results confirm that GALE function is essential in developing animals; Drosophila lacking GALE die as embryos but are rescued by the expression of a human GALE transgene. Larvae in which GALE has been conditionally knocked down die within days of GALE loss. Conditional knockdown and transgene expression studies further demonstrate that GALE expression in the gut primordium and Malpighian tubules is both necessary and sufficient for survival. Finally, like patients with generalized epimerase deficiency galactosemia, Drosophila with partial GALE loss survive in the absence of galactose but succumb in development if exposed to dietary galactose. These data establish the utility of the fly model of GALE deficiency and set the stage for future studies to define the mechanism(s) and modifiers of outcome in epimerase deficiency galactosemia.

  16. A Novel Bacterial Enzyme with d-Glucuronyl C5-epimerase Activity*

    Science.gov (United States)

    Raedts, John; Lundgren, Magnus; Kengen, Servé W. M.; Li, Jin-Ping; van der Oost, John

    2013-01-01

    Glycosaminoglycans are biologically active polysaccharides that are found ubiquitously in the animal kingdom. The biosynthesis of these complex polysaccharides involves complicated reactions that turn the simple glycosaminoglycan backbone into highly heterogeneous structures. One of the modification reactions is the epimerization of d-glucuronic acid to its C5-epimer l-iduronic acid, which is essential for the function of heparan sulfate. Although l-iduronic acid residues have been shown to exist in polysaccharides of some prokaryotes, there has been no experimental evidence for the existence of a prokaryotic d-glucuronyl C5-epimerase. This work for the first time reports on the identification of a bacterial enzyme with d-glucuronyl C5-epimerase activity. A gene of the marine bacterium Bermanella marisrubri sp. RED65 encodes a protein (RED65_08024) of 448 amino acids that has an overall 37% homology to the human d-glucuronic acid C5-epimerase. Alignment of this peptide with the human and mouse sequences revealed a 60% similarity at the carboxyl terminus. The recombinant protein expressed in Escherichia coli showed epimerization activity toward substrates generated from heparin and the E. coli K5 capsular polysaccharide, thereby providing the first evidence for bacterial d-glucuronyl C5-epimerase activity. These findings may eventually be used for modification of mammalian glycosaminoglycans. PMID:23824188

  17. Intramolecular epistasis and the evolution of a new enzymatic function.

    Directory of Open Access Journals (Sweden)

    Sajid Noor

    Full Text Available Atrazine chlorohydrolase (AtzA and its close relative melamine deaminase (TriA differ by just nine amino acid substitutions but have distinct catalytic activities. Together, they offer an informative model system to study the molecular processes that underpin the emergence of new enzymatic function. Here we have constructed the potential evolutionary trajectories between AtzA and TriA, and characterized the catalytic activities and biophysical properties of the intermediates along those trajectories. The order in which the nine amino acid substitutions that separate the enzymes could be introduced to either enzyme, while maintaining significant catalytic activity, was dictated by epistatic interactions, principally between three amino acids within the active site: namely, S331C, N328D and F84L. The mechanistic basis for the epistatic relationships is consistent with a model for the catalytic mechanisms in which protonation is required for hydrolysis of melamine, but not atrazine.

  18. Carbohydrate Utilization in Streptococcus thermophilus : Characterization of the Genes for Aldose 1-Epimerase (Mutarotase) and UDPglucose 4-Epimerase

    NARCIS (Netherlands)

    Poolman, Bert; Royer, Theresa J.; Mainzer, Stanley E.; Schmidt, Brian F.

    1990-01-01

    The complete nucleotide sequences of the genes encoding aldose 1-epimerase (mutarotase) (galM) and UDPglucose 4-epimerase (galE) and flanking regions of Streptococcus thermophilus have been determined. Both genes are located immediately upstream of the S. thermophilus lac operon. To facilitate the

  19. Redox control of enzymatic functions: The electronics of life's circuitry.

    Science.gov (United States)

    Bonini, Marcelo G; Consolaro, Marcia E L; Hart, Peter C; Mao, Mao; de Abreu, Andre Luelsdorf Pimenta; Master, Alyssa M

    2014-03-26

    The field of redox biology has changed tremendously over the past 20 years. Formerly regarded as bi-products of the aerobic metabolism exclusively involved in tissue damage, reactive oxygen species (ROS) are now recognized as active participants of cell signaling events in health and in disease. In this sense, ROS and the more recently defined reactive nitrogen species (RNS) are, just like hormones and second messengers, acting as fundamental orchestrators of cell signaling pathways. The chemical modification of enzymes by ROS and RNS (that result in functional enzymatic alterations) accounts for a considerable fraction of the transient and persistent perturbations imposed by variations in oxidant levels. Upregulation of ROS and RNS in response to stress is a common cellular response that foments adaptation to a variety of physiologic alterations (hypoxia, hyperoxia, starvation, and cytokine production). Frequently, these are beneficial and increase the organisms' resistance against subsequent acute stress (preconditioning). Differently, the sustained ROS/RNS-dependent rerouting of signaling produces irreversible alterations in cellular functioning, often leading to pathogenic events. Thus, the duration and reversibility of protein oxidations define whether complex organisms remain "electronically" healthy. Among the 20 essential amino acids, four are particularly susceptible to oxidation: cysteine, methionine, tyrosine, and tryptophan. Here, we will critically review the mechanisms, implications, and repair systems involved in the redox modifications of these residues in proteins while analyzing well-characterized prototypic examples. Occasionally, we will discuss potential consequences of amino acid oxidation and speculate on the biologic necessity for such events in the context of adaptative redox signaling. © 2014 IUBMB Life, 2014. © 2014 International Union of Biochemistry and Molecular Biology.

  20. Energy Landscape of Alginate-Epimerase Interactions Assessed by Optical Tweezers and Atomic Force Microscopy.

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    Armend Gazmeno Håti

    Full Text Available Mannuronan C-5 epimerases are a family of enzymes that catalyze epimerization of alginates at the polymer level. This group of enzymes thus enables the tailor-making of various alginate residue sequences to attain various functional properties, e.g. viscosity, gelation and ion binding. Here, the interactions between epimerases AlgE4 and AlgE6 and alginate substrates as well as epimerization products were determined. The interactions of the various epimerase-polysaccharide pairs were determined over an extended range of force loading rates by the combined use of optical tweezers and atomic force microscopy. When studying systems that in nature are not subjected to external forces the access to observations obtained at low loading rates, as provided by optical tweezers, is a great advantage since the low loading rate region for these systems reflect the properties of the rate limiting energy barrier. The AlgE epimerases have a modular structure comprising both A and R modules, and the role of each of these modules in the epimerization process were examined through studies of the A- module of AlgE6, AlgE6A. Dynamic strength spectra obtained through combination of atomic force microscopy and the optical tweezers revealed the existence of two energy barriers in the alginate-epimerase complexes, of which one was not revealed in previous AFM based studies of these complexes. Furthermore, based on these spectra estimates of the locations of energy transition states (xβ, lifetimes in the absence of external perturbation (τ0 and free energies (ΔG# were determined for the different epimerase-alginate complexes. This is the first determination of ΔG# for these complexes. The values determined were up to 8 kBT for the outer barrier, and smaller values for the inner barriers. The size of the free energies determined are consistent with the interpretation that the enzyme and substrate are thus not tightly locked at all times but are able to relocate

  1. Functional palm oil-based margarine by enzymatic interesterification

    DEFF Research Database (Denmark)

    Ibrahim, Nuzul Amri Bin; Xu, Xuebing

    Palm stearin, palm kernel and fish oils were blended to a various composition ratios and enzymatically interesterified by Lipozyme TL IM lipase (Thermomyces lanuginosa) using a continuous packed bed reactor. The ratio of the oils ranged from 60-90%, 10-40% and 0-10% respectively. The enzyme...

  2. 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.

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

    Science.gov (United States)

    Amidi, Shervine; Amidi, Afshine; Vlachakis, Dimitrios; Paragios, Nikos; Zacharaki, Evangelia I

    2017-01-01

    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.

  4. Precision Synthesis of Functional Polysaccharide Materials by Phosphorylase-Catalyzed Enzymatic Reactions

    Directory of Open Access Journals (Sweden)

    Jun-ichi Kadokawa

    2016-04-01

    Full Text Available In this review article, the precise synthesis of functional polysaccharide materials using phosphorylase-catalyzed enzymatic reactions is presented. This particular enzymatic approach has been identified as a powerful tool in preparing well-defined polysaccharide materials. Phosphorylase is an enzyme that has been employed in the synthesis of pure amylose with a precisely controlled structure. Similarly, using a phosphorylase-catalyzed enzymatic polymerization, the chemoenzymatic synthesis of amylose-grafted heteropolysaccharides containing different main-chain polysaccharide structures (e.g., chitin/chitosan, cellulose, alginate, xanthan gum, and carboxymethyl cellulose was achieved. Amylose-based block, star, and branched polymeric materials have also been prepared using this enzymatic polymerization. Since phosphorylase shows a loose specificity for the recognition of substrates, different sugar residues have been introduced to the non-reducing ends of maltooligosaccharides by phosphorylase-catalyzed glycosylations using analog substrates such as α-d-glucuronic acid and α-d-glucosamine 1-phosphates. By means of such reactions, an amphoteric glycogen and its corresponding hydrogel were successfully prepared. Thermostable phosphorylase was able to tolerate a greater variance in the substrate structures with respect to recognition than potato phosphorylase, and as a result, the enzymatic polymerization of α-d-glucosamine 1-phosphate to produce a chitosan stereoisomer was carried out using this enzyme catalyst, which was then subsequently converted to the chitin stereoisomer by N-acetylation. Amylose supramolecular inclusion complexes with polymeric guests were obtained when the phosphorylase-catalyzed enzymatic polymerization was conducted in the presence of the guest polymers. Since the structure of this polymeric system is similar to the way that a plant vine twines around a rod, this polymerization system has been named

  5. Glucose level determination with a multi-enzymatic cascade reaction in a functionalized glass chip.

    Science.gov (United States)

    Costantini, Francesca; Tiggelaar, Roald; Sennato, Simona; Mura, Francesco; Schlautmann, Stefan; Bordi, Federico; Gardeniers, Han; Manetti, Cesare

    2013-09-07

    In this work we show the functionalization of the interior of microfluidic glass chips with poly(2-hydroxyethyl methacrylate) polymer brushes as anchors for co-immobilization of the enzymes glucose-oxidase and horseradish peroxidase. The formation of the brush layer and subsequent immobilization of these enzymes have been characterized on flat surfaces by atomic force microscopy and Fourier transform infrared spectroscopy, and studied inside glass chips by field emission scanning microscopy. Enzyme-functionalized glass chips have been applied for performing a multi-enzymatic cascade reaction for the fast (20 s) determination of glucose in human blood samples and the result is in excellent agreement with values obtained from the conventional hospital laboratory. The limit of detection of this bi-enzymatic method is 60 μM. With the advantages of high selectivity and reproducibility, this functionalization method can be used for improving the efficiency of glucose sensors.

  6. Energy Landscape of Alginate-Epimerase Interactions Assessed by Optical Tweezers and Atomic Force Microscopy

    Science.gov (United States)

    Håti, Armend Gazmeno; Aachmann, Finn Lillelund; Stokke, Bjørn Torger; Skjåk-Bræk, Gudmund; Sletmoen, Marit

    2015-01-01

    Mannuronan C-5 epimerases are a family of enzymes that catalyze epimerization of alginates at the polymer level. This group of enzymes thus enables the tailor-making of various alginate residue sequences to attain various functional properties, e.g. viscosity, gelation and ion binding. Here, the interactions between epimerases AlgE4 and AlgE6 and alginate substrates as well as epimerization products were determined. The interactions of the various epimerase–polysaccharide pairs were determined over an extended range of force loading rates by the combined use of optical tweezers and atomic force microscopy. When studying systems that in nature are not subjected to external forces the access to observations obtained at low loading rates, as provided by optical tweezers, is a great advantage since the low loading rate region for these systems reflect the properties of the rate limiting energy barrier. The AlgE epimerases have a modular structure comprising both A and R modules, and the role of each of these modules in the epimerization process were examined through studies of the A- module of AlgE6, AlgE6A. Dynamic strength spectra obtained through combination of atomic force microscopy and the optical tweezers revealed the existence of two energy barriers in the alginate-epimerase complexes, of which one was not revealed in previous AFM based studies of these complexes. Furthermore, based on these spectra estimates of the locations of energy transition states (xβ), lifetimes in the absence of external perturbation (τ0) and free energies (ΔG#) were determined for the different epimerase–alginate complexes. This is the first determination of ΔG# for these complexes. The values determined were up to 8 kBT for the outer barrier, and smaller values for the inner barriers. The size of the free energies determined are consistent with the interpretation that the enzyme and substrate are thus not tightly locked at all times but are able to relocate. Together with the

  7. Phenotypic characterization and functional analysis of human tumor immune infiltration after mechanical and enzymatic disaggregation.

    Science.gov (United States)

    Grange, Cécile; Létourneau, Jason; Forget, Marie-Andrée; Godin-Ethier, Jessica; Martin, Jocelyne; Liberman, Moishe; Latour, Mathieu; Widmer, Hugues; Lattouf, Jean-Baptiste; Piccirillo, Ciriaco A; Cailhier, Jean-François; Lapointe, Réjean

    2011-09-30

    Multi-parametric flow cytometry analysis is a reliable method for phenotypic and functional characterization of tumor infiltrating immune cells (TIIC). The isolation of infiltrating leukocytes from solid tumors can be achieved through various methods which can be both enzymatic and mechanical; however, these methods may alter cell biology. The aim of this study was to compare the effects of three tissue disaggregation techniques on TIIC biology in breast, kidney and lung tumor specimens. We therefore compared two enzymatic treatments using either collagenase type IA alone or in combination with collagenase type IV and DNase I type II, and one mechanical system (Medimachine™). We evaluated the impact of treatments on cell viability, surface marker integrity and proliferative capacity. We show that cell viability was not significantly altered by treatments. However, enzymatic treatments decreased cell proliferation; specifically collagenases and DNase provoked a significant decrease in detection of surface markers such as CD4, CD8, CD45RA and CD14, indicating that results of phenotypic studies employing these techniques could be affected. In conclusion, mechanical tissue disaggregation by Medimachine™ appears to be optimal to maintain phenotypic and functional TIIC features. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Hydrophobic surface functionalization of lignocellulosic jute fabrics by enzymatic grafting of octadecylamine.

    Science.gov (United States)

    Dong, Aixue; Fan, Xuerong; Wang, Qiang; Yu, Yuanyuan; Cavaco-Paulo, Artur

    2015-08-01

    Enzymatic grafting of synthetic molecules onto lignins provides a mild and eco-friendly alternative for the functionalization of lignocellulosic materials. In this study, laccase-mediated grafting of octadecylamine (OA) onto lignin-rich jute fabrics was investigated for enhancing the surface hydrophobicity. First, the lignins in jute fabrics were isolated and analyzed in the macromolecular level by MALDI-TOF MS, (1)H NMR, (13)C NMR, and HSQC-NMR. Then, the surface of jute fabrics was characterized by FT-IR, XPS, and SEM. Subsequently, the nitrogen content of jute fabrics was determined by the micro-Kjeldahl method, and the grafting percentage (Gp) and grafting efficiency (GE) of the enzymatic reaction were calculated. Finally, the surface hydrophobicity of the jute fabrics was estimated by contact angle and wetting time measurements. The results indicate that the OA monomers were successfully grafted onto the lignin moieties on the jute fiber surface by laccase with Gp and GE values of 0.712% and 10.571%, respectively. Moreover, the modified jute fabrics via OA-grafting showed an increased wetting time of 18.5 min and a contact angle of 116.72°, indicating that the surface hydrophobicity of the jute fabrics increased after the enzymatic grafting modification with hydrophobic OA molecules. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Selection and validation of enzymatic activities as functional markers in wood biotechnology and fungal ecology.

    Science.gov (United States)

    Mathieu, Yann; Gelhaye, Eric; Dumarçay, Stéphane; Gérardin, Philippe; Harvengt, Luc; Buée, Marc

    2013-02-15

    The dead wood and forest soils are sources of diversity and under-explored fungal strains with biotechnological potential, which require to be studied. Numerous enzymatic tests have been proposed to investigate the functional potential of the soil microbial communities or to test the functional abilities of fungal strains. Nevertheless, the diversity of these functional markers and their relevance in environmental studies or biotechnological screening does still have not been demonstrated. In this work, we assessed ten different extracellular enzymatic activities involved in the wood decaying process including β-etherase that specifically cleaves the β-aryl ether linkages in the lignin polymer. For this purpose, a collection of 26 fungal strains, distributed within three ecological groups (white, brown and soft rot fungi), has been used. Among the ten potential functional markers, the combinatorial use of only six of them allowed separation between the group of white and soft rot fungi from the brown rot fungi. Moreover, our results suggest that extracellular β-etherase is a rare and dispensable activity among the wood decay fungi. Finally, we propose that this set of markers could be useful for the analysis of fungal communities in functional and environmental studies, and for the selection of strains with biotechnological interests. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Enzymatic synthesis and in vitro evaluation of folate-functionalized liposomes

    Directory of Open Access Journals (Sweden)

    Guo B

    2017-06-01

    Full Text Available Bohong Guo, Danqiao Xu, Xiaohong Liu, Jun Yi Department of Pharmaceutics, Guangdong Pharmaceutical University, Guangzhou, China Abstract: In this study, folate–poly(ethylene glycol3400–cholesterol conjugates (FA–PEG–Chol were enzymatically synthesized in one step and incorporated into liposomes to prepare folate (FA-functionalized liposomes for targeted drug delivery. The FA-functionalized liposomes loaded with betulinic acid (BA (FA-L-BA were prepared by thin lipid film method. The FA-L-BA was characterized by their morphology, particle size, zeta potential, encapsulation efficiency (EE, stability, cell cytotoxicity and cellular uptake. The average size of FA-L-BA was 222±8 nm. The spherical particles exhibited a negative electrical charge of -20.12±1.45 mV and high EE of 91.61%±1.16%. The liposomes were taken up selectively by HepG2 cells. FA-L-BA showed enhanced cytotoxicity (50% inhibitory concentration [IC50] =63.07±2.22 µg/mL compared to nontargeted control normal liposomes loaded with BA (L-BA; IC50 =93.14±2.19 µg/mL in HepG2 cells in vitro. In addition, FA-functionalized liposomes loaded with Ir-1 (FA-L-Ir-1 showed significantly higher cellular uptake in HepG2 cells compared to nontargeted control normal liposomes loaded with Ir-1 (L-Ir-1. This novel approach for the liposomes surface modified with FA by a one-step enzymatic amidation was expected to provide potential application as a drug carrier for active targeted delivery to tumor cells. Keywords: enzymatic synthesis, liposomes, folate, surface modification, betulinic acid

  11. Enzymatic Synthesis of Amino Acids Endcapped Polycaprolactone: A Green Route Towards Functional Polyesters.

    Science.gov (United States)

    Duchiron, Stéphane W; Pollet, Eric; Givry, Sébastien; Avérous, Luc

    2018-01-30

    ε-caprolactone (CL) has been enzymatically polymerized using α-amino acids based on sulfur (methionine and cysteine) as (co-)initiators and immobilized lipase B of Candida antarctica (CALB) as biocatalyst. In-depth characterizations allowed determining the corresponding involved mechanisms and the polymers thermal properties. Two synthetic strategies were tested, a first one with direct polymerization of CL with the native amino acids and a second one involving the use of an amino acid with protected functional groups. The first route showed that mainly polycaprolactone (PCL) homopolymer could be obtained and highlighted the lack of reactivity of the unmodified amino acids due to poor solubility and affinity with the lipase active site. The second strategy based on protected cysteine showed higher monomer conversion, with the amino acids acting as (co-)initiators, but their insertion along the PCL chains remained limited to chain endcapping. These results thus showed the possibility to synthesize enzymatically polycaprolactone-based chains bearing amino acids units. Such cysteine endcapped PCL materials could then find application in the biomedical field. Indeed, subsequent functionalization of these polyesters with drugs or bioactive molecules can be obtained, by derivatization of the amino acids, after removal of the protecting group.

  12. Biofabricated film with enzymatic and redox-capacitor functionalities to harvest and store electrons.

    Science.gov (United States)

    Liba, Benjamin D; Kim, Eunkyoung; Martin, Alexandra N; Liu, Yi; Bentley, William E; Payne, Gregory F

    2013-03-01

    Exciting opportunities in bioelectronics will be facilitated by materials that can bridge the chemical logic of biology and the digital logic of electronics. Here we report the fabrication of a dual functional hydrogel film that can harvest electrons from its chemical environment and store these electrons by switching the film's redox-state. The hydrogel scaffold was formed by the anodic deposition of the aminopolysaccharide chitosan. Electron-harvesting function was conferred by co-depositing the enzyme glucose dehydrogenase (GDH) with chitosan. GDH catalyzes the transfer of electrons from glucose to the soluble redox-shuttle NADP(+). Electron-storage function was conferred by the redox-active food phenolic chlorogenic acid (CA) that was enzymatically grafted to the chitosan scaffold using tyrosinase. The grafted CA undergoes redox-cycling reactions with NADPH resulting in the net transfer of electrons to the film where they are stored in the reduced state of CA. The individual and dual functionalities of these films were demonstrated experimentally. There are three general conclusions from this proof-of-concept study. First, enzymatically-grafted catecholic moieties confer redox-capacitor function to the chitosan scaffold. Second, biological materials (i.e. chitosan and CA) and mechanisms (i.e. tyrosinase-mediated grafting) allow the reagentless fabrication of functional films that should be environmentally-friendly, safe and potentially even edible. Finally, the film's ability to mediate the transfer of electrons from a biological metabolite to an electrode suggests an approach to bridge the chemical logic of biology with the digital logic of electronics.

  13. Uncovering Biphasic Catalytic Mode of C5-epimerase in Heparan Sulfate Biosynthesis*

    Science.gov (United States)

    Sheng, Juzheng; Xu, Yongmei; Dulaney, Steven B.; Huang, Xuefei; Liu, Jian

    2012-01-01

    Heparan sulfate (HS), a highly sulfated polysaccharide, is biosynthesized through a pathway involving several enzymes. C5-epimerase (C5-epi) is a key enzyme in this pathway. C5-epi is known for being a two-way catalytic enzyme, displaying a “reversible” catalytic mode by converting a glucuronic acid to an iduronic acid residue, and vice versa. Here, we discovered that C5-epi can also serve as a one-way catalyst to convert a glucuronic acid to an iduronic acid residue, displaying an “irreversible” catalytic mode. Our data indicated that the reversible or irreversible catalytic mode strictly depends on the saccharide substrate structures. The biphasic mode of C5-epi offers a novel mechanism to regulate the biosynthesis of HS with the desired biological functions. PMID:22528493

  14. PDB-UF: database of predicted enzymatic functions for unannotated protein structures from structural genomics

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    Rychlewski Leszek

    2006-02-01

    Full Text Available Abstract Background The number of protein structures from structural genomics centers dramatically increases in the Protein Data Bank (PDB. Many of these structures are functionally unannotated because they have no sequence similarity to proteins of known function. However, it is possible to successfully infer function using only structural similarity. Results Here we present the PDB-UF database, a web-accessible collection of predictions of enzymatic properties using structure-function relationship. The assignments were conducted for three-dimensional protein structures of unknown function that come from structural genomics initiatives. We show that 4 hypothetical proteins (with PDB accession codes: 1VH0, 1NS5, 1O6D, and 1TO0, for which standard BLAST tools such as PSI-BLAST or RPS-BLAST failed to assign any function, are probably methyltransferase enzymes. Conclusion We suggest that the structure-based prediction of an EC number should be conducted having the different similarity score cutoff for different protein folds. Moreover, performing the annotation using two different algorithms can reduce the rate of false positive assignments. We believe, that the presented web-based repository will help to decrease the number of protein structures that have functions marked as "unknown" in the PDB file. Availability http://paradox.harvard.edu/PDB-UF and http://bioinfo.pl/PDB-UF

  15. Linking Microbial Enzymatic Activities and Functional Diversity of Soil around Earthworm Burrows and Casts.

    Science.gov (United States)

    Lipiec, Jerzy; Frąc, Magdalena; Brzezińska, Małgorzata; Turski, Marcin; Oszust, Karolina

    2016-01-01

    The aim of this work was to evaluate the effect of earthworms (Lumbricidae) on the enzymatic activity and microbial functional diversity in the burrow system [burrow wall (BW) 0-3 mm, transitional zone (TZ) 3-7 mm, bulk soil (BS) > 20 mm from the BW] and cast aggregates of a loess soil under a pear orchard. The dehydrogenase, β-glucosidase, protease, alkaline phosphomonoesterase, and acid phosphomonoesterase enzymes were assessed using standard methods. The functional diversity (catabolic potential) was assessed using the Average Well Color Development and Richness Index following the community level physiological profiling from Biolog Eco Plates. All measurements were done using soil from each compartment immediately after in situ sampling in spring. The enzymatic activites including dehydrogenase, protease, β-glucosidase and alkaline phosphomonoesterase were appreciably greater in the BW or casts than in BS and TZ. Conversely, acid phosphomonoesterase had the largest value in the BS. Average Well Color Development in both the TZ and the BS (0.98-0.94 A590 nm) were more than eight times higher than in the BWs and casts. The lowest richness index in the BS (15 utilized substrates) increased by 86-113% in all the other compartments. The PC1 in principal component analysis mainly differentiated the BWs and the TZ. Utilization of all substrate categories was the lowest in the BS. The PC2 differentiated the casts from the other compartments. The enhanced activity of a majority of the enzymes and increased microbial functional diversity in most earthworm-influenced compartments make the soils less vulnerable to degradation and thus increases the stability of ecologically relevant processes in the orchard ecosystem.

  16. Sol-Gel Derived Adsorbents with Enzymatic and Complexonate Functions for Complex Water Remediation.

    Science.gov (United States)

    Pogorilyi, Roman P; Pylypchuk, Ievgen; Melnyk, Inna V; Zub, Yurii L; Seisenbaeva, Gulaim A; Kessler, Vadim G

    2017-09-28

    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.

  17. 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.

  18. Linking microbial enzymatic activities and functional diversity of soil around earthworm burrows and casts

    Directory of Open Access Journals (Sweden)

    Jerzy Lipiec

    2016-08-01

    Full Text Available The aim of this work was to evaluate the effect of earthworms (Lumbricidae on the enzymatic activity and microbial functional diversity in the burrow system (burrow wall 0–3 mm, transitional zone 3–7 mm, bulk soil >20 mm from the burrow wall and cast aggregates of a loess soil under a pear orchard. The dehydrogenase, β-glucosidase, protease, alkaline phosphomonoesterase, and acid phosphomonoesterase enzymes were assessed using standard methods. The functional diversity (catabolic potential was assessed using the Average Well Color Development and Richness Index following the community level physiological profiling from Biolog Eco Plates. All measurements were done using soil from each compartment immediately after in situ sampling in spring. The enzymatic activites including dehydrogenase, protease, β-glucosidase and alkaline phosphomonoesterase were appreciably greater in the burrow wall or casts than in bulk soil and transitional zone. Conversely, acid phosphomonoesterase had the largest value in the bulk soil. Average Well Color Development in both the transitional zone and the bulk soil (0.98-0.94 A590nm were more than eight times higher than in the burrow walls and casts. The lowest richness index in the bulk soil (15 utilized substrates increased by 86-113% in all the other compartments. The PC1 in principal component analysis (PCA mainly differentiated the burrow walls and the transitional zone. Utilization of all substrate categories was the lowest in the bulk soil. The PC2 differentiated the casts from the other compartments. The enhanced activity of a majority of the enzymes and increased microbial functional diversity in most earthworm-influenced compartments make the soils less vulnerable to degradation and thus increases the stability of ecologically relevant processes in the orchard ecosystem.

  19. 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.

  20. An Orthogonal D2 O-Based Induction System that Provides Insights into d-Amino Acid Pattern Formation by Radical S-Adenosylmethionine Peptide Epimerases.

    Science.gov (United States)

    Morinaka, Brandon I; Verest, Marjan; Freeman, Michael F; Gugger, Muriel; Piel, Jörn

    2017-01-16

    Radical S-adenosyl methionine peptide epimerases (RSPEs) are an enzyme family that accomplishes regiospecific and irreversible introduction of multiple d-configured residues into ribosomally encoded peptides. Collectively, RSPEs can generate diverse epimerization patterns in a wide range of substrates. Previously, the lack of rapid methods to localize epimerized residues has impeded efforts to investigate the function and applicative potential of RSPEs. An efficient mass spectrometry-based assay is introduced that permits characterization of products generated in E. coli. Applying this to a range of non-natural peptide-epimerase combinations, it is shown that the d-amino acid pattern is largely but not exclusively dictated by the core peptide sequence, while the epimerization order is dependent on the enzyme-leader pair. RSPEs were found to be highly promiscuous, which allowed for modular introduction of peptide segments with defined patterns. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Characterization of functionalized multiwalled carbon nanotubes for use in an enzymatic sensor.

    Science.gov (United States)

    Guadarrama-Fernández, Leonor; Chanona-Pérez, Jorge; Manzo-Robledo, Arturo; Calderón-Domínguez, Georgina; Martínez-Rivas, Adrián; Ortiz-López, Jaime; Vargas-García, Jorge Roberto

    2014-10-01

    Carbon nanotubes (CNT) have proven to be materials with great potential for the construction of biosensors. Development of fast, simple, and low cost biosensors to follow reactions in bioprocesses, or to detect food contaminants such as toxins, chemical compounds, and microorganisms, is presently an important research topic. This report includes microscopy and spectroscopy to characterize raw and chemically modified multiwall carbon nanotubes (MWCNTs) synthesized by chemical vapor deposition with the intention of using them as the active transducer in bioprocessing sensors. MWCNT were simultaneously purified and functionalized by an acid mixture involving HNO3-H2SO4 and amyloglucosidase attached onto the chemically modified MWCNT surface. A 49.0% decrease in its enzymatic activity was observed. Raw, purified, and enzyme-modified MWCNTs were analyzed by scanning and transmission electron microscopy and Raman and X-ray photoelectron spectroscopy. These studies confirmed purification and functionalization of the CNTs. Finally, cyclic voltammetry electrochemistry was used for electrical characterization of CNTs, which showed promising results that can be useful for construction of electrochemical biosensors applied to biological areas.

  2. Analysis of substructural variation in families of enzymatic proteins with applications to protein function prediction

    Directory of Open Access Journals (Sweden)

    Fofanov Viacheslav Y

    2010-05-01

    Full Text Available Abstract Background Structural variations caused by a wide range of physico-chemical and biological sources directly influence the function of a protein. For enzymatic proteins, the structure and chemistry of the catalytic binding site residues can be loosely defined as a substructure of the protein. Comparative analysis of drug-receptor substructures across and within species has been used for lead evaluation. Substructure-level similarity between the binding sites of functionally similar proteins has also been used to identify instances of convergent evolution among proteins. In functionally homologous protein families, shared chemistry and geometry at catalytic sites provide a common, local point of comparison among proteins that may differ significantly at the sequence, fold, or domain topology levels. Results This paper describes two key results that can be used separately or in combination for protein function analysis. The Family-wise Analysis of SubStructural Templates (FASST method uses all-against-all substructure comparison to determine Substructural Clusters (SCs. SCs characterize the binding site substructural variation within a protein family. In this paper we focus on examples of automatically determined SCs that can be linked to phylogenetic distance between family members, segregation by conformation, and organization by homology among convergent protein lineages. The Motif Ensemble Statistical Hypothesis (MESH framework constructs a representative motif for each protein cluster among the SCs determined by FASST to build motif ensembles that are shown through a series of function prediction experiments to improve the function prediction power of existing motifs. Conclusions FASST contributes a critical feedback and assessment step to existing binding site substructure identification methods and can be used for the thorough investigation of structure-function relationships. The application of MESH allows for an automated

  3. Analysis of substructural variation in families of enzymatic proteins with applications to protein function prediction.

    Science.gov (United States)

    Bryant, Drew H; Moll, Mark; Chen, Brian Y; Fofanov, Viacheslav Y; Kavraki, Lydia E

    2010-05-11

    Structural variations caused by a wide range of physico-chemical and biological sources directly influence the function of a protein. For enzymatic proteins, the structure and chemistry of the catalytic binding site residues can be loosely defined as a substructure of the protein. Comparative analysis of drug-receptor substructures across and within species has been used for lead evaluation. Substructure-level similarity between the binding sites of functionally similar proteins has also been used to identify instances of convergent evolution among proteins. In functionally homologous protein families, shared chemistry and geometry at catalytic sites provide a common, local point of comparison among proteins that may differ significantly at the sequence, fold, or domain topology levels. This paper describes two key results that can be used separately or in combination for protein function analysis. The Family-wise Analysis of SubStructural Templates (FASST) method uses all-against-all substructure comparison to determine Substructural Clusters (SCs). SCs characterize the binding site substructural variation within a protein family. In this paper we focus on examples of automatically determined SCs that can be linked to phylogenetic distance between family members, segregation by conformation, and organization by homology among convergent protein lineages. The Motif Ensemble Statistical Hypothesis (MESH) framework constructs a representative motif for each protein cluster among the SCs determined by FASST to build motif ensembles that are shown through a series of function prediction experiments to improve the function prediction power of existing motifs. FASST contributes a critical feedback and assessment step to existing binding site substructure identification methods and can be used for the thorough investigation of structure-function relationships. The application of MESH allows for an automated, statistically rigorous procedure for incorporating structural

  4. Mutations causing Greenberg dysplasia but not Pelger anomaly uncouple enzymatic from structural functions of a nuclear membrane protein.

    OpenAIRE

    Clayton, P; Fischer, B; Mann, A.; Mansour, S.; Rossier, E; Van der Veen, M.; C. Lang; Baasanjav, S.; Kieslich, M.; Brossuleit, K.; Gravemann, S; Schnipper, N.; Karbasyian, M.; Demuth, I.; Zwerger, M.

    2010-01-01

    The lamin B receptor (LBR) is an inner nuclear membrane protein with a structural function interacting with chromatin and lamins, and an enzymatic function as a sterol reductase. Heterozygous LBR mutations cause nuclear hyposegmentation in neutrophils (Pelger anomaly), while homozygous mutations cause prenatal death with skeletal defects and abnormal sterol metabolism (Greenberg dysplasia). It has remained unclear whether the lethality in Greenberg dysplasia is due to cholesterol defects or a...

  5. The In Vitro Effects of Enzymatic Digested Gliadin on the Functionality of the Autophagy Process

    Directory of Open Access Journals (Sweden)

    Federico Manai

    2018-02-01

    Full Text Available Gliadin, the alcohol-soluble protein fraction of wheat, contains the factor toxic for celiac disease (CD, and its toxicity is not reduced by digestion with gastro-pancreatic enzymes. Importantly, it is proved that an innate immunity to gliadin plays a key role in the development of CD. The immune response induces epithelial stress and reprograms intraepithelial lymphocytes into natural killer (NK-like cells, leading to enterocyte apoptosis and an increase in epithelium permeability. In this contribution, we have reported that in Caco-2 cells the administration of enzymatically digested gliadin (PT-gliadin reduced significantly the expression of the autophagy-related marker LC3-II. Furthermore, electron and fluorescent microscope analysis suggested a compromised functionality of the autophagosome apparatus. The rescue of the dysregulated autophagy process, along with a reduction of PT-gliadin toxicity, was obtained with a starvation induction protocol and by 3-methyladenine administration, while rapamycin, a well-known autophagy inducer, did not produce a significant improvement in the clearance of extra- and intra-cellular fluorescent PT-gliadin amount. Altogether, our results highlighted the possible contribution of the autophagy process in the degradation and in the reduction of extra-cellular release of gliadin peptides and suggest novel molecular targets to counteract gliadin-induced toxicity in CD.

  6. Drosophila immunity: analysis of PGRP-SB1 expression, enzymatic activity and function.

    Directory of Open Access Journals (Sweden)

    Anna Zaidman-Rémy

    Full Text Available Peptidoglycan is an essential and specific component of the bacterial cell wall and therefore is an ideal recognition signature for the immune system. Peptidoglycan recognition proteins (PGRPs are conserved from insects to mammals and able to bind PGN (non-catalytic PGRPs and, in some cases, to efficiently degrade it (catalytic PGRPs. In Drosophila, several non-catalytic PGRPs function as selective peptidoglycan receptors upstream of the Toll and Imd pathways, the two major signalling cascades regulating the systemic production of antimicrobial peptides. Recognition PGRPs specifically activate the Toll pathway in response to Lys-type peptidoglycan found in most Gram-positive bacteria and the Imd pathway in response to DAP-type peptidoglycan encountered in Gram-positive bacilli-type bacteria and in Gram-negative bacteria. Catalytic PGRPs on the other hand can potentially reduce the level of immune activation by scavenging peptidoglycan. In accordance with this, PGRP-LB and PGRP-SC1A/B/2 have been shown to act as negative regulators of the Imd pathway. In this study, we report a biochemical and genetic analysis of PGRP-SB1, a catalytic PGRP. Our data show that PGRP-SB1 is abundantly secreted into the hemolymph following Imd pathway activation in the fat body, and exhibits an enzymatic activity towards DAP-type polymeric peptidoglycan. We have generated a PGRP-SB1/2 null mutant by homologous recombination, but its thorough phenotypic analysis did not reveal any immune function, suggesting a subtle role or redundancy of PGRP-SB1/2 with other molecules. Possible immune functions of PGRP-SB1 are discussed.

  7. Entamoeba histolytica: molecular characterization of an aldose 1-epimerase (mutarotase).

    Science.gov (United States)

    Villalobo, Eduardo; Wender, Nomy; Mirelman, David

    2005-07-01

    In cells, the alpha-anomers of aldoses are the preferred metabolizable substrates, while beta-anomers of aldoses play their role in glycan structure. In the cytoplasm, alpha- and beta-anomers of aldoses interconvert through the enzyme termed aldose 1-epimerase or mutarotase (EC 5.1.3.3). We have identified a mutarotase gene in Entamoeba histolytica, the causative agent of non-bacterial dysentery in humans. Cloning and characterization of this gene in two strains of the parasite (HM-1:IMSS and Rahman) that differ in their pathogenicity, revealed that the sequence is identical in both strains. A recombinant E. histolytica mutarotase was produced as well as specific antibodies that recognized a 38 kDa protein in trophozoite lysates of both strains. Mutarotase activity was observed with the recombinant protein as well as in lysates of both HM-1:IMSS and Rahman, the former exhibiting a slightly higher mutarotase activity. Finally, we have shown by complementation that overexpression of the E. histolytica mutarotase in a mutarotase defective Escherichia coli strain restores the ability of these bacteria to grow in minimal medium with phenyl-beta-galactopyranoside as the sole carbon source.

  8. Comparison of Dynamics of Wildtype and V94M Human UDP-Galactose 4-Epimerase – A computational perspective on severe Epimerase-deficiency Galactosemia

    Science.gov (United States)

    Timson, David J.; Lindert, Steffen

    2013-01-01

    UDP-galactose 4′-epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose, an important step in galactose catabolism. Type III galactosemia, an inherited metabolic disease, is associated with mutations in human GALE. The V94M mutation has been associated with a very severe form of type III galactosemia. While a variety of structural and biochemical studies have been reported that elucidate differences between the wildtype and this mutant form of human GALE, little is known about the dynamics of the protein and how mutations influence structure and function. We performed molecular dynamics simulations on the wildtype and V94M enzyme in different states of substrate and cofactor binding. In the mutant, the average distance between the substrate and both a key catalytic residue (Tyr-157) and the enzyme-bound NAD+ cofactor and the active site dynamics are altered making substrate binding slightly less stable. However, overall stability or dynamics of the protein is not altered. This is consistent with experimental findings that the impact is largely on the turnover number (kcat), with less substantial effects on Km. Active site fluctuations were found to be correlated in enzyme with substrate bound to just one of the subunits in the homodimer suggesting inter-subunit communication. Greater active site loop mobility in human GALE compared to the equivalent loop in Escherichia coli GALE explains why the former can catalyze the interconversion of UDP-N-acetylgalactosemine and UDP-N-acetylglucosamine while the bacterial enzyme cannot. This work illuminates molecular mechanisms of disease and may inform the design of small molecule therapies for type III galactosemia. PMID:23732289

  9. Comparison of dynamics of wildtype and V94M human UDP-galactose 4-epimerase-A computational perspective on severe epimerase-deficiency galactosemia.

    Science.gov (United States)

    Timson, David J; Lindert, Steffen

    2013-09-10

    UDP-galactose 4'-epimerase (GALE) catalyzes the interconversion of UDP-galactose and UDP-glucose, an important step in galactose catabolism. Type III galactosemia, an inherited metabolic disease, is associated with mutations in human GALE. The V94M mutation has been associated with a very severe form of type III galactosemia. While a variety of structural and biochemical studies have been reported that elucidate differences between the wildtype and this mutant form of human GALE, little is known about the dynamics of the protein and how mutations influence structure and function. We performed molecular dynamics simulations on the wildtype and V94M enzyme in different states of substrate and cofactor binding. In the mutant, the average distance between the substrate and both a key catalytic residue (Tyr157) and the enzyme-bound NAD+ cofactor and the active site dynamics are altered making substrate binding slightly less stable. However, overall stability or dynamics of the protein is not altered. This is consistent with experimental findings that the impact is largely on the turnover number (kcat), with less substantial effects on Km. Active site fluctuations were found to be correlated in enzyme with substrate bound to just one of the subunits in the homodimer suggesting inter-subunit communication. Greater active site loop mobility in human GALE compared to the equivalent loop in Escherichia coli GALE explains why the former can catalyze the interconversion of UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine while the bacterial enzyme cannot. This work illuminates molecular mechanisms of disease and may inform the design of small molecule therapies for type III galactosemia. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Enzymatic Specific Production and Chemical Functionalization of Phenylpropanone Platform Monomers from Lignin.

    Science.gov (United States)

    Ohta, Yukari; Hasegawa, Ryoichi; Kurosawa, Kanako; Maeda, Allyn H; Koizumi, Toshio; Nishimura, Hiroshi; Okada, Hitomi; Qu, Chen; Saito, Kaori; Watanabe, Takashi; Hatada, Yuji

    2017-01-20

    Enzymatic catalysis is an ecofriendly strategy for the production of high-value low-molecular-weight aromatic compounds from lignin. Although well-definable aromatic monomers have been obtained from synthetic lignin-model dimers, enzymatic-selective synthesis of platform monomers from natural lignin has not been accomplished. In this study, we successfully achieved highly specific synthesis of aromatic monomers with a phenylpropane structure directly from natural lignin using a cascade reaction of β-O-4-cleaving bacterial enzymes in one pot. Guaiacylhydroxylpropanone (GHP) and the GHP/syringylhydroxylpropanone (SHP) mixture are exclusive monomers from lignin isolated from softwood (Cryptomeria japonica) and hardwood (Eucalyptus globulus). The intermediate products in the enzymatic reactions show the capacity to accommodate highly heterologous substrates at the substrate-binding sites of the enzymes. To demonstrate the applicability of GHP as a platform chemical for bio-based industries, we chemically generate value-added GHP derivatives for bio-based polymers. Together with these chemical conversions for the valorization of lignin-derived phenylpropanone monomers, the specific and enzymatic production of the monomers directly from natural lignin is expected to provide a new stream in "white biotechnology" for sustainable biorefineries. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  11. Purification and characterization of methylmalonyl-CoA epimerase from Propionibacterium shermanii.

    Science.gov (United States)

    Leadlay, P F

    1981-01-01

    Methylmalonyl-CoA epimerase, which specifically interconverts the (2R)- and (2S)- epimers of methylmalonyl-CoA, was purified 95-fold from Propionibacterium shermanii by a new method that affords apparently homogeneous enzyme, in 80-100mg quantities, in yields representing about 40% of the activity in cell-free extracts. The specific activity of the purified enzyme, 10.1 mukat/mg, is much greater than previously reported. Native methylmalonyl-CoA epimerase has Mr about 33000, and apparently consists of two identical subunits. The purified enzyme is stable indefinitely when stored at -20 degrees C and pH 8.5, but contrary to previous reports it is not unusually acid-stable. The activity of methylmalonyl-CoA epimerase is increased by Co2+, and to a smaller extent by Ni2+, Mn2+ and Zn2+. Images Fig. 2. PMID:7325964

  12. Radical S-adenosyl methionine epimerases: regioselective introduction of diverse D-amino acid patterns into peptide natural products.

    Science.gov (United States)

    Morinaka, Brandon I; Vagstad, Anna L; Helf, Maximilian J; Gugger, Muriel; Kegler, Carsten; Freeman, Michael F; Bode, Helge B; Piel, Jörn

    2014-08-04

    PoyD is a radical S-adenosyl methionine epimerase that introduces multiple D-configured amino acids at alternating positions into the highly complex marine peptides polytheonamide A and B. This novel post-translational modification contributes to the ability of the polytheonamides to form unimolecular minimalistic ion channels and its cytotoxic activity at picomolar levels. Using a genome mining approach we have identified additional PoyD homologues in various bacteria. Three enzymes were expressed in E. coli with their cognate as well as engineered peptide precursors and shown to introduce diverse D-amino acid patterns into all-L peptides. The data reveal a family of architecturally and functionally distinct enzymes that exhibit high regioselectivity, substrate promiscuity, and irreversible action and thus provide attractive opportunities for peptide engineering. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Novel cellobiose 2-epimerases for the production of epilactose from milk ultrafiltrate containing lactose.

    Science.gov (United States)

    Krewinkel, Manuel; Kaiser, Jana; Merz, Michael; Rentschler, Eva; Kuschel, Beatrice; Hinrichs, Jörg; Fischer, Lutz

    2015-06-01

    A selected number of enzymes have recently been assigned to the emerging class of cellobiose 2-epimerases (CE). All CE convert lactose to the rare sugar epilactose, which is regarded as a new prebiotic. Within this study, the gene products of 2 potential CE genes originating from the mesophilic bacteria Cellulosilyticum lentocellum and Dysgonomonas gadei were recombinantly produced in Escherichia coli and purified by chromatography. The enzymes have been identified as novel CE by sequence analysis and biochemical characterizations. The biochemical characterizations included the determination of the molecular weight, the substrate spectrum, and the kinetic parameters, as well as the pH and temperature profiles in buffer and food matrices. Both identified CE epimerize cellobiose and lactose into the C2 epimerization products glucosylmannose and epilactose, respectively. The epimerization activity for lactose was maximal at pH 8.0 or 7.5 and 40°C in defined buffer systems for the CE from C. lentocellum and the CE from D. gadei, respectively. In addition, biotransformations of the foodstuff milk ultrafiltrate containing lactose were demonstrated. The CE from D. gadei was produced in a stirred-tank reactor (12 L) and purified using an automatic system. Enzyme production and purification in this scale indicates that a future upscaling of CE production is possible. The bioconversions of lactose in milk ultrafiltrate were carried out either in a batch process or in a continuously operated enzyme membrane reactor (EMR) process. Both processes ran at an industrially relevant low temperature of 8°C to reduce undesirable microbial growth. The enzyme was reasonably active at the low process temperature because the CE originated from a mesophilic organism. An epilactose yield of 29.9% was achieved in the batch process within 28 h of operation time. In the continuous EMR process, the epilactose yield in the product stream was lower, at 18.5%. However, the enzyme productivity

  14. Mutations causing Greenberg dysplasia but not Pelger anomaly uncouple enzymatic from structural functions of a nuclear membrane protein.

    Science.gov (United States)

    Clayton, Peter; Fischer, Björn; Mann, Anuska; Mansour, Sahar; Rossier, Eva; Veen, Markus; Lang, Christine; Baasanjav, Sevjidmaa; Kieslich, Moritz; Brossuleit, Katja; Gravemann, Sophia; Schnipper, Nele; Karbasyian, Mohsen; Demuth, Ilja; Zwerger, Monika; Vaya, Amparo; Utermann, Gerd; Mundlos, Stefan; Stricker, Sigmar; Sperling, Karl; Hoffmann, Katrin

    2010-01-01

    The lamin B receptor (LBR) is an inner nuclear membrane protein with a structural function interacting with chromatin and lamins, and an enzymatic function as a sterol reductase. Heterozygous LBR mutations cause nuclear hyposegmentation in neutrophils (Pelger anomaly), while homozygous mutations cause prenatal death with skeletal defects and abnormal sterol metabolism (Greenberg dysplasia). It has remained unclear whether the lethality in Greenberg dysplasia is due to cholesterol defects or altered nuclear morphology.To answer this question we characterized two LBR missense mutations and showed that they cause Greenberg dysplasia. Both mutations affect residues that are evolutionary conserved among sterol reductases. In contrast to wildtype LBR, both mutations failed to rescue C14 sterol reductase deficient yeast, indicating an enzymatic defect. We found no Pelger anomaly in the carrier parent excluding marked effects on nuclear structure. We studied Lbr in mouse embryos and demonstrate expression in skin and the developing skeletal system consistent with sites of histological changes in Greenberg dysplasia. Unexpectedly we found in disease-relevant cell types not only nuclear but also cytoplasmatic LBR localization. The cytoplasmatic LBR staining co-localized with ER-markers and is thus consistent with the sites of endogeneous sterol synthesis. We conclude that LBR missense mutations can abolish sterol reductase activity, causing lethal Greenberg dysplasia but not Pelger anomaly. The findings separate the metabolic from the structural function and indicate that the sterol reductase activity is essential for human intrauterine development.

  15. Purification, crystallization and preliminary crystallographic study of an IDS-epimerase from Agrobacterium tumefaciens BY6

    Energy Technology Data Exchange (ETDEWEB)

    Bäuerle, Bettina [Institute of Microbiology, University of Stuttgart, 70569 Stuttgart (Germany); Sandalova, Tatyana; Schneider, Gunter [Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17177 Stockholm (Sweden); Rieger, Paul-Gerhard, E-mail: pg.rieger@imb.uni-stuttgart.de [Institute of Microbiology, University of Stuttgart, 70569 Stuttgart (Germany)

    2006-08-01

    This is the first report of the crystallization of an IDS-epimerase from A. tumefaciens BY6 and its l-selenomethionine derivative. The initial degradation of all stereoisomers of the complexing agent iminodisuccinate (IDS) is enabled by an epimerase in the bacterial strain Agrobacterium tumefaciens BY6. This protein was produced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method. Crystals of IDS-epimerase were obtained under several conditions. The best diffracting crystals were grown in 22% PEG 3350, 0.2 M (NH{sub 4}){sub 2}SO{sub 4} and 0.1 M bis-Tris propane pH 7.2 at 293 K. These crystals belong to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 55.4, b = 104.2, c = 78.6 Å, β = 103.3°, and diffracted to 1.7 Å resolution. They contain two protein molecules per asymmetric unit. In order to solve the structure using the MAD phasing method, crystals of the l-selenomethionine-substituted epimerase were grown in the presence of 20% PEG 3350, 0.2 M Na{sub 2}SO{sub 4} and 0.1 M bis-Tris propane pH 8.5.

  16. A Case Study of Monozygotic Twins Apparently Homozygous for a Novel Variant of UDP-Galactose 4'-epimerase (GALE) : A Complex Case of Variant GALE.

    Science.gov (United States)

    Liu, Ying; Bentler, Kristi; Coffee, Bradford; Chhay, Juliet S; Sarafoglou, Kyriakie; Fridovich-Keil, Judith L

    2013-01-01

    Epimerase deficiency galactosemia is an autosomal recessive disorder that results from partial impairment of UDP-galactose 4'-epimerase (GALE), the third enzyme in the Leloir pathway of galactose metabolism. Clinical severity of epimerase deficiency ranges from potentially lethal to apparently benign, likely reflecting the extent of GALE enzyme impairment, among other factors. We report here a case study of monozygotic twins identified by newborn screening with elevated total galactose and normal galactose-1P uridylyltransferase (GALT). Follow-up testing revealed partial impairment of GALE in hemolysates but near-normal activity in lymphoblasts; molecular testing identified a missense substitution, R220W, apparently in the homozygous state. The twins were treated with dietary galactose restriction for the first 18 months of life. During this time, independent testing revealed concurrent diagnoses of Williams Syndrome in both twins, and cytomegalovirus (CMV) infection in one. Expression studies of R220W-hGALE in a null-background strain of Saccharomyces cerevisiae demonstrated a very limited impairment of V (max) for UDP-galactose (UDP-Gal) and K (m) for UDP-N-acetylgalactosamine (UDP-GalNAc), but a galactose challenge in vivo failed to uncover any evidence of impaired Leloir function. Similarly, both twins demonstrated normal hemolysate galactose-1-phosphate (Gal-1P) levels following normalization of their diets at 18 months of age. While these studies cannot rule out a negative consequence from some cryptic GALE impairment in a specific tissue or developmental stage, they suggest that the substitution, R220W, is mild to neutral, so that any GALE impairment in these twins is likely to be peripheral and therefore unlikely to be the cause of the negative outcomes observed.

  17. Awakening sleeping beauty: production of propionic acid in Escherichia coli through the sbm operon requires the activity of a methylmalonyl-CoA epimerase.

    Science.gov (United States)

    Gonzalez-Garcia, Ricardo Axayacatl; McCubbin, Tim; Wille, Annalena; Plan, Manuel; Nielsen, Lars Keld; Marcellin, Esteban

    2017-07-17

    Propionic acid is used primarily as a food preservative with smaller applications as a chemical building block for the production of many products including fabrics, cosmetics, drugs, and plastics. Biological production using propionibacteria would be competitive against chemical production through hydrocarboxylation of ethylene if native producers could be engineered to reach near-theoretical yield and good productivity. Unfortunately, engineering propionibacteria has proven very challenging. It has been suggested that activation of the sleeping beauty operon in Escherichia coli is sufficient to achieve propionic acid production. Optimising E. coli production should be much easier than engineering propionibacteria if tolerance issues can be addressed. Propionic acid is produced in E. coli via the sleeping beauty mutase operon under anaerobic conditions in rich medium via amino acid degradation. We observed that the sbm operon enhances amino acids degradation to propionic acid and allows E. coli to degrade isoleucine. However, we show here that the operon lacks an epimerase reaction that enables propionic acid production in minimal medium containing glucose as the sole carbon source. Production from glucose can be restored by engineering the system with a methylmalonyl-CoA epimerase from Propionibacterium acidipropionici (0.23 ± 0.02 mM). 1-Propanol production was also detected from the promiscuous activity of the native alcohol dehydrogenase (AdhE). We also show that aerobic conditions are favourable for propionic acid production. Finally, we increase titre 65 times using a combination of promoter engineering and process optimisation. The native sbm operon encodes an incomplete pathway. Production of propionic acid from glucose as sole carbon source is possible when the pathway is complemented with a methylmalonyl-CoA epimerase. Although propionic acid via the restored succinate dissimilation pathway is considered a fermentative process, the engineered pathway

  18. Effects of different bulking agents on the maturity, enzymatic activity, and microbial community functional diversity of kitchen waste compost.

    Science.gov (United States)

    Wang, Xiaojuan; Zhang, Wenwei; Gu, Jie; Gao, Hua; Qin, Qingjun

    2016-10-01

    Aerobic composting is an effective method for the disposal and utilization of kitchen waste. However, the addition of a bulking agent is necessary during kitchen waste composting because of its high moisture content and low C/N ratio. In order to select a suitable bulking agent, we investigated the influence of leaf litter (LL), sawdust (SD), and wheat straw (WS) on the enzymatic activity, microbial community functional diversity, and maturity indices during the kitchen waste composting process. The results showed that the addition of WS yielded the highest maturity (the C/N ratio decreased from 25 to 13, T value = 0.5, and germination index (GI) = 114.7%), whereas the compost containing SD as a bulking agent had the lowest maturity (GI = 32.4%). The maximum cellulase and urease activities were observed with the WS treatment on day 8, whereas the SD treatment had the lowest cellulase activity and the LL treatment had the lowest urease activity. The compost temperature and microbial activity (as the average well color development) showed that bulking the composts with SD prolonged the composting process. The diversity index based on the community-level physiological profile showed that the composts bulked with LL and WS had greater microbial community functional diversity compared with those bulked with SD. Thus, the maturity indexes and enzymatic activities suggest that WS is a suitable bulking agent for use in kitchen waste composting systems.

  19. A rice plastidial nucleotide sugar epimerase is involved in galactolipid biosynthesis and improves photosynthetic efficiency.

    Directory of Open Access Journals (Sweden)

    Chunlai Li

    2011-07-01

    Full Text Available Photosynthesis is the final determinator for crop yield. To gain insight into genes controlling photosynthetic capacity, we selected from our large T-DNA mutant population a rice stunted growth mutant with decreased carbon assimilate and yield production named photoassimilate defective1 (phd1. Molecular and biochemical analyses revealed that PHD1 encodes a novel chloroplast-localized UDP-glucose epimerase (UGE, which is conserved in the plant kingdom. The chloroplast localization of PHD1 was confirmed by immunoblots, immunocytochemistry, and UGE activity in isolated chloroplasts, which was approximately 50% lower in the phd1-1 mutant than in the wild type. In addition, the amounts of UDP-glucose and UDP-galactose substrates in chloroplasts were significantly higher and lower, respectively, indicating that PHD1 was responsible for a major part of UGE activity in plastids. The relative amount of monogalactosyldiacylglycerol (MGDG, a major chloroplast membrane galactolipid, was decreased in the mutant, while the digalactosyldiacylglycerol (DGDG amount was not significantly altered, suggesting that PHD1 participates mainly in UDP-galactose supply for MGDG biosynthesis in chloroplasts. The phd1 mutant showed decreased chlorophyll content, photosynthetic activity, and altered chloroplast ultrastructure, suggesting that a correct amount of galactoglycerolipids and the ratio of glycolipids versus phospholipids are necessary for proper chloroplast function. Downregulated expression of starch biosynthesis genes and upregulated expression of sucrose cleavage genes might be a result of reduced photosynthetic activity and account for the decreased starch and sucrose levels seen in phd1 leaves. PHD1 overexpression increased photosynthetic efficiency, biomass, and grain production, suggesting that PHD1 plays an important role in supplying sufficient galactolipids to thylakoid membranes for proper chloroplast biogenesis and photosynthetic activity. These

  20. Biochemical studies on WbcA, a sugar epimerase from Yersinia enterocolitica.

    Science.gov (United States)

    Salinger, Ari J; Brown, Haley A; Thoden, James B; Holden, Hazel M

    2015-10-01

    Yersinia enterocolitica is a Gram-negative bacterium that causes yersiniosis, a zoonotic disease affecting the gastrointestinal tract of humans, cattle, and pigs, among others. The lipopolysaccharide of Y. enterocolitica O:8 contains an unusual sugar, 6-deoxy-d-gulose, which requires four enzymes for its biosynthesis. Here, we describe a combined structural and functional investigation of WbcA, which catalyzes the third step in the pathway, namely an epimerization about the C-3' carbon of a CDP-linked sugar. The structure of WbcA was determined to 1.75-Å resolution, and the model was refined to an overall R-factor of 19.5%. The fold of WbcA places it into the well-defined cupin superfamily of sugar epimerases. Typically, these enzymes contain both a conserved histidine and a tyrosine residue that play key roles in catalysis. On the basis of amino acid sequence alignments, it was anticipated that the "conserved" tyrosine had been replaced with a cysteine residue in WbcA (Cys 133), and indeed this was the case. However, what was not anticipated was the fact that another tyrosine residue (Tyr 50) situated on a neighboring β-strand moved into the active site. Site-directed mutant proteins were subsequently constructed and their kinetic properties analyzed to address the roles of Cys 133 and Tyr 50 in WbcA catalysis. This study emphasizes the continuing need to experimentally verify assumptions that are based solely on bioinformatics approaches. © 2015 The Protein Society.

  1. Enzymatic hydrolysis of rice dreg protein: effects of enzyme type on the functional properties and antioxidant activities of recovered proteins.

    Science.gov (United States)

    Zhao, Qiang; Xiong, Hua; Selomulya, Cordelia; Chen, Xiao Dong; Zhong, Honglan; Wang, Shenqi; Sun, Wenjing; Zhou, Qiang

    2012-10-01

    The effects of various proteases on the formation and characteristics of rice dreg protein hydrolysates (RDPHs) were investigated. Enzymatic hydrolysis of often under-utilised rice dreg protein (RDP) with different enzymes studied here was found to significantly improve protein content and solubility. RDPHs prepared by alkaline protease showed better protein recovery, producing higher protein content with much smaller peptides, while hydrolysates generated by Protamex showed the highest antioxidant activities with more than 80% solubility over a wide pH range. The results indicated that the type of protease greatly influenced the molecular weight and amino acid residue composition of RDPH. The enzyme type also determined the functional properties and antioxidant activity of the recovered proteins. It was found that an optimum allocation of alkaline protease in addition to the Neutrase enzyme could be an appropriate strategy to produce RDPH with desirable functionalities, antioxidant properties, and low salt content. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Purification, crystallization and preliminary X-ray diffraction studies of d-tagatose 3-epimerase from Pseudomonas cichorii

    Energy Technology Data Exchange (ETDEWEB)

    Yoshida, Hiromi; Yamada, Mitsugu [Molecular Structure Research Group, Information Technology Center and Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793 (Japan); Nishitani, Takeyori; Takada, Goro; Izumori, Ken [Department of Biochemistry and Food Science, Faculty of Agriculture and Rare Sugar Research Center, Kagawa University, Miki-cho, Kagawa 761-0795 (Japan); Kamitori, Shigehiro, E-mail: kamitori@med.kagawa-u.ac.jp [Molecular Structure Research Group, Information Technology Center and Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793 (Japan)

    2007-02-01

    Recombinant d-tagatose 3-epimerase from P. cichorii was purified and crystallized. Diffraction data were collected to 2.5 Å resolution. d-Tagatose 3-epimerase (D-TE) from Pseudomonas cichorii catalyzes the epimerization of various ketohexoses at the C3 position. The epimerization of d-psicose has not been reported with epimerases other than P. cichorii D-TE and d-psicose 3-epimerase from Agrobacterium tumefaciens. Recombinant P. cichorii D-TE has been purified and crystallized. Crystals of P. cichorii D-TE were obtained by the sitting-drop method at room temperature. The crystal belongs to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 76.80, b = 94.92, c = 91.73 Å, β = 102.82°. Diffraction data were collected to 2.5 Å resolution. The asymmetric unit is expected to contain four molecules.

  3. Efficient chemo-enzymatic gluten detoxification: reducing toxic epitopes for celiac patients improving functional properties

    Science.gov (United States)

    Ribeiro, Miguel; Nunes, Fernando M.; Guedes, Sofia; Domingues, Pedro; Silva, Amélia M.; Carrillo, Jose Maria; Rodriguez-Quijano, Marta; Branlard, Gérard; Igrejas, Gilberto

    2015-01-01

    Protein engineering of gluten, the exogenous effector in celiac disease, seeking its detoxification by selective chemical modification of toxic epitopes is a very attractive strategy and promising technology when compared to pharmacological treatment or genetic engineering of wheat. Here we present a simple and efficient chemo-enzymatic methodology that decreases celiac disease toxic epitopes of gluten proteins improving its technological value through microbial transglutaminase-mediated transamidation of glutamine with n-butylamine under reducing conditions. First, we found that using low concentrations of amine-nucleophile under non-reducing conditions, the decrease in toxic epitopes is mainly due to transglutaminase-mediated cross-linking. Second, using high amine nucleophile concentrations protein cross-linking is substantially reduced. Third, reducing conditions increase 7-fold the transamidation reaction further decreasing toxic epitopes amount. Fourth, using n-butylamine improves gluten hydrophobicity that strengthens the gluten network. These results open the possibility of tailoring gluten for producing hypoallergenic flours while still taking advantage of the unique viscoelastic properties of gluten. PMID:26691232

  4. Enzymatic synthesis of chlorogenic acid glucoside using dextransucrase and its physical and functional properties.

    Science.gov (United States)

    Nam, Seung-Hee; Ko, Jin-A; Jun, Woojin; Wee, Young-Jung; Walsh, Marie K; Yang, Kwang-Yeol; Choi, Jin-Ho; Eun, Jon-Bang; Choi, Jeong; Kim, Young-Min; Han, Songhee; Nguyen, Thi Thanh Hanh; Kim, Doman

    2017-12-01

    Chlorogenic acid, a major polyphenol in edible plants, possesses strong antioxidant activity, anti-lipid peroxidation and anticancer effects. It used for industrial applications; however, this is limited by its instability to heat or light. In this study, we for the first time synthesized chlorogenic acid glucoside (CHG) via transglycosylation using dextransucrase from Leuconostoc mesenteroides and sucrose. CHG was purified and its structure determined by nuclear magnetic resonance and matrix-associated laser desorption ionization-time-of-flight mass spectroscopy. The production yield of CHG was 44.0% or 141mM, as determined by response surface methodology. CHG possessed a 65% increased water solubility and 2-fold browning resistance while it displayed stronger inhibition of lipid peroxidation and of colon cancer cell growth by MTT assay, compared to chlorogenic acid. Therefore, this study may expand the industrial applications of chlorogenic acid as water-soluble or browning resistant compound (CHG) through enzymatic glycosylation. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Functional tailor-made polyesters via chemical and enzymatic catalysis and their applications as biomaterials

    OpenAIRE

    Vaida, Cristian

    2011-01-01

    This thesis deals with the preparation of novel functional (co)polyesters and copolyester resins based on e-caprolactone and functional e-caprolactones as well as (co)polyesters based on w-pentadecalactone and functional macrocyclic lactones or functional e-caprolactones. The characteristics of these polyesters were evaluated with respect to their molecular weight, molecular weight distribution, composition, microstructure, and concentration of the functional groups. The degradation of polyes...

  6. Autotaxin (ATX): a multi-functional and multi-modular protein possessing enzymatic lysoPLD activity and matricellular properties.

    Science.gov (United States)

    Yuelling, Larra M; Fuss, Babette

    2008-09-01

    Recent studies have established that autotaxin (ATX), also known as phosphodiesterase Ialpha/autotaxin (PD-Ialpha/ATX) or (ecto)nucleotide pyrophosphatase/phosphodiesterase 2 [(E)NPP2], represents a multi-functional and multi-modular protein. ATX was initially thought to function exclusively as a phosphodiesterase/pyrophosphatase. However, it has become apparent that this enzymatically active site, which is ultimately responsible for ATX's originally discovered property of tumor cell motility stimulation, mediates the conversion of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA). In addition, a separate functionally active domain, here referred to as the Modulator of Oligodendrocyte Remodeling and Focal adhesion Organization (MORFO) domain, was discovered in studies analyzing the role of ATX during the differentiation of myelinating cells of the central nervous system (CNS), namely oligodendrocytes. This novel domain was found to mediate anti-adhesive, i.e. matricellular, properties and to promote morphological maturation of oligodendrocytes. In this review, we summarize our current understanding of ATX's structure-function domains and discuss their contribution to the presently known main functional roles of ATX.

  7. Pseudomonas aeruginosa C5-mannuronan epimerase: steady-state kinetics and characterization of the product.

    Science.gov (United States)

    Jerga, Agoston; Raychaudhuri, Aniruddha; Tipton, Peter A

    2006-01-17

    Alginate is a major constituent of mature biofilms produced by Pseudomonas aeruginosa. The penultimate step in the biosynthesis of alginate is the conversion of some beta-D-mannuronate residues in the polymeric substrate polymannuronan to alpha-L-guluronate residues in a reaction catalyzed by C5-mannuronan epimerase. Specificity studies conducted with size-fractionated oligomannuronates revealed that the minimal substrate contained nine monosaccharide residues. The maximum velocity of the reaction increased from 0.0018 to 0.0218 s(-1) as the substrate size increased from 10 to 20 residues, and no additional increase in kcat was observed for substrates up to 100 residues in length. The Km decreased from 80 microM for a substrate containing fewer than 15 residues to 4 microM for a substrate containing more than 100 residues. In contrast to C5-mannuronan epimerases that have been characterized in other bacterial species, P. aeruginosa C5-mannuronan epimerase does not require Ca2+ for activity, and the Ca2+-alginate complex is not a substrate for the enzyme. Analysis of the purified, active enzyme by inductively coupled plasma-emission spectroscopy revealed that no metals were present in the protein. The pH dependence of the kinetic parameters revealed that three residues on the enzyme which all have a pKa of approximately 7.6 must be protonated for catalysis to occur. The composition of the polymeric product of the epimerase reaction was analyzed by 1H NMR spectroscopy, which revealed that tracts of adjacent guluronate residues were readily formed. The reaction reached an apparent equilibrium when the guluronate composition of the polymer was 75%.

  8. Pseudomonas aeruginosa C5-Mannuronan Epimerase: Steady-State Kinetics and Characterization of the Product†

    Science.gov (United States)

    Jerga, Agoston; Raychaudhuri, Aniruddha; Tipton, Peter A.

    2008-01-01

    Alginate is a major constituent of mature biofilms produced by Pseudomonas aeruginosa. The penultimate step in the biosynthesis of alginate is the conversion of some β-D-mannuronate residues in the polymeric substrate polymannuronan to α-L-guluronate residues in a reaction catalyzed by C5-mannuronan epimerase. Specificity studies conducted with size-fractionated oligomannuronates revealed that the minimal substrate contained 9 monosaccharide residues. The maximum velocity of the reaction increased from 0.0018 s−1 to 0.0218 s−1 as the substrate size increased from 10 to 20 residues, and no additional increase in kcat was observed for substrates up to 100 residues in length. The Km decreased from 80 μM for substrate containing fewer than 15 residues to 4 μM for substrate containing over 100 residues. In contrast to C5-mannuronan epimerases that have been characterized in other bacterial species, P. aeruginosa C5-mannuronan epimerase does not require Ca2+ for activity, and the Ca2+-alginate complex is not a substrate for the enzyme. Analysis of purified, active enzyme by inductively coupled plasma-emission spectroscopy revealed that no metals were present in the protein. The pH dependence of the kinetic parameters revealed that 3 residues on the enzyme which all have a pKa of about 7.6 must be protonated for catalysis to occur. The composition of the polymeric product of the epimerase reaction was analyzed by 1H-NMR spectroscopy, which revealed that tracts of adjacent guluronate residues were readily formed. The reaction reached an apparent equilibrium when the guluronate composition of the polymer was 75%. PMID:16401084

  9. Phosphomolybdic acid functionalized graphene loading copper nanoparticles modified electrodes for non-enzymatic electrochemical sensing of glucose

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Jiaoyan; Cao, Xiyue [College of Chemistry and Chemical Engineering, Shandong Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, Collaborative Innovation Center for Marine Biomass Fiber Materials and Textiles, Laboratory of Fiber Materials and Modern Textile, The Growing Base for State Key Laboratory, Qingdao University, Qingdao 266071 (China); Xia, Jianfei, E-mail: xiajianfei@126.com [College of Chemistry and Chemical Engineering, Shandong Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, Collaborative Innovation Center for Marine Biomass Fiber Materials and Textiles, Laboratory of Fiber Materials and Modern Textile, The Growing Base for State Key Laboratory, Qingdao University, Qingdao 266071 (China); Gong, Shida [College of Chemistry and Chemical Engineering, Shandong Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, Collaborative Innovation Center for Marine Biomass Fiber Materials and Textiles, Laboratory of Fiber Materials and Modern Textile, The Growing Base for State Key Laboratory, Qingdao University, Qingdao 266071 (China); Wang, Zonghua, E-mail: wangzonghua@qdu.edu.cn [College of Chemistry and Chemical Engineering, Shandong Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, Collaborative Innovation Center for Marine Biomass Fiber Materials and Textiles, Laboratory of Fiber Materials and Modern Textile, The Growing Base for State Key Laboratory, Qingdao University, Qingdao 266071 (China); Lu, Lin [College of Chemistry and Chemical Engineering, Shandong Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, Collaborative Innovation Center for Marine Biomass Fiber Materials and Textiles, Laboratory of Fiber Materials and Modern Textile, The Growing Base for State Key Laboratory, Qingdao University, Qingdao 266071 (China); Zibo Normal College, Zibo, Shandong 255100 (China)

    2016-08-31

    A sensitive non-enzymatic glucose electrochemical biosensor (Cu/PMo{sub 12}-GR/GCE) was developed based on the combination of copper nanoparticles (CuNPs) and phosphomolybdic acid functionalized graphene (PMo{sub 12}-GR). PMo{sub 12}-GR films were modified on the surface of glassy carbon electrode (GCE) through electrostatic self-assembly with the aid of poly diallyl dimethyl ammonium chloride (PDDA). Then CuNPs were successfully decorated onto the PMo{sub 12}-GR modified GCE through electrodeposition. The morphology of Cu/PMo{sub 12}-GR/GCE was characterized by scanning electron microscope (SEM). Cyclic voltammetry (CV) and chronoamperometry were used to investigate the electrochemical performances of the biosensor. The results indicated that the modified electrode displayed a synergistic effect of PMo{sub 12}-GR sheets and CuNPs towards the electro-oxidation of glucose in the alkaline solution. At the optimal detection potential of 0.50 V, the response towards glucose presented a linear response ranging from 0.10 μM to 1.0 mM with a detection limit of 3.0 × 10{sup −2} μM (S/N = 3). In addition, Cu/PMo{sub 12}-GR/GCE possessed a high selectivity, good reproducibility, excellent stability and acceptable recovery, which indicating the potential application in clinical field. - Highlights: • Cu/PMo{sub 12}-GR/GCE as a non-enzymatic glucose electrochemical sensor. • PMo{sub 12} is efficient for the uniform growth of Cu-NPs and electron transport. • The sensor exhibits good sensitivity and specificity towards glucose.

  10. Microbial functional diversity and enzymatic activity of soil degraded by sulphur mining reclaimed with various waste

    Science.gov (United States)

    Joniec, Jolanta; Frąc, Magdalena

    2017-10-01

    The aim of the study was to evaluate microbial functional diversity based on community level physiological profiling and β-glucosidase activity changes in soil degraded by sulphur mining and subjected to reclamation with various waste. The experiment was set up in the area of the former `Jeziórko' Sulphur Mine (Poland), on a soilless substrate with a particle size distribution of slightly loamy sand. The experimental variants included the application of post-flotation lime, sewage sludge and mineral wool. The analyses of soil samples included the assessment of the following microbiological indices: β-glucosidase activity and functional diversity average well color development and richness). The results indicate that sewage sludge did not exert a significant impact on the functional diversity of microorganisms present in the reclaimed soil. In turn, the application of other types of waste contributed to a significant increase in the parameters of total metabolic activity and functional diversity of the reclaimed soil. However, the temporal analysis of the metabolic profile of soil microorganisms demonstrated that a single application of waste did not yield a durable, stable metabolic profile in the reclaimed soil. Still, there was an increase in β-glucosidase activity, especially in objects treated with sewage sludge.

  11. Glucose level determination with a multi-enzymatic cascade reaction in a functionalized glass chip

    NARCIS (Netherlands)

    Costantini, F.; Tiggelaar, Roald M.; Sennato, S.; Mura, F.; Schlautmann, Stefan; Bordi, F.; Gardeniers, Johannes G.E.; Manetti, C.

    2013-01-01

    In this work we show the functionalization of the interior of microfluidic glass chips with poly(2-hydroxyethyl methacrylate) polymer brushes as anchors for co-immobilization of the enzymes glucose-oxidase and horseradish peroxidase. The formation of the brush layer and subsequent immobilization of

  12. From metagenomic gene discovery to enzymatic breakdown of crosslinks in agricultural fibers for functional products

    Science.gov (United States)

    From the rumen microflora, more than twenty novel genes involved in the hydrolysis of glucuronoarabinoxylans have been discovered and isolated. The specific genes functioning in the breakdown of crosslinkages have been cloned and expressed in E. coli, and the active enzymes purified and extensively ...

  13. Enzymatic Oxidation of Cholesterol: Properties and Functional Effects of Cholestenone in Cell Membranes

    DEFF Research Database (Denmark)

    Neuvonen, M.; Manna, M.; Mokkila, S.

    2014-01-01

    of cholestenone using simulations and cell biological experiments and assessed the functional effects of cholestenone in human cells. Atomistic simulations predicted that cholestenone reduces membrane order, undergoes faster flip-flop and desorbs more readily from membranes than cholesterol. In primary human...

  14. Enzymatic oxidation of cholesterol: properties and functional effects of cholestenone in cell membranes.

    Directory of Open Access Journals (Sweden)

    Maarit Neuvonen

    Full Text Available Bacterial cholesterol oxidase is commonly used as an experimental tool to reduce cellular cholesterol content. That the treatment also generates the poorly degradable metabolite 4-cholesten-3-one (cholestenone has received less attention. Here, we investigated the membrane partitioning of cholestenone using simulations and cell biological experiments and assessed the functional effects of cholestenone in human cells. Atomistic simulations predicted that cholestenone reduces membrane order, undergoes faster flip-flop and desorbs more readily from membranes than cholesterol. In primary human fibroblasts, cholestenone was released from membranes to physiological extracellular acceptors more avidly than cholesterol, but without acceptors it remained in cells over a day. To address the functional effects of cholestenone, we studied fibroblast migration during wound healing. When cells were either cholesterol oxidase treated or part of cellular cholesterol was exchanged for cholestenone with cyclodextrin, cell migration during 22 h was markedly inhibited. Instead, when a similar fraction of cholesterol was removed using cyclodextrin, cells replenished their cholesterol content in 3 h and migrated similarly to control cells. Thus, cholesterol oxidation produces long-term functional effects in cells and these are in part due to the generated membrane active cholestenone.

  15. Enzymatic Synthesis, Amplification, and Application of DNA with a Functionalized Backbone.

    Science.gov (United States)

    Chen, Tingjian; Romesberg, Floyd E

    2017-11-06

    The ability to amplify DNA along with its unprecedented sequence control has led to its use for different applications, but all are limited by the properties available to natural nucleotides. We previously reported the evolution of polymerase SFM4-3, which better tolerates 2'-modified substrates. To explore the utility of SFM4-3, we now report the characterization of its recognition of substrates with 2'-azido, 2'-chloro, 2'-amino, or arabinose sugars. We find that SFM4-3 can efficiently synthesize polymers composed of these nucleotides, and most interestingly, that SFM4-3 can also PCR amplify these modified oligonucleotides. When combined with post-amplification modification, the latter allows for the exponential amplification of polymers that may be functionalized with desired moieties arrayed in a controlled fashion, the utility of which we demonstrate with extensive small molecule functionalization and the production and initial characterization of a novel DNA hydrogel. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Non-enzymatic glucose sensing properties of MoO3 nanorods: experimental and density functional theory investigations

    Science.gov (United States)

    Sharma, Maneesha; Gangan, Abhijeet; Chakraborty, Brahmananda; Sekhar Rout, Chandra

    2017-11-01

    We report the growth of monoclinic MoO3 nanorods by a simple and highly reproducible hydrothermal method. Structural and morphological studies provide significant insights about the phase and crystalline structure of the synthesized samples. Further, the non-enzymatic glucose sensing properties were investigated and the MoO3 nanorods exhibited a sensitivity of 15.4 µA µM‑1 cm‑2 in the 5–175 µM linear range. Also, a quick response time of 8 s towards glucose molecules was observed, exhibiting an excellent electrochemical activity. We have also performed density functional theory (DFT) simulations to qualitatively support our experimental observations by investigating the interactions and charge-transfer mechanism of glucose on MoO3. There is a strong interaction between glucose and the MoO3 surface due to charge transfer from a bonded O atom of glucose to a Mo atom of MoO3 resulting in a strong hybridization between the p orbital of O and d orbital of Mo. Thus, the MoO3 nanorod-based electrodes are found to be good glucose sensing materials for practical industrial applications.

  17. Phosphomolybdic acid functionalized graphene loading copper nanoparticles modified electrodes for non-enzymatic electrochemical sensing of glucose.

    Science.gov (United States)

    Xu, Jiaoyan; Cao, Xiyue; Xia, Jianfei; Gong, Shida; Wang, Zonghua; Lu, Lin

    2016-08-31

    A sensitive non-enzymatic glucose electrochemical biosensor (Cu/PMo12-GR/GCE) was developed based on the combination of copper nanoparticles (CuNPs) and phosphomolybdic acid functionalized graphene (PMo12-GR). PMo12-GR films were modified on the surface of glassy carbon electrode (GCE) through electrostatic self-assembly with the aid of poly diallyl dimethyl ammonium chloride (PDDA). Then CuNPs were successfully decorated onto the PMo12-GR modified GCE through electrodeposition. The morphology of Cu/PMo12-GR/GCE was characterized by scanning electron microscope (SEM). Cyclic voltammetry (CV) and chronoamperometry were used to investigate the electrochemical performances of the biosensor. The results indicated that the modified electrode displayed a synergistic effect of PMo12-GR sheets and CuNPs towards the electro-oxidation of glucose in the alkaline solution. At the optimal detection potential of 0.50 V, the response towards glucose presented a linear response ranging from 0.10 μM to 1.0 mM with a detection limit of 3.0 × 10(-2) μM (S/N = 3). In addition, Cu/PMo12-GR/GCE possessed a high selectivity, good reproducibility, excellent stability and acceptable recovery, which indicating the potential application in clinical field. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Valorisation of tuna processing waste biomass for recovery of functional and antioxidant peptides using enzymatic hydrolysis and membrane fractionation process.

    Science.gov (United States)

    Saidi, Sami; Ben Amar, Raja

    2016-10-01

    The enzymatic hydrolysis using Prolyve BS coupled to membrane process (Ultrafiltration (UF) and nanofiltration (NF)) is a means of biotransformation of tuna protein waste to Tuna protein hydrolysate (TPH) with higher added values. This method could be an effective solution for the production of bioactive compounds used in various biotechnological applications and minimizing the pollution problems generated by the seafood processing industries. The amino acid composition, functional and antioxidant properties of produced TPH were evaluated. The results show that the glutamic acid, aspartic acid, glycine, alaline, valine and leucine were the major amino acids detected in the TPH profile. After membrane fractionation process, those major amino acids were concentrated in the NF retentate (NFR). The NFR and NF permeate (NFP) have a higher protein solubility (>95 %) when compared to TPH (80 %). Higher oil and water binding capacity were observed in TPH and higher emulsifying and foam stability was found in UF retentate. The NFP showed the highest DPPH radical scavenging activity (65 %). The NFR contained antioxidant amino acid (30.3 %) showed the highest superoxide radical and reducing power activities. The TPH showed the highest iron chelating activity (75 %) compared to other peptide fractions. The effect of the membrane fractionation on the molecular weight distribution of the peptide and their bioactivities was underlined. We concluded that the TPH is a valuable source of bioactive peptides and their peptide fractions may serve as useful ingredients for application in food industry and formulation of nutritional products.

  19. Enzymatic electrodes nanostructured with functionalized carbon nanotubes for biofuel cell applications.

    Science.gov (United States)

    Nazaruk, E; Sadowska, K; Biernat, J F; Rogalski, J; Ginalska, G; Bilewicz, R

    2010-10-01

    Nanostructured bioelectrodes were designed and assembled into a biofuel cell with no separating membrane. The glassy carbon electrodes were modified with mediator-functionalized carbon nanotubes. Ferrocene (Fc) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS) bound chemically to the carbon nanotubes were found useful as mediators of the enzyme catalyzed electrode processes. Glucose oxidase from Aspergillus niger AM-11 and laccase from Cerrena unicolor C-139 were incorporated in a liquid-crystalline matrix-monoolein cubic phase. The carbon nanotubes-nanostructured electrode surface was covered with the cubic phase film containing the enzyme and acted as the catalytic surface for the oxidation of glucose and reduction of oxygen. Thanks to the mediating role of derivatized nanotubes the catalysis was almost ten times more efficient than on the GCE electrodes: catalytic current of glucose oxidation was 1 mA cm(-2) and oxygen reduction current exceeded 0.6 mA cm(-2). The open circuit voltage of the biofuel cell was 0.43 V. Application of carbon nanotubes increased the maximum power output of the constructed biofuel cell to 100 μW cm(-2) without stirring of the solution which was ca. 100 times more efficient than using the same bioelectrodes without nanotubes on the electrode surface.

  20. Enzymatic decontamination

    Directory of Open Access Journals (Sweden)

    Edyta Prusińska-Kurstak

    2014-12-01

    Full Text Available [b]Abstract[/b]. This paper is devoted to the methods of decontamination of weapons of mass destruction (biological and chemical, based on the use of protein catalysts of chemical reactions — enzymes. This paper presents the possibility of using enzymes to neutralize the harmful and destructive to the environment and human chemicals used in weapons of mass destruction. The mechanism of the enzymatic reaction is showed. These are the possibilities of using lysozyme as destructor dangerous bacteria (E. coli, anthrax Bacillus anthracis and their spores. The advantages and disadvantages of chemical and enzymatic methods of decontamination have been compared. It was found that under certain conditions the enzymes can be an alternative to chemical methods of decontamination of weapons of mass destruction.[b]Keywords[/b]: decontamination, weapons of mass destruction, enzymes

  1. UDP-galactose 4-epimerase from Escherichia coli: existence of a catalytic monomer.

    Science.gov (United States)

    Nayar, S; Bhattacharyya, D

    1997-06-16

    UDP-galactose 4-epimerase from Escherichia coli is a homodimer of molecular mass 39 kDa/subunit and requires NAD as a co-factor. X-ray crystallographic studies indicate two pyridine nucleotide co-factor-binding sites of the dimeric molecule situated in a symmetry-oriented manner. Size-exclusion HPLC of an equilibrium intermediate at 3 M urea suggests a monomeric holoenzyme structure that is catalytically active. Ultracentrifugal studies of the native enzyme in a 5-20% sucrose gradient at low protein concentration also indicate existence of a catalytic monomer. The monomer resembles the dimeric protein in stability and most of its physico-chemical properties.

  2. A non-enzymatic function of Golgi glycosyltransferases: mediation of Golgi fragmentation by interaction with non-muscle myosin IIA.

    Science.gov (United States)

    Petrosyan, Armen; Cheng, Pi-Wan

    2013-06-01

    The Golgi apparatus undergoes morphological changes under stress or malignant transformation, but the precise mechanisms are not known. We recently showed that non-muscle myosin IIA (NMIIA) binds to the cytoplasmic tail of Core 2 N-acetylglucosaminyltransferase mucus-type (C2GnT-M) and transports it to the endoplasmic reticulum for recycling. Here, we report that Golgi fragmentation induced by brefeldin A (BFA) or coatomer protein (β-COP) knockdown (KD) in Panc1-bC2GnT-M (c-Myc) cells is accompanied by the increased association of NMIIA with C2GnT-M and its degradation by proteasomes. Golgi fragmentation is prevented by inhibition or KD of NMIIA. Using multiple approaches, we have shown that the speed of BFA-induced Golgi fragmentation is positively correlated with the levels of this enzyme in the Golgi. The observation is reproduced in LNCaP cells which express high levels of two endogenous glycosyltransferases--C2GnT-L and β-galactoside α2,3 sialyltransferase 1. NMIIA is found to form complexes with these two enzymes but not Golgi matrix proteins. The KD of both enzymes or the prevention of Golgi glycosyltransferases from exiting endoplasmic reticulum reduced Golgi-associated NMIIA and decreased the BFA-induced fragmentation. Interestingly, the fragmented Golgi detected in colon cancer HT-29 cells can be restored to a compact morphology after inhibition or KD of NMIIA. The Golgi disorganization induced by the microtubule or actin destructive agent is NMIIA-independent and does not affect the levels of glycosyltransferases. We conclude that NMIIA interacts with Golgi residential but not matrix proteins, and this interaction is responsible for Golgi fragmentation induced by β-COP KD or BFA treatment. This is a novel non-enzymatic function of Golgi glycosyltransferases.

  3. A non-enzymatic function of Golgi glycosyltransferases: Mediation of Golgi fragmentation by interaction with non-muscle myosin IIA

    Science.gov (United States)

    Petrosyan, Armen; Cheng, Pi-Wan

    2013-01-01

    The Golgi apparatus undergoes morphological changes under stress or malignant transformation, but the precise mechanisms are not known. We recently showed that non-muscle myosin IIA (NMIIA) binds to the cytoplasmic tail of Core 2 N-acetylglucosaminyltransferase mucus-type (C2GnT-M) and transports it to the endoplasmic reticulum for recycling. Here, we report that Golgi fragmentation induced by brefeldin A (BFA) or coatomer protein (β-COP) knockdown (KD) in Panc1-bC2GnT-M (c-Myc) cells is accompanied by the increased association of NMIIA with C2GnT-M and its degradation by proteasomes. Golgi fragmentation is prevented by inhibition or KD of NMIIA. Using multiple approaches, we have shown that the speed of BFA-induced Golgi fragmentation is positively correlated with the levels of this enzyme in the Golgi. The observation is reproduced in LNCaP cells which express high levels of two endogenous glycosyltransferases—C2GnT-L and β-galactoside α2,3 sialyltransferase 1. NMIIA is found to form complexes with these two enzymes but not Golgi matrix proteins. The KD of both enzymes or the prevention of Golgi glycosyltransferases from exiting endoplasmic reticulum reduced Golgi-associated NMIIA and decreased the BFA-induced fragmentation. Interestingly, the fragmented Golgi detected in colon cancer HT-29 cells can be restored to a compact morphology after inhibition or KD of NMIIA. The Golgi disorganization induced by the microtubule or actin destructive agent is NMIIA-independent and does not affect the levels of glycosyltransferases. We conclude that NMIIA interacts with Golgi residential but not matrix proteins, and this interaction is responsible for Golgi fragmentation induced by β-COP KD or BFA treatment. This is a novel non-enzymatic function of Golgi glycosyltransferases. PMID:23396488

  4. Evolution of land plant genes encoding L-Ala-D/L-Glu epimerases (AEEs) via horizontal gene transfer and positive selection.

    Science.gov (United States)

    Yang, Zefeng; Wang, Yifan; Zhou, Yong; Gao, Qingsong; Zhang, Enying; Zhu, Lei; Hu, Yunyun; Xu, Chenwu

    2013-03-01

    The L-Ala-D/L-Glu epimerases (AEEs), a subgroup of the enolase superfamily, catalyze the epimerization of L-Ala-D/L-Glu and other dipeptides in bacteria and contribute to the metabolism of the murein peptide of peptidoglycan. Although lacking in peptidoglycan, land plants possess AEE genes that show high similarity to those in bacteria. Similarity searches revealed that the AEE gene is ubiquitous in land plants, from bryophytas to angiosperms. However, other eukaryotes, including green and red algae, do not contain genes encoding proteins with an L-Ala-D/L-Glu_epimerase domain. Homologs of land plant AEE genes were found to only be present in prokaryotes, especially in bacteria. Phylogenetic analysis revealed that the land plant AEE genes formed a monophyletic group with some bacterial homologs. In addition, land plant AEE proteins showed the highest similarity with these bacterial homologs and shared motifs only conserved in land plant and these bacterial AEEs. Integrated information on the taxonomic distribution, phylogenetic relationships and sequence similarity of the AEE proteins revealed that the land plant AEE genes were acquired from bacteria through an ancient horizontal gene transfer (HGT) event. Further evidence revealed that land plant AEE genes had undergone positive selection and formed the main characteristics of exon/intron structures through gaining some introns during the initially evolutionary period in the ancestor of land plants. The results of this study clearly demonstrated that the ancestor of land plants acquired an AEE gene from bacteria via an ancient HGT event. Other findings illustrated that adaptive evolution through positive selection has contributed to the functional adaptation and fixation of this gene in land plants.

  5. Evolution of land plant genes encoding L-Ala-D/L-Glu epimerases (AEEs) via horizontal gene transfer and positive selection

    Science.gov (United States)

    2013-01-01

    Background The L-Ala-D/L-Glu epimerases (AEEs), a subgroup of the enolase superfamily, catalyze the epimerization of L-Ala-D/L-Glu and other dipeptides in bacteria and contribute to the metabolism of the murein peptide of peptidoglycan. Although lacking in peptidoglycan, land plants possess AEE genes that show high similarity to those in bacteria. Results Similarity searches revealed that the AEE gene is ubiquitous in land plants, from bryophytas to angiosperms. However, other eukaryotes, including green and red algae, do not contain genes encoding proteins with an L-Ala-D/L-Glu_epimerase domain. Homologs of land plant AEE genes were found to only be present in prokaryotes, especially in bacteria. Phylogenetic analysis revealed that the land plant AEE genes formed a monophyletic group with some bacterial homologs. In addition, land plant AEE proteins showed the highest similarity with these bacterial homologs and shared motifs only conserved in land plant and these bacterial AEEs. Integrated information on the taxonomic distribution, phylogenetic relationships and sequence similarity of the AEE proteins revealed that the land plant AEE genes were acquired from bacteria through an ancient horizontal gene transfer (HGT) event. Further evidence revealed that land plant AEE genes had undergone positive selection and formed the main characteristics of exon/intron structures through gaining some introns during the initially evolutionary period in the ancestor of land plants. Conclusions The results of this study clearly demonstrated that the ancestor of land plants acquired an AEE gene from bacteria via an ancient HGT event. Other findings illustrated that adaptive evolution through positive selection has contributed to the functional adaptation and fixation of this gene in land plants. PMID:23452519

  6. Chemical mechanism and specificity of the C5-mannuronan epimerase reaction.

    Science.gov (United States)

    Jerga, Agoston; Stanley, Matthew D; Tipton, Peter A

    2006-08-01

    C5-mannuronan epimerase catalyzes the formation of alpha-L-guluronate residues from beta-D-mannuronate residues in the synthesis of the linear polysaccharide alginate. The reaction requires the abstraction of a proton from C5 of the residue undergoing epimerization followed by re-protonation on the opposite face. Rapid-mixing chemical quench experiments were conducted to determine the nature of the intermediate formed upon proton abstraction in the reaction catalyzed by the enzyme from Pseudomonas aeruginosa. Colorimetric and HPLC analysis of quenched samples indicated that shortened oligosaccharides containing an unsaturated sugar residue form as transient intermediates in the epimerization reaction. This suggests that the carbanion is stabilized by glycal formation, concomitant with cleavage of the glycosidic bond between the residue undergoing epimerization and the adjacent residue. The time dependence of glycal formation suggested that slow steps flank the chemical steps in the catalytic cycle. Solvent isotope effects on V and V/K were unity, consistent with a catalytic cycle in which chemistry is not rate-limiting. The specificity of the epimerase with regard to neighboring residues was examined, and it was determined that the enzyme showed no bias for mannuronate residues adjacent to guluronates versus those adjacent to mannuronates. Proton abstraction and sugar epimerization were irreversible. Existing guluronate residues already present in the polysaccharide were not converted to mannuronates, nor was incorporation of solvent deuterium into existing mannuronates observed.

  7. Structure-function relationships of bacterial and enzymatically produced reuterans and dextran in sourdough bread baking application.

    Science.gov (United States)

    Chen, Xiao Yan; Levy, Clemens; Gänzle, Michael G

    2016-12-19

    Exopolysaccharides from lactic acid bacteria may improve texture and shelf life of bread. The effect of exopolysaccharides on bread quality, however, depends on properties of the EPS and the EPS producing strain. This study investigated structure-function relationships of EPS in baking application. The dextransucrase DsrM and the reuteransucrase GtfA were cloned from Weissella cibaria 10M and Lactobacillus reuteri TMW1.656, respectively, and heterologously expressed in Escherichia coli. Site-directed mutagenesis of GtfA was generates reuterans with different glycosidic bonds. NMR spectrum indicated reuteranPI, reuteranNS and reuteranPINS produced by GtfA-V1024P:V1027I, GtfA-S1135N:A1137S and GtfA-V1024P:V1027I:S1135N:A1137S, respectively, had a higher proportion of α-(1→4) linkages when compared to reuteran. ReuteranNS has the lowest molecular weight as measured by asymmetric flow-field-flow fractionation. The reuteransucrase negative mutant L. reuteri TMW1.656ΔgtfA was generated as EPS-negative derivative of L. reuteri TMW1.656. Cell counts, pH, and organic acid levels of sourdough fermented with L. reuteri TMW1.656 and TMW1.656ΔgtfA were comparable. Reuteran produced by L. reuteri TMW1.656 during growth in sourdough and reuteran produced ex situ by GtfA-ΔN had comparable effects on bread volume and crumb hardness. Enzymatically produced dextran improved volume and texture of wheat bread, and of bread containing 20% rye flour. ReuteranNS but not reuteranPI or reuteran was as efficient as dextran in enhancing wheat bread volume and texture. Overall, reuteran linkage type and molecular weight are determinants of EPS effects on bread quality. This study established a valuable method to elucidate structure-function relationships of glucans in baking applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Optimization of the xylan degradation activity of monolithic enzymatic membranes as a function of their composition using design of experiments.

    Science.gov (United States)

    Cano, Angels; Moschou, Elizabeth A; Daunert, Sylvia; Coello, Jordi; Palet, Cristina

    2006-10-01

    The aim of this work was the development and optimization of enzymatic monolithic membranes with high catalytic activity for the degradation of xylan into xylooligosaccharides. The chemometric tool design of experiments has been utilized here for the first time for the optimization of the enzymatic activity of the monolithic membranes based on their constituents. The effect of three process variables, including the amount of various monomer contents and the porogenic solvents ratio, has been studied on the enzymatic activity of the resulted membranes. The experimental design chosen was a central face centred with six central points in order to obtain an orthogonal model, with the precision of the results being independent of the range of values considered for each parameter. The software Modde(c) 6.0 from Umetrics(c) was used to build and analyze the results of the experimental design using partial least squares regression. The optimization of the suggested model provided the best membrane composition to achieve maximum enzymatic activity, which can be related to the amount of enzyme immobilized on the monolithic membrane. The predictive capacity of the model was evaluated performing additional experiments.

  9. Asymmetric transformation of ß- and γ-functionalized alcohols : Study of combined ruthenium-catalyzed racemization and enzymatic resolution

    OpenAIRE

    Träff, Annika

    2011-01-01

    The major part of this thesis describes the asymmetric synthesis of β- and γ-amino alcohols through the combination of ruthenium catalyzed racemization and enzymatic kinetic resolution. The dynamic kinetic resolution, DKR, protocol for chlorohydrins was improved by employing Bäckvall’s catalyst, which is a base activated racemization catalyst, in combination with Burkholderia cepacia lipase. These optimized conditions broadened the substrate scope and improved the yields and ee’s of the obtai...

  10. Expression, crystallization and preliminary characterization of methylmalonyl coenzyme A epimerase from Propionibacterium shermanii.

    Science.gov (United States)

    McCarthy, A A; Baker, H M; Shewry, S C; Kagawa, T F; Saafi, E; Patchett, M L; Baker, E N

    2001-05-01

    Methylmalonyl-CoA epimerase (MMCE) is an enzyme that interconverts the R and S epimers of methylmalonyl-CoA in the pathway that links propionyl-CoA with succinyl-CoA. This is used for both biosynthetic and degradative processes, including the breakdown of odd-numbered fatty acids and some amino acids. The enzyme has been expressed in Escherichia coli both as the native enzyme and as its selenomethionine (SeMet) derivative. Crystals of both forms have been obtained by vapour diffusion using monomethylether PEG 2000 as precipitant. The native MMCE crystals are orthorhombic, with unit-cell parameters a = 56.0, b = 114.0, c = 156.0 A, and the SeMet-MMCE crystals are monoclinic, with unit-cell parameters a = 43.6, b = 78.6, c = 89.4 A, beta = 92.0 degrees; both diffract to better than 2.8 A resolution.

  11. Rv3634c from Mycobacterium tuberculosis H37Rv encodes an enzyme with UDP-Gal/Glc and UDP-GalNAc 4-epimerase activities.

    Directory of Open Access Journals (Sweden)

    Peehu Pardeshi

    Full Text Available A bioinformatics study revealed that Mycobacterium tuberculosis H37Rv (Mtb contains sequence homologs of Campylobacter jejuni protein glycosylation enzymes. The ORF Rv3634c from Mtb was identified as a sequence homolog of C. jejuni UDP-Gal/GalNAc 4-epimerase. This study reports the cloning of Rv3634c and its expression as an N-terminal His-tagged protein. The recombinant protein was shown to have UDP-Gal/Glc 4-epimerase activity by GOD-POD assay and by reverse phase HPLC. This enzyme was shown to have UDP-GalNAc 4-epimerase activity also. Residues Ser121, Tyr146 and Lys150 were shown by site-directed mutagenesis to be important for enzyme activity. Mutation of Ser121 and Tyr146 to Ala and Phe, respectively, led to complete loss of activity whereas mutation of Lys150 to Arg led to partial loss of activity. There were no gross changes in the secondary structures of any of these three mutants. These results suggest that Ser121 and Tyr146 are essential for epimerase activity of Rv3634c. UDP-Gal/Glc 4-epimerases from other organisms also have a catalytic triad consisting of Ser, Tyr and Lys. The triad carries out proton transfer from nucleotide sugar to NAD+ and back, thus effecting the epimerization of the substrate. Addition of NAD+ to Lys150 significantly abrogates the loss of activity, suggesting that, as in other epimerases, NAD+ is associated with Rv3634c.

  12. Evidence that the fadB gene of the fadAB operon of Escherichia coli encodes 3-hydroxyacyl-coenzyme A (CoA) epimerase, delta 3-cis-delta 2-trans-enoyl-CoA isomerase, and enoyl-CoA hydratase in addition to 3-hydroxyacyl-CoA dehydrogenase.

    Science.gov (United States)

    Yang, S Y; Li, J M; He, X Y; Cosloy, S D; Schulz, H

    1988-01-01

    Genetic complementation of a mutant defective in fatty acid oxidation (fadAB) with plasmids containing DNA inserts from the fadAB region of the Escherichia coli genome was studied. The mutant containing the hybrid plasmid with a 5.2-kilobase (kb) PstI-SalI fragment was found to overproduce 3-hydroxyacyl-coenzyme A (CoA) epimerase and delta 3-cis-delta 2-trans-enoyl-CoA isomerase as well as three other beta-oxidation enzymes by 16- to 18-fold compared with the wild-type parental strain LE392. The purification of a fully functional multienzyme complex of fatty acid oxidation from the transformant ultimately established that the 5.2-kb DNA fragment contained an entire fadAB operon. Since immunotitration of cell extracts with antibodies against the fatty acid oxidation complex proved that all 3-hydroxyacyl-CoA epimerase and delta 3-cis-delta 2-trans-enoyl-CoA isomerase activities were associated with the complex, no genetic loci other than the fadAB operon encoded these two enzymes. Moreover, the binding of antibodies caused parallel inhibition of four component enzymes, whereas 3-ketoacyl-CoA thiolase activity was slightly increased. These findings support the suggestion that the epimerase and isomerase as well as enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase are located on the same polypeptide. The results of this study, together with published data (S.-Y. Yang and H. Schulz, J. Biol. Chem. 258:9780-9785, 1983), lead to the conclusion that 3-hydroxyacyl-CoA epimerase, delta 3-cis-delta 2-trans-enoyl-CoA isomerase, and enoyl-CoA hydratase in addition to 3-hydroxyacyl-CoA dehydrogenase are encoded by the fadB gene. Images PMID:3286611

  13. Altered cofactor binding affects stability and activity of human UDP-galactose 4′-epimerase: implications for type III galactosemia

    Science.gov (United States)

    McCorvie, Thomas J.; Liu, Ying; Frazer, Andrew; Gleason, Tyler J.; Fridovich-Keil, Judith L.; Timson, David J.

    2012-01-01

    Deficiency of UDP-galactose 4′-epimerase is implicated in type III galactosemia. Two variants, p.K161N-hGALE and p.D175N-hGALE, have been previously found in combination with other alleles in patients with a mild form of the disease. Both variants were studied in vivo and in vitro and showed different levels of impairment. p.K161N-hGALE was severely impaired with substantially reduced enzymatic activity, increased thermal stability, reduced cofactor binding and inability to rescue the galactose-sensitivity of gal10-null yeast. Interestingly p.K161N-hGALE showed less impairment of activity with UDP-N-acetylgalactosamine in comparison to UDP-galactose. Differential scanning fluorimetry revealed that p.K161N-hGALE was more stable than the wild-type protein and only changed stability in the presence of UDP-N-acetylglucosamine and NAD+. p.D175N-hGALE essentially rescued the galactose-sensitivity of gal10-null yeast, was less stable than the wild-type protein but showed increased stability in the presence of substrates and cofactor. We postulate that p.K161N-hGALE causes its effects by abolishing an important interaction between the protein and the cofactor, whereas p.D175N-hGALE is predicted to remove a stabilizing salt bridge between the ends of two α-helices that contain residues that interact with NAD+. These results suggest that the cofactor binding is dynamic and that its loss results in significant structural changes that may be important in disease causation. PMID:22613355

  14. UDP-N-Acetylglucosamine 4-Epimerase Activity Indicates the Presence of N-Acetylgalactosamine in Exopolysaccharides of Streptococcus thermophilus Strains

    Science.gov (United States)

    Degeest, Bart; Vaningelgem, Frederik; Laws, Andrew P.; De Vuyst, Luc

    2001-01-01

    The monomer composition of the exopolysaccharides (EPS) produced by Streptococcus thermophilus LY03 and S. thermophilus Sfi20 were evaluated by high-pressure liquid chromatography with amperometric detection and nuclear magnetic resonance spectroscopy. Both strains produced the same EPS composed of galactose, glucose, and N-acetylgalactosamine. Further, it was demonstrated that the activity of the precursor-producing enzyme UDP-N-acetylglucosamine 4-epimerase, converting UDP-N-acetylglucosamine into UDP-N-acetylgalactosamine, is responsible for the presence of N-acetylgalactosamine in the EPS repeating units of both strains. The activity of UDP-N-acetylglucosamine 4-epimerase was higher in both S. thermophilus strains than in a non-EPS-producing control strain. However, the level of this activity was not correlated with EPS yields, a result independent of the carbohydrate source applied in the fermentation process. On the other hand, both the amounts of EPS and the carbohydrate consumption rates were influenced by the type of carbohydrate source used during S. thermophilus Sfi20 fermentations. A correlation between activities of the enzymes α-phosphoglucomutase, UDP-glucose pyrophosphorylase, and UDP-galactose 4-epimerase and EPS yields was seen. These experiments confirm earlier observed results for S. thermophilus LY03, although S. thermophilus Sfi20 preferentially consumed glucose for EPS production instead of lactose in contrast to the former strain. PMID:11525994

  15. Structural Basis for Substrate Specificity in Phosphate Binding (beta/alpha)8-Barrels: D-Allulose 6-Phosphate 3-Epimerase from Escherichia coli K-12

    Energy Technology Data Exchange (ETDEWEB)

    Chan,K.; Fedorov, A.; Almo, S.; Gerlt, J.

    2008-01-01

    Enzymes that share the ({beta}/{alpha})8-barrel fold catalyze a diverse range of reactions. Many utilize phosphorylated substrates and share a conserved C-terminal ({beta}/a)2-quarter barrel subdomain that provides a binding motif for the dianionic phosphate group. We recently reported functional and structural studies of d-ribulose 5-phosphate 3-epimerase (RPE) from Streptococcus pyogenes that catalyzes the equilibration of the pentulose 5-phosphates d-ribulose 5-phosphate and d-xylulose 5-phosphate in the pentose phosphate pathway [J. Akana, A. A. Fedorov, E. Fedorov, W. R. P. Novack, P. C. Babbitt, S. C. Almo, and J. A. Gerlt (2006) Biochemistry 45, 2493-2503]. We now report functional and structural studies of d-allulose 6-phosphate 3-epimerase (ALSE) from Escherichia coli K-12 that catalyzes the equilibration of the hexulose 6-phosphates d-allulose 6-phosphate and d-fructose 6-phosphate in a catabolic pathway for d-allose. ALSE and RPE prefer their physiological substrates but are promiscuous for each other's substrate. The active sites (RPE complexed with d-xylitol 5-phosphate and ALSE complexed with d-glucitol 6-phosphate) are superimposable (as expected from their 39% sequence identity), with the exception of the phosphate binding motif. The loop following the eighth {beta}-strand in ALSE is one residue longer than the homologous loop in RPE, so the binding site for the hexulose 6-phosphate substrate/product in ALSE is elongated relative to that for the pentulose 5-phosphate substrate/product in RPE. We constructed three single-residue deletion mutants of the loop in ALSE, ?T196, ?S197 and ?G198, to investigate the structural bases for the differing substrate specificities; for each, the promiscuity is altered so that d-ribulose 5-phosphate is the preferred substrate. The changes in kcat/Km are dominated by changes in kcat, suggesting that substrate discrimination results from differential transition state stabilization. In both ALSE and RPE, the

  16. Association between plasma soluble RAGE and renal function is unaffected by medication usage and enzymatic antioxidants in chronic kidney disease with type 2 diabetes.

    Science.gov (United States)

    Wong, Foo Nian; Tan, Jin Ai Mary Anne; Keng, Tee Chau; Ng, Kok Peng; Chua, Kek Heng; Kuppusamy, Umah Rani

    2016-01-30

    This study aimed to investigate the relationship between soluble RAGE and estimated glomerular filtration rate (eGFR) in patients with chronic kidney disease (CKD) after controlling for the potential confounding factors such as medication usage and enzymatic antioxidants. A total of 222 CKD patients whose eGFR is less than 60ml/min/1.73m(2) and 111 non-CKD individuals were recruited. The study subjects were classified based on their diabetes status. The plasma glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities as well as plasma soluble RAGE level were measured. The plasma GPx and SOD activities were significantly lower and the plasma soluble RAGE level was significantly higher in the CKD patients than in the non-CKD individuals, regardless of the diabetes status. Soluble RAGE was significantly correlated with eGFR in both diabetic CKD (D-CKD) and non-diabetic CKD (ND-CKD) patients. The association between soluble RAGE and eGFR remained largely unaffected by the confounding factors in D-CKD patients. However, the confounding effect of enzymatic antioxidants in the relationship between eGFR and soluble RAGE was observed in ND-CKD patients. The increased plasma level of soluble RAGE is a better indicator of renal function decline in diabetic CKD patients instead of non-diabetic CKD patients. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Acetolactate synthase (AlsS) in Bacillus licheniformis WX-02: enzymatic properties and efficient functions for acetoin/butanediol and L-valine biosynthesis.

    Science.gov (United States)

    Huo, Yanli; Zhan, Yangyang; Wang, Qin; Li, Shunyi; Yang, Shihui; Nomura, Christopher T; Wang, Changjun; Chen, Shouwen

    2018-01-01

    Acetolactate synthase catalyzes two molecules of pyruvates to form α-acetolactate, which is further converted to acetoin and 2,3-butanediol. In this study, by heterologous expression in Escherichia coli, the enzymatic properties of acetolactate synthase (AlsS) from Bacillus licheniformis WX-02 were characterized. Its K m and k cat for pyruvate were 3.96 mM and 514/s, respectively. It has the optimal activity at pH 6.5, 37 °C and was feedback inhibited by L-valine, L-leucine and L-isoleucine. Furthermore, the alsS-deficient strain could not produce acetoin, 2,3-butanediol, and L-valine, while the complementary strain was able to restore these capacities. The alsS overexpressing strain produced higher amounts of acetoin/2,3-butanediol (57.06 g/L) and L-valine (2.68 mM), which were 10.90 and 92.80% higher than those of the control strain, respectively. This is the first report regarding the in-depth understanding of AlsS enzymatic properties and its functions in B. licheniformis, and overexpression of AlsS can effectively improve acetoin/2,3-butanediol and L-valine production in B. licheniformis. We envision that this AlsS can also be applied in the improvement of acetoin/2,3-butanediol and L-valine production in other microbes.

  18. Structure, kinetic characterization and subcellular localization of the two ribulose 5-phosphate epimerase isoenzymes from Trypanosoma cruzi.

    Directory of Open Access Journals (Sweden)

    Soledad Natalia Gonzalez

    Full Text Available The enzyme of the pentose phosphate pathway (PPP ribulose-5-phosphate-epimerase (RPE is encoded by two genes present in the genome of Trypanosoma cruzi CL Brener clone: TcRPE1 and TcRPE2. Despite high sequence similarity at the amino acid residue level, the recombinant isoenzymes show a strikingly different kinetics. Whereas TcRPE2 follows a typical michaelian behavior, TcRPE1 shows a complex kinetic pattern, displaying a biphasic curve, suggesting the coexistence of -at least- two kinetically different molecular forms. Regarding the subcellular localization in epimastigotes, whereas TcRPE1 is a cytosolic enzyme, TcRPE2 is localized in glycosomes. To our knowledge, TcRPE2 is the first PPP isoenzyme that is exclusively localized in glycosomes. Over-expression of TcRPE1, but not of TcRPE2, significantly reduces the parasite doubling time in vitro, as compared with wild type epimastigotes. Both TcRPEs represent single domain proteins exhibiting the classical α/β TIM-barrel fold, as expected for enzymes with this activity. With regard to the architecture of the active site, all the important amino acid residues for catalysis -with the exception of M58- are also present in both TcRPEs models. The superimposition of the binding pocket of both isoenzyme models shows that they adopt essentially identical positions in the active site with a residue specific RMSD < 2Å, with the sole exception of S12, which displays a large deviation (residue specific RMSD: 11.07 Å. Studies on the quaternary arrangement of these isoenzymes reveal that both are present in a mixture of various oligomeric species made up of an even number of molecules, probably pointing to the dimer as their minimal functional unit. This multiplicity of oligomeric species has not been reported for any of the other RPEs studied so far and it might bear implications for the regulation of TcRPEs activity, although further investigation will be necessary to unravel the physiological

  19. D-ribulose-5-phosphate 3-epimerase: Cloning and heterologous expression of the spinach gene, and purification and characterization of the recombinant enzyme

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Y.R. [Univ. of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, Oak Ridge, TN (United States); Hartman, F.C.; Lu, T.Y.S.; Larimer, F.W. [Oak Ridge National Lab., TN (United States)

    1998-09-01

    The authors have achieved, to their knowledge, the first high-level heterologous expression of the gene encoding D-ribulose-5-phosphate 3-epimerase from any source, thereby permitting isolation and characterization of the epimerase as found in photosynthetic organisms. The extremely labile recombinant spinach (Spinacia oleracea L.) enzyme was stabilized by DL-{alpha}-glycerophosphate or ethanol and destabilized by D-ribulose-5-phosphate or 2-mercaptoethanol. Despite this lability, the unprecedentedly high specific activity of the purified material indicates that the structural integrity of the enzyme is maintained throughout isolation. Ethylenediaminetetraacetate and divalent metal cations did not affect epimerase activity, thereby excluding a requirement for the latter in catalysis. As deduced from the sequence of the cloned spinach gene and the electrophoretic mobility under denaturing conditions of the purified recombinant enzyme, its 25-kD subunit size was about the same as that of the corresponding epimerases of yeast and mammals. However, in contrast to these other species, the recombinant spinach enzyme was octameric rather than dimeric, as assessed by gel filtration and polyacrylamide gel electrophoresis under nondenaturing conditions. Western-blot analyses with antibodies to the purified recombinant enzyme confirmed that the epimerase extracted from spinach leaves is also octameric.

  20. High content screening identifies decaprenyl-phosphoribose 2' epimerase as a target for intracellular antimycobacterial inhibitors.

    Directory of Open Access Journals (Sweden)

    Thierry Christophe

    2009-10-01

    Full Text Available A critical feature of Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB, is its ability to survive and multiply within macrophages, making these host cells an ideal niche for persisting microbes. Killing the intracellular tubercle bacilli is a key requirement for efficient tuberculosis treatment, yet identifying potent inhibitors has been hampered by labor-intensive techniques and lack of validated targets. Here, we present the development of a phenotypic cell-based assay that uses automated confocal fluorescence microscopy for high throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages. Screening a library of 57,000 small molecules led to the identification of 135 active compounds with potent intracellular anti-mycobacterial efficacy and no host cell toxicity. Among these, the dinitrobenzamide derivatives (DNB showed high activity against M. tuberculosis, including extensively drug resistant (XDR strains. More importantly, we demonstrate that incubation of M. tuberculosis with DNB inhibited the formation of both lipoarabinomannan and arabinogalactan, attributable to the inhibition of decaprenyl-phospho-arabinose synthesis catalyzed by the decaprenyl-phosphoribose 2' epimerase DprE1/DprE2. Inhibition of this new target will likely contribute to new therapeutic solutions against emerging XDR-TB. Beyond validating the high throughput/content screening approach, our results open new avenues for finding the next generation of antimicrobials.

  1. Post-translational modification of ribosomally synthesized peptides by a radical SAM epimerase in Bacillus subtilis

    Science.gov (United States)

    Benjdia, Alhosna; Guillot, Alain; Ruffié, Pauline; Leprince, Jérôme; Berteau, Olivier

    2017-07-01

    Ribosomally synthesized peptides are built out of L-amino acids, whereas D-amino acids are generally the hallmark of non-ribosomal synthetic processes. Here we show that the model bacterium Bacillus subtilis is able to produce a novel type of ribosomally synthesized and post-translationally modified peptide that contains D-amino acids, and which we propose to call epipeptides. We demonstrate that a two [4Fe-4S]-cluster radical S-adenosyl-L-methionine (SAM) enzyme converts L-amino acids into their D-counterparts by catalysing Cα-hydrogen-atom abstraction and using a critical cysteine residue as the hydrogen-atom donor. Unexpectedly, these D-amino acid residues proved to be essential for the activity of a peptide that induces the expression of LiaRS, a major component of the bacterial cell envelope stress-response system. Present in B. subtilis and in several members of the human microbiome, these epipeptides and radical SAM epimerases broaden the landscape of peptidyl structures accessible to living organisms.

  2. Modelling Tethered Enzymatic Reactions

    Science.gov (United States)

    Solis Salas, Citlali; Goyette, Jesse; Coker-Gordon, Nicola; Bridge, Marcus; Isaacson, Samuel; Allard, Jun; Maini, Philip; Dushek, Omer

    Enzymatic reactions are key to cell functioning, and whilst much work has been done in protein interaction in cases where diffusion is possible, interactions of tethered proteins are poorly understood. Yet, because of the large role cell membranes play in enzymatic reactions, several reactions may take place where one of the proteins is bound to a fixed point in space. We develop a model to characterize tethered signalling between the phosphatase SHP-1 interacting with a tethered, phosphorylated protein. We compare our model to experimental data obtained using surface plasmon resonance (SPR). We show that a single SPR experiment recovers 5 independent biophysical/biochemical constants. We also compare the results between a three dimensional model and a two dimensional model. The work gives the opportunity to use known techniques to learn more about signalling processes, and new insights into how enzyme tethering alters cellular signalling. With support from the Mexican Council for Science and Technology (CONACyT), the Public Education Secretariat (SEP), and the Mexican National Autonomous University's Foundation (Fundacion UNAM).

  3. Optimization of extraction efficiency by shear emulsifying assisted enzymatic hydrolysis and functional properties of dietary fiber from deoiled cumin (Cuminum cyminum L.).

    Science.gov (United States)

    Ma, Mengmei; Mu, Taihua; Sun, Hongnan; Zhang, Miao; Chen, Jingwang; Yan, Zhibin

    2015-07-15

    This study evaluated the optimal conditions for extracting dietary fiber (DF) from deoiled cumin by shear emulsifying assisted enzymatic hydrolysis (SEAEH) using the response surface methodology. Fat adsorption capacity (FAC), glucose adsorption capacity (GAC), and bile acid retardation index (BRI) were measured to evaluate the functional properties of the extracted DF. The results revealed that the optimal extraction conditions included an enzyme to substrate ratio of 4.5%, a reaction temperature of 57 °C, a pH value of 7.7, and a reaction time of 155 min. Under these conditions, DF extraction efficiency and total dietary fiber content were 95.12% and 84.18%, respectively. The major components of deoiled cumin DF were hemicellulose (37.25%) and cellulose (33.40%). FAC and GAC increased with decreasing DF particle size (51-100 μm), but decreased with DF particle sizes <26 μm; BRI increased with decreasing DF particle size. The results revealed that SEAEH is an effective method for extracting DF. DF with particle size 26-51 μm had improved functional properties. Copyright © 2015. Published by Elsevier Ltd.

  4. Enzymatic modification of bacterial exopolysaccharides : xanthan lyase as a tool for structural and functional modification of xanthan

    NARCIS (Netherlands)

    Ruijssenaars, H.J.

    2001-01-01

    Bacterial extracellular polysaccharides (EPSs) can be applied, e.g., in foods, as a thickener or stabilizer. The functional properties that make a polysaccharide suitable for such applications are largely determined by the primary structure, i.e., the sugar composition, the linkage types

  5. Impact of variety type and particle size distribution on starch enzymatic hydrolysis and functional properties of tef flours.

    Science.gov (United States)

    Abebe, Workineh; Collar, Concha; Ronda, Felicidad

    2015-01-22

    Tef grain is becoming very attractive in the Western countries since it is a gluten-free grain with appreciated nutritional advantages. However there is little information of its functional properties and starch digestibility and how they are affected by variety type and particle size distribution. This work evaluates the effect of the grain variety and the mill used on tef flour physico-chemical and functional properties, mainly derived from starch behavior. In vitro starch digestibility of the flours by Englyst method was assessed. Two types of mills were used to obtain whole flours of different granulation. Rice and wheat flours were analyzed as references. Protein molecular weight distribution and flour structure by SEM were also analyzed to justify some of the differences found among the cereals studied. Tef cultivar and mill type exhibited important effect on granulation, bulking density and starch damage, affecting the processing performance of the flours and determining the hydration and pasting properties. The color was darker although one of the white varieties had a lightness near the reference flours. Different granulation of tef flour induced different in vitro starch digestibility. The disc attrition mill led to higher starch digestibility rate index and rapidly available glucose, probably as consequence of a higher damaged starch content. The results confirm the adequacy of tef flour as ingredient in the formulation of new cereal based foods and the importance of the variety and the mill on its functional properties. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Functionalized graphene oxide-polypyrrole-chitosan (fGO-PPy-CS) modified screen-printed electrodes for non-enzymatic hydrogen peroxide detection

    Science.gov (United States)

    Akhtar, Muhammad Asim; Hayat, Akhtar; Iqbal, Naseer; Marty, Jean Louis; Nawaz, Mian Hasnain

    2017-10-01

    Functionalized graphene oxide (fGO) was synthesized and subsequently used for synthesis of nanocomposite with polypyrrole (PPy) and chitosan (CS) to give fGO-PPy-CS nanocomposites. Screen-printed carbon electrodes were then modified with these nanocomposites to construct an unprecedented hydrogen peroxide (H2O2) sensor. Cyclic voltammetry (CV) and amperometric response demonstrated that the composite materials hold potential for electrocataytic reduction towards H2O2. Under optimal experimental conditions, the amperometric response of the fGO-PPy-CS sensor was linearly proportional to H2O2 in the range of 2.5-200 μM with a detection limit of 1.95 μM (S/ N = 3). The present study provides new opportunities for fGO-PPy-CS-modified electrodes to probe the improved non-enzymatic detection of H2O2. It further broadens the applications of graphene-based conducting composites in the field of sensors and biosensors for diverse applications. [Figure not available: see fulltext.

  7. Altered cofactor binding affects stability and activity of human UDP-galactose 4′-epimerase: Implications for type III galactosemia

    National Research Council Canada - National Science Library

    McCorvie, Thomas J; Liu, Ying; Frazer, Andrew; Gleason, Tyler J; Fridovich-Keil, Judith L; Timson, David J

    2012-01-01

    Deficiency of UDP-galactose 4'-epimerase is implicated in type III galactosemia. Two variants, p.K161N-hGALE and p.D175N-hGALE, have been previously found in combination with other alleles in patients with a mild form of the disease...

  8. Degradation-by-design: Surface modification with functional substrates that enhance the enzymatic degradation of carbon nanotubes.

    Science.gov (United States)

    Sureshbabu, Adukamparai Rajukrishnan; Kurapati, Rajendra; Russier, Julie; Ménard-Moyon, Cécilia; Bartolini, Isacco; Meneghetti, Moreno; Kostarelos, Kostas; Bianco, Alberto

    2015-12-01

    Biodegradation of carbon-based nanomaterials has been pursued intensively in the last few years, as one of the most crucial issues for the design of safe, clinically relevant conjugates for biomedical applications. In this paper it is demonstrated that specific functional molecules can enhance the catalytic activity of horseradish peroxidase (HRP) and xanthine oxidase (XO) for the degradation of carbon nanotubes. Two different azido coumarins and one cathecol derivative are linked to multi-walled carbon nanotubes (MWCNTs). These molecules are good reducing substrates and strong redox mediators to enhance the catalytic activity of HRP. XO, known to metabolize various molecules mainly in the mammalian liver, including human, was instead used to test the biodegradability of MWCNTs modified with an azido purine. The products of the biodegradation process are characterized by transmission electron microscopy and Raman spectroscopy. The results indicate that coumarin and catechol moieties have enhanced the biodegradation of MWCNTs compared to oxidized nanotubes, likely due to the capacity of these substrates to better interact with and activate HRP. Although azido purine-MWCNTs are degraded less effectively by XO than oxidized nanotubes, the data uncover the importance of XO in the biodegradation of carbon-nanomaterials leading to their better surface engineering for biomedical applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. The Interaction of UDP-N-Acetylglucosamine 2-Epimerase/N-Acetylmannosamine Kinase (GNE) and Alpha-Actinin 2 Is Altered in GNE Myopathy M743T Mutant.

    Science.gov (United States)

    Harazi, Avi; Becker-Cohen, Michal; Zer, Hagit; Moshel, Ofra; Hinderlich, Stephan; Mitrani-Rosenbaum, Stella

    2017-05-01

    UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is the gene mutated in GNE myopathy. In an attempt to elucidate GNE functions that could account for the muscle pathophysiology of this disorder, the interaction of GNE with α-actinins has been investigated. Surface plasmon resonance and microscale thermophoresis analysis revealed, that in vitro, GNE interacts with α-actinin 2, and that this interaction has a 10-fold higher affinity compared to the GNE-α-actinin 1 interaction. Further, GNE carrying the M743T mutation, the most frequent mutation in GNE myopathy, has a 10-fold lower binding affinity to α-actinin 2 than intact GNE. It is possible that this decrease eventually affects the interaction, thus causing functional imbalance of this complex in skeletal muscle that could contribute to the myopathy phenotype. In vivo, using bi-molecular fluorescent complementation, we show the specific binding of the two proteins inside the intact cell, in a unique interaction pattern between the two partners. This interaction is disrupted in the absence of the C-terminal calmodulin-like domain of α-actinin 2, which is altered in α-actinin 1. Moreover, the binding of GNE to α-actinin 2 prevents additional binding of α-actinin 1 but not vice versa. These results suggest that the interaction between GNE and α-actinin 1 and α-actinin 2 occur at different sites in the α-actinin molecules and that for α-actinin 2 the interaction site is located at the C-terminus of the protein.

  10. Protein similarity networks reveal relationships among sequence, structure, and function within the Cupin superfamily.

    Science.gov (United States)

    Uberto, Richard; Moomaw, Ellen W

    2013-01-01

    The cupin superfamily is extremely diverse and includes catalytically inactive seed storage proteins, sugar-binding metal-independent epimerases, and metal-dependent enzymes possessing dioxygenase, decarboxylase, and other activities. Although numerous proteins of this superfamily have been structurally characterized, the functions of many of them have not been experimentally determined. We report the first use of protein similarity networks (PSNs) to visualize trends of sequence and structure in order to make functional inferences in this remarkably diverse superfamily. PSNs provide a way to visualize relatedness of structure and sequence among a given set of proteins. Structure- and sequence-based clustering of cupin members reflects functional clustering. Networks based only on cupin domains and networks based on the whole proteins provide complementary information. Domain-clustering supports phylogenetic conclusions that the N- and C-terminal domains of bicupin proteins evolved independently. Interestingly, although many functionally similar enzymatic cupin members bind the same active site metal ion, the structure and sequence clustering does not correlate with the identity of the bound metal. It is anticipated that the application of PSNs to this superfamily will inform experimental work and influence the functional annotation of databases.

  11. Protein similarity networks reveal relationships among sequence, structure, and function within the Cupin superfamily.

    Directory of Open Access Journals (Sweden)

    Richard Uberto

    Full Text Available The cupin superfamily is extremely diverse and includes catalytically inactive seed storage proteins, sugar-binding metal-independent epimerases, and metal-dependent enzymes possessing dioxygenase, decarboxylase, and other activities. Although numerous proteins of this superfamily have been structurally characterized, the functions of many of them have not been experimentally determined. We report the first use of protein similarity networks (PSNs to visualize trends of sequence and structure in order to make functional inferences in this remarkably diverse superfamily. PSNs provide a way to visualize relatedness of structure and sequence among a given set of proteins. Structure- and sequence-based clustering of cupin members reflects functional clustering. Networks based only on cupin domains and networks based on the whole proteins provide complementary information. Domain-clustering supports phylogenetic conclusions that the N- and C-terminal domains of bicupin proteins evolved independently. Interestingly, although many functionally similar enzymatic cupin members bind the same active site metal ion, the structure and sequence clustering does not correlate with the identity of the bound metal. It is anticipated that the application of PSNs to this superfamily will inform experimental work and influence the functional annotation of databases.

  12. APPLICATION OF ENZYMATIC MEDICATIONS IN PEDIATRICS

    Directory of Open Access Journals (Sweden)

    O.S. Gundobina

    2008-01-01

    Full Text Available The article considers one of the topical issues of children's gas troenterology — disturbance of the pancreas functioning, its possible reasons and clinical manifestations, as well as diagnostics and treatment issues. The article considers the requirements for enzymatic medications to efficiently correct the excretory insufficiency of the pancreas. The authors describe a high performance medication for this case, demonstrate its features.Key words: excretory insufficiency pancreas, treatment, enzymatic medications, children.

  13. In silico prediction of the effects of mutations in the human UDP-galactose 4'-epimerase gene: towards a predictive framework for type III galactosemia.

    Science.gov (United States)

    McCorvie, Thomas J; Timson, David J

    2013-07-25

    The enzyme UDP-galactose 4'-epimerase (GALE) catalyses the reversible epimerisation of both UDP-galactose and UDP-N-acetyl-galactosamine. Deficiency of the human enzyme (hGALE) is associated with type III galactosemia. The majority of known mutations in hGALE are missense and private thus making clinical guidance difficult. In this study a bioinformatics approach was employed to analyse the structural effects due to each mutation using both the UDP-glucose and UDP-N-acetylglucosamine bound structures of the wild-type protein. Changes to the enzyme's overall stability, substrate/cofactor binding and propensity to aggregate were also predicted. These predictions were found to be in good agreement with previous in vitro and in vivo studies when data was available and allowed for the differentiation of those mutants that severely impair the enzyme's activity against UDP-galactose. Next this combination of techniques were applied to another twenty-six reported variants from the NCBI dbSNP database that have yet to be studied to predict their effects. This identified p.I14T, p.R184H and p.G302R as likely severely impairing mutations. Although severely impaired mutants were predicted to decrease the protein's stability, overall predicted stability changes only weakly correlated with residual activity against UDP-galactose. This suggests other protein functions such as changes in cofactor and substrate binding may also contribute to the mechanism of impairment. Finally this investigation shows that this combination of different in silico approaches is useful in predicting the effects of mutations and that it could be the basis of an initial prediction of likely clinical severity when new hGALE mutants are discovered. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Glutamate dehydrogenase (RocG) in Bacillus licheniformis WX-02: Enzymatic properties and specific functions in glutamic acid synthesis for poly-γ-glutamic acid production.

    Science.gov (United States)

    Tian, Guangming; Wang, Qin; Wei, Xuetuan; Ma, Xin; Chen, Shouwen

    2017-04-01

    Poly-γ-glutamic acid (γ-PGA), a natural biopolymer, is widely used in cosmetics, medicine, food, water treatment, and agriculture owing to its features of moisture sequestration, cation chelation, non-toxicity and biodegradability. Intracellular glutamic acid, the substrate of γ-PGA, is a limiting factor for high yield in γ-PGA production. Bacillus subtilis and Bacillus licheniformis are both important γ-PGA producing strains, and B. subtilis synthesizes glutamic acid in vivo using the unique GOGAT/GS pathway. However, little is known about the glutamate synthesis pathway in B. licheniformis. The aim of this work was to characterize the glutamate dehydrogenase (RocG) in glutamic acid synthesis from B. licheniformis with both in vivo and in vitro experiments. By re-directing the carbon flux distribution, the rocG gene deletion mutant WX-02ΔrocG produced intracellular glutamic acid with a concentration of 90ng/log(CFU), which was only 23.7% that of the wild-type WX-02 (380ng/log(CFU)). Furthermore, the γ-PGA yield of mutant WX-02ΔrocG was 5.37g/L, a decrease of 45.3% compared to the wild type (9.82g/L). In vitro enzymatic assays of RocG showed that RocG has higher affinity for 2-oxoglutarate than glutamate, and the glutamate synthesis rate was far above degradation. This is probably the first study to reveal the glutamic acid synthesis pathway and the specific functions of RocG in B. licheniformis. The results indicate that γ-PGA production can be enhanced through improving intracellular glutamic acid synthesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. DOTA-Functionalized Polylysine: A High Number of DOTA Chelates Positively Influences the Biodistribution of Enzymatic Conjugated Anti-Tumor Antibody chCE7agl

    OpenAIRE

    Gr?nberg, J?rgen; Jeger, Simone; Sarko, Dikran; Dennler, Patrick; Zimmermann, Kurt; Mier, Walter; Schibli, Roger

    2013-01-01

    Site-specific enzymatic reactions with microbial transglutaminase (mTGase) lead to a homogenous species of immunoconjugates with a defined ligand/antibody ratio. In the present study, we have investigated the influence of different numbers of 1,4,7,10-tetraazacyclododecane-N-N'-N''-N'''-tetraacetic acid (DOTA) chelats coupled to a decalysine backbone on the in vivo behavior of the chimeric monoclonal anti-L1CAM antibody chCE7agl. The enzymatic conjugation of (DOTA)1-decalysine, (DOTA)3-decaly...

  16. Enzymatic desulfurization of coal

    Energy Technology Data Exchange (ETDEWEB)

    Marquis, J.K. (Boston Univ., MA (United States). School of Medicine); Kitchell, J.P. (Holometrix, Inc., Cambridge, MA (United States))

    1988-12-15

    Our current efforts to develop clean coal technology emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes or commercially available enzymes. Our work is focused on the treatment of model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix.

  17. Identification and characterization of bifunctional proline racemase/hydroxyproline epimerase from archaea: discrimination of substrates and molecular evolution.

    Directory of Open Access Journals (Sweden)

    Seiya Watanabe

    Full Text Available Proline racemase (ProR is a member of the pyridoxal 5'-phosphate-independent racemase family, and is involved in the Stickland reaction (fermentation in certain clostridia as well as the mechanisms underlying the escape of parasites from host immunity in eukaryotic Trypanosoma. Hydroxyproline epimerase (HypE, which is in the same protein family as ProR, catalyzes the first step of the trans-4-hydroxy-L-proline metabolism of bacteria. Their substrate specificities were previously considered to be very strict, in spite of similarities in their structures and catalytic mechanisms, and no racemase/epimerase from the ProR superfamily has been found in archaea. We here characterized the ProR-like protein (OCC_00372 from the hyperthermophilic archaeon, Thermococcus litoralis (TlProR. This protein could reversibly catalyze not only the racemization of proline, but also the epimerization of 4-hydroxyproline and 3-hydroxyproline with similar kinetic constants. Among the four (putative ligand binding sites, one amino acid substitution was detected between TlProR (tryptophan at the position of 241 and natural ProR (phenylalanine. The W241F mutant showed a significant preference for proline over hydroxyproline, suggesting that this (hydrophobic and bulky tryptophan residue played an importance role in the recognition of hydroxyproline (more hydrophilic and bulky than proline, and substrate specificity for hydroxyproline was evolutionarily acquired separately between natural HypE and ProR. A phylogenetic analysis indicated that such unique broad substrate specificity was derived from an ancestral enzyme of this superfamily.

  18. 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.

  19. Proton transfer in methylmalonyl-CoA epimerase from Propionibacterium shermanii. Studies with specifically tritiated (2R)-methylmalonyl-CoA as substrate.

    Science.gov (United States)

    Leadlay, P F; Fuller, J Q

    1983-01-01

    (2R)-Methyl[2-3H]malonyl-CoA was used as the substrate for methylmalonyl-CoA epimerase from Propionibacterium shermanii, under conditions where the (2S)-methylmalonyl-CoA product was removed enzymically as fast as it was formed, and the fate of the label was monitored at different extents of reaction. Very little, if any, tritium is found attached to the C-2 position in the (2S)-epimer product (isolated as propionyl-CoA). Evidently, the hydrogen atom of the new C-H bond in the product is essentially solvent-derived. The rate of tritium release into the solvent is lower than the rate of product formation, and shows a primary kinetic tritium-isotope effect on kcat./Km of 2.3 +/- 0.1. The specific radioactivity of the remaining substrate rises slowly during the epimerase-catalysed reaction, and this provides an independent estimate of the primary kinetic tritium-isotope effect on kcat./Km of 1.6 +/- 0.5. These results, taken together, indicate that the mechanistic pathway of the epimerase-catalysed reaction resembles that established for proline racemase [Cardinale & Abeles, (1968) Biochemistry 7, 3970-3978], in which two enzyme bases are involved in catalysis. One base removes the proton from the substrate, the second provides the new proton, and there is no fast isotopic exchange between enzyme-bound intermediates and solvent protons. PMID:6311169

  20. Enzymatic Modifications of Polysaccharides

    Science.gov (United States)

    Polysaccharides are often modified chemically in order to improve its properties or to impart specific characteristics. Indeed quite a few commercial products are based on modified polysaccharides. In this talk, I shall describe a new set of modified polysaccharides based on enzymatic reactions. ...

  1. 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

  2. Chemo-enzymatic synthesis of a series of 2,4-syn-functionalized (S)-glutamate analogues: new insight into the structure-activity relation of ionotropic glutamate receptor subtypes 5, 6, and 7

    DEFF Research Database (Denmark)

    Sagot, Emanuelle; Pickering, Darryl S; Pu, Xiaosui

    2008-01-01

    ( S)-Glutamic acid (Glu) is the major excitatory neurotransmitter in the central nervous system (CNS) activating the plethora of ionotropic Glu receptors (iGluRs) and metabotropic Glu receptors (mGluRs). In this paper, we present a chemo-enzymatic strategy for the enantioselective synthesis of five...... new Glu analogues 2a- f ( 2d is exempt) holding a functionalized substituent in the 4-position. Nine Glu analogues 2a- j are characterized pharmacologically at native 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), kainic acid (KA), and N-methyl- d-aspartic acid (NMDA) receptors...

  3. Methods for Improving Enzymatic Trans-glycosylation for Synthesis of Human Milk Oligosaccharide Biomimetics

    DEFF Research Database (Denmark)

    Zeuner, Birgitte; Jers, Carsten; Mikkelsen, Jørn Dalgaard

    2014-01-01

    Recently, significant progress has been made within enzymatic synthesis of biomimetic, functional glycans, including, for example, human milk oligosaccharides. These compounds are mainly composed of N-acetylglucosamine, fucose, sialic acid, galactose, and glucose, and their controlled enzymatic...

  4. 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)

  5. 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.

  6. ATP-ases of synaptic plasma membranes in striatum: enzymatic systems for synapses functionality by in vivo administration of L-acetylcarnitine in relation to Parkinson's Disease.

    Science.gov (United States)

    Villa, R F; Ferrari, F; Gorini, A

    2013-09-17

    The maximum rate (Vmax) of some enzymatic activities related to energy consumption was evaluated in synaptic plasma membranes from rat brain striatum, the synaptic energy state being a crucial factor in neurodegenerative diseases etiopathogenesis. Two types of synaptic plasma membranes were isolated from rats subjected to in vivo treatment with L-acetylcarnitine at two different doses (30 and 60 mg × kg(-1) i.p., 28 days, 5 days/week). The following enzyme activities were evaluated: acetylcholinesterase (AChE); Na(+), K(+), Mg(2+)-ATP-ase; ouabain insensitive Mg(2+)-ATP-ase; Na(+), K(+)-ATP-ase; direct Mg(2+)-ATP-ase; Ca(2+), Mg(2+)-ATP-ase; and low- and high-affinity Ca(2+)-ATP-ase. In control (vehicle-treated) animals, enzymatic activities are differently expressed in synaptic plasma membranes type I (SPM1) with respect to synaptic plasma membranes type II (SPM2), the evaluated enzymatic activities being higher in SPM2. Subchronic treatment with L-acetylcarnitine decreased AChE on SPM1 and SPM2 at the dose of 30 mg × kg(-1). Pharmacological treatment decreased ouabain insensitive Mg(2+)-ATP-ase activity and high affinity Ca(2+)-ATP-ase activity at the doses of 30 and 60 mg × kg(-1) respectively on SPM1, while it decreased Na(+), K(+)-ATP-ase, direct Mg(2+)-ATP-ase and Ca(2+), Mg(2+)-ATP-ase activities at the dose of 30 mg × kg(-1) on SPM2. These results suggest that the sensitivity to drug treatment is different between these two populations of synaptic plasma membranes from the striatum, confirming the micro-heterogeneity of these subfractions, possessing different metabolic machinery with respect to energy consumption and utilization and the regional selective effect of L-acetylcarnitine on cerebral tissue, depending on the considered area. The drug potential effect at the synaptic level in Parkinson's Disease neuroprotection is also discussed with respect to acetylcholine and energy metabolism. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights

  7. Enzymatic cascade bioreactor

    Energy Technology Data Exchange (ETDEWEB)

    Simmons, Blake A. (San Francisco, CA); Volponi, Joanne V. (Livermore, CA); Ingersoll, David (Albuquerque, NM); Walker, Andrew (Woodinville, WA)

    2007-09-04

    Disclosed is an apparatus and method for continuously converting sucrose to .beta.-D-glucose. The method comprises a three stage enzymatic reactor in which an aqueous solution of sucrose is first converted into a solution of fructose and .alpha.-D-glucose by passing it through a porous, packed column containing an inert media on which invertase is immobilized. This solution is then sent through a second packed column containing glucose isomerase and finally a third packed column containing mutarotase. Solution temperature and pH are adjusted to maximize glucose output.

  8. Enzymatic Hydrolysis of Lignocelluloses

    DEFF Research Database (Denmark)

    Kolasa, Marta; Ahring, Birgitte Kiær; Lübeck, Peter Stephensen

    2010-01-01

    bonds. Cellulose can be degraded to simple sugar components by means of enzymatic hydrolysis. However, due to its complex, crystalline structure it is difficult to break it down and the cooperative action of a variety of cellulolytic enzymes is necessary. Fungi are known to have potential in production...... source. By means of degenerate PCR, specific genes, homologous to the genes of previously classified glycoside hydrolases from CAZY database, are searched for in selected strains of Aspergillus sp., Trichoderma sp. and Penicillium sp. Both methods are anticipated to facilitate identification of target...

  9. DOTA-functionalized polylysine: a high number of DOTA chelates positively influences the biodistribution of enzymatic conjugated anti-tumor antibody chCE7agl.

    Directory of Open Access Journals (Sweden)

    Jürgen Grünberg

    Full Text Available Site-specific enzymatic reactions with microbial transglutaminase (mTGase lead to a homogenous species of immunoconjugates with a defined ligand/antibody ratio. In the present study, we have investigated the influence of different numbers of 1,4,7,10-tetraazacyclododecane-N-N'-N''-N'''-tetraacetic acid (DOTA chelats coupled to a decalysine backbone on the in vivo behavior of the chimeric monoclonal anti-L1CAM antibody chCE7agl. The enzymatic conjugation of (DOTA1-decalysine, (DOTA3-decalysine or (DOTA5-decalysine to the antibody heavy chain (via Gln295/297 gave rise to immunoconjugates containing two, six or ten DOTA moieties respectively. Radiolabeling of the immunoconjugates with (177Lu yielded specific activities of approximately 70 MBq/mg, 400 MBq/mg and 700 MBq/mg with increasing numbers of DOTA chelates. Biodistribution experiments in SKOV3ip human ovarian cancer cell xenografts demonstrated a high and specific accumulation of radioactivity at the tumor site for all antibody derivatives with a maximal tumor accumulation of 43.6±4.3% ID/g at 24 h for chCE7agl-[(DOTA-decalysine]2, 30.6±12.0% ID/g at 24 h for chCE7agl-[(DOTA3-decalysine]2 and 49.9±3.1% ID/g at 48 h for chCE7agl-[(DOTA5-decalysine]2. The rapid elimination from the blood of chCE7agl-[(DOTA-decalysine]2 (1.0±0.1% ID/g at 24 h is associated with a high liver accumulation (23.2±4.6% ID/g at 24 h. This behavior changed depending on the numbers of DOTA moieties coupled to the decalysine peptide with a slower blood clearance (5.1±1.0 (DOTA3 versus 11.7±1.4% ID/g (DOTA5, p<0.005 at 24 h and lower radioactivity levels in the liver (21.4±3.4 (DOTA3 versus 5.8±0.7 (DOTA5, p<0.005 at 24 h. We conclude that the site-specific and stoichiometric uniform conjugation of the highly DOTA-substituted decalysine ((DOTA5-decalysine to an anti-tumor antibody leads to the formation of immunoconjugates with high specific activity and excellent in vivo behavior and is a valuable option for

  10. Investigation of enzymatic hydrolysis conditions on the properties of protein hydrolysate from fish muscle (Collichthys niveatus) and evaluation of its functional properties.

    Science.gov (United States)

    Shen, Qing; Guo, Rui; Dai, Zhiyuan; Zhang, Yanping

    2012-05-23

    This study was carried out to investigate the enzymatic hydrolysis conditions on the properties of protein hydrolysate from fish muscle of the marine fish species Collichthys niveatus. About 160 fish samples were tested, and the analyzed fish species was found to be a lean fish with low fat (1.77 ± 0.01%) and high protein (16.76 ± 1.21%). Fish muscle of C. niveatus was carefully collected and hydrolyzed with four commercial enzymes: Alcalase, Neutrase, Protamex, and Flavourzyme under the conditions recommended by the manufacturers. Among the tested proteases, Neutrase catalyzed the hydrolysis process most effectively since the hydrolysate generated by Neutrase has the highest content of sweet and umami taste amino acids (SUA). The effect of hydrolysis conditions was further optimized using response surface methodology (RSM), and the optimum values for temperature, pH, and enzyme/substrate ratio (E/S ratio) were found to be 40.7 °C, 7.68, and 0.84%, respectively. Finally, the amino acid composition of the hydrolysate was analyzed by AccQ·Tag derivatization and HPLC-PDA determination. Major amino acids of the muscle of C. niveatus were threonine, glutamic acid, phenyalanine, tryptophan, and lysine, accounting for respectively 10.92%, 10.85%, 10.79%, 9.86%, and 9.76% of total amino acid content. The total content of essential amino acids was 970.7 ng·mL(-1), while that of nonessential amino acids was 709.1 ng·mL(-1). The results suggest that the fish muscle and its protein hydrolysate from C. niveatus provide a versatile supply of the benefits and can be incorporated as supplements in health-care foods.

  11. UDP-galactose 4'-epimerase activities toward UDP-Gal and UDP-GalNAc play different roles in the development of Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Jennifer M I Daenzer

    Full Text Available In both humans and Drosophila melanogaster, UDP-galactose 4'-epimerase (GALE catalyzes two distinct reactions, interconverting UDP-galactose (UDP-gal and UDP-glucose (UDP-glc in the final step of the Leloir pathway of galactose metabolism, and also interconverting UDP-N-acetylgalactosamine (UDP-galNAc and UDP-N-acetylglucosamine (UDP-glcNAc. All four of these UDP-sugars serve as vital substrates for glycosylation in metazoans. Partial loss of GALE in humans results in the spectrum disorder epimerase deficiency galactosemia; partial loss of GALE in Drosophila melanogaster also results in galactose-sensitivity, and complete loss in Drosophila is embryonic lethal. However, whether these outcomes in both humans and flies result from loss of one GALE activity, the other, or both has remained unknown. To address this question, we uncoupled the two activities in a Drosophila model, effectively replacing the endogenous dGALE with prokaryotic transgenes, one of which (Escherichia coli GALE efficiently interconverts only UDP-gal/UDP-glc, and the other of which (Plesiomonas shigelloides wbgU efficiently interconverts only UDP-galNAc/UDP-glcNAc. Our results demonstrate that both UDP-gal and UDP-galNAc activities of dGALE are required for Drosophila survival, although distinct roles for each activity can be seen in specific windows of developmental time or in response to a galactose challenge. By extension, these data also suggest that both activities might play distinct and essential roles in humans.

  12. Proton transfer in methylmalonyl-CoA epimerase from Propionibacterium shermanii. The reaction of (2R)-methylmalonyl-CoA in tritiated water.

    Science.gov (United States)

    Fuller, J Q; Leadlay, P F

    1983-01-01

    The reaction catalysed by methylmalonyl-CoA epimerase from Propionibacterium shermanii was studied in tritiated water, in the direction with (2R)-methylmalonyl-CoA as substrate, under 'irreversible' conditions. After partial reaction, even when most of the substrate had been converted into product (isolated as propionyl-CoA) essentially no solvent tritium appeared in residual (2R)-methylmalonyl-CoA. The product, however, did contain tritium, and the specific radioactivity of the (2S)-epimer was deduced to be 0.33 times that of the solvent. These results provide further support for the mechanism proposed for the epimerase-catalysed reaction in the accompanying paper [Leadlay & Fuller (1983) Biochem. J. 213, 635-642], in which two enzyme bases act respectively as proton donor and acceptor. The observed low discrimination against solvent tritium entering the product can be accounted for by a mechanism in which the release of product is slow, and the re-protonation step on the enzyme is reversible, without leading to isotopic exchange with the solvent. PMID:6311170

  13. Production of d-psicose from d-glucose by co-expression of d-psicose 3-epimerase and xylose isomerase.

    Science.gov (United States)

    Chen, Xiaoyan; Wang, Wen; Xu, Jingliang; Yuan, Zhenhong; Yuan, Tao; Zhang, Yu; Liang, Cuiyi; He, Minchao; Guo, Ying

    2017-10-01

    d-Psicose has been drawing increasing attention in recent years because of its medical and health applications. The production of d-psicose from d-glucose requires the co-expression and synergistic action of xylose isomerase and d-psicose 3-epimerase. To co-express these genes, vector pET-28a(+)-dual containing two T7 promoters and RBS sites and an Multiple Cloning Sites was constructed using the Escherichia coli expression plasmid pET-28a(+). The xylose isomerase gene from E. coli MG1665 and the d-psicose 3-epimerase gene from Agrobacterium tumefaciens CGMCC 1.1488 were cloned and co-expressed in E. coli BL21(DE3). After 24h incubation with the dual enzyme system at 40°C, the sugar conversion ratio from d-glucose to d-psicose reached 10%. The optimal conditions were 50°C, pH 7.5 with Co2+ and Mg2+. The d-psicose yields from sugarcane bagasse and microalgae hydrolysate were 1.42 and 1.69g/L, respectively. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. 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...... of the obtained products and were correlated to properties of the starch substrates. It was found that the obtained products differed depending on both the conditions used and the properties of the starch. Products of starch from certain origins completely lost their granular structure during the enzyme treatment...

  15. Model-enabled gene search (MEGS) allows fast and direct discovery of enzymatic and transport gene functions in the marine bacterium Vibrio fischeri.

    Science.gov (United States)

    Pan, Shu; Nikolakakis, Kiel; Adamczyk, Paul A; Pan, Min; Ruby, Edward G; Reed, Jennifer L

    2017-06-16

    Whereas genomes can be rapidly sequenced, the functions of many genes are incompletely or erroneously annotated because of a lack of experimental evidence or prior functional knowledge in sequence databases. To address this weakness, we describe here a model-enabled gene search (MEGS) approach that (i) identifies metabolic functions either missing from an organism's genome annotation or incorrectly assigned to an ORF by using discrepancies between metabolic model predictions and experimental culturing data; (ii) designs functional selection experiments for these specific metabolic functions; and (iii) selects a candidate gene(s) responsible for these functions from a genomic library and directly interrogates this gene's function experimentally. To discover gene functions, MEGS uses genomic functional selections instead of relying on correlations across large experimental datasets or sequence similarity as do other approaches. When applied to the bioluminescent marine bacterium Vibrio fischeri, MEGS successfully identified five genes that are responsible for four metabolic and transport reactions whose absence from a draft metabolic model of V. fischeri caused inaccurate modeling of high-throughput experimental data. This work demonstrates that MEGS provides a rapid and efficient integrated computational and experimental approach for annotating metabolic genes, including those that have previously been uncharacterized or misannotated. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. 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.

  17. Homogeneous, Heterogeneous, and Enzymatic Catalysis.

    Science.gov (United States)

    Oyama, S. Ted; Somorjai, Gabor A.

    1988-01-01

    Discusses three areas of catalysis: homegeneous, heterogeneous, and enzymatic. Explains fundamentals and economic impact of catalysis. Lists and discusses common industrial catalysts. Provides a list of 107 references. (MVL)

  18. Identification of an evolutionary conserved structural loop that is required for the enzymatic and biological function of tryptophan 2,3-dioxygenase

    NARCIS (Netherlands)

    Michels, Helen; Seinstra, Renee I.; Uitdehaag, Joost C. M.; Koopman, Mandy; van Faassen, Martijn; Martineau, Celine N.; Kema, Ido P.; Buijsman, Rogier; Nollen, Ellen A. A.

    2016-01-01

    The enzyme TDO (tryptophan 2,3-dioxygenase; TDO-2 in Caenorhabditis elegans) is a potential therapeutic target to cancer but is also thought to regulate proteotoxic events seen in the progression of neurodegenerative diseases. To better understand its function and develop specific compounds that

  19. QM/MM Geometry Optimization on Extensive Free-Energy Surfaces for Examination of Enzymatic Reactions and Design of Novel Functional Properties of Proteins

    Science.gov (United States)

    Hayashi, Shigehiko; Uchida, Yoshihiro; Hasegawa, Taisuke; Higashi, Masahiro; Kosugi, Takahiro; Kamiya, Motoshi

    2017-05-01

    Many remarkable molecular functions of proteins use their characteristic global and slow conformational dynamics through coupling of local chemical states in reaction centers with global conformational changes of proteins. To theoretically examine the functional processes of proteins in atomic detail, a methodology of quantum mechanical/molecular mechanical (QM/MM) free-energy geometry optimization is introduced. In the methodology, a geometry optimization of a local reaction center is performed with a quantum mechanical calculation on a free-energy surface constructed with conformational samples of the surrounding protein environment obtained by a molecular dynamics simulation with a molecular mechanics force field. Geometry optimizations on extensive free-energy surfaces by a QM/MM reweighting free-energy self-consistent field method designed to be variationally consistent and computationally efficient have enabled examinations of the multiscale molecular coupling of local chemical states with global protein conformational changes in functional processes and analysis and design of protein mutants with novel functional properties.

  20. QM/MM Geometry Optimization on Extensive Free-Energy Surfaces for Examination of Enzymatic Reactions and Design of Novel Functional Properties of Proteins.

    Science.gov (United States)

    Hayashi, Shigehiko; Uchida, Yoshihiro; Hasegawa, Taisuke; Higashi, Masahiro; Kosugi, Takahiro; Kamiya, Motoshi

    2017-05-05

    Many remarkable molecular functions of proteins use their characteristic global and slow conformational dynamics through coupling of local chemical states in reaction centers with global conformational changes of proteins. To theoretically examine the functional processes of proteins in atomic detail, a methodology of quantum mechanical/molecular mechanical (QM/MM) free-energy geometry optimization is introduced. In the methodology, a geometry optimization of a local reaction center is performed with a quantum mechanical calculation on a free-energy surface constructed with conformational samples of the surrounding protein environment obtained by a molecular dynamics simulation with a molecular mechanics force field. Geometry optimizations on extensive free-energy surfaces by a QM/MM reweighting free-energy self-consistent field method designed to be variationally consistent and computationally efficient have enabled examinations of the multiscale molecular coupling of local chemical states with global protein conformational changes in functional processes and analysis and design of protein mutants with novel functional properties.

  1. A Lanthipeptide-like N-Terminal Leader Region Guides Peptide Epimerization by Radical SAM Epimerases: Implications for RiPP Evolution.

    Science.gov (United States)

    Fuchs, Sebastian W; Lackner, Gerald; Morinaka, Brandon I; Morishita, Yohei; Asai, Teigo; Riniker, Sereina; Piel, Jörn

    2016-09-26

    Ribosomally synthesized and posttranslationally modified peptide natural products (RiPPs) exhibit diverse structures and bioactivities and are classified into distinct biosynthetic families. A recently reported family is the proteusins, with the prototype members polytheonamides being generated by almost 50 maturation steps, including introduction of d-residues at multiple positions by an unusual radical SAM epimerase. A region in the protein-like N-terminal leader of proteusin precursors is identified that is crucial for epimerization. It resembles a precursor motif previously shown to mediate interaction in thioether bridge-formation in class I lanthipeptide biosynthesis. Beyond this region, similarities were identified between proteusin and further RiPP families, including class I lanthipeptides. The data suggest that common leader features guide distinct maturation types and that nitrile hydratase-like enzymes are ancestors of several RiPP classes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Functional Role of Lanthanides in Enzymatic Activity and Transcriptional Regulation of Pyrroloquinoline Quinone-Dependent Alcohol Dehydrogenases in Pseudomonas putida KT2440

    Directory of Open Access Journals (Sweden)

    Matthias Wehrmann

    2017-06-01

    Full Text Available The oxidation of alcohols and aldehydes is crucial for detoxification and efficient catabolism of various volatile organic compounds (VOCs. Thus, many Gram-negative bacteria have evolved periplasmic oxidation systems based on pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs that are often functionally redundant. Here we report the first description and characterization of a lanthanide-dependent PQQ-ADH (PedH in a nonmethylotrophic bacterium based on the use of purified enzymes from the soil-dwelling model organism Pseudomonas putida KT2440. PedH (PP_2679 exhibits enzyme activity on a range of substrates similar to that of its Ca2+-dependent counterpart PedE (PP_2674, including linear and aromatic primary and secondary alcohols, as well as aldehydes, but only in the presence of lanthanide ions, including La3+, Ce3+, Pr3+, Sm3+, or Nd3+. Reporter assays revealed that PedH not only has a catalytic function but is also involved in the transcriptional regulation of pedE and pedH, most likely acting as a sensory module. Notably, the underlying regulatory network is responsive to as little as 1 to 10 nM lanthanum, a concentration assumed to be of ecological relevance. The present study further demonstrates that the PQQ-dependent oxidation system is crucial for efficient growth with a variety of volatile alcohols. From these results, we conclude that functional redundancy and inverse regulation of PedE and PedH represent an adaptive strategy of P. putida KT2440 to optimize growth with volatile alcohols in response to the availability of different lanthanides.

  3. Enzymatic labeling of proteins: techniques and approaches.

    Science.gov (United States)

    Rashidian, Mohammad; Dozier, Jonathan K; Distefano, Mark D

    2013-08-21

    Site-specific modification of proteins is a major challenge in modern chemical biology due to the large number of reactive functional groups typically present in polypeptides. Because of its importance in biology and medicine, the development of methods for site-specific modification of proteins is an area of intense research. Selective protein modification procedures have been useful for oriented protein immobilization, for studies of naturally occurring post-translational modifications, for creating antibody–drug conjugates, for the introduction of fluorophores and other small molecules on to proteins, for examining protein structure, folding, dynamics, and protein–protein interactions, and for the preparation of protein–polymer conjugates. One of the most important approaches for protein labeling is to incorporate bioorthogonal functionalities into proteins at specific sites via enzymatic reactions. The incorporated tags then enable reactions that are chemoselective, whose functional groups not only are inert in biological media, but also do not occur natively in proteins or other macromolecules. This review article summarizes the enzymatic strategies, which enable site-specific functionalization of proteins with a variety of different functional groups. The enzymes covered in this review include formylglycine generating enzyme, sialyltransferases, phosphopantetheinyltransferases, O-GlcNAc post-translational modification, sortagging, transglutaminase, farnesyltransferase, biotin ligase, lipoic acid ligase, and N-myristoyltransferase.

  4. Effect of refined functional carbohydrates from enzymatically hydrolyzed yeast on the presence of Salmonella spp. in the ceca of broiler breeder females.

    Science.gov (United States)

    Walker, G K; Jalukar, S; Brake, J

    2017-08-01

    Broiler breeders hatched from Salmo-nella negative grandparents received either zero or 50 g/MT of refined functional carbohydrates (RFC) in their diets from d of placement to end of lay. There were no other treatments used. Pullets and cockerels were reared separately in an enclosed litter-floor house to 21 wk of age when 28 randomly selected pullets from each diet were transferred to individual cages for an additional 14 d before they were killed, and their ceca were excised aseptically and tested for Salmonella spp. The remaining birds were transferred to a two-thirds slat and one-third litter curtain-sided laying house. There were 8 pens of 60 to 65 females and 8 to 18 males, depending upon flock age and housing type, fed each diet, and there was no effort made to isolate pens from typical daily foot traffic between pens. At 51 wk of age, male progeny broiler chicks were hatched and received either zero or 50 g/MT of RFC to complete a 2 × 2 design with 4 replicate pens of 12 males per interaction. All broilers were tested for cecal Salmonella spp. at 34 d of age. Ceca were collected from 30 breeder hens from each treatment at 64 wk of age and tested for Salmonella spp. Of the ceca sampled at 23 wk from the control pullets, 71.4% were found to contain Salmonella spp., while none of the ceca from the RFC pullets tested positive. Of the ceca sampled from the control hens at 64 wk, 40% were found to contain Salmonella spp., while none of the ceca from the RFC hens tested positive. Salmonella spp. was isolated from broilers in one pen of the control broilers that were also progeny of control breeders out of 4 replicates but not from any pens in which the breeders had been fed RFC. These data demonstrated that RFC reduced natural Salmonella spp. colonization of broiler breeder hen and broiler progeny ceca during a complete production cycle. © 2017 Poultry Science Association Inc.

  5. 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.

  6. Enzymatic conversion of carbon dioxide.

    Science.gov (United States)

    Shi, Jiafu; Jiang, Yanjun; Jiang, Zhongyi; Wang, Xueyan; Wang, Xiaoli; Zhang, Shaohua; Han, Pingping; Yang, Chen

    2015-10-07

    With the continuous increase in fossil fuels consumption and the rapid growth of atmospheric CO2 concentration, the harmonious state between human and nature faces severe challenges. Exploring green and sustainable energy resources and devising efficient methods for CO2 capture, sequestration and utilization are urgently required. Converting CO2 into fuels/chemicals/materials as an indispensable element for CO2 capture, sequestration and utilization may offer a win-win strategy to both decrease the CO2 concentration and achieve the efficient exploitation of carbon resources. Among the current major methods (including chemical, photochemical, electrochemical and enzymatic methods), the enzymatic method, which is inspired by the CO2 metabolic process in cells, offers a green and potent alternative for efficient CO2 conversion due to its superior stereo-specificity and region/chemo-selectivity. Thus, in this tutorial review, we firstly provide a brief background about enzymatic conversion for CO2 capture, sequestration and utilization. Next, we depict six major routes of the CO2 metabolic process in cells, which are taken as the inspiration source for the construction of enzymatic systems in vitro. Next, we focus on the state-of-the-art routes for the catalytic conversion of CO2 by a single enzyme system and by a multienzyme system. Some emerging approaches and materials utilized for constructing single-enzyme/multienzyme systems to enhance the catalytic activity/stability will be highlighted. Finally, a summary about the current advances and the future perspectives of the enzymatic conversion of CO2 will be presented.

  7. 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.

  8. Protein Similarity Networks Reveal Relationships among Sequence, Structure, and Function within the Cupin Superfamily

    OpenAIRE

    Richard Uberto; Moomaw, Ellen W.

    2013-01-01

    The cupin superfamily is extremely diverse and includes catalytically inactive seed storage proteins, sugar-binding metal-independent epimerases, and metal-dependent enzymes possessing dioxygenase, decarboxylase, and other activities. Although numerous proteins of this superfamily have been structurally characterized, the functions of many of them have not been experimentally determined. We report the first use of protein similarity networks (PSNs) to visualize trends of sequence and structur...

  9. Highly Stable Foams from Block Oligomers Synthesized by Enzymatic Reactions

    NARCIS (Netherlands)

    Sagis, L.M.C.; Boeriu, C.G.; Frissen, A.E.; Schols, H.A.; Wierenga, P.A.

    2008-01-01

    We have synthesized a new amphiphilic block oligomer by the enzymatic linking of a fatty acid (lauric acid) to a fructan oligomer (inulin) and tested the functionality of this carbohydrate derivative in foam stabilization. The structure of the modified oligosaccharide was found to be

  10. Novel process for producing 6-deoxy monosaccharides from l-fucose by coupling and sequential enzymatic method.

    Science.gov (United States)

    Shompoosang, Sirinan; Yoshihara, Akihide; Uechi, Keiko; Asada, Yasuhiko; Morimoto, Kenji

    2016-01-01

    We biosynthesized 6-deoxy-L-talose, 6-deoxy-L-sorbose, 6-deoxy-L-gulose, and 6-deoxy-L-idose, which rarely exist in nature, from L-fucose by coupling and sequential enzymatic reactions. The first product, 6-deoxy-L-talose, was directly produced from L-fucose by the coupling reactions of immobilized D-arabinose isomerase and immobilized L-rhamnose isomerase. In one-pot reactions, the equilibrium ratio of L-fucose, L-fuculose, and 6-deoxy-L-talose was 80:9:11. In contrast, 6-deoxy-L-sorbose, 6-deoxy-L-gulose, and 6-deoxy-L-idose were produced from L-fucose by sequential enzymatic reactions. D-Arabinose isomerase converted L-fucose into L-fuculose with a ratio of 88:12. Purified L-fuculose was further epimerized into 6-deoxy-L-sorbose by D-allulose 3-epimerase with a ratio of 40:60. Finally, purified 6-deoxy-L-sorbose was isomerized into both 6-deoxy-L-gulose with an equilibrium ratio of 40:60 by L-ribose isomerase, and 6-deoxy-L-idose with an equilibrium ratio of 73:27 by D-glucose isomerase. Based on the amount of L-fucose used, the production yields of 6-deoxy-L-talose, 6-deoxy-L-sorbose, 6-deoxy-L-gulose, and 6-deoxy-L-idose were 7.1%, 14%, 2%, and 2.4%, respectively. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  11. 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.

  12. Enzymatic synthesis of designer lipids

    Directory of Open Access Journals (Sweden)

    Devi B.L.A. Prabhavathi

    2008-05-01

    Full Text Available Even though natural oils and fats play an important role in human nutrition, its excessive intake became major cause for so many health related problems and hence designer lipids came into focus. Designed or structured lipids are nothing but tailor-made oils and fats with improved physical and organoleptic properties to enhance the role of fats and oils in food, nutrition, and health applications. These designer lipids can be produced by chemical- or enzymatic (interesterification reactions and genetic engineering of oilseed crops. This review gives a general idea about the enzymatic modifications of natural lipids and their derivatives for the preparation of designer lipids. The commercialization outlook, food, nutritional and pharmaceutical applications of designer lipids are also briefly discussed.

  13. Enzymatic synthesis of designer lipids

    OpenAIRE

    Devi B.L.A. Prabhavathi; Zhang Hong; Damstrup Marianne L.; Guo Zheng; Zhang Long; Lue Bena-Marie; Xu Xuebing

    2008-01-01

    Even though natural oils and fats play an important role in human nutrition, its excessive intake became major cause for so many health related problems and hence designer lipids came into focus. Designed or structured lipids are nothing but tailor-made oils and fats with improved physical and organoleptic properties to enhance the role of fats and oils in food, nutrition, and health applications. These designer lipids can be produced by chemical- or enzymatic (inter)esterification reactions ...

  14. The metastability of human UDP-galactose 4'-epimerase (GALE) is increased by variants associated with type III galactosemia but decreased by substrate and cofactor binding.

    Science.gov (United States)

    Pey, Angel L; Padín-Gonzalez, Esperanza; Mesa-Torres, Noel; Timson, David J

    2014-11-15

    Type III galactosemia is an inherited disease caused by mutations which affect the activity of UDP-galactose 4'-epimerase (GALE). We evaluated the impact of four disease-associated variants (p.N34S, p.G90E, p.V94M and p.K161N) on the conformational stability and dynamics of GALE. Thermal denaturation studies showed that wild-type GALE denatures at temperatures close to physiological, and disease-associated mutations often reduce GALE's thermal stability. This denaturation is under kinetic control and results partly from dimer dissociation. The natural ligands, NAD(+) and UDP-glucose, stabilize GALE. Proteolysis studies showed that the natural ligands and disease-associated variations affect local dynamics in the N-terminal region of GALE. Proteolysis kinetics followed a two-step irreversible model in which the intact protein is cleaved at Ala38 forming a long-lived intermediate in the first step. NAD(+) reduces the rate of the first step, increasing the amount of undigested protein whereas UDP-glucose reduces the rate of the second step, increasing accumulation of the intermediate. Disease-associated variants affect these rates and the amounts of protein in each state. Our results also suggest communication between domains in GALE. We hypothesize that, in vivo, concentrations of natural ligands modulate GALE stability and that it should be possible to discover compounds which mimic the stabilising effects of the natural ligands overcoming mutation-induced destabilization. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Characterization of a radical S-adenosyl-L-methionine epimerase, NeoN, in the last step of neomycin B biosynthesis.

    Science.gov (United States)

    Kudo, Fumitaka; Hoshi, Shota; Kawashima, Taiki; Kamachi, Toshiaki; Eguchi, Tadashi

    2014-10-01

    The last step of neomycin biosynthesis is the epimerization at C-5‴ of neomycin C to give neomycin B. A candidate enzyme responsible for the epimerization was a putative radical S-adenosyl-L-methionine (SAM) enzyme, NeoN, which is uniquely encoded in the neomycin biosynthetic gene cluster and remained an unassigned protein in the neomycin biosynthesis. The reconstituted and reduced NeoN showed the expected epimerization activity in the presence of SAM. In the epimerization, 1 equiv of SAM was consumed to convert neomycin C into neomycin B. The site of neomycin C reactive toward epimerization was clearly confirmed to be C-5‴ by detecting the incorporation of a deuterium atom from the deuterium oxide-based buffer solution. Further, alanine scanning of the NeoN cysteine residues revealed that C249 is a critical amino acid residue that provides a hydrogen atom to complete the epimerization. Furthermore, electron paramagnetic resonance analysis of the C249A variant in the presence of SAM and neomycin C revealed that a radical intermediate is generated at the C-5‴ of neomycin C. Therefore, the present study clearly illustrates that the epimerization of neomycin C to neomycin B is catalyzed by a unique radical SAM epimerase NeoN with a radical reaction mechanism.

  16. Production of d-psicose from d-fructose by whole recombinant cells with high-level expression of d-psicose 3-epimerase from Agrobacterium tumefaciens.

    Science.gov (United States)

    Park, Chang-Su; Park, Chul-Soon; Shin, Kyung-Chul; Oh, Deok-Kun

    2016-02-01

    The specific activity of recombinant Escherichia coli cells expressing the double-site variant (I33L-S213C) d-psicose 3-epimerase (DPEase) from Agrobacterium tumefaciens was highest at 24 h of cultivation time in Terrific Broth (TB) medium among the media tested. The contents of crude protein and DPEase in recombinant cells at 24 h were 37.0 and 8.6% (w/w), respectively, indicating that the enzyme was highly expressed. The reaction conditions for the production of d-psicose from d-fructose by whole recombinant cells with the highest specific activity were optimal at 60°C, pH 8.5, 4 g/l cells, and 700 g/l d-fructose. Under these conditions, whole recombinant cells produced 230 g/l d-psicose after 40 min, with a conversion yield of 33% (w/w), a volumetric productivity of 345 g/l/h, and a specific productivity of 86.2 g/g/h. These are the highest conversion yield and volumetric and specific productivities of d-psicose from d-fructose by cells reported thus far. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  17. "Unknown genome" proteomics: a new NADP-dependent epimerase/dehydratase revealed by N-terminal sequencing, inverted PCR, and high resolution mass spectrometry.

    Science.gov (United States)

    Simeonova, Diliana Dancheva; Susnea, Iuliana; Moise, Adrian; Schink, Bernhard; Przybylski, Michael

    2009-01-01

    We present here a new approach that enabled the identification of a new protein from a bacterial strain with unknown genomic background using a combination of inverted PCR with degenerate primers derived from N-terminal protein sequences and high resolution peptide mass determination of proteolytic digests from two-dimensional electrophoretic separation. Proteins of the sulfate-reducing bacterium Desulfotignum phosphitoxidans specifically induced in the presence of phosphite were separated by two-dimensional gel electrophoresis as a series of apparent soluble and membrane-bound isoforms with molecular masses of approximately 35 kDa. Inverted PCR based on N-terminal sequences and high resolution peptide mass fingerprinting by Fourier transform-ion cyclotron resonance mass spectrometry provided the identification of a new NAD(P) epimerase/dehydratase by specific assignment of peptide masses to a single ORF, excluding other possible ORF candidates. The protein identification was ascertained by chromatographic separation and sequencing of internal proteolytic peptides. Metal ion affinity isolation of tryptic peptides and high resolution mass spectrometry provided the identification of five phosphorylations identified in the domains 23-47 and 91-118 of the protein. In agreement with the phosphorylations identified, direct molecular weight determination of the soluble protein eluted from the two-dimensional gels by mass spectrometry provided a molecular mass of 35,400 Da, which is consistent with an average degree of three phosphorylations.

  18. D-Allulose Production from D-Fructose by Permeabilized Recombinant Cells of Corynebacterium glutamicum Cells Expressing D-Allulose 3-Epimerase Flavonifractor plautii.

    Directory of Open Access Journals (Sweden)

    Chul-Soon Park

    Full Text Available A d-allulose 3-epimerase from Flavonifractor plautii was cloned and expressed in Escherichia coli and Corynebacterium glutamicum. The maximum activity of the enzyme purified from recombinant E. coli cells was observed at pH 7.0, 65°C, and 1 mM Co2+ with a half-life of 40 min at 65°C, Km of 162 mM, and kcat of 25280 1/s. For increased d-allulose production, recombinant C. glutamicum cells were permeabilized via combined treatments with 20 mg/L penicillin and 10% (v/v toluene. Under optimized conditions, 10 g/L permeabilized cells produced 235 g/L d-allulose from 750 g/L d-fructose after 40 min, with a conversion rate of 31% (w/w and volumetric productivity of 353 g/L/h, which were 1.4- and 2.1-fold higher than those obtained for nonpermeabilized cells, respectively.

  19. Preparation, properties, and uses of enzymatic milk protein hydrolysates.

    Science.gov (United States)

    Abd El-Salam, M H; El-Shibiny, S

    2017-04-13

    Enzymatic hydrolysis of milk proteins has been a subject of numerous research studies and patents. The driving force for these studies has been the increased utilization of milk proteins. The industrial uses of milk proteins are based on their unique composition, functionality, and nutritive values. The diversity of milk protein fraction, the large number of proteinases, and controlled hydrolysis conditions used resulted in the preparation of hydrolysates suitable for several purposes. Enzymatic hydrolysis of milk proteins modifies the techno-functional and biofunctional properties of hydrolysates depending on the enzyme(s) and hydrolysis conditions used. Milk protein hydrolysates (MPH) are used commonly in normal and clinical nutrition and as a functional food ingredient. In the present review, emphasis has been made to highlight methods applied for the preparation of MPH, and the functional properties and utilization of the obtained hydrolysates.

  20. Enzymatic degradation of multiwalled carbon nanotubes.

    Science.gov (United States)

    Zhao, Yong; Allen, Brett L; Star, Alexander

    2011-09-01

    Because of their unique properties, carbon nanotubes and, in particular, multiwalled carbon nanotubes (MWNTs) have been used for the development of advanced composite and catalyst materials. Despite their growing commercial applications and increased production, the potential environmental and toxicological impacts of MWNTs are not fully understood; however, many reports suggest that they may be toxic. Therefore, a need exists to develop protocols for effective and safe degradation of MWNTs. In this article, we investigated the effect of chemical functionalization of MWNTs on their enzymatic degradation with horseradish peroxidase (HRP) and hydrogen peroxide (H(2)O(2)). We investigated HRP/H(2)O(2) degradation of purified, oxidized, and nitrogen-doped MWNTs and proposed a layer-by-layer degradation mechanism of nanotubes facilitated by side wall defects. These results provide a better understanding of the interaction between HRP and carbon nanotubes and suggest an eco-friendly way of mitigating the environmental impact of nanotubes. © 2011 American Chemical Society

  1. Enzymatic activity of myccorrhizal fungi

    Directory of Open Access Journals (Sweden)

    Roman Pachlewski

    2014-11-01

    Full Text Available The investigations included assays of enzymatic activity of ectomycorrhizal fungi from the genera: Amanita, Cenococcum, Coltricia, Hebeloma, Lactarius, Rhizopogon, Russula, Suillus, Tricholoma and the pine ectendomycorrhizal strain MrgX. Among the 22 investigated strains of fungi 18 could decompose starch, 14 urea, 11 asparagine, 7 protein, 6 pectin and 3 ce1lulose. The most varied enzyme activities were found in Amanita muscaria, A. verna, Hebeloma, mesophaeum, ectendomycorrhizal isolate MrgX, Rhizopogon luteolus and Suillus bovinus, the highest cellolotytic activity was shown by the ectendomycorrhizal strain.

  2. Enzymatic synthesis of structured lipids.

    Science.gov (United States)

    Iwasaki, Yugo; Yamane, Tsuneo

    2004-01-01

    Structured lipids (SLs) are defined as lipids that are modified chemically or enzymatically in order to change their structure. This review deals with structured triacylglycerols (STGs) and structured phospholipids (SPLs). The most typical STGs are MLM-type STGs, having medium chain fatty acids (FAs) at the 1- and 3-positions and a long chain fatty acid at the 2- position. MLM-type STGs are synthesized by: 1) 1,3-position-specific lipase-catalyzed acyl exchange of TG with FA or with FA ethylester (FAEt); 2) 1,3-position-specific lipase-catalyzed acylation of glycerol with FA, giving symmetric 1,3-diacyl-sn-glycerol, followed by chemical acylation at the sn-2 position, and; 3) 1,3-position-specific lipase-catalyzed deacylation of TG, giving 2-monoacylglycerol, followed by reacylation at the 1- and 3-positions with FA or with (FAEt). Enzymatic preparation of SPLs requires: 1) acyl group modification, and 2) head group modification of phospholipids. Acyl group modification is performed using lipases or phospholipase A2-mediated transesterification or ester synthesis to introduce arbitrary fatty acid to phospholipids. Head group modification is carried out by phospholipase D-catalyzed transphosphatidylation. A wide range of compounds can be introduced into the polar head of phospholipids, making it possible to prepare various SPLs.

  3. The Synthesis of N-Acetyllactosamine Functionalized Dendrimers, and the Functionalization of Silica Surfaces Using Tunable Dendrons and beta-Cyclodextrins

    Science.gov (United States)

    Ennist, Jessica Helen

    Galectin-3 is beta-galactoside binding protein which is found in many healthy cells. In cancer, the galectin-3/tumor-associated Thomsen-Friedenreich antigen (TF antigen) interaction has been implicated in heterotypic and homotypic cellular adhesion and apoptotic signaling pathways. However, a stronger mechanistic understanding of the role of galectin-3 in these processes is needed. N-acetyllactosamine (LacNAc) is a non-native ligand for galectin-3 which binds with comparable affinity to the TF antigen and therefore an important ligand to study galectin-3 mediated processes. To study galectin-3 mediated homotypic cellular aggregation, four generations of polyamidoamine (PAMAM) dendrimers were functionalized with N-acetyllactosamine using a four-step chemoenzymatic route. The enzymatic step controlled the regiochemistry of the galactose addition to N-acetylglucosamine functionalized dendrimers using a recombinant beta-1,4-Galactosyltransferase-/UDP-4'-Gal Epimerase Fusion Protein (lgtB-galE). Homotypic cellular aggregation, which is promoted by the presence of galectin-3 as it binds to glycosides at the cell surface, was studied using HT-1080 fibrosarcoma, A549 lung, and DU-145 prostate cancer cell lines. In the presence of small LacNAc functionalized PAMAM dendrimers, galectin-3 induced cancer cellular aggregation was inhibited. However, the larger glycodendrimers induced homotypic cellular aggregation. Additionally, novel poly(aryl ether) dendronized silica surfaces designed for reversible adsorbtion of targeted analytes were synthesized, and characterization using X-ray Photoelectron Spectroscopy (XPS) was performed. Using a Cu(I) mediated cycloaddition "click" reaction, beta-cyclodextrin was appended to dendronized surfaces via triazole formation and also to a non-dendronized surface for comparison purposes. First generation G(1) dendrons have more than 6 times greater capacity to adsorb targeted analytes than slides functionalized with monomeric beta

  4. Production of d-allulose from d-glucose by Escherichia coli transformant cells co-expressing d-glucose isomerase and d-psicose 3-epimerase genes.

    Science.gov (United States)

    Zhang, Wenli; Li, Hao; Jiang, Bo; Zhang, Tao; Mu, Wanmeng

    2017-08-01

    d-Allulose is a novel and low-calorie rare monosaccharide that is a C-3 epimer of d-fructose. Because of its excellent physiological properties and commercial potential, d-allulose has attracted researchers' interests. Based on the Izumoring strategy, d-allulose is converted from d-fructose by d-psicose 3-epimerase (DPEase), while d-fructose is converted from d-glucose by d-glucose isomerase (GIase). In this study, we created a cellular system capable of converting d-glucose to d-allulose in a one-step process that co-expressed the GIase from Acidothermus cellulolyticus and the DPEase from Dorea sp. CAG. The co-expression plasmid pETDuet-Dosp-DPE/Acce-GI was generated and transformed into Escherichia coli BL21(DE3) cells. The recombinant co-expression cells exhibited maximum catalytic activity at pH 6.5 and 75 °C. These cells were thermostable at less than 60 °C. The addition of Co 2+ significantly increased the catalytic activity by 10.8-fold. When the reaction equilibrium was reached, the ratio of d-glucose, d-fructose and d-allulose was approximately 6.5:7:3, respectively. A recombinant co-expression strain that catalysed the bioconversion of d-allulose from d-glucose in a one-step process was created and characterised. When adding 500 g L -1 d-glucose as a substrate, 204.3 g L -1 d-fructose and 89.1 g L -1 d-allulose were produced. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  5. 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.

  6. Enzymatic reactivity of glucose oxidase confined in nanochannels.

    Science.gov (United States)

    Yu, Jiachao; Zhang, Yuanjian; Liu, Songqin

    2014-05-15

    The construction of nanodevices coupled with an integrated real-time detection system for evaluation of the function of biomolecules in biological processes, and enzymatic reaction kinetics occurring at the confined space or interface is a significant challenge. In this work, a nanochannel-enzyme system in which the enzymatic reaction could be investigated with an electrochemical method was constructed. The model system was established by covalently linking glucose oxidase (GOD) onto the inner wall of the nanochannels of the porous anodic alumina (PAA) membrane. An Au disc was attached at the end of the nanochannels of the PAA membrane as the working electrode for detection of H2O2 product of enzymatic reaction. The effects of ionic strength, amount of immobilized enzyme and pore diameter of the nanochannels on the enzymatic reaction kinetics were illustrated. The GOD confined in nanochannels showed high stability and reactivity. Upon addition of glucose to the nanochannel-enzyme system, the current response had a calibration range span from 0.005 to 2 mM of glucose concentration. The apparent Michaelis-Menten constant (K(m)(app)) of GOD confined in nanochannel was 0.4 mM. The presented work provided a platform for real-time monitoring of the enzyme reaction kinetics confined in nanospaces. Such a nanochannel-enzyme system could also help design future biosensors and enzyme reactors with high sensitivity and efficiency. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. 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.

  8. Enzymatic acylglycerol synthesis in membrane reactor systems

    NARCIS (Netherlands)

    Padt, van der A.

    1993-01-01

    Up till twenty years ago, only chemical modifications of agricultural oils for novel uses were studied. Because of the instability of various fatty acids, enzymatic biomodifications can have advantages above the chemical route. Nowadays, enzymatic catalysis can be used for the modification

  9. Enzymatic Self-Assembly of Nanostructures for Theranostics

    OpenAIRE

    Yue Chen, Gaolin Liang

    2012-01-01

    Self-assembly of small molecules or macromolecules through non-covalent or covalent bonds to build up supramolecular nanostructures is a prevalent and important process in nature. While most chemists use small molecules to assemble nanostructures with physical or chemical perturbations, nature adopts enzymes to catalyze the reaction to assemble biological, functional nanostructures with high efficiency and specificity. Although enzymatic self-assembly of nanostructures has been remained chall...

  10. Enzymatic self-assembly of nanostructures for theranostics.

    Science.gov (United States)

    Chen, Yue; Liang, Gaolin

    2012-01-01

    Self-assembly of small molecules or macromolecules through non-covalent or covalent bonds to build up supramolecular nanostructures is a prevalent and important process in nature. While most chemists use small molecules to assemble nanostructures with physical or chemical perturbations, nature adopts enzymes to catalyze the reaction to assemble biological, functional nanostructures with high efficiency and specificity. Although enzymatic self-assembly of nanostructures has been remained challenging for chemists, there are still a few examples of using important enzymes to initiate the self-assembly of nanostructures for diagnosis or therapy of certain diseases because down-regulation or overexpression of certain enzymes always associates with abnormalities of tissues/organs or diseases in living body. Herein, we introduce the concept of enzymatic self-assembly and illustrate the design and application of enzyme-catalyzed or -regulated formation of nanostructures for theranostics.

  11. Two-Photon Small Molecule Enzymatic Probes.

    Science.gov (United States)

    Qian, Linghui; Li, Lin; Yao, Shao Q

    2016-04-19

    Enzymes are essential for life, especially in the development of disease and on drug effects, but as we cannot yet directly observe the inside interactions and only partially observe biochemical outcomes, tools "translating" these processes into readable information are essential for better understanding of enzymes as well as for developing effective tools to fight against diseases. Therefore, sensitive small molecule probes suitable for direct in vivo monitoring of enzyme activities are ultimately desirable. For fulfilling this desire, two-photon small molecule enzymatic probes (TSMEPs) producing amplified fluorescent signals based on enzymatic conversion with better photophysical properties and deeper penetration in intact tissues and whole animals have been developed and demonstrated to be powerful in addressing the issues described above. Nonetheless, currently available TSMEPs only cover a small portion of enzymes despite the distinct advantages of two-photon fluorescence microscopy. In this Account, we would like to share design principles for TSMEPs as potential indicators of certain pathology-related biomarkers together with their applications in disease models to inspire more elegant work to be done in this area. Highlights will be addressed on how to equip two-photon fluorescent probes with features amenable for direct assessment of enzyme activities in complex pathological environments. We give three recent examples from our laboratory and collaborations in which TSMEPs are applied to visualize the distribution and activity of enzymes at cellular and organism levels. The first example shows that we could distinguish endogenous phosphatase activity in different organelles; the second illustrates that TSMEP is suitable for specific and sensitive detection of a potential Parkinson's disease marker (monoamine oxidase B) in a variety of biological systems from cells to patient samples, and the third identifies that TSMEPs can be applied to other enzyme

  12. Enzymatically degradable mussel-inspired adhesive hydrogel.

    Science.gov (United States)

    Brubaker, Carrie E; Messersmith, Phillip B

    2011-12-12

    Mussel-inspired adhesive hydrogels represent innovative candidate medical sealants or glues. In the present work, we describe an enzyme-degradable mussel-inspired adhesive hydrogel formulation, achieved by incorporating minimal elastase substrate peptide Ala-Ala into the branched poly(ethylene glycol) (PEG) macromonomer structure. The system takes advantage of neutrophil elastase expression upregulation and secretion from neutrophils upon recruitment to wounded or inflamed tissue. By integrating adhesive degradation behaviors that respond to cellular cues, we expand the functional range of our mussel-inspired adhesive hydrogel platforms. Rapid (adhesion of the proteolytically active, catechol-terminated precursor macromonomer was achieved by addition of sodium periodate oxidant. Rheological analysis and equilibrium swelling studies demonstrated that the hydrogel is appropriate for soft tissue-contacting applications. Notably, hydrogel storage modulus (G') achieved values on the order of 10 kPa, and strain at failure exceeded 200% strain. Lap shear testing confirmed the material's adhesive behavior (shear strength: 30.4 ± 3.39 kPa). Although adhesive hydrogel degradation was not observed during short-term (27 h) in vitro treatment with neutrophil elastase, in vivo degradation proceeded over several months following dorsal subcutaneous implantation in mice. This work represents the first example of an enzymatically degradable mussel-inspired adhesive and expands the potential biomedical applications of this family of materials.

  13. Nanocrystal Bioassembly: Asymmetry, Proximity, and Enzymatic Manipulation

    Energy Technology Data Exchange (ETDEWEB)

    Claridge, Shelley A. [Univ. of California, Berkeley, CA (United States)

    2008-05-01

    Research at the interface between biomolecules and inorganic nanocrystals has resulted in a great number of new discoveries. In part this arises from the synergistic duality of the system: biomolecules may act as self-assembly agents for organizing inorganic nanocrystals into functional materials; alternatively, nanocrystals may act as microscopic or spectroscopic labels for elucidating the behavior of complex biomolecular systems. However, success in either of these functions relies heavily uponthe ability to control the conjugation and assembly processes.In the work presented here, we first design a branched DNA scaffold which allows hybridization of DNA-nanocrystal monoconjugates to form discrete assemblies. Importantly, the asymmetry of the branched scaffold allows the formation of asymmetric2assemblies of nanocrystals. In the context of a self-assembled device, this can be considered a step toward the ability to engineer functionally distinct inputs and outputs.Next we develop an anion-exchange high performance liquid chromatography purification method which allows large gold nanocrystals attached to single strands of very short DNA to be purified. When two such complementary conjugates are hybridized, the large nanocrystals are brought into close proximity, allowing their plasmon resonances to couple. Such plasmon-coupled constructs are of interest both as optical interconnects for nanoscale devices and as `plasmon ruler? biomolecular probes.We then present an enzymatic ligation strategy for creating multi-nanoparticle building blocks for self-assembly. In constructing a nanoscale device, such a strategy would allow pre-assembly and purification of components; these constructs can also act as multi-label probes of single-stranded DNA conformational dynamics. Finally we demonstrate a simple proof-of-concept of a nanoparticle analog of the polymerase chain reaction.

  14. 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).

  15. Nanostructured Metal Oxides Based Enzymatic Electrochemical Biosensors

    OpenAIRE

    Ansari, Anees A.; Alhoshan, M.; Alsalhi, M.S.; Aldwayyan, A.S.

    2010-01-01

    The unique electrocatalytic properties of the metal oxides and the ease of metal oxide nanostructured fabrication make them extremely interesting materials for electrochemical enzymatic biosensor applications. The application of nanostructured metal oxides in such sensing devices has taken off rapidly and will surely continue to expand. This article provides a review on current research status of electrochemical enzymatic biosensors based on various new types of nanostructured metal oxides su...

  16. [Enzymatic properties in muscle membranes].

    Science.gov (United States)

    Kursky, M D; Grigoryeva, V A

    1975-01-01

    A study in the enzymatic properties of muscle membranes established that sarcolemma of the rabbit skeletal muscles contains the Ca2+-ATPase system which does not require Mg2+ for manifestation of ions activity. By some kinetic properties it differs from ATPase of myosin. The complex Ca-ATP2+ is a substrate of Ca2+-ATPase. Ions of a series of bivalent metals inhibit the latter as well as the passive transport of Ca2+, that may evidence for a definite relation of Ca2+-ATPase with Ca+2 transport in skeletal muscles. Acetyl cholinesterase and AMP-aminohydrolase are strongly bound with the sarcolemma. The sarcolemma structural organization is shown to play a certain role in manifestation of their activity. On the basis of the data obtained when studying the activity in the ATPase systems and dynamics of formation and decay of the intermediate phosphorylated product in the microsomal fraction of cow and rabbit myometrium certain peculiarities are established for the active mechanisms of Ca2+ transport in smooth muscles. A problem is under discussion on the possible active participation of sarcolemma in regulation of Ca2+ concentration in the smooth muscle cells. Two ATPase systems, Mg2+-dependent and Mg2+-dependent Ca2+ activated are found in nuclei; the role of lipids of the skeletal muscles in manifestation of their activity is studied. AMP-amino hydrolase properties are characterized for different areas of the sarcoplasmatic reticulum membranes. The model of E-avitaminous muscular distrophy was used to show disturbances in the structure of sarcolemma and membranes of the sarcoplasmatic reticulum which are accompanied by changes in their ATPase and Ca2+-transporting properties.

  17. Expansion of the mammalian 3 beta-hydroxysteroid dehydrogenase/plant dihydroflavonol reductase superfamily to include a bacterial cholesterol dehydrogenase, a bacterial UDP-galactose-4-epimerase, and open reading frames in vaccinia virus and fish lymphocystis disease virus.

    Science.gov (United States)

    Baker, M E; Blasco, R

    1992-04-13

    Mammalian 3 beta-hydroxysteroid dehydrogenase and plant dihydroflavonol reductases are descended from a common ancestor. Here we present evidence that Nocardia cholesterol dehydrogenase, E. coli UDP-galactose-4 epimerase, and open reading frames in vaccinia virus and fish lymphocystis disease virus are homologous to 3 beta-hydroxysteroid dehydrogenase and dihydroflavonol reductase. Analysis of a multiple alignment of these sequences indicates that viral ORFs are most closely related to the mammalian 3 beta-hydroxysteroid dehydrogenases. The ancestral protein of this superfamily is likely to be one that metabolized sugar nucleotides. The sequence similarity between 3 beta-hydroxysteroid dehydrogenase and the viral ORFs is sufficient to suggest that these ORFs have an activity that is similar to 3 beta-hydroxysteroid dehydrogenase or cholesterol dehydrogenase, although the putative substrates are not yet known.

  18. Swimming training attenuates oxidative damage and increases enzymatic but not non-enzymatic antioxidant defenses in the rat brain.

    Science.gov (United States)

    Nonato, L F; Rocha-Vieira, E; Tossige-Gomes, R; Soares, A A; Soares, B A; Freitas, D A; Oliveira, M X; Mendonça, V A; Lacerda, A C; Massensini, A R; Leite, H R

    2016-09-29

    Although it is well known that physical training ameliorates brain oxidative function after injuries by enhancing the levels of neurotrophic factors and oxidative status, there is little evidence addressing the influence of exercise training itself on brain oxidative damage and data is conflicting. This study investigated the effect of well-established swimming training protocol on lipid peroxidation and components of antioxidant system in the rat brain. Male Wistar rats were randomized into trained (5 days/week, 8 weeks, 30 min; n=8) and non-trained (n=7) groups. Forty-eight hours after the last session of exercise, animals were euthanized and the brain was collected for oxidative stress analysis. Swimming training decreased thiobarbituric acid reactive substances (TBARS) levels (Pbrain non-enzymatic total antioxidant capacity, estimated by FRAP (ferric-reducing antioxidant power) assay (P>0.05). Moreover, the swimming training promoted metabolic adaptations, such as increased maximal workload capacity (Ptraining are key factors in promoting brain resistance. In conclusion, swimming training attenuated oxidative damage and increased enzymatic antioxidant but not non-enzymatic status in the rat brain.

  19. Enzymatic polymerization of bio-based monomers for applications in hydrogels and coatings

    DEFF Research Database (Denmark)

    Hoffmann, Christian; Nguyen, Hiep Dinh; Storgaard, Thomas

    of the enzymatic catalysts that can provide control over polymer structure in functional polymers. Lipase catalyzed polymerizations (specifically CALB) has been applied to prepare functional polyesters and to evaluate the possibilities of using less stable bio-based monomers such as itaconic acid or its...

  20. Enzymatic sequencing of partially acetylated chitosan oligomers.

    Science.gov (United States)

    Hamer, Stefanie Nicole; Moerschbacher, Bruno Maria; Kolkenbrock, Stephan

    2014-06-17

    Chitosan oligosaccharides have diverse biological activities with potentially valuable applications, for example, in the fields of medicine and agriculture. These functionalities are thought to depend on their degree of polymerization and acetylation, and possibly on specific patterns of acetylation. Chitosan oligomers with fully defined architecture are difficult to produce, and their complete analysis is demanding. Analysis is typically done using MS or NMR, requiring access to expensive infrastructure, and yielding unequivocal results only in the case of rather small oligomers. We here describe a simple and cost-efficient method for the sequencing of μg amounts of chitosan oligosaccharides which is based on the sequential action of two recombinant glycosidases, namely an exo-β-N-acetylhexosaminidase (GlcNAcase) from Bacillus subtilis 168 and an exo-β-d-glucosaminidase (GlcNase) from Thermococcus kodakarensis KOD1. Starting from the non-reducing end, GlcNAcase and GlcNase specifically remove N-acetyl glucosamine (A) and glucosamine (D) units, respectively. By the sequential addition and removal of these enzymes in an alternating way followed by analysis of the products using high-performance thin-layer chromatography, the sequence of chitosan oligosaccharides can be revealed. Importantly, both enzymes work under identical conditions so that no buffer exchange is required between steps, and the enzyme can be removed conveniently using simple ultra-filtration devices. As proof-of-principle, the method was used to sequence the product of enzymatic deacetylation of chitin pentamer using a recombinant chitin deacetylase from Vibrio cholerae which specifically removes the acetyl group from the second unit next to the non-reducing end of the substrate, yielding mono-deacetylated pentamer with the sequence ADAAA. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Non-Enzymatic biopolymerization reactions supported by heterogeneous media

    DEFF Research Database (Denmark)

    Monnard, Pierre-Alain

    2011-01-01

    Heterogeneous media, such as micro-structured aqueous environments, could offer an alternative approach to the synthesis of biopolymers with novel functions. Structured media are here defined as specialized, self-assembled structures that are formed, e.g, by amphiphiles, such as liposomes, emulsion...... or unavailable in bulk aqueous phases. Reactions can then proceed which do not readily occur in homogeneous solutions. To gauge the potential of this idea, we have investigated the non-enzymatic polymerization of RNA from monomers in the presence of various catalysts....

  2. Enzymatic desulfurization of coal: Third quarterly report

    Energy Technology Data Exchange (ETDEWEB)

    Marquis, Judith K. [School of Medicine, Boston Univ., MA (United States); Kitchell, Judith P. [Holometrix, Inc., Cambridge, Massachusetts (United States)

    1989-03-14

    Our current efforts to develop clean coal technology emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes or commercially available enzymes. Our work is focused on the treatment of ''model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix. In this quarter we obtained important results both with the development of our understanding of the enzyme reaction systems and also with the microbial work at Woods Hole. 12 figs., 11 tabs.

  3. Comparison of Enzymatic and Ultrasonic Extraction of Albumin from Defatted Pumpkin (Cucurbita pepo Seed Powder

    Directory of Open Access Journals (Sweden)

    Gia Loi Tu

    2015-01-01

    Full Text Available In this study, ultrasound- and enzyme-assisted extractions of albumin (water-soluble protein group from defatted pumpkin (Cucurbita pepo seed powder were compared. Both advanced extraction techniques strongly increased the albumin yield in comparison with conventional extraction. The extraction rate was two times faster in the ultrasonic extraction than in the enzymatic extraction. However, the maximum albumin yield was 16 % higher when using enzymatic extraction. Functional properties of the pumpkin seed albumin concentrates obtained using the enzymatic, ultrasonic and conventional methods were then evaluated. Use of hydrolase for degradation of cell wall of the plant material did not change the functional properties of the albumin concentrate in comparison with the conventional extraction. The ultrasonic extraction enhanced water-holding, oil-holding and emulsifying capacities of the pumpkin seed albumin concentrate, but slightly reduced the foaming capacity, and emulsion and foam stability.

  4. 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 l...

  5. pH & Rate of Enzymatic Reactions.

    Science.gov (United States)

    Clariana, Roy B.

    1991-01-01

    A quantitative and inexpensive way to measure the rate of enzymatic reaction is provided. The effects of different pH levels on the reaction rate of an enzyme from yeast are investigated and the results graphed. Background information, a list of needed materials, directions for preparing solutions, procedure, and results and discussion are…

  6. Enzymatic hydrolysis - present status and future developments

    Energy Technology Data Exchange (ETDEWEB)

    Linko, M.

    1983-01-01

    The environmental conditions for efficient cellulose and scylanate production with Trichoderma reesei have been studied. An interesting new aproach is cellulose production by Trichoderma reesei immobilized on K-carageenan. Hexoses and pentoses are produceed by the enzymatic hydrolysis of cellulose and hemicellulose and used for ethanol fermentation.

  7. Enhancement of enzymatic adipyl-7-ADCA hydrolysis

    NARCIS (Netherlands)

    Schroën, C.G.P.H.; Kroon, P.J.; Vanderlaan, J.M.; Janssen, A.E.M.; Tramper, J.

    2002-01-01

    We studied enzymatic adipyl-7-ADCA hydrolysis as a new process for the production of 7-aminodeacetoxycephalosporanic acid (7-ADCA), one of the building blocks for cephalosporin antibiotics like cephalexin and cefadroxil. Adipyl-7-ADCA hydrolysis carried out with immobilised glutaryl acylase was

  8. 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

  9. HAEMATOLOGICAL INDICES AND ENZYMATIC BIOMAKER OF ...

    African Journals Online (AJOL)

    AGROSEARCH UIL

    out on the fish to ascertain the normal range of blood parameter, find out the variation with age, sex, season, and determine the effects of disease condition on the fish. This study is aimed at assessing the enzymatic biomarkers and haematological indices of Tilapia specie (Sarotherodon melanotheron) of the Lagos lagoon.

  10. Starch facilitates enzymatic wheat gluten hydrolysis

    NARCIS (Netherlands)

    Hardt, N.A.; Boom, R.M.; Goot, van der A.J.

    2015-01-01

    Wheat gluten can be hydrolyzed by either using (vital) wheat gluten or directly from wheat flour. This study investigates the influence of the presence of starch, the main component of wheat, on enzymatic wheat gluten hydrolysis. Wheat gluten present in wheat flour (WFG) and vital wheat gluten (VWG)

  11. 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...

  12. Biochemical control of CARM1 enzymatic activity by phosphorylation.

    Science.gov (United States)

    Feng, Qin; He, Bin; Jung, Sung-Yun; Song, Yongcheng; Qin, Jun; Tsai, Sophia Y; Tsai, Ming-Jer; O'Malley, Bert W

    2009-12-25

    Coactivator-associated arginine methyltransferase 1 (CARM1) is a dual functional coregulator that facilitates transcription initiation by methylation of Arg(17) and Arg(26) of histone H3 and also dictates the subsequent coactivator complex disassembly by methylation of the steroid receptor coactivator family coactivators and p300/cAMP-response element-binding protein-binding protein. However, the regulation of CARM1 enzymatic activity and substrate specificity remains largely unknown. In this study, we report that CARM1 function is regulated by phosphorylation at Ser(217), a residue completely conserved in the type I protein arginine methyltransferase (PRMT) family of enzymes. Comparative analysis of the published CARM1 crystal structures reveals that the hydroxyl group of Ser(217) forms a strong hydrogen bond with the carbonyl oxygen atom of Tyr(154) to lock the cofactor S-adenosylmethionine inside the binding cavity. Phosphorylation of Ser(217) disrupts this hydrogen bond and subsequently abolishes S-adenosylmethionine binding and its methyltransferase activity. Importantly, Tyr(154) is also conserved in the type I PRMT family of enzymes, suggesting a general role of this hydrogen bond in maintaining the holo structure of the type I PRMT catalytic domain. Moreover, we found that phosphorylation at Ser(217) also promoted CARM1 cytoplasmic localization and that this translocation occurred mainly during mitosis. We propose that phosphorylation at Ser(217) serves as a molecular switch for controlling CARM1 enzymatic activity during the cell cycle.

  13. 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.

  14. Enzymatic induction of supramolecular order and bioactivity

    Science.gov (United States)

    Yang, Chengbiao; Ren, Xinrui; Ding, Dan; Wang, Ling; Yang, Zhimou

    2016-05-01

    We showed in this study that enzymatic triggering is a totally different pathway for the preparation of self-assembling nanomaterials to the heating-cooling process. Because the molecules were under lower energy levels and the molecular conformation was more ordered during the enzymatic triggeration under mild conditions, nanomaterials with higher supramolecular order could be obtained through biocatalytic control. In this study, nanoparticles were obtained by an enzymatic reaction and nanofibers were observed through the heating-cooling process. We observed a distinct trough at 318 nm from the CD spectrum of a particle sample but not a fiber sample, suggesting the long range arrangement of molecules and helicity in the nanoparticles. The nanoparticles with higher supramolecular order possessed much better potency as a protein vaccine adjuvant because it accelerated the DC maturation and elicited stronger T-cells cytokine production than the nanofibers. Our study demonstrated that biocatalytic triggering is a useful method for preparing supramolecular nanomaterials with higher supramolecular order and probably better bioactivity.We showed in this study that enzymatic triggering is a totally different pathway for the preparation of self-assembling nanomaterials to the heating-cooling process. Because the molecules were under lower energy levels and the molecular conformation was more ordered during the enzymatic triggeration under mild conditions, nanomaterials with higher supramolecular order could be obtained through biocatalytic control. In this study, nanoparticles were obtained by an enzymatic reaction and nanofibers were observed through the heating-cooling process. We observed a distinct trough at 318 nm from the CD spectrum of a particle sample but not a fiber sample, suggesting the long range arrangement of molecules and helicity in the nanoparticles. The nanoparticles with higher supramolecular order possessed much better potency as a protein vaccine

  15. Protein cluster formation during enzymatic cross-linking of globular proteins

    NARCIS (Netherlands)

    Saricay, Y.; Dhayal, S.K.; Wierenga, P.A.; Vries, de R.J.

    2012-01-01

    Work on enzymatic cross-linking of globular food proteins has mainly focused on food functional effects such as improvements of gelation and enhanced stabilization of emulsions and foams, and on the detailed biochemical characterization of the cross-linking chemistry. What is still lacking is a

  16. Enzymatic degradation of plant cell wall polysaccharides: the kinetic effect of competitive adsorption

    DEFF Research Database (Denmark)

    Bergsøe, Merete Norsker; Bloch, Line; Adler-Nissen, Jens

    1999-01-01

    Insoluble potato dietary fibre, isolated from potato pulp, can be enzymatically hydrolysed with the pectolytic enzyme preparation Pectinex Ultra SP from Novo Nordisk A/S, in order to produce soluble fibre. The soluble fibre has valuable functional properties for the food industry. Cloned monocomp...

  17. Enzymatic Synthesis of Amylose Brushes Revisited : Details from X-Ray Photoelectron Spectroscopy and Spectroscopic Ellipsometry

    NARCIS (Netherlands)

    Mazzocchetti, Laura; Tsoufis, Theodorus; Rudolf, Petra; Loos, Katja

    The successful synthesis of amylose brushes via enzymatic ‘‘grafting from’’ polymerization and the detailed characterization of all synthetic steps by X-ray photoelectron spectroscopy (XPS) and spectroscopic ellipsometry measurements are reported. Au and Si surfaces are amino-functionalized with

  18. Enzymatic detection of formalin-fixed museum specimens for DNA analysis and enzymatic maceration of formalin-fixed specimens

    DEFF Research Database (Denmark)

    Sørensen, Margrethe; Redsted Rasmussen, Arne; Simonsen, Kim Pilkjær

    2016-01-01

    Abstract.—A simple enzymatic screening method has been developed to detect whether a tissue sample has been preserved with formalin or with ethanol only because such a method is a useful tool for predicting the quality of genetic test results. The method is based on enzymatic digestion at 55 C at...... be macerated by enzymatic digestion under alkaline conditions at 55 C....

  19. Association genetics in Solanum tuberosum provides new insights into potato tuber bruising and enzymatic tissue discoloration.

    Science.gov (United States)

    Urbany, Claude; Stich, Benjamin; Schmidt, Lysann; Simon, Ludwig; Berding, Hergen; Junghans, Holger; Niehoff, Karl-Heinz; Braun, Alexander; Tacke, Eckhard; Hofferbert, Hans-Rheinhardt; Lübeck, Jens; Strahwald, Josef; Gebhardt, Christiane

    2011-01-05

    Most agronomic plant traits result from complex molecular networks involving multiple genes and from environmental factors. One such trait is the enzymatic discoloration of fruit and tuber tissues initiated by mechanical impact (bruising). Tuber susceptibility to bruising is a complex trait of the cultivated potato (Solanum tuberosum) that is crucial for crop quality. As phenotypic evaluation of bruising is cumbersome, the application of diagnostic molecular markers would empower the selection of low bruising potato varieties. The genetic factors and molecular networks underlying enzymatic tissue discoloration are sparsely known. Hitherto there is no association study dealing with tuber bruising and diagnostic markers for enzymatic discoloration are rare. The natural genetic diversity for bruising susceptibility was evaluated in elite middle European potato germplasm in order to elucidate its molecular basis. Association genetics using a candidate gene approach identified allelic variants in genes that function in tuber bruising and enzymatic browning. Two hundred and five tetraploid potato varieties and breeding clones related by descent were evaluated for two years in six environments for tuber bruising susceptibility, specific gravity, yield, shape and plant maturity. Correlations were found between different traits. In total 362 polymorphic DNA fragments, derived from 33 candidate genes and 29 SSR loci, were scored in the population and tested for association with the traits using a mixed model approach, which takes into account population structure and kinship. Twenty one highly significant (p enzymatic tissue discoloration and tuber bruising. Their validation and characterization will increase the knowledge about the underlying biological processes.

  20. Evaluation of the effects of isolated lignin on enzymatic hydrolysis of cellulose.

    Science.gov (United States)

    Zhang, Hongdan; Wu, Shubin; Xie, Jun

    2017-06-01

    The different physical and chemical properties of lignin might have various effects on the enzymatic hydrolysis of lignocellulosic substrates. In this study, the influence of lignin on enzymatic digestibility of cellulose was assessed. Addition of 20% (4g/L) isolated enzymatic lignin (lignin 2 and 3) and kraft lignin (lignin 4) resulted in 5-20% drop of glucose yield, depending on lignin sources. The inhibitory effect of lignin was abated as the enzyme loading increased from 10 to 20FPU/g dry substrate. However, the increasing lignin amount to 40% (8g/L) did not appear to further decrease the cellulose hydrolysis efficiency. Ethanol lignin (lignin 1) and calcium lignosulfonate (lignin 5) had no negative effect on the enzymatic hydrolysis of cellulose at cellulase loading of 10 or 20FPU/g dry substrate, the increasing lignin content to 40% presented 6.2% increase of glucose yield. The results indicated that different lignin had significantly influence on the enzymatic hydrolysis, which was confirmed by analysis in chemical composition, elemental analysis, functionality, and thermogravimetry. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Localized cranial hyperostosis of meningiomas: a result of neoplastic enzymatic activity?

    DEFF Research Database (Denmark)

    Heick, A.; Mosdal, C.; Klinken, Leif

    1993-01-01

    Neuropathology, alkaline phosphatase, cranial hyperostosis, meningioma, ossifying enzymatic activity......Neuropathology, alkaline phosphatase, cranial hyperostosis, meningioma, ossifying enzymatic activity...

  2. Enzymatic interesterification of vegetable oil/ fish oil blend for margarine production

    DEFF Research Database (Denmark)

    Ibrahim, Nuzul Amri Bin; Xu, Xuebing

    In margarine formulation, oils of different melting points are blended to make a product that is spreadable at room temperature. Usually, the blend would be subjected to modification process, either by interesterification (chemical or enzymatic) or partial hydrogenation in order to achieve...... the desired properties. In this study, palm stearin (PS), palm kernel oil (PKO) and fish oil (FO) are blended and modified by enzymatic interesterification. PS functioned as the hard stock, PKO as the soft oil and FO as a source for eicosapentaenoic acid (EPA)/ docosahexaenoic acid (DHA). The purpose...... they are consumed as a quick source of energy. The remaining 2-monoacyl- glycerol becomes a source of essential fatty acid, after being absorbed through the intestinal wall. This would enhance the nutritional value of the enzymatically interesterified product. However, the incorporation of FO into the blend would...

  3. Non-enzymatic amperometric glucose biosensor based on nickel hexacyanoferrate nanoparticle film modified electrodes.

    Science.gov (United States)

    Wang, Xiaoyan; Zhang, Yun; Banks, Craig E; Chen, Qiyuan; Ji, Xiaobo

    2010-07-01

    A non-enzymatic amperometric glucose biosensor based on the modification of functional nickel hexacyanoferrate nanoparticles was prepared via electrochemical deposition. The electrochemical deposition of the nickel hexacyanoferrate nanoparticles was obtained by potential cycling in a solution containing nickel (II) and hexacyanoferrate (III) producing a modified surface with a high degree of uniformity. The modified electrode is exemplified towards the non-enzymatic sensing of glucose where using cyclic voltammetry and amperometry, low micro-molar up to milli-molar glucose concentrations are readily detectable. The non-enzymatic sensing of glucose also shows a modest selectivity over ascorbic acid. This platform offers a novel route for glucose sensors with wide analytical applications. 2010 Elsevier B.V. All rights reserved.

  4. Enzymatic degradation of polycaprolactone-gelatin blend

    Science.gov (United States)

    Banerjee, Aditi; Chatterjee, Kaushik; Madras, Giridhar

    2015-04-01

    Blends of polycaprolactone (PCL), a synthetic polymer and gelatin, natural polymer offer a optimal combination of strength, water wettability and cytocompatibility for use as a resorbable biomaterial. The enzymatic degradation of PCL, gelatin and PCL-gelatin blended films was studied in the presence of lipase (Novozym 435, immobilized) and lysozyme. Novozym 435 degraded the PCL films whereas lysozyme degraded the gelatin. Though Novozym 435 and lysozyme individually could degrade PCL-gelatin blended films, the combination of these enzymes showed the highest degradation of these blended films. Moreover, the enzymatic degradation was much faster when fresh enzymes were added at regular intervals. The changes in physico-chemical properties of polymer films due to degradation were studied by scanning electron microscopy, Fourier transform infrared spectroscopy and differential scanning calorimetry. These results have important implications for designing resorbable biomedical implants.

  5. Enzymatic hydrolysis of corn bran arabinoxylan

    DEFF Research Database (Denmark)

    Agger, Jane

    This thesis concerns enzymatic hydrolysis of corn bran arabinoxylan. The work has focused on understanding the composition and structure of corn bran with specific interest in arabinoxylan with the main purpose of targeting enzymatic hydrolysis for increased yields. Corn bran has been used...... as a model substrate because it represents a readily available agroindustrial side product with upgrading potentials. Corn bran originates from the wet-milling process in corn starch processing, is the outmost layers of the corn kernel and is particularly rich in pentose monosaccharides comprising the major...... components of arabinoxylan. Corn bran is one of the most recalcitrant cereal byproducts with arabinoxylans of particular heterogeneous nature. It is also rich in feruloyl derived substitutions, which are responsible for extensive cross-linking between arabinoxylan molecules and thereby participate...

  6. Production of MAG via enzymatic glycerolysis

    Science.gov (United States)

    Jamlus, Norul Naziraa Ahmad; Derawi, Darfizzi; Salimon, Jumat

    2015-09-01

    Enzymatic glycerolysis of a medium chain methyl ester, methyl laurate was performed using lipase Candida antarctica (Novozyme 435) for 6 hours at 55°C. The percentage of components mixture of product were determined by using gas chromatography technique. The enzymatic reaction was successfully produced monolaurin (45.9 %), dilaurin (47.1 %) and trilaurin (7.0 %) respectively. Thin layer chromatography (TLC) plate also showed a good separation of component spots. Fourier transformation infra-red (FTIR) spectrum showed the presence of ester carbonyl at wavenumber 1739.99 cm-1 and hydrogen bonded O-H at 3512.03 cm-1. The product is potentially to be used as emulsifier and additive in food industry, pharmaceutical, as well as antibacterial.

  7. 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...... 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....... Critical to the project is to develop a control methodology to optimize the productivity of biodiesel production (e.g. the dosing of alcohol to minimize catalyst deactivation, minimization of waste and delivering consistent product quality meeting specifications). For production of biodiesel (BD) via...

  8. Enzymatic cybernetics: an unpublished work by Jacques Monod.

    Science.gov (United States)

    Gayon, Jean

    2015-06-01

    In 1959, Jacques Monod wrote a manuscript entitled Cybernétique enzymatique [Enzymatic cybernetics]. Never published, this unpublished manuscript presents a synthesis of how Monod interpreted enzymatic adaptation just before the publication of the famous papers of the 1960s on the operon. In addition, Monod offers an example of a philosophy of biology immersed in scientific investigation. Monod's philosophical thoughts are classified into two categories, methodological and ontological. On the methodological side, Monod explicitly hints at his preferences regarding the scientific method in general: hypothetical-deductive method, and use of theoretical models. He also makes heuristic proposals regarding molecular biology: the need to analyse the phenomena in question at the level of individual cells, and the dual aspect of all biological explanation, functional and evolutionary. Ontological issues deal with the notions of information and genetic determinism, "cellular memory", the irrelevance of the notion of "living matter", and the usefulness of a cybernetic comprehension of molecular biology. Copyright © 2015 Académie des sciences. Published by Elsevier SAS. All rights reserved.

  9. Investigation of bi-enzymatic reactor based on hybrid monolith with nanoparticles embedded and its proteolytic characteristics.

    Science.gov (United States)

    Shangguan, Lulu; Zhang, Lingyi; Xiong, Zhichao; Ren, Jun; Zhang, Runsheng; Gao, Fangyuan; Zhang, Weibing

    2015-04-03

    The bottom-up strategy of proteomic profiling study based on mass spectrometer (MS) has drawn high attention. However, conventional solution-based digestion could not satisfy the demands of highly efficient and complete high throughput proteolysis of complex samples. We proposed a novel bi-enzymatic reactor by immobilizing two different enzymes (trypsin/chymotrypsin) onto a mixed support of hybrid organic-inorganic monolith with SBA-15 nanoparticles embedded. Typsin and chymotrypsin were crossly immobilized onto the mixed support by covalent bonding onto the monolith with glutaraldehyde as bridge reagent and chelation via copper ion onto the nanoparticles, respectively. Compared with single enzymatic reactors, the bi-enzymatic reactor improved the overall functional analysis of membrane proteins of rat liver by doubling the number of identified peptides (from 1184/1010 with trypsin/chymotrypsin enzymatic reactors to 2891 with bi-enzymatic reactor), which led to more proteins identified with deep coverage (from 452/336 to 620); the efficiency of the bi-enzymatic reactor is also better than that of solution-based tandem digestion, greatly shorting the digestion time from 24h to 50s. Moreover, more transmembrane proteins were identified by bi-enzymatic reactor (106) compared with solution-based tandem digestion (95) with the same two enzymes and enzymatic reactors with single enzyme immobilized (75 with trypsin and 66 with chymotrypsin). The proteolytic characteristics of the bi-enzymatic reactors were evaluated by applying them to digestion of rat liver proteins. The reactors showed good digestion capability for proteins with different hydrophobicity and molecular weight. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. 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.

  11. Microbial Enzymatic Degradation of Biodegradable Plastics.

    Science.gov (United States)

    Roohi; Bano, Kulsoom; Kuddus, Mohammed; Zaheer, Mohammed R; Zia, Qamar; Khan, Mohammed F; Ashraf, Ghulam Md; Gupta, Anamika; Aliev, Gjumrakch

    2017-01-01

    The renewable feedstock derived biodegradable plastics are important in various industries such as packaging, agricultural, paper coating, garbage bags and biomedical implants. The increasing water and waste pollution due to the available decomposition methods of plastic degradation have led to the emergence of biodegradable plastics and biological degradation with microbial (bacteria and fungi) extracellular enzymes. The microbes utilize biodegradable polymers as the substrate under starvation and in unavailability of microbial nutrients. Microbial enzymatic degradation is suitable from bioremediation point of view as no waste accumulation occurs. It is important to understand the microbial interaction and mechanism involved in the enzymatic degradation of biodegradable plastics under the influence of several environmental factors such as applied pH, thermo-stability, substrate molecular weight and/or complexity. To study the surface erosion of polymer film is another approach for hydrolytic degradation characteristion. The degradation of biopolymer is associated with the production of low molecular weight monomer and generation of carbon dioxide, methane and water molecule. This review reported the degradation study of various existing biodegradable plastics along with the potent degrading microbes (bacteria and fungi). Patents available on plastic biodegradation with biotechnological significance is also summarized in this paper. This paper assesses that new disposal technique should be adopted for the degradation of polymers and further research is required for the economical production of biodegradable plastics along with their enzymatic degradation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  12. Enzymatic glycoprotein synthesis: Preparation of ribonuclease glycoforms via enzymatic glycopeptide condensation and glycosylation

    Energy Technology Data Exchange (ETDEWEB)

    Witte, K.; Sears, P.; Martin, R.; Wong, C.H. [Scripps Research Inst., La Jolla, CA (United States)

    1997-03-05

    In order to study the effects carbohydrates have on glycoprotein structure and funciton, it is imperative to be able to synthesize the appropriate natural and non-natural glycoprotein variants in a single form. Because the available in vivo techniques provide only heterogeneous mixtures of different glycoforms, enzymatic in vitro methodologies have been pursued. Using the N-glycoprotein RNase B as a model system, the oligosaccharide was removed leaving only the N-acetylglucosamine as a `tag` to the site of glycosylation. Glycosyltransferases were then used to build a unique carbohydrate moiety. A new RNase glycoform containing the branched oligosccharide, sialyl Lewis X or the Hg derivative, was synthesized enzymatically to demonstrate the feasibility of the method. In addition, the monoglycosylated protein was digested into several smaller pieces by subtilisin BPN`. These fragments were religated by subtilisin 8397 to the full length RNase by addition glycerol; this method points to a new chemical-enzymatic process for the synthesis of glycoproteins using synthetic peptides and glycopeptides as substrates for enzymatic ligation followed by further enzymatic glycosylations. 29 refs., 6 figs.

  13. 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.

  14. 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.

  15. Enzymatic processes in alternative reaction media: a mini review

    Directory of Open Access Journals (Sweden)

    Mansour Ghaffari-Moghaddam

    2015-08-01

    Full Text Available Biocatalysis is a growing field in the production of fine chemicals and will most probably increase its share in the future. Enzymatic reactions are carried out under mild conditions, i.e., non-toxic solvents, low temperature and pressure, which eliminates most environmental drawbacks associated with conventional production methods. The superiority of chemo-, regio- and enantioselectivity of enzymes exhibit significant advantages over conventional catalysts for production of fine chemicals, flavors, fragrances, agrochemicals and pharmaceuticals. Enzymes can function both in aqueous and non-aqueous solvents. As a result of the growing scientific and industrial interest towards green chemistry, green solvent systems, which are mainly water, supercritical fluids, ionic liquids, fluorinated solvents, and solvent-free systems have become more popular in biocatalysis. However, the activity and selectivity of an enzyme is heavily dependent on solvent properties. In this review, various green solvents were classified and some of their influential features on enzyme activity were discussed.

  16. DEXTRINIZED SYRUPS OBTAINING THROUGH THE ENZYMATIC HYDROLYSIS OF SORGHUM STARCH

    Directory of Open Access Journals (Sweden)

    Leyanis Rodríguez Rodríguez

    2015-10-01

    Full Text Available The main objective of this work was the production of syrups dextrinized by enzymatic hydrolysis of starch red sorghum CIAPR-132 using α-amylase on solutions at different concentrations, with different concentrations of enzyme and enzyme hydrolysis time. The response variable was the dextrose equivalent in each obtained syrup (ED using the modified Lane-Eynon method. In some of the experiments, we used a full factorial design 23 and in others we worked with intermediate concentration and higher hydrolysis time with different levels of enzyme. The obtained products were syrups dextrinized ED between 10,25 and 33,97% (values we can find within the established ones for these types of syrups, which can be used for their functional properties as intermediates syrups or as raw material for different processes of the food industry. This allows you to set a pattern for the use of sorghum feedstock in unconventional obtaining products from its starch.

  17. Oxidative enzymatic gelation of sugar beet pectin for emulsion stabilization

    DEFF Research Database (Denmark)

    Abang Zaidel, Dayang Norulfairuz; Meyer, Anne S.

    2013-01-01

    Pectin from sugar beet is derived from the sugar beet pulp residue which results when sugar beets are processed for sucrose extraction. The sugar beet pectin has poor gelationability by the classic divalentcation molecular mechanism because of a relatively high acetylation degree and short...... polygalacturonate backbone chain length. However, due to the feruloyl-substitutions on the side chains, the sugar beet pectic polysaccharides can be cross-linked via enzyme catalyzed oxidation. The enzyme kinetics and functionality of such oxidativelycross-linked sugar beet pectin, in relation to stabilizing...... emulsions has recently been investigated in model food emulsions. This paper reviews the pectin chemistry, enzymatic oxidative gelation mechanisms, interaction mechanisms of the sugar beet pectin with the emulsion droplets and explores how the gelation affects the rheology and stability of emulsion systems...

  18. Enzymatic gelation of sugar beet pectin in food products

    DEFF Research Database (Denmark)

    Bergsøe, Merete Norsker; Jensen, Mette; Adler-Nissen, Jens

    2000-01-01

    Sugar beet pectin is a food ingredient with specific functional properties. It may form gels by an oxidative cross-linking of ferulic acid. In the present study, the gel forming properties of three oxidative enzymes were examined in different food relevant conditions. The enzymes chosen were two...... laccases and one peroxidase. The textural properties of the produced gels were measured on a texture analyser. The influence of sugar, salt and protein were analysed. Finally, the enzymatic gelation was studied in three food products with added sugar beet pectin. These were black currant juice, milk...... and chopped heat-treated meat emulsion. The addition of salt resulted in softer, less stiff and chewy, and less adhesive gels. Generally speaking, sugar addition increased the hardness but at high concentration the gels were very brittle. However, Young's modulus was lower in gels containing sugar than...

  19. Sulfate radicals enable a non-enzymatic Krebs cycle precursor

    OpenAIRE

    Keller, Markus A; Kampjut, Domen; Harrison, Stuart A.; Ralser, Markus

    2017-01-01

    The evolutionary origins of the tricarboxylic acid cycle (TCA), or Krebs cycle, are so far unclear. Despite a few years ago, the existence of a simple non-enzymatic Krebs-cycle catalyst has been dismissed ?as an appeal to magic?, citrate and other intermediates have meanwhile been discovered on a carbonaceous meteorite and do interconvert non-enzymatically. To identify the non-enzymatic Krebs cycle catalyst, we used combinatorial, quantitative high-throughput metabolomics to systematically sc...

  20. Evaluation of an enzymatic method for determining creatinine in plasma.

    OpenAIRE

    Crocker, H; Shephard, M D; White, G H

    1988-01-01

    An enzymatic kit method for the determination of plasma creatinine was optimised for use with a centrifugal analyser and its performance characteristics and practicability compared with an end point and a kinetic Jaffé-based method. The enzymatic method exhibited several advantages over Jaffé-based methods--namely, smaller sample size, rapid sample throughput (200 per hour), and improved specificity. Glucose, acetoacetate, and cefoxitin did not interfere with the enzymatic method, although bi...

  1. 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...... 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....

  2. Fluorometric enzymatic assay of L-arginine

    Science.gov (United States)

    Stasyuk, Nataliya; Gayda, Galina; Yepremyan, Hasmik; Stepien, Agnieszka; Gonchar, Mykhailo

    2017-01-01

    The enzymes of L-arginine (further - Arg) metabolism are promising tools for elaboration of selective methods for quantitative Arg analysis. In our study we propose an enzymatic method for Arg assay based on fluorometric monitoring of ammonia, a final product of Arg splitting by human liver arginase I (further - arginase), isolated from the recombinant yeast strain, and commercial urease. The selective analysis of ammonia (at 415 nm under excitation at 360 nm) is based on reaction with o-phthalaldehyde (OPA) in the presence of sulfite in alkali medium: these conditions permit to avoid the reaction of OPA with any amino acid. A linearity range of the fluorometric arginase-urease-OPA method is from 100 nM to 6 μМ with a limit of detection of 34 nM Arg. The method was used for the quantitative determination of Arg in the pooled sample of blood serum. The obtained results proved to be in a good correlation with the reference enzymatic method and literature data. The proposed arginase-urease-OPA method being sensitive, economical, selective and suitable for both routine and micro-volume formats, can be used in clinical diagnostics for the simultaneous determination of Arg as well as urea and ammonia in serum samples.

  3. Effects of enzymatic hydrolysis on lentil allergenicity.

    Science.gov (United States)

    Cabanillas, Beatriz; Pedrosa, Mercedes M; Rodríguez, Julia; González, Angela; Muzquiz, Mercedes; Cuadrado, Carmen; Crespo, Jesús F; Burbano, Carmen

    2010-09-01

    Enzymatic hydrolysis and further processing are commonly used to produce hypoallergenic dietary products derived from different protein sources, such as cow's milk. Lentils and chickpeas seem to be an important cause of IgE-mediated hypersensitivity in the Mediterranean area and India. Some studies have investigated the effects of enzymatic treatments on the in vitro immunological reactivity of members of the Leguminosae family, such as soybean, chickpea, lentil, and lupine. Nevertheless, there are only a few studies carried out to evaluate the effect on IgE reactivity of these food-hydrolysis products with sera from patients with well-documented allergy to these foods. In this study, lentil protein extract was hydrolyzed by sequential action of an endoprotease (Alcalase) and an exoprotease (Flavourzyme). Immunoreactivity to raw and hydrolyzed lentil extract was evaluated by means of IgE immunoblotting and ELISA using sera from five patients with clinical allergy to lentil. The results indicated that sequential hydrolysis of lentil results in an important proteolytic destruction of IgE-binding epitopes shown by in vitro experiments. However, some allergenic proteins were still detected by sera from four out of five patients in the last step of sequential hydrolyzation.

  4. Palm date fibers: analysis and enzymatic hydrolysis.

    Science.gov (United States)

    Shafiei, Marzieh; Karimi, Keikhosro; Taherzadeh, Mohammad J

    2010-11-01

    Waste palm dates were subjected to analysis for composition and enzymatic hydrolysis of their flesh fibers. The fruit contained 32% glucose and 30% fructose, while the water-insoluble fibers of its flesh consisted of 49.9% lignin and 20.9% polysaccharides. Water-insoluble fibers were settled to 55% of its initial volume in 12 h. The presence of skin and flesh colloidal fibers results in high viscosity and clogging problems during industrial processes. The settling velocity of the fibers was improved by enzymatic hydrolysis. Hydrolysis resulted in 84.3% conversion of the cellulosic part of the fibers as well as reducing the settling time to 10 minutes and the final settled volume to 4% of the initial volume. It implies easier separation of the fibers and facilitates fermentation processes in the corresponding industries. Two kinds of high- and low-lignin fibers were identified from the water-insoluble fibers. The high-lignin fibers (75% lignin) settled easily, while the low-lignin fibers (41.4% lignin) formed a slurry suspension which settled very slowly. The hydrophilicity of these low-lignin fibers is the major challenge of the industrial processes.

  5. Stability of Enzymatic Biosensors for Wearable Applications.

    Science.gov (United States)

    Sonawane, Apurva; Manickam, Pandiaraj; Bhansali, Shekhar

    2017-05-19

    Technological evolution in wearable sensors is accounting for major growth and transformation in multitude of industries ranging from healthcare to computing & informatics to communication and biomedical sciences. The major driver for this transformation is the new-found ability to continuously monitor and analyze the patients' physiology in patients' natural setting. Numerous wearable sensors are already on the market and are summarized. Most of the current technologies have focused on electro-physiological, electro-mechanical or acoustic measurements. Wearable bio-chemical sensing devices are in their infancy. Traditional challenges in biochemical sensing such as reliability, repeatability, stability, and drift are amplified in wearable sensing systems due to variabilities in operating environment, sample/sensor handling and motion artifacts. Enzymatic sensing technologies, due to reduced fluidic challenges continue to be forerunners for translation into wearable sensors. This paper reviews the recent developments in wearable enzymatic sensors. The wearable sensors have been classified in three major groups based on sensor embodiment and placement relative to the human body: (i) On-body, (ii) Clothing/textile-based biosensors and (iii) Biosensor accessories. The sensors, which come in the forms of stickers, tattoos are categorized as on-body biosensors. The fabric-based biosensor comes in different models such as smart-shirts, socks, gloves and smart undergarments with printed sensors for continuous monitoring.

  6. Palm Date Fibers: Analysis and Enzymatic Hydrolysis

    Directory of Open Access Journals (Sweden)

    Mohammad J. Taherzadeh

    2010-11-01

    Full Text Available Waste palm dates were subjected to analysis for composition and enzymatic hydrolysis of their flesh fibers. The fruit contained 32% glucose and 30% fructose, while the water-insoluble fibers of its flesh consisted of 49.9% lignin and 20.9% polysaccharides. Water-insoluble fibers were settled to 55% of its initial volume in 12 h. The presence of skin and flesh colloidal fibers results in high viscosity and clogging problems during industrial processes. The settling velocity of the fibers was improved by enzymatic hydrolysis. Hydrolysis resulted in 84.3% conversion of the cellulosic part of the fibers as well as reducing the settling time to 10 minutes and the final settled volume to 4% of the initial volume. It implies easier separation of the fibers and facilitates fermentation processes in the corresponding industries. Two kinds of high- and low-lignin fibers were identified from the water-insoluble fibers. The high-lignin fibers (75% lignin settled easily, while the low-lignin fibers (41.4% lignin formed a slurry suspension which settled very slowly. The hydrophilicity of these low-lignin fibers is the major challenge of the industrial processes.

  7. 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.)

  8. Enzymatically Controlled Vacancies in Nanoparticle Crystals

    Energy Technology Data Exchange (ETDEWEB)

    Barnaby, Stacey N.; Ross, Michael B.; Thaner, Ryan V.; Lee, Byeongdu; Schatz, George C.; Mirkin, Chad A.

    2016-08-01

    In atomic systems, the mixing of metals results in distinct phase behavior that depends on the identity and bonding characteristics of the atoms. In nanoscale systems, the use of oligonucleotides as programmable “bonds” that link nanoparticle “atoms” into superlattices allows for the decoupling of atom identity and bonding. While much research in atomic systems is dedicated to understanding different phase behavior of mixed metals, it is not well understood on the nanoscale how changes in the nanoscale “bond” affect the phase behavior of nanoparticle crystals. In this work, the identity of the atom is kept the same but the chemical nature of the bond is altered, which is not possible in atomic systems, through the use of DNA and RNA bonding elements. These building blocks assemble into single crystal nanoparticle superlattices with mixed DNA and RNA bonding elements throughout. The nanoparticle crystals can be dynamically changed through the selective and enzymatic hydrolysis of the RNA bonding elements, resulting in superlattices that retain their crystalline structure and habit, while incorporating up to 35% random vacancies generated from the nanoparticles removed. Therefore, the bonding elements of nanoparticle crystals can be enzymatically and selectively addressed without affecting the nature of the atom.

  9. 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.

  10. Laccase immobilization on enzymatically functionalized polyamide 6,6 fibres

    OpenAIRE

    Silva, Carla Manuela Pereira Marinho da; Silva, Carla J. S. M.; Zille, Andrea; Gübitz, Georg M.; Paulo, Artur Cavaco

    2007-01-01

    Polyamide matrices, such as membranes, gels and non-wovens, have been applied as supports for enzyme immobilization, although in literature the enzyme immobilization on woven nylon matrices is rarely reported. In this work, a protocol for a Trametes hirsuta laccase immobilization using woven polyamide 6,6 (nylon) was developed. A 24 full factorial design was used to study the influence of pH, spacer (1,6-hexanediamine), enzyme and crosslinker concentration on the efficiency of immobilization....

  11. 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.

  12. Subcellular localization of Arabidopsis arogenate dehydratases suggests novel and non-enzymatic roles

    Science.gov (United States)

    Bross, Crystal D.; Howes, Travis R.; Abolhassani Rad, Sara; Kljakic, Ornela

    2017-01-01

    Abstract Arogenate dehydratases (ADTs) catalyze the final step in phenylalanine biosynthesis in plants. The Arabidopsis thaliana genome encodes a family of six ADTs capable of decarboxylating/dehydrating arogenate into phenylalanine. Using cyan fluorescent protein (CFP)-tagged proteins, the subcellular localization patterns of all six A. thaliana ADTs were investigated in intact Nicotiana benthamiana and A. thaliana leaf cells. We show that A. thaliana ADTs localize to stroma and stromules (stroma-filled tubules) of chloroplasts. This localization pattern is consistent with the enzymatic function of ADTs as many enzymes required for amino acid biosynthesis are primarily localized to chloroplasts, and stromules are thought to increase metabolite transport from chloroplasts to other cellular compartments. Furthermore, we provide evidence that ADTs have additional, non-enzymatic roles. ADT2 localizes in a ring around the equatorial plane of chloroplasts or to a chloroplast pole, which suggests that ADT2 is a component of the chloroplast division machinery. In addition to chloroplasts, ADT5 was also found in nuclei, again suggesting a non-enzymatic role for ADT5. We also show evidence that ADT5 is transported to the nucleus via stromules. We propose that ADT2 and ADT5 are moonlighting proteins that play an enzymatic role in phenylalanine biosynthesis and a second role in chloroplast division or transcriptional regulation, respectively. PMID:28338876

  13. Accessibility of Enzymatically Delignified Bambusa bambos for Efficient Hydrolysis at Minimum Cellulase Loading: An Optimization Study

    Directory of Open Access Journals (Sweden)

    Arindam Kuila

    2011-01-01

    Full Text Available In the present investigation, Bambusa bambos was used for optimization of enzymatic pretreatment and saccharification. Maximum enzymatic delignification achieved was 84%, after 8 h of incubation time. Highest reducing sugar yield from enzyme-pretreated Bambusa bambos was 818.01 mg/g dry substrate after 8 h of incubation time at a low cellulase loading (endoglucanase, β-glucosidase, exoglucanase, and xylanase were 1.63 IU/mL, 1.28 IU/mL, 0.08 IU/mL, and 47.93 IU/mL, respectively. Enzyme-treated substrate of Bambusa bambos was characterized by analytical techniques such as Fourier transformed infrared spectroscopy (FTIR, X-ray diffraction (XRD, and scanning electron microscopy (SEM. The FTIR spectrum showed that the absorption peaks of several functional groups were decreased after enzymatic pretreatment. XRD analysis indicated that cellulose crystallinity of enzyme-treated samples was increased due to the removal of amorphous lignin and hemicelluloses. SEM image showed that surface structure of Bambusa bambos was distorted after enzymatic pretreatment.

  14. 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

  15. Stochastic ensembles, conformationally adaptive teamwork, and enzymatic detoxification.

    Science.gov (United States)

    Atkins, William M; Qian, Hong

    2011-05-17

    It has been appreciated for a long time that enzymes exist as conformational ensembles throughout multiple stages of the reactions they catalyze, but there is renewed interest in the functional implications. The energy landscape that results from conformationlly diverse poteins is a complex surface with an energetic topography in multiple dimensions, even at the transition state(s) leading to product formation, and this represents a new paradigm. At the same time there has been renewed interest in conformational ensembles, a new paradigm concerning enzyme function has emerged, wherein catalytic promiscuity has clear biological advantages in some cases. "Useful", or biologically functional, promiscuity or the related behavior of "multifunctionality" can be found in the immune system, enzymatic detoxification, signal transduction, and the evolution of new function from an existing pool of folded protein scaffolds. Experimental evidence supports the widely held assumption that conformational heterogeneity promotes functional promiscuity. The common link between these coevolving paradigms is the inherent structural plasticity and conformational dynamics of proteins that, on one hand, lead to complex but evolutionarily selected energy landscapes and, on the other hand, promote functional promiscuity. Here we consider a logical extension of the overlap between these two nascent paradigms: functionally promiscuous and multifunctional enzymes such as detoxification enzymes are expected to have an ensemble landscape with more states accessible on multiple time scales than substrate specific enzymes. Two attributes of detoxification enzymes become important in the context of conformational ensembles: these enzymes metabolize multiple substrates, often in substrate mixtures, and they can form multiple products from a single substrate. These properties, combined with complex conformational landscapes, lead to the possibility of interesting time-dependent, or emergent

  16. Enzymatic Halogenation and Dehalogenation Reactions: Pervasive and Mechanistically Diverse.

    Science.gov (United States)

    Agarwal, Vinayak; Miles, Zachary D; Winter, Jaclyn M; Eustáquio, Alessandra S; El Gamal, Abrahim A; Moore, Bradley S

    2017-04-26

    Naturally produced halogenated compounds are ubiquitous across all domains of life where they perform a multitude of biological functions and adopt a diversity of chemical structures. Accordingly, a diverse collection of enzyme catalysts to install and remove halogens from organic scaffolds has evolved in nature. Accounting for the different chemical properties of the four halogen atoms (fluorine, chlorine, bromine, and iodine) and the diversity and chemical reactivity of their organic substrates, enzymes performing biosynthetic and degradative halogenation chemistry utilize numerous mechanistic strategies involving oxidation, reduction, and substitution. Biosynthetic halogenation reactions range from simple aromatic substitutions to stereoselective C-H functionalizations on remote carbon centers and can initiate the formation of simple to complex ring structures. Dehalogenating enzymes, on the other hand, are best known for removing halogen atoms from man-made organohalogens, yet also function naturally, albeit rarely, in metabolic pathways. This review details the scope and mechanism of nature's halogenation and dehalogenation enzymatic strategies, highlights gaps in our understanding, and posits where new advances in the field might arise in the near future.

  17. Integrated reactor concepts for the enzymatic kinetic synthesis of cephalexin

    NARCIS (Netherlands)

    Schroën, C.G.P.H.; Nierstrasz, V.A.; Bosma, R.; Kroon, P.J.; Tjeerdsma, P.S.; DeVroom, E.; VanderLaan, J.M.; Moody, H.M.; Beeftink, H.H.; Janssen, A.E.M.; Tramper, J.

    2002-01-01

    Integrated process concepts for enzymatic cephalexin synthesis were investigated by our group, and this article focuses on the integration of reactions and product removal during the reactions. The last step in cephalexin production is the enzymatic kinetic coupling of activated phenylglycine

  18. Enzymatic browning and its control in fresh-cut produce

    Science.gov (United States)

    Enzymatic browning of damaged tissues of fruits and vegetables during postharvest handling and processing degrades the sensory properties and nutritional value and discourages the consumer purchase of fresh-cut products. Consequently, enzymatic browning results in significant economic losses for the...

  19. Phyto-agglutinin, total proteins and amino assimilating enzymatic ...

    African Journals Online (AJOL)

    ... the amino assimilating (glutamine synthetase) enzymatic activity. Both of KK-1 and Hassan-2K cultivars extract showed highly phyto-agglutination of human erythrocytes with reproductive organs and other tissues, which represents the presence of potent lectins (phyto-agglutinin). The amino assimilating enzymatic activity ...

  20. Soil bacterial flora and enzymatic activities in zinc and lead ...

    African Journals Online (AJOL)

    JTEkanem

    Abstract. Soil bacterial flora and enzymatic activities in lead and zinc contaminated soil of Ishiagu, ... concentration. This showed that the higher the heavy metal concentration the lower the enzymatic activities. Urease, dehydrogenase activity, hydrogen peroxidase and polyphenol ..... high concentrations and even inhibit the.

  1. Colors as catalysts in enzymatic reactions.

    Science.gov (United States)

    Azeemi, Samina T Yousuf; Raza, Syed Mohsin; Yasinzai, Masoom

    2008-12-01

    We studied the effects of visible range irradiation (in vitro) on the enzyme solutions (glucose oxidase, cholesterol oxidase + cholesterol esterase and lipase) in order to infer the changes produced in the human body after chromotherapy. The glucose oxidase showed enhanced activity to the color purple (464 nm), while the activity of the other enzymes, cholesterol esterase + cholesterol oxidase and lipase, increased when exposed to dark violet (400 nm). Purple is being used in conventional chromotherapy for diabetes, as supported by the experimental observation in which purple enhanced the activity of enzymes responsible for the oxidation of glucose. Specific wavelengths regulate living processes by acting as catalysts in enzyme activity, while some wavelengths may reduce enzyme activity. The irradiation of specific wavelengths effect enzymatic processes, which as a consequence, accelerated biochemical reactions. This particular frequency when provided to the enzymes (in vitro) lead to changes which may well be occurring in vivo.

  2. Isothermal calorimetry on enzymatic biodiesel production

    DEFF Research Database (Denmark)

    Fjerbæk, Lene

    2008-01-01

    information about effects taking place when using lipases immobilized on an inert carrier for transesterification of a triglyceride and an alcohol as for biodiesel production. The biodiesel is produced by rapeseed oil and methanol as well as ethanol and a commercial biocatalyst Novozym 435 from Novozymes...... containing a Candida Antarctica B lipase immobilized on an acrylic resin. The reaction investigated is characterized by immiscible liquids (oil, methanol, glycerol and biodiesel) and enzymes imm. on an inert carrier during reaction, which allows several effects to take place that during normal reaction...... conditions can not be elucidated. These effects have been observed with isothermal calorimetry bringing forth new information about the reaction of enzymes catalyzing transesterification. Enzymatic biodiesel production has until now not been investigated with isothermal microcalorimetry, but the results...

  3. Structure of the enzymatically synthesized fructan inulin

    Energy Technology Data Exchange (ETDEWEB)

    Heyer, A.G.; Schroeer, B. [Max-Planck-Institut fuer Molekulare Pflanzenphysiologie, Karl-Liebknecht-Str. 25, 14476 Golm (Germany); Radosta, S. [Fraunhofer-Institut fuer Angewandte Polymerforschung, Postfach 126, 14504 Teltow (Germany); Wolff, D.; Czapla, S.; Springer, J. [Technische Universitaet Berlin, FG Makromolekulare Chemie, Str. des 17. Juni 135, 10623 Berlin (Germany)

    1998-12-15

    Construction, purification and characterization of a fusion protein of maltose-binding protein of Escherichia coli and the fructosyltransferase of Streptococcus mutans is described. With the purified protein, in vitro synthesis of inulin was performed. The obtained polysaccharide was characterized by high-performance size-exclusion chromatography (HPSEC) and static light scattering (SLS) in dilute aqueous and dimethyl sulfoxide solution. For all samples very high molecular weights between 60x10{sup 6} and 90x10{sup 6} g/mol and a remarkable small polydispersity index of 1.1 have been determined. Small root-mean-square radii of gyration point to a compact conformation in dilute solution. No difference between native and enzymatically synthesized inulin was observed by X-ray powder diffraction and thermoanalysis of solid samples. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

  4. Additives enhancing enzymatic hydrolysis of lignocellulosic biomass.

    Science.gov (United States)

    Rocha-Martín, Javier; Martinez-Bernal, Claudio; Pérez-Cobas, Yolanda; Reyes-Sosa, Francisco Manuel; García, Bruno Díez

    2017-11-01

    Linked to the development of cellulolytic enzyme cocktails from Myceliophthora thermophila, we studied the effect of different additives on the enzymatic hydrolysis yield. The hydrolysis of pretreated corn stover (PCS), sugar cane straw (PSCS) and microcrystalline cellulose (Avicel) was performed under industrial conditions using high solid loadings, limited mixing, and low enzyme dosages. The addition of polyethylene glycol (PEG4000) allowed to increase the glucose yields by 10%, 7.5%, and 32%, respectively in the three materials. PEG4000 did not have significant effect on the stability of the main individual enzymes but increased beta-glucosidase and endoglucanase activity by 20% and 60% respectively. Moreover, the presence of PEG4000 accelerated cellulase-catalyzed hydrolysis reducing up to 25% the liquefaction time. However, a preliminary economical assessment concludes that even with these improvements, a lower contribution of PEG4000 to the 2G bioethanol production costs would be needed to reach commercial feasibility. Copyright © 2017. Published by Elsevier Ltd.

  5. Analog noise reduction in enzymatic logic gates.

    Science.gov (United States)

    Melnikov, Dmitriy; Strack, Guinevere; Pita, Marcos; Privman, Vladimir; Katz, Evgeny

    2009-07-30

    In this work, we demonstrate both experimentally and theoretically that the analog noise generation by a single enzymatic logic gate can be dramatically reduced to yield gate operation with virtually no input noise amplification. We demonstrate that when a cosubstrate with a much smaller affinity than the primary substrate is used, a negligible increase in the noise output from the logic gate is obtained, as compared to the input noise level. Our general theoretical conclusions were confirmed by experimental realizations of the AND logic gate based on the enzyme horseradish peroxidase using hydrogen peroxide as the substrate, with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) or ferrocyanide as cosubstrates with vastly different rate constants.

  6. Enzymatic assay for methotrexate in erythrocytes

    DEFF Research Database (Denmark)

    Schrøder, H; Heinsvig, E M

    1985-01-01

    Methotrexate (MTX) accumulates in erythrocytes in MTX-treated patients. We present a modified enzymatic assay measuring MTX concentrations between 10 and 60 nmol/l in erythrocytes, adapted for a centrifugal analyser (Cobas Bio). About 40 patient's samples could be analysed within 1 h. The detection...... limit was 3 nmol/l. Within run and between-run precision was 7.4% and 13.5% for control 10 nmol/l and 1.2% and 3.2% for control 50 nmol/l. Recovery was 85-115% of MTX added to haemolysed erythrocytes. We found the method useful for pharmacokinetic studies of MTX in erythrocytes in MTX-treated patients...

  7. Enzyme orientation for direct electron transfer in an enzymatic fuel cell with alcohol oxidase and laccase electrodes.

    Science.gov (United States)

    Arrocha, Andrés A; Cano-Castillo, Ulises; Aguila, Sergio A; Vazquez-Duhalt, Rafael

    2014-11-15

    A new full enzymatic fuel cell was built and characterized. Both enzymatic electrodes were molecularly oriented to enhance the direct electron transfer between the enzyme active site and the electrode surface. The anode consisted in immobilized alcohol oxidase on functionalized carbon nanotubes with 4-azidoaniline, which acts as active-site ligand to orientate the enzyme molecule. The cathode consisted of immobilized laccase on functionalized graphite electrode with 4-(2-aminoethyl) benzoic acid. The enzymatic fuel cell reaches 0.5 V at open circuit voltage with both, ethanol and methanol, while in short circuit the highest current intensity of 250 μA cm(-2) was obtained with methanol. Concerning the power density, the methanol was the best substrate reaching 60 μW cm(-2), while with ethanol 40 μW cm(-2) was obtained. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Dual modification of starch via partial enzymatic hydrolysis in the granular state and subsequent hydroxypropylation.

    Science.gov (United States)

    Karim, A A; Sufha, E H; Zaidul, I S M

    2008-11-26

    The effect of enzymatic pretreatment on the degree of corn and mung bean starch derivatization by propylene oxide was investigated. The starch was enzymatically treated in the granular state with a mixture of fungal alpha-amylase and glucoamylase at 35 degrees C for 16 h and then chemically modified to produce enzyme-hydrolyzed-hydroxypropyl (HP) starch. Partial enzyme hydrolysis of starch in the granular state appeared to enhance the subsequent hydroxypropylation, as judged from the significant increase in the molar substitution. A variable degree of granule modification was obtained after enzyme hydrolysis, and one of the determinants of the modification degree appeared to be the presence of natural pores in the granules. Enzyme-hydrolyzed-HP starch exhibited significantly different functional properties compared to hydroxypropyl starch prepared from untreated (native) starch. It is evident that the dual modification of starch using this approach provides a range of functional properties that can be customized for specific applications.

  9. Synthesis of monoacylglycerols by enzymatic methods

    Directory of Open Access Journals (Sweden)

    Bradić Milena R.

    2010-01-01

    Full Text Available Monoacylglycerols are non-ionic surfactants widely used in the food industry. They are also important in cosmetic and pharmaceutical industries as drug carriers and for the consistency improvements in creams and lotions. Current process for their production is based on the glycerolysis of natural fats and oils in the presence of inorganic catalysts at temperatures higher than 220 oC. The major drawbacks of this process include high-energy consumption, low yield, and poor product quality. The use of lipases for the monoacylglycerols production offers environmental advantages and a reduction in energy consumption. Besides, the same surfactants prepared by the enzymatic synthesis may be labeled as “natural”. Recent progress in the application of highly-stable lipases in the organic solvents offers the possibility of employing various methods to the enzyme-catalyzed synthesis of monoacylglycerols, such as selective hydrolysis of fats and oils using 1,3-regiospecific lipases, the esterification of glycerol with fatty acids and the glycerolysis of fats or oils. In this review, different reaction systems such as aqueous-organic two-phase systems, microemulsions and reverse micelles systems, anhydrous organic solvents, solvent-free systems with free or immobilized lipases, as well as the use of two-phase membrane reactor systems are presented. We discuss some of the key factors, such as the control of water content, removing of the products from reaction system, and the effects of solvent on the lipase activity and selectivity, that must be addressed in order to obtain an efficient reaction system with high yields of monoacylglycerols. Engineering of the enzymatic monoacylglycerols synthesis processes requires also optimization of other factors as: molar ratio of substrates, temperature, type of lipase immobilization and supports (if any, reactor design and operating regime.

  10. The Enzymatic Oxidation of Graphene Oxide

    Science.gov (United States)

    Kotchey, Gregg P.; Allen, Brett L.; Vedala, Harindra; Yanamala, Naveena; Kapralov, Alexander A.; Tyurina, Yulia Y.; Klein-Seetharaman, Judith; Kagan, Valerian E.; Star, Alexander

    2011-01-01

    Two-dimensional graphitic carbon is a new material with many emerging applications, and studying its chemical properties is an important goal. Here, we reported a new phenomenon – the enzymatic oxidation of a single layer of graphitic carbon by horseradish peroxidase (HRP). In the presence of low concentrations of hydrogen peroxide (~40 µM), HRP catalyzed the oxidation of graphene oxide, which resulted in the formation of holes on its basal plane. During the same period of analysis, HRP failed to oxidize chemically reduced graphene oxide (RGO). The enzymatic oxidation was characterized by Raman, UV-Vis, EPR and FT-IR spectroscopy, TEM, AFM, SDS-PAGE, and GC-MS. Computational docking studies indicated that HRP was preferentially bound to the basal plane rather than the edge for both graphene oxide and RGO. Due to the more dynamic nature of HRP on graphene oxide, the heme active site of HRP was in closer proximity to graphene oxide compared to RGO, thereby facilitating the oxidation of the basal plane of graphene oxide. We also studied the electronic properties of the reduced intermediate product, holey reduced graphene oxide (hRGO), using field-effect transistor (FET) measurements. While RGO exhibited a V-shaped transfer characteristic similar to a single layer of graphene that was attributed to its zero band gap, hRGO demonstrated a p-type semiconducting behavior with a positive shift in the Dirac points. This p-type behavior rendered hRGO, which can be conceptualized as interconnected graphene nanoribbons, as a potentially attractive material for FET sensors. PMID:21344859

  11. The enzymatic oxidation of graphene oxide.

    Science.gov (United States)

    Kotchey, Gregg P; Allen, Brett L; Vedala, Harindra; Yanamala, Naveena; Kapralov, Alexander A; Tyurina, Yulia Y; Klein-Seetharaman, Judith; Kagan, Valerian E; Star, Alexander

    2011-03-22

    Two-dimensional graphitic carbon is a new material with many emerging applications, and studying its chemical properties is an important goal. Here, we reported a new phenomenon--the enzymatic oxidation of a single layer of graphitic carbon by horseradish peroxidase (HRP). In the presence of low concentrations of hydrogen peroxide (∼40 μM), HRP catalyzed the oxidation of graphene oxide, which resulted in the formation of holes on its basal plane. During the same period of analysis, HRP failed to oxidize chemically reduced graphene oxide (RGO). The enzymatic oxidation was characterized by Raman, ultraviolet-visible, electron paramagnetic resonance, Fourier transform infrared spectroscopy, transmission electron microscopy, atomic force microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gas chromatography-mass spectrometry. Computational docking studies indicated that HRP was preferentially bound to the basal plane rather than the edge for both graphene oxide and RGO. Owing to the more dynamic nature of HRP on graphene oxide, the heme active site of HRP was in closer proximity to graphene oxide compared to RGO, thereby facilitating the oxidation of the basal plane of graphene oxide. We also studied the electronic properties of the reduced intermediate product, holey reduced graphene oxide (hRGO), using field-effect transistor (FET) measurements. While RGO exhibited a V-shaped transfer characteristic similar to a single layer of graphene that was attributed to its zero band gap, hRGO demonstrated a p-type semiconducting behavior with a positive shift in the Dirac points. This p-type behavior rendered hRGO, which can be conceptualized as interconnected graphene nanoribbons, as a potentially attractive material for FET sensors.

  12. Enzymatic Hydrolysis of Hydrotropic Pulps at Different Substrate Loadings.

    Science.gov (United States)

    Denisova, Marina N; Makarova, Ekaterina I; Pavlov, Igor N; Budaeva, Vera V; Sakovich, Gennady V

    2016-03-01

    Enzymatic hydrolysis of cellulosic raw materials to produce nutrient broths for microbiological synthesis of ethanol and other valuable products is an important field of modern biotechnology. Biotechnological processing implies the selection of an effective pretreatment technique for raw materials. In this study, the hydrotropic treatment increased the reactivity of the obtained substrates toward enzymatic hydrolysis by 7.1 times for Miscanthus and by 7.3 times for oat hulls. The hydrotropic pulp from oat hulls was more reactive toward enzymatic hydrolysis compared to that from Miscanthus, despite that the substrates had similar compositions. As the initial substrate loadings were raised during enzymatic hydrolysis of the hydrotropic Miscanthus and oat hull pulps, the concentration of reducing sugars increased by 34 g/dm(3) and the yield of reducing sugars decreased by 31 %. The findings allow us to predict the efficiency of enzymatic hydrolysis of hydrotropic pulps from Miscanthus and oat hulls when scaling up the process by volume.

  13. EFFECT OF ENZYMATIC TREATMENTS ON CARBOHYDRATE MATRICES TOWARDS HEALTHY GLUTEN FREE FOODS APPLICATION

    OpenAIRE

    DURÁ DE MIGUEL, ÁNGELA

    2017-01-01

    Starch is the major energy reserve in plants, extensively present in many food and non-food applications, and is one of the most abundant carbohydrates in human diet. In addition to starch native form currently used as a raw material for industrial applications, modified starches have become very attractive to develop numerous products that have greatly expanded starch use and utility. Enzymatic modifications are carried out to enhance starch functionality with the aim of overcoming technolog...

  14. SELECTION THE SOURCE OF FUCOIDAN AND OPTIMIZATION OF ITS ENZYMATIC HYDROLYSIS

    Directory of Open Access Journals (Sweden)

    Y. S. Novikova

    2015-01-01

    Full Text Available The development of biotechnology fucose and fucooligosaccharides from vegetable raw materials is of particular interest, due to the wide range of biological activity fucose. The fucose is an important carbohydrate component of immunoglobulins and performs important functions in the biological processes of ontogenesis, cell differentiation as well as in the reproductive processes in vertebrates and in the formation of immunity. The fucose and its polymers have prebiotic effects, fucooligosaccharides have also exhibit antioxidant activity. One of the promising methods of obtaining fucose and fucooligosaccharides is an enzymatic hydrolysis of fucoidan vegetable raw materials. The most accessible and inexpensive plant raw materials to extract fucoidan are the brown algae. Literary analysis showed that the algae Fucus vesiculosis are characterized by the highest content of fucoidan, the chemical structure of which is a homopolymer of fucose with a predominance of 1,3 glycoside bonds. It was investigated the hydrolysis of fucoidan algae Fucus vesiculosis by immobilized enzyme preparation α-L-fucosidase for determine the optimal conditions of the enzymatic process. The efficiency of enzymatic degradation of fucoidan depends on several factors, the most important of which are the amount of the enzyme preparation, the pH of the reaction medium and the temperature at which the hydrolysis takes place, as well as its duration. Were determined the optimal process parameters of the enzymatic hydrolysis of fucoidan algal Fucus vesiculosis: the amount of the enzyme preparation of 6 U / g, the temperature 50 ° C, pH 7.0, the hydrolysis time 5 hours. Under these conditions, the degree of hydrolysis of fucoidan was 83-85%, which shows prospects of the enzymatic hydrolysis for obtaining of fucose from vegetable raw materials.

  15. Association genetics in Solanum tuberosum provides new insights into potato tuber bruising and enzymatic tissue discoloration

    Directory of Open Access Journals (Sweden)

    Tacke Eckhard

    2011-01-01

    Full Text Available Abstract Background Most agronomic plant traits result from complex molecular networks involving multiple genes and from environmental factors. One such trait is the enzymatic discoloration of fruit and tuber tissues initiated by mechanical impact (bruising. Tuber susceptibility to bruising is a complex trait of the cultivated potato (Solanum tuberosum that is crucial for crop quality. As phenotypic evaluation of bruising is cumbersome, the application of diagnostic molecular markers would empower the selection of low bruising potato varieties. The genetic factors and molecular networks underlying enzymatic tissue discoloration are sparsely known. Hitherto there is no association study dealing with tuber bruising and diagnostic markers for enzymatic discoloration are rare. Results The natural genetic diversity for bruising susceptibility was evaluated in elite middle European potato germplasm in order to elucidate its molecular basis. Association genetics using a candidate gene approach identified allelic variants in genes that function in tuber bruising and enzymatic browning. Two hundred and five tetraploid potato varieties and breeding clones related by descent were evaluated for two years in six environments for tuber bruising susceptibility, specific gravity, yield, shape and plant maturity. Correlations were found between different traits. In total 362 polymorphic DNA fragments, derived from 33 candidate genes and 29 SSR loci, were scored in the population and tested for association with the traits using a mixed model approach, which takes into account population structure and kinship. Twenty one highly significant (p Conclusions The observed trait correlations and associated marker fragments provide new insight in the molecular basis of bruising susceptibility and its natural variation. The markers diagnostic for increased or decreased bruising susceptibility will facilitate the combination of superior alleles in breeding programs. In

  16. Prevention of enzymatic browning of yacon flour by the combined use of anti-browning agents and the study of its chemical composition

    OpenAIRE

    Oscar Romero Lopes Rodrigues; Eduardo Ramirez Asquieri; Daniela Castilho Orsi

    2014-01-01

    Yacon roots present functional properties because of the high levels of fructooligosaccharides (FOS), which are considered as prebiotic fibers. In addition, yacon roots are rich in phenolic compounds. During the processing of yacon, the freshly cut surface undergoes rapid enzymatic browning. Control of enzymatic browning during processing is very important to preserve the appearance of yacon flour. In this study, it was evaluated the combined effect of anti-browning agents (ascorbic acid, cit...

  17. [Role of CO-binding cytochrome c in enzymatic oxidation of methane by the bacterium Methylococcus capsulatus].

    Science.gov (United States)

    Gvozdev, R I; Nikonova, E L; Piliashenko-Novokhatnyi, A I; Shushenacheva, E V; Grigorian, A N

    1982-07-01

    The cytochrome c spectrally related to cco cytochromes has been isolated and purified from the methane-oxidizing bacterium Methylococcus capsulatus. The cytochrome binds CO but does not bind other substrates of methane monooxygenase, does not activate the methane monooxygenase reaction and is not a component of methane monooxygenase. In the methanol dehydrogenase enzymatic system cytochrome cco functions as electron acceptor. A possible role of cytochrome cco as electron carrier intermediate in the sequence of the dehydrogenase and oxidase enzymatic systems of M. capsulatus is discussed.

  18. Hybrid model for an enzymatic reactor: hydrolysis of cheese whey proteins by alcalase immobilized in agarose gel particles.

    Science.gov (United States)

    Sousa, Ruy; Resende, Mariam M; Giordano, Raquel L C; Giordano, Roberto C

    2003-01-01

    Cheese whey proteolysis, carried out by immobilized enzymes, can either change or evidence functional properties of the produced peptides, increasing the potential applications of this byproduct of the dairy industry. Optimization and scale-up of the enzymatic reactor relies on its mathematical model-a set of mass balance equations, with reaction rates usually given by Michaelis-Menten-like kinetics; no information about the distribution of peptides' molecular sizes is supplied. In this article, a hybrid model of a batch enzymatic reactor is presented, consisting of differential mass balances coupled to a "neural-kinetic model," which provides the molecular weight distributions of the resulting peptides.

  19. Genetic Screening Identifies Cyanogenesis-Deficient Mutants of Lotus japonicus and Reveals Enzymatic Specificity in Hydroxynitrile Glucoside Metabolism

    DEFF Research Database (Denmark)

    Takos, A.; Lai, D.; Mikkelsen, L.

    2010-01-01

    Cyanogenesis, the release of hydrogen cyanide from damaged plant tissues, involves the enzymatic degradation of amino acid-derived cyanogenic glucosides (alpha-hydroxynitrile glucosides) by specific beta-glucosidases. Release of cyanide functions as a defense mechanism against generalist herbivores...

  20. Enzymatic isolation of viable human odontoblasts.

    Science.gov (United States)

    Cuffaro, H M; Pääkkönen, V; Tjäderhane, L

    2016-05-01

    To improve an enzymatic method previously used for isolation of rat odontoblasts to isolate viable mature human odontoblasts. Collagenase I, collagenase I/hyaluronidase mixture and hyaluronidase were used to extract mature human odontoblasts from the pulp chamber. Detachment of odontoblasts from dentine was determined with field emission scanning electron microscopy (FESEM) and to analyse the significance of differences in tubular diameter, and the t-test was used. MTT-reaction was used to analyse cell viability, and nonparametric Kruskal-Wallis and Mann-Whitney post hoc tests were used to analyse the data. Immunofluorescent staining of dentine sialoprotein (DSP), aquaporin-4 (AQP4) and matrix metalloproteinase-20 (MMP-20) and quantitative PCR (qPCR) of dentine sialophosphoprotein (DSPP) were used to confirm the odontoblastic nature of the cells. MTT-reaction and FESEM demonstrated collagenase I/hyaluronidase resulted in more effective detachment and higher viability than collagenase I alone. Hyaluronidase alone was not able to detach odontoblasts. Immunofluorescence revealed the typical odontoblastic-morphology with one process, and DSP, AQP4 and MMP-20 were detected. Quantitative PCR of DSPP confirmed that the isolated cells expressed this odontoblast-specific gene. The isolation of viable human odontoblasts was successful. The cells demonstrated morphology typical for odontoblasts and expressed characteristic odontoblast-type genes and proteins. This method will enable new approaches, such as apoptosis analysis, for studies using fully differentiated odontoblasts. © 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  1. Enzymatic conversion of sucrose to hydrogen

    Energy Technology Data Exchange (ETDEWEB)

    Woodward, J.; Orr, M. [Oak Ridge National Lab., TN (United States). Chemical Technology Div.

    1998-11-01

    The enzymatic conversion of sugars to hydrogen could be a promising method for alternative fuel production. Maple tree sap is a source of environmental sugar (e.g., sucrose) that has the potential to be converted into hydrogen using the enzymes invertase, glucose dehydrogenase (GDH), hydrogenase, and glucose isomerase (GI) and the cofactor NADP{sup +}/NADPH. The kinetics of hydrogen production have been studied, and optimal conditions for hydrogen production are described. At low initial sucrose concentrations, in the absence of glucose isomerase, stoichiometric yields of mol of H{sub 2}/mol of sucrose were achieved. At higher sucrose concentrations, the yield of hydrogen declined so that at an initial sucrose concentration of 292 mM only 7% yield of hydrogen was obtained. The reason for this low yield was studied and shown not to be caused by enzyme inactivation or a pH drop during the reaction but due to an instability of the cofactor NADP{sup +}. Although gluconic and inhibited both NADPH production and oxidation of GDH and hydrogenase, respectively, it was not the major cause of NADP{sup +} instability. Fructose was also shown to be converted to hydrogen if GI was present in the reaction mixture. Also, by starting with sucrose, 1.34 mol of H{sub 2}/mol of sucrose was obtained if GI was present in the reaction mixture.

  2. Enzymatic descemetic lamellar keratoplasty: pilot study.

    Science.gov (United States)

    Bonci, Paolo; Bonci, Paola; Della Valle, Vincenzo; Fanini, Francesca; Nardi-Pantoli, Angela

    2010-01-01

    This was a pilot study to evaluate the efficacy of a new surgical technique of descemetic anterior lamellar keratoplasty (DALK) with the help of enzymatic hyaluronidase solution. We selected 10 patients, 6 male and 4 female, with surgical keratoconus, with a mean age of 45+/-16 years (range 29-61), with best-corrected visual acuity (BCVA) Keratron Scout, Optikon 2000, Rome, Italy). The parameters were evaluated monthly, with a maximum follow-up of 8 months. There were no perforations and minimal complications (second anterior chamber in 4 eyes) on the first day, which spontaneously resolved after 7 days. Two patients required resuturing because of loose suture on the second postoperative day. The visual recovery was comparable to that obtained by most authors after DALK. The final mean BCVA was 20/25. There was a drop in endothelial cells of approximately 10.1% after 7 months. The mean central astigmatism at the end of the follow-up was 2 diopters (+/-0.88). The enzyme application to the recipient cornea makes the stromal dissection easier, but other studies are needed about type and concentration of the enzyme, and the application times.

  3. Enzymatic hydrolysis of biomass from wood.

    Science.gov (United States)

    Álvarez, Consolación; Reyes-Sosa, Francisco Manuel; Díez, Bruno

    2016-03-01

    Current research and development in cellulosic ethanol production has been focused mainly on agricultural residues and dedicated energy crops such as corn stover and switchgrass; however, woody biomass remains a very important feedstock for ethanol production. The precise composition of hemicellulose in the wood is strongly dependent on the plant species, therefore different types of enzymes are needed based on hemicellulose complexity and type of pretreatment. In general, hardwood species have much lower recalcitrance to enzymes than softwood. For hardwood, xylanases, beta-xylosidases and xyloglucanases are the main hemicellulases involved in degradation of the hemicellulose backbone, while for softwood the effect of mannanases and beta-mannosidases is more relevant. Furthermore, there are different key accessory enzymes involved in removing the hemicellulosic fraction and increasing accessibility of cellulases to the cellulose fibres improving the hydrolysis process. A diversity of enzymatic cocktails has been tested using from low to high densities of biomass (2-20% total solids) and a broad range of results has been obtained. The performance of recently developed commercial cocktails on hardwoods and softwoods will enable a further step for the commercialization of fuel ethanol from wood. © 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  4. Method for the enzymatic production of hydrogen

    Science.gov (United States)

    Woodward, J.; Mattingly, S.M.

    1999-08-24

    The present invention is an enzymatic method for producing hydrogen comprising the steps of: (a) forming a reaction mixture within a reaction vessel comprising a substrate capable of undergoing oxidation within a catabolic reaction, such as glucose, galactose, xylose, mannose, sucrose, lactose, cellulose, xylan and starch; the reaction mixture also comprising an amount of glucose dehydrogenase in an amount sufficient to catalyze the oxidation of the substrate, an amount of hydrogenase sufficient to catalyze an electron-requiring reaction wherein a stoichiometric yield of hydrogen is produced, an amount of pH buffer in an amount sufficient to provide an environment that allows the hydrogenase and the glucose dehydrogenase to retain sufficient activity for the production of hydrogen to occur and also comprising an amount of nicotinamide adenine dinucleotide phosphate sufficient to transfer electrons from the catabolic reaction to the electron-requiring reaction; (b) heating the reaction mixture at a temperature sufficient for glucose dehydrogenase and the hydrogenase to retain sufficient activity and sufficient for the production of hydrogen to occur, and heating for a period of time that continues until the hydrogen is no longer produced by the reaction mixture, wherein the catabolic reaction and the electron-requiring reactions have rates of reaction dependent upon the temperature; and (c) detecting the hydrogen produced from the reaction mixture. 8 figs.

  5. Enzymatic Purification of Microplastics in Environmental Samples.

    Science.gov (United States)

    Löder, Martin G J; Imhof, Hannes K; Ladehoff, Maike; Löschel, Lena A; Lorenz, Claudia; Mintenig, Svenja; Piehl, Sarah; Primpke, Sebastian; Schrank, Isabella; Laforsch, Christian; Gerdts, Gunnar

    2017-12-19

    Micro-Fourier transform infrared (micro-FTIR) spectroscopy and Raman spectroscopy enable the reliable identification and quantification of microplastics (MPs) in the lower micron range. Since concentrations of MPs in the environment are usually low, the large sample volumes required for these techniques lead to an excess of coenriched organic or inorganic materials. While inorganic materials can be separated from MPs using density separation, the organic fraction impedes the ability to conduct reliable analyses. Hence, the purification of MPs from organic materials is crucial prior to conducting an identification via spectroscopic techniques. Strong acidic or alkaline treatments bear the danger of degrading sensitive synthetic polymers. We suggest an alternative method, which uses a series of technical grade enzymes for purifying MPs in environmental samples. A basic enzymatic purification protocol (BEPP) proved to be efficient while reducing 98.3 ± 0.1% of the sample matrix in surface water samples. After showing a high recovery rate (84.5 ± 3.3%), the BEPP was successfully applied to environmental samples from the North Sea where numbers of MPs range from 0.05 to 4.42 items m-3. Experiences with different environmental sample matrices were considered in an improved and universally applicable version of the BEPP, which is suitable for focal plane array detector (FPA)-based micro-FTIR analyses of water, wastewater, sediment, biota, and food samples.

  6. Aqueous enzymatic extraction of Moringa oleifera oil.

    Science.gov (United States)

    Mat Yusoff, Masni; Gordon, Michael H; Ezeh, Onyinye; Niranjan, Keshavan

    2016-11-15

    This paper reports on the extraction of Moringa oleifera (MO) oil by using aqueous enzymatic extraction (AEE) method. The effect of different process parameters on the oil recovery was discovered by using statistical optimization, besides the effect of selected parameters on the formation of its oil-in-water cream emulsions. Within the pre-determined ranges, the use of pH 4.5, moisture/kernel ratio of 8:1 (w/w), and 300stroke/min shaking speed at 40°C for 1h incubation time resulted in highest oil recovery of approximately 70% (goil/g solvent-extracted oil). These optimized parameters also result in a very thin emulsion layer, indicating minute amount of emulsion formed. Zero oil recovery with thick emulsion were observed when the used aqueous phase was re-utilized for another AEE process. The findings suggest that the critical selection of AEE parameters is key to high oil recovery with minimum emulsion formation thereby lowering the load on the de-emulsification step. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Enzymatic Systems for Cellulose Acetate Degradation

    Directory of Open Access Journals (Sweden)

    Oskar Haske-Cornelius

    2017-09-01

    Full Text Available Cellulose acetate (CA-based materials, like cigarette filters, contribute to landscape pollution challenging municipal authorities and manufacturers. This study investigates the potential of enzymes to degrade CA and to be potentially incorporated into the respective materials, enhancing biodegradation. Deacetylation studies based on Liquid Chromatography-Mass Spectrometry-Time of Flight (LC-MS-TOF, High Performance Liquid Chromatography (HPLC, and spectrophotometric analysis showed that the tested esterases were able to deacetylate the plasticizer triacetin (glycerol triacetate and glucose pentaacetate (cellulose acetate model compound. The most effective esterases for deacetylation belong to the enzyme family 2 (AXE55, AXE 53, GAE, they deacetylated CA with a degree of acetylation of up to 1.8. A combination of esterases and cellulases showed synergistic effects, the absolute glucose recovery for CA 1.8 was increased from 15% to 28% when an enzymatic deacetylation was performed. Lytic polysaccharide monooxygenase (LPMO, and cellobiohydrolase were able to cleave cellulose acetates with a degree of acetylation of up to 1.4, whereas chitinase showed no activity. In general, the degree of substitution, chain length, and acetyl group distribution were found to affect CA degradation. This study shows that, for a successful enzyme-based deacetylation system, a cocktail of enzymes, which will randomly cleave and generate shorter CA fragments, is the most suitable.

  8. Total enzymatic synthesis of cholecystokinin CCK-5.

    Science.gov (United States)

    Xiang, H; Xiang, G Y; Lu, Z M; Guo, L; Eckstein, H

    2004-08-01

    This paper describes the enzymatic synthesis of the C-terminal fragment H-Gly-Trp-Met-Asp-Phe-NH2 of cholecystokinin. Immobilized enzymes were used for the formation of all peptide bonds except thermolysin. Beginning the synthesis with phenylacetyl (PhAc) glycine carboxamidomethyl ester (OCam) and H-Trp-OMe by using immobilized papain as biocatalyst in buffered ethyl acetate, the dipeptide methyl ester was then coupled directly with Met-OEt.HCl by alpha-chymotrypsin/Celite 545 in a solvent free system. For the 3+2 coupling PhAc-Gly-Trp-Met-OEt had to be converted into its OCam ester. The other fragment H-Asp(OMe)-Phe-NH2 resulted from the coupling of Cbo-Asp(OMe)-OH with H-Phe-NH2.HCl and thermolysin as catalyst, followed by catalytic hydrogenation. Finally PhAc-Gly-Trp-Met-Asp-Phe-NH2 was obtained in a smooth reaction from PhAc-Gly-Trp-Met-OCam and H-Asp(OMe)-Phe-NH2 with alpha-chymotrypsin/Celite 545 in acetonitrile, followed by basic hydrolysis of the beta-methyl ester. The PhAc-group is removed with penicillin G amidase and CCK-5 is obtained in an overall isolated yield of 19.6%.

  9. O2 Reduction in Enzymatic Biofuel Cells.

    Science.gov (United States)

    Mano, Nicolas; de Poulpiquet, Anne

    2017-09-20

    Catalytic four-electron reduction of O2 to water is one of the most extensively studied electrochemical reactions due to O2 exceptional availability and high O2/H2O redox potential, which may in particular allow highly energetic reactions in fuel cells. To circumvent the use of expensive and inefficient Pt catalysts, multicopper oxidases (MCOs) have been envisioned because they provide efficient O2 reduction with almost no overpotential. MCOs have been used to elaborate enzymatic biofuel cells (EBFCs), a subclass of fuel cells in which enzymes replace the conventional catalysts. A glucose/O2 EBFC, with a glucose oxidizing anode and a O2 reducing MCO cathode, could become the in vivo source of electricity that would power sometimes in the future integrated medical devices. This review covers the challenges and advances in the electrochemistry of MCOs and their use in EBFCs with a particular emphasis on the last 6 years. First basic features of MCOs and EBFCs are presented. Clues provided by electrochemistry to understand these enzymes and how they behave once connected at electrodes are described. Progresses realized in the development of efficient biocathodes for O2 reduction relying both on direct and mediated electron transfer mechanism are then discussed. Some implementations in EBFCs are finally presented.

  10. The non-enzymatic reduction of azo dyes by flavin and nicotinamide cofactors under varying conditions.

    Science.gov (United States)

    Morrison, Jessica M; John, Gilbert H

    2013-10-01

    Azo dyes are ubiquitous in products and often become environmental pollutants due to their anthropogenic nature. Azoreductases are enzymes which are present within many bacteria and are capable of breaking down the azo dyes via reduction of the azo bond. Often, though, carcinogenic aromatic amines are formed as metabolites and are of concern to humans. Azoreductases function via an oxidation-reduction reaction and require cofactors (a nicotinamide cofactor and sometimes a flavin cofactor) to perform their function. Non-enzymatic reduction of azo dyes in the absence of an azoreductase enzyme has been suggested in previous studies, but has never been studied in detail in terms of varying cofactor combinations, different oxygen states or pHs, nor has the enzymatic reduction been compared to azoreduction in terms of dye reduction or metabolites produced, which was the aim of this study. Reduction of azo dyes by different cofactor combinations was found to occur under both aerobic and anaerobic conditions and under physiologically-relevant pHs to produce the same metabolites as an azoreductase. Our results show that, in some cases, the non-enzymatic reduction by the cofactors was found to be equal to that seen with the azoreductase, suggesting that all dye reduction in these cases is due to the cofactors themselves. This study details the importance of the use of a cofactor-only control when studying azoreductase enzymes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  11. Effects of surface proteins and lipids on molecular structure, thermal properties, and enzymatic hydrolysis of rice starch

    Directory of Open Access Journals (Sweden)

    Pan HU

    Full Text Available Abstract Rice starches with different amylose contents were treated with sodium dodecyl sulfate (SDS to deplete surface proteins and lipids, and the changes in molecular structure, thermal properties, and enzymatic hydrolysis were evaluated. SDS treatment did not significantly change the molecular weight distribution, crystalline structure, short-range ordered degree, and gelatinization properties of starch, but significantly altered the pasting properties and increased the swelling power of starch. The removal of surface proteins and lipids increased the enzymatic hydrolysis and in vitro digestion of starch. The influences of removing surface proteins and lipids from starch on swelling power, pasting properties, and enzymatic hydrolysis were different among the various starches because of the differences in molecular structures of different starch styles. The aforementioned results indicated that removing the surface proteins and lipids from starch did not change the molecular structure but had significant effects on some functional properties.

  12. Fuzzy logic feedback control for fed-batch enzymatic hydrolysis of lignocellulosic biomass.

    Science.gov (United States)

    Tai, Chao; Voltan, Diego S; Keshwani, Deepak R; Meyer, George E; Kuhar, Pankaj S

    2016-06-01

    A fuzzy logic feedback control system was developed for process monitoring and feeding control in fed-batch enzymatic hydrolysis of a lignocellulosic biomass, dilute acid-pretreated corn stover. Digested glucose from hydrolysis reaction was assigned as input while doser feeding time and speed of pretreated biomass were responses from fuzzy logic control system. Membership functions for these three variables and rule-base were created based on batch hydrolysis data. The system response was first tested in LabVIEW environment then the performance was evaluated through real-time hydrolysis reaction. The feeding operations were determined timely by fuzzy logic control system and efficient responses were shown to plateau phases during hydrolysis. Feeding of proper amount of cellulose and maintaining solids content was well balanced. Fuzzy logic proved to be a robust and effective online feeding control tool for fed-batch enzymatic hydrolysis.

  13. Networked enzymatic logic gates with filtering: new theoretical modeling expressions and their experimental application.

    Science.gov (United States)

    Privman, Vladimir; Zavalov, Oleksandr; Halámková, Lenka; Moseley, Fiona; Halámek, Jan; Katz, Evgeny

    2013-12-05

    We report the first study of a network of connected enzyme-catalyzed reactions, with added chemical and enzymatic processes that incorporate the recently developed biochemical filtering steps into the functioning of this biocatalytic cascade. New theoretical expressions are derived to allow simple, few-parameter modeling of network components concatenated in such cascades, both with and without filtering. The derived expressions are tested against experimental data obtained for the realized network's responses, measured optically, to variations of its input chemicals' concentrations with and without filtering processes. We also describe how the present modeling approach captures and explains several observations and features identified in earlier studies of enzymatic processes when they were considered as potential network components for multistep information/signal processing systems.

  14. Epimerase (Msed_0639) and Mutase (Msed_0638 and Msed_2055) Convert (S)-Methylmalonyl-Coenzyme A (CoA) to Succinyl-CoA in the Metallosphaera sedula 3-Hydroxypropionate/4-Hydroxybutyrate Cycle

    Science.gov (United States)

    Han, Yejun; Hawkins, Aaron S.; Adams, Michael W. W.

    2012-01-01

    Crenarchaeotal genomes encode the 3-hydroxypropionate/4-hydroxybutyrate (3-HP/4-HB) cycle for carbon dioxide fixation. Of the 13 enzymes putatively comprising the cycle, several of them, including methylmalonyl-coenzyme A (CoA) epimerase (MCE) and methylmalonyl-CoA mutase (MCM), which convert (S)-methylmalonyl-CoA to succinyl-CoA, have not been confirmed and characterized biochemically. In the genome of Metallosphaera sedula (optimal temperature [Topt], 73°C), the gene encoding MCE (Msed_0639) is adjacent to that encoding the catalytic subunit of MCM-α (Msed_0638), while the gene for the coenzyme B12-binding subunit of MCM (MCM-β) is located remotely (Msed_2055). The expression of all three genes was significantly upregulated under autotrophic compared to heterotrophic growth conditions, implying a role in CO2 fixation. Recombinant forms of MCE and MCM were produced in Escherichia coli; soluble, active MCM was produced only if MCM-α and MCM-β were coexpressed. MCE is a homodimer and MCM is a heterotetramer (α2β2) with specific activities of 218 and 2.2 μmol/min/mg, respectively, at 75°C. The heterotetrameric MCM differs from the homo- or heterodimeric orthologs in other organisms. MCE was activated by divalent cations (Ni2+, Co2+, and Mg2+), and the predicted metal binding/active sites were identified through sequence alignments with less-thermophilic MCEs. The conserved coenzyme B12-binding motif (DXHXXG-SXL-GG) was identified in M. sedula MCM-β. The two enzymes together catalyzed the two-step conversion of (S)-methylmalonyl-CoA to succinyl-CoA, consistent with their proposed role in the 3-HP/4-HB cycle. Based on the highly conserved occurrence of single copies of MCE and MCM in Sulfolobaceae genomes, the M. sedula enzymes are likely to be representatives of these enzymes in the 3-HP/4-HB cycle in crenarchaeal thermoacidophiles. PMID:22752162

  15. Effects of agitation on particle-size distribution and enzymatic hydrolysis of pretreated spruce and giant reed.

    Science.gov (United States)

    Kadić, Adnan; Palmqvist, Benny; Lidén, Gunnar

    2014-01-01

    Mixing is an energy demanding process which has been previously shown to affect enzymatic hydrolysis. Concentrated biomass slurries are associated with high and non-Newtonian viscosities and mixing in these systems is a complex task. Poor mixing can lead to mass and/or heat transfer problems as well as inhomogeneous enzyme distribution, both of which can cause possible yield reduction. Furthermore the stirring energy dissipation may impact the particle size which in turn may affect the enzymatic hydrolysis. The objective of the current work was to specifically quantify the effects of mixing on particle-size distribution (PSD) and relate this to changes in the enzymatic hydrolysis. Two rather different materials were investigated, namely pretreated Norway spruce and giant reed. Changes in glucan hydrolysis and PSD were measured as a function of agitation during enzymatic hydrolysis at fiber loadings of 7 or 13% water-insoluble solids (WIS). Enzymatic conversion of pretreated spruce was strongly affected by agitation rates at the higher WIS content. However, at low WIS content the agitation had almost no effect on hydrolysis. There was some effect of agitation on the hydrolysis of giant reed at high WIS loading, but it was smaller than that for spruce, and there was no measurable effect at low WIS loading. In the case of spruce, intense agitation clearly affected the PSD and resulted in a reduced mean particle size, whereas for giant reed the decrease in particle size was mainly driven by enzymatic action. However, the rate of enzymatic hydrolysis was not increased after size reduction by agitation. The impact of agitation on the enzymatic hydrolysis clearly depends not only on feedstock but also on the solids loading. Agitation was found to affect the PSD differently for the examined pretreated materials spruce and giant reed. The fact that the reduced mean particle diameter could not explain the enhanced hydrolysis rates found for spruce at an elevated agitation

  16. Effects of agitation on particle-size distribution and enzymatic hydrolysis of pretreated spruce and giant reed

    Science.gov (United States)

    2014-01-01

    Background Mixing is an energy demanding process which has been previously shown to affect enzymatic hydrolysis. Concentrated biomass slurries are associated with high and non-Newtonian viscosities and mixing in these systems is a complex task. Poor mixing can lead to mass and/or heat transfer problems as well as inhomogeneous enzyme distribution, both of which can cause possible yield reduction. Furthermore the stirring energy dissipation may impact the particle size which in turn may affect the enzymatic hydrolysis. The objective of the current work was to specifically quantify the effects of mixing on particle-size distribution (PSD) and relate this to changes in the enzymatic hydrolysis. Two rather different materials were investigated, namely pretreated Norway spruce and giant reed. Results Changes in glucan hydrolysis and PSD were measured as a function of agitation during enzymatic hydrolysis at fiber loadings of 7 or 13% water-insoluble solids (WIS). Enzymatic conversion of pretreated spruce was strongly affected by agitation rates at the higher WIS content. However, at low WIS content the agitation had almost no effect on hydrolysis. There was some effect of agitation on the hydrolysis of giant reed at high WIS loading, but it was smaller than that for spruce, and there was no measurable effect at low WIS loading. In the case of spruce, intense agitation clearly affected the PSD and resulted in a reduced mean particle size, whereas for giant reed the decrease in particle size was mainly driven by enzymatic action. However, the rate of enzymatic hydrolysis was not increased after size reduction by agitation. Conclusions The impact of agitation on the enzymatic hydrolysis clearly depends not only on feedstock but also on the solids loading. Agitation was found to affect the PSD differently for the examined pretreated materials spruce and giant reed. The fact that the reduced mean particle diameter could not explain the enhanced hydrolysis rates found for

  17. Modelling of the enzymatic kinetically controlled synthesis of cephalexin

    NARCIS (Netherlands)

    Schroën, C.G.P.H.; Fretz, C.B.; Bruin, de V.H.; Berendsen, W.; Moody, H.M.; Roos, E.C.; Roon, van J.L.; Kroon, P.J.; Strubel, M.; Janssen, A.E.M.; Tramper, J.

    2002-01-01

    In this study the influence of diffusion limitation on enzymatic kinetically controlled cephalexin synthesis from phenylglycine amide and 7-aminodeacetoxycephalosporinic acid (7-ADCA) was investigated systematically. It was found that if diffusion limitation occurred, both the synthesis/hydrolysis

  18. Multicompartment Artificial Organelles Conducting Enzymatic Cascade Reactions inside Cells

    DEFF Research Database (Denmark)

    Gallardo, Maria Godoy; Labay, Cédric Pierre; Trikalitis, Vasileios

    2017-01-01

    capsosome system, which consists of multiple liposomes and fluorescent gold nanoclusters embedded within a polymer carrier capsule. We subsequently demonstrate that encapsulated enzymes preserve their activity intracellularly, allowing for controlled enzymatic cascade reaction within a host cell....

  19. Enzymatic hydrolysis of defatted mackerel protein with low bitter taste

    Science.gov (United States)

    Hou, Hu; Li, Bafang; Zhao, Xue

    2011-03-01

    Ultrasound-assisted solvent extraction was confirmed as a novel, effective method for separating lipid from mackerel protein, resulting in a degreasing rate (DR) of 95% and a nitrogen recovery (NR) of 88.6%. To obtain protein hydrolysates with high nitrogen recovery and low bitter taste, enzymatic hydrolysis was performed using eight commercially available proteases. It turned out that the optimum enzyme was the `Mixed enzymes for animal proteolysis'. An enzyme dosage of 4%, a temperature of 50°, and a hydrolysis time of 300 min were found to be the optimum conditions to obtain high NR (84.28%) and degree of hydrolysis (DH, 16.18%) by orthogonal experiments. Glutamic acid was the most abundant amino acid of MDP (defatted mackerel protein) and MDPH (defatted mackerel protein hydrolysates). Compared with the FAO/WHO reference protein, the essential amino acid chemical scores (CS) were greater than 1.0 (1.0-1.7) in MDPH, which is reflective of high nutritional value. This, coupled with the light color and slight fishy odor, indicates that MDPH would potentially have a wide range of applications such as nutritional additives, functional ingredients, and so on.

  20. Enzymatic synthesis of phytosteryl lipoate and its antioxidant properties.

    Science.gov (United States)

    Wang, Huiqi; Jia, Chengsheng; Xia, Xue; Karangwa, Eric; Zhang, Xiaoming

    2018-02-01

    In this work, an enzymatic route for synthesizing phytosteryl lipoate was successfully set up for the first time. The structure of final product phytosteryl lipoate was determined by Fourier Transform Infrared (FTIR), Mass Spectrometry (MS) and Nuclear Magnetic Resonance (NMR). The maximum conversion of 71.2% was achieved when the following conditions were employed: 150mmol/L phytosterol, 1: 2.5M ratio of phytosterol to lipoic acid, 10g/L of 4Å molecular sieves and 60g/L Candida rugosa in 2-methyl-2-butanol/n-hexane (1:1, v/v) at 55°C for 96h. The physicochemical properties including solubility and antioxidant ability of phytosteryl lipoate in oil were assessed. The results revealed that phytosteryl lipoate possessed over twice as much oil solubility as free phytosterol and also showed better antioxidant ability. Investigation on its biological functions will be the main object in the future work. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Novel flavonolignan hybrid antioxidants: From enzymatic preparation to molecular rationalization.

    Science.gov (United States)

    Vavříková, Eva; Křen, Vladimír; Jezova-Kalachova, Lubica; Biler, Michal; Chantemargue, Benjamin; Pyszková, Michaela; Riva, Sergio; Kuzma, Marek; Valentová, Kateřina; Ulrichová, Jitka; Vrba, Jiří; Trouillas, Patrick; Vacek, Jan

    2017-02-15

    A series of antioxidants was designed and synthesized based on conjugation of the hepatoprotective flavonolignan silybin with l-ascorbic acid, trolox alcohol or tyrosol via a C12 aliphatic linker. These hybrid molecules were prepared from 12-vinyl dodecanedioate-23-O-silybin using the enzymatic regioselective acylation procedure with Novozym 435 (lipase B) or with lipase PS. Voltammetric analyses showed that the silybin-ascorbic acid conjugate exhibited excellent electron donating ability, in comparison to the other conjugates. Free radical scavenging, antioxidant activities and cytoprotective action were evaluated. The silybin-ascorbic acid hybrid exhibited the best activities (IC50 = 30.2 μM) in terms of lipid peroxidation inhibition. The promising protective action of the conjugate against lipid peroxidation can be attributed to modulated electron transfer abilities of both the silybin and ascorbate moieties, but also to the hydrophobic C12 linker facilitating membrane insertion. This was supported experimentally and theoretically by density functional theory (DFT) and molecular dynamics (MD) calculations. The results presented here can be used in the further development of novel multipotent antioxidants and cytoprotective agents, in particular for substances acting at an aqueous/lipid interface. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  2. Redox Regulation of Inflammatory Processes Is Enzymatically Controlled

    Directory of Open Access Journals (Sweden)

    Inken Lorenzen

    2017-01-01

    Full Text Available Redox regulation depends on the enzymatically controlled production and decay of redox active molecules. NADPH oxidases, superoxide dismutases, nitric oxide synthases, and others produce the redox active molecules superoxide, hydrogen peroxide, nitric oxide, and hydrogen sulfide. These react with target proteins inducing spatiotemporal modifications of cysteine residues within different signaling cascades. Thioredoxin family proteins are key regulators of the redox state of proteins. They regulate the formation and removal of oxidative modifications by specific thiol reduction and oxidation. All of these redox enzymes affect inflammatory processes and the innate and adaptive immune response. Interestingly, this regulation involves different mechanisms in different biological compartments and specialized cell types. The localization and activity of distinct proteins including, for instance, the transcription factor NFκB and the immune mediator HMGB1 are redox-regulated. The transmembrane protein ADAM17 releases proinflammatory mediators, such as TNFα, and is itself regulated by a thiol switch. Moreover, extracellular redox enzymes were shown to modulate the activity and migration behavior of various types of immune cells by acting as cytokines and/or chemokines. Within this review article, we will address the concept of redox signaling and the functions of both redox enzymes and redox active molecules in innate and adaptive immune responses.

  3. Controlling enzymatic activity by immobilization on graphene oxide

    Science.gov (United States)

    Bolibok, Paulina; Wiśniewski, Marek; Roszek, Katarzyna; Terzyk, Artur P.

    2017-04-01

    In this study, graphene oxide (GO) has been applied as a matrix for enzyme immobilization. The protein adsorption capacity of GO is much higher than of other large surface area carbonaceous materials. Its structure and physicochemical properties are reported beneficial also for enzymatic activity modifications. The experimental proof was done here that GO-based biocatalytic systems with immobilized catalase are modifiable in terms of catalyzed reaction kinetic constants. It was found that activity and stability of catalase, considered here as model enzyme, closely depend on enzyme/GO ratio. The changes in kinetic parameters can be related to secondary structure alterations. The correlation between enzyme/GO ratio and kinetic and structure parameters is reported for the first time and enables the conscious control of biocatalytic processes and their extended applications. The biological activity of obtained biocatalytic systems was confirmed in vitro by the use of functional test. The addition of immobilized catalase improved the cells' viability after they were exposed to hydrogen peroxide and tert-butyl-hydroperoxide used as source of reactive oxygen species.

  4. Enzymatic Synthesis of Sucrose Polyester as Food Emulsifier Compound

    Directory of Open Access Journals (Sweden)

    Sri Handayani

    2012-12-01

    Full Text Available Sucrose polyester (SPE is a carbohydrate ester compound that has diverse functions, from surfactant to low-calorie food products. Sucrose fatty acid ester with the degree of substitution 1-3 can be used as emulsifier in foods and cosmetics. The enzymatic synthesis of sucrose polyesters can be carried out using lipase in organic solvent and contain small amount of water. In these studies sucrose esters were synthesized by esterification reaction between sucrose with fatty acids from coconut and palm oil using Candida rugosa lipase in n-hexane. Optimization esterification reaction carried out for parameters of incubation time, temperature, and the ratio of the substrate. The optimum incubation time is at 18 hours for coconut oil and 12 hours palm oil, the optimum temperature is 30 oC for coconut and palm oil, and the mole ratio of fatty acid to sucrose is 40:1 for coconut oil and 64:1 for palm oil. Esterification products were characterized by FT-IR. The FT-IR spectrum showed the ester bond was formed as indicated by the wave number 1739.79/cm. Esterification products have 2 substitution degrees.

  5. Phenol removal through combined biological and enzymatic treatments

    OpenAIRE

    Bevilaqua J.V.; Cammarota M.C.; Freire D.M.G.; Sant?Anna Jr G.L.

    2002-01-01

    This work studies the use of biological and combined biological/enzymatic treatments in phenol degradation. The systems studied were conventional batch aerobic biological followed or preceded by enzymatic treatment. Tyrosinase extracted from the mushroom Agaricus bispora was employed. Biological treatment efficiently degraded effluents containing up to 420 mg.L-1 of phenol, removing 97% of the COD and 99% of the phenol in 48-hour batches. Alterations in phenol concentration intake reduced tre...

  6. Interaction of Solvents and Mechanical Pretreatment with Enzymatic Lignocellulose Hydrolysis

    OpenAIRE

    Wang, Yumei

    2017-01-01

    Renewable plant biomass is considered as an alternative raw material for the production of fuels and chemicals instead of decreasing fossil resources. Enzymatic hydrolysis of pretreated lignocellulosic material to produce high sugar concentrations is one important step in a bio-refinery process, and can be operated under moderate conditions without by-products. However, the efficiency of the enzymatic hydrolysis is hindered by lignocellulose recalcitrance. Therefore, pretreatments of the biom...

  7. Kinetics of enzymatic hydrolysis of methyl ricinoleate

    Directory of Open Access Journals (Sweden)

    Neeharika, T. S.V.R.

    2015-12-01

    Full Text Available 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 Candida antarctica Lipase B was carried out in this study by varying reaction temperature (40–60 °C and enzyme concentration (2–5%. The optimal conditions were found to be 6 h reaction time, temperature 60°C, buffer to methyl ricinoleate ratio 2:1(v/w and 4% enzyme concentration to achieve a maximum conversion of 98.5%. A first order reversible reaction kinetic model was proposed to describe this reaction and a good agreement was observed between the experimental data and the model values. The effect of temperature on the forward reaction rate constant was determined by fitting data to the Arrhenius equation. The activation energy for forward reaction was found to be 14.69 KJ·mol−1.El ácido ricinoleico es un hidroxiácido insaturado que se produce naturalmente en el aceite de ricino en proporciones de hasta el 85–90%. El ácido ricinoleico es una materia prima con gran potencial y tiene aplicaciones en revestimientos, formulaciones lubricantes y en áreas farmacéuticas. Para la preparación del ácido ricinoleico se prefiere la hidrólisis enzimática del aceite de ricino a la hidrólisis convencional, para evitar la formación de estólidos. En este estudio se llevó a cabo la cinética de la hidrólisis enzimática del ricinoleato de metilo en presencia de lipasa de Candida antarctica B mediante la variación de la temperatura de reacción (40–60 °C y la concentración de la enzima (2–5%. Las condiciones óptimas de la reacción para

  8. Performance of glucose/O2 enzymatic fuel cell based on supporting electrodes over-coated by polymer-nanogold particle composite with entrapped enzymes

    Science.gov (United States)

    Huo, W. S.; Zeng, H.; Yang, Y.; Zhang, Y. H.

    2017-03-01

    Enzymatic electrodes over-coated by thin film of nano-composite made up of polymer and functionalized nano-gold particle was prepared. Glucose/O2 membrane-free enzymatic fuel cell based on nano-composite based electrodes with incorporated glucose oxidase and laccase was assembled. This enzymatic fuel cell exhibited high energy out-put density even when applied in human serum. Catalytic cycle involved in enzymatic fuel cell was limited by oxidation of glucose occurred on bioanode resulting from impact of sophisticated interaction between active site in glucose oxidase and nano-gold particle on configuration of redox center of enzyme molecule which crippled catalytic efficiency of redox protein.

  9. Volatile Compound, Physicochemical, and Antioxidant Properties of Beany Flavor-Removed Soy Protein Isolate Hydrolyzates Obtained from Combined High Temperature Pre-Treatment and Enzymatic Hydrolysis

    Science.gov (United States)

    Yoo, Sang-Hun; Chang, Yoon Hyuk

    2016-01-01

    The present study investigated the volatile compound, physicochemical, and antioxidant properties of beany flavor-removed soy protein isolate (SPI) hydrolyzates produced by combined high temperature pre-treatment and enzymatic hydrolysis. Without remarkable changes in amino acid composition, reductions of residual lipoxygenase activity and beany flavor-causing volatile compounds such as hexanol, hexanal, and pentanol in SPI were observed after combined heating and enzymatic treatments. The degree of hydrolysis, emulsion capacity and stability, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity, and superoxide radical scavenging activity of SPI were significantly increased, but the magnitudes of apparent viscosity, consistency index, and dynamic moduli (G′, G″) of SPI were significantly decreased after the combined heating and enzymatic treatments. Based on these results, it was suggested that the enzymatic hydrolysis in combination with high temperature pre-treatment may allow for the production of beany flavor-removed SPI hydrolyzates with superior emulsifying and antioxidant functionalities. PMID:28078256

  10. Chemical and Enzymatic Strategies for Bacterial and Mammalian Cell Surface Engineering.

    Science.gov (United States)

    Bi, Xiaobao; Yin, Juan; Chen Guanbang, Ashley; Liu, Chuan-Fa

    2018-01-17

    Cell surface serves important functions such as the regulation of cell-cell and cell-environment interactions. The understanding and manipulation of the cell surface is important for a wide range of fundamental studies of cellular behavior and for biotechnological and medical applications. With the rapid advance of biology, chemistry and materials science, many strategies have been developed for the functionalization of bacterial and mammalian cell surfaces. Here, we review the recent development of chemical and enzymatic approaches to cell surface engineering with particular emphasis on discussing the advantages and limitations of each of these strategies. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Enzymatic reduction of azo and indigoid compounds.

    Science.gov (United States)

    Pricelius, S; Held, C; Murkovic, M; Bozic, M; Kokol, V; Cavaco-Paulo, A; Guebitz, G M

    2007-11-01

    A customer- and environment-friendly method for the decolorization azo dyes was developed. Azoreductases could be used both to bleach hair dyed with azo dyes and to reduce dyes in vat dyeing of textiles. A new reduced nicotinamide adenine dinucleotide-dependent azoreductase of Bacillus cereus, which showed high potential for reduction of these dyes, was purified using a combination of ammonium sulfate precipitation and chromatography and had a molecular mass of 21.5 kDa. The optimum pH of the azoreductase depended on the substrate and was within the range of pH 6 to 7, while the maximum temperature was reached at 40 degrees C. Oxygen was shown to be an alternative electron acceptor to azo compounds and must therefore be excluded during enzymatic dye reduction. Biotransformation of the azo dyes Flame Orange and Ruby Red was studied in more detail using UV-visible spectroscopy, high-performance liquid chromatography, and mass spectrometry (MS). Reduction of the azo bonds leads to cleavage of the dyes resulting in the cleavage product 2-amino-1,3 dimethylimidazolium and N approximately 1 approximately ,N approximately 1 approximately -dimethyl-1,4-benzenediamine for Ruby Red, while only the first was detected for Flame Orange because of MS instability of the expected 1,4-benzenediamine. The azoreductase was also found to reduce vat dyes like Indigo Carmine (C.I. Acid Blue 74). Hydrogen peroxide (H(2)O(2)) as an oxidizing agent was used to reoxidize the dye into the initial form. The reduction and oxidation mechanism of Indigo Carmine was studied using UV-visible spectroscopy.

  12. Role of substrate unbinding in Michaelis-Menten enzymatic reactions.

    Science.gov (United States)

    Reuveni, Shlomi; Urbakh, Michael; Klafter, Joseph

    2014-03-25

    The Michaelis-Menten equation provides a hundred-year-old prediction by which any increase in the rate of substrate unbinding will decrease the rate of enzymatic turnover. Surprisingly, this prediction was never tested experimentally nor was it scrutinized using modern theoretical tools. Here we show that unbinding may also speed up enzymatic turnover--turning a spotlight to the fact that its actual role in enzymatic catalysis remains to be determined experimentally. Analytically constructing the unbinding phase space, we identify four distinct categories of unbinding: inhibitory, excitatory, superexcitatory, and restorative. A transition in which the effect of unbinding changes from inhibitory to excitatory as substrate concentrations increase, and an overlooked tradeoff between the speed and efficiency of enzymatic reactions, are naturally unveiled as a result. The theory presented herein motivates, and allows the interpretation of, groundbreaking experiments in which existing single-molecule manipulation techniques will be adapted for the purpose of measuring enzymatic turnover under a controlled variation of unbinding rates. As we hereby show, these experiments will not only shed first light on the role of unbinding but will also allow one to determine the time distribution required for the completion of the catalytic step in isolation from the rest of the enzymatic turnover cycle.

  13. Enzymatically induced motion at nano- and micro-scales

    Science.gov (United States)

    Gáspár, Szilveszter

    2014-06-01

    In contrast to adenosine triphosphate (ATP)-dependent motor enzymes, other enzymes are little-known as ``motors'' or ``pumps'', that is, for their ability to induce motion. The enhanced diffusive movement of enzyme molecules, the self-propulsion of enzyme-based nanomotors, and liquid pumping with enzymatic micropumps were indeed only recently reported. Enzymatically induced motion can be achieved in mild conditions and without the use of external fields. It is thus better suited for use in living systems (from single-cell to whole-body) than most other ways to achieve motion at small scales. Enzymatically induced motion is thus not only new but also important. Therefore, the present work reviews the most significant discoveries in enzymatically induced motion. As we will learn, freely diffusing enzymes enhance their diffusive movement by nonreciprocal conformational changes which parallel their catalytic cycles. Meanwhile, enzyme-modified nano- and micro-objects turn chemical energy into kinetic energy through mechanisms such as bubble recoil propulsion, self-electrophoresis, and self-diffusiophoresis. Enzymatically induced motion of small objects ranges from enhanced diffusive movement to directed motion at speeds as high as 1 cm s-1. In spite of the progress made in understanding how the energy of enzyme reactions is turned into motion, most enzymatically powered devices remain inefficient and need improvements before we will witness their application in real world environments.

  14. Enhanced enzymatic conversion with freeze pretreatment of rice straw

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Ken-Lin; Thitikorn-amorn, Jitladda; Ou, Bay-Ming; Chen, Shan-He; Huang, Po-Jung [Institute of Biological Chemistry and Genomics Research Center Academia Sinica, Nankang, Taipei 115 (China); Hsieh, Jung-Feng [Department of Food Science, Fu Jen Catholic University, Xin Zhuang, Taipei 242 (China); Ratanakhanokchai, Khanok [School of Bioresources and Technology, King Mongkut' s University of Technology Thonburi, Bangkok 10150 (Thailand); Chen, Shui-Tein [Institute of Biological Chemistry and Genomics Research Center Academia Sinica, Nankang, Taipei 115 (China); Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei 106 (China)

    2011-01-15

    Production of bioethanol by the conversion of lignocellulosic waste has attracted much interest in recent years, because of its low cost and great potential availability. The pretreatment process is important for increasing the enzymatic digestibility of lignocellulosic materials. Enzymatic conversion with freeze pretreatment of rice straw was evaluated in this study. The freeze pretreatment was found to significantly increase the enzyme digestibility of rice straw from 48% to 84%. According to the results, enzymatic hydrolysis of unpretreated rice straw with 150 U cellulase and 100 U xylanase for 48 h yielded 226.77 g kg{sup -1} and 93.84 g kg{sup -1} substrate-reducing sugars respectively. However, the reducing sugar yields from freeze pretreatment under the same conditions were 417.27 g kg{sup -1} and 138.77 g kg{sup -1} substrate, respectively. In addition, hydrolyzates analysis showed that the highest glucose yield obtained during the enzymatic hydrolysis step in the present study was 371.91 g kg{sup -1} of dry rice straw, following pretreatment. Therefore, the enhanced enzymatic conversion with freeze pretreatment of rice straw was observed in this study. This indicated that freeze pretreatment was highly effective for enzymatic hydrolysis and low environmental impact. (author)

  15. Enzymatic Catalysis at Interfaces—Heterophase Systems as Substrates for Enzymatic Action

    Directory of Open Access Journals (Sweden)

    Katharina Landfester

    2013-04-01

    Full Text Available Several important enzymatic reactions occurring in nature, such as, e.g., the digestion of fat, proceed only at the interface of two immiscible phases. Typically, these systems consist of an organic substrate, dispersed in an aqueous continuous phase, with a specialized enzyme capable of working at the interface. For adopting such a system for organic synthesis, a stable heterophase system with a large interfacial area is required. These prerequisites can be found in so-called miniemulsions. Such liquid-liquid heterophase systems feature droplets with sizes smaller than 500 nm, and more importantly, these emulsions do not suffer from Ostwald ripening, as conventional emulsions do. Consequently, the droplets show long-term stability, even throughout reactions conducted in the droplets. In this review, we will briefly discuss the physicochemical background of miniemulsions, provide a comprehensive overview of the enzymatically catalyzed reactions conducted in miniemulsions and, as data are available, to compare the most important features to conventional systems, as reverse microemulsions, (macroemulsions and solvent-based systems.

  16. 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 reactions of these

  17. Low enzymatic activity haplotypes of the human catechol-O-methyltransferase gene: enrichment for marker SNPs.

    Directory of Open Access Journals (Sweden)

    Andrea G Nackley

    Full Text Available Catechol-O-methyltransferase (COMT is an enzyme that plays a key role in the modulation of catechol-dependent functions such as cognition, cardiovascular function, and pain processing. Three common haplotypes of the human COMT gene, divergent in two synonymous and one nonsynonymous (val(158met position, designated as low (LPS, average (APS, and high pain sensitive (HPS, are associated with experimental pain sensitivity and risk of developing chronic musculoskeletal pain conditions. APS and HPS haplotypes produce significant functional effects, coding for 3- and 20-fold reductions in COMT enzymatic activity, respectively. In the present study, we investigated whether additional minor single nucleotide polymorphisms (SNPs, accruing in 1 to 5% of the population, situated in the COMT transcript region contribute to haplotype-dependent enzymatic activity. Computer analysis of COMT ESTs showed that one synonymous minor SNP (rs769224 is linked to the APS haplotype and three minor SNPs (two synonymous: rs6267, rs740602 and one nonsynonymous: rs8192488 are linked to the HPS haplotype. Results from in silico and in vitro experiments revealed that inclusion of allelic variants of these minor SNPs in APS or HPS haplotypes did not modify COMT function at the level of mRNA folding, RNA transcription, protein translation, or enzymatic activity. These data suggest that neutral variants are carried with APS and HPS haplotypes, while the high activity LPS haplotype displays less linked variation. Thus, both minor synonymous and nonsynonymous SNPs in the coding region are markers of functional APS and HPS haplotypes rather than independent contributors to COMT activity.

  18. 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/O2 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.

  19. Inhibition of enzymatic browning in actual food systems by the Maillard reaction products.

    Science.gov (United States)

    Mogol, Burçe Ataç; Yildirim, Asli; Gökmen, Vural

    2010-12-01

    The Maillard reaction occurring between amino acids and sugars produces neo-formed compounds having certain levels of antioxidant activity depending on the reaction conditions and the type of reactants. The objective of this study was to investigate enzymatic browning inhibition capacity of Maillard reaction products (MRPs) formed from different amino acids including arginine (Arg), histidine (His), lysine (Lys) and proline (Pro). The inhibitory effects of the MRPs on polyphenol oxidase (PPO) were determined. The total antioxidant capacity (TAC) of MRPs derived from different amino acids were in the order Arg > His > Lys > Pro. The TAC and PPO inhibition of MRPs were evaluated as a function of temperature (80-120 °C), time (1-6 h) and pH (2-12). Arg-Glc and His-Glc MRPs exhibited strong TAC and PPO inhibition. Increasing temperature (up to 100 °C) and time also increased TAC and PPO inhibition. Kinetics analysis indicated a mixed type inhibition of PPO by MRPs. The results indicate that the MRPs derived from Arg and His under certain reaction conditions significantly prevent enzymatic browning in actual food systems. The intermediate compounds capable of preventing enzymatic browning are reductones and dehydroreductones, as confirmed by liquid chromatographic-mass spectrometric analyses. Copyright © 2010 Society of Chemical Industry.

  20. Enzymatic mechanisms regulating protein S-nitrosylation: implications in health and disease.

    Science.gov (United States)

    Anand, Puneet; Stamler, Jonathan S

    2012-03-01

    Nitric oxide participates in cellular signal transduction largely through S-nitrosylation of allosteric and active-site cysteine thiols within proteins, forming S-nitroso-proteins (SNO-proteins). S-nitrosylation of proteins has been demonstrated to affect a broad range of functional parameters including enzymatic activity, subcellular localization, protein-protein interactions, and protein stability. Analogous to other ubiquitous posttranslational modifications that are regulated enzymatically, including phosphorylation and ubiquitinylation, accumulating evidence suggests the existence of enzymatic mechanisms for regulating protein S-nitrosylation. In particular, studies have led to the identification of multiple enzymes (nitrosylases and denitrosylases) that participate in targeted S-nitrosylation or denitrosylation of proteins in physiological settings. Nitrosylases are best characterized in the context of transnitrosylation in which a SNO-protein transfers an NO group to an acceptor protein (Cys-to-Cys transfer), but examples of transnitrosylation catalyzed by metalloproteins (Metal-to-Cys transfer) also exist. By contrast, denitrosylases remove the NO group from SNO-proteins, ultimately using reducing equivalents derived from NADH or NADPH. Here, we focus on the recent discoveries of nitrosylases and denitrosylases and the notion that their aberrant activities may play roles in health and disease.

  1. 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.

  2. Effects of ultrasound irradiation on enzymatic hydrolysis of protein and application for the determination of tetracyclines in complex matrices.

    Science.gov (United States)

    Zhou, Qian; Wang, Hong; Zhu, Kuanzheng; Zhu, Lihua; Zhou, Shengyin; Peng, Jin; Lu, Xiya

    2017-10-01

    The widespread use of tetracyclines (TCs) in food animals has led to concerns regarding unsafe residue levels in feed, food and manure. The determination of TCs in such matrices suffers from interference by the interactions between proteins and TCs. Three deproteination methods were compared in this study. In contrast with acid deproteination, which caused a large loss of TCs due to the strong adsorption of TCs on protein precipitates, a normal enzymatic hydrolysis was confirmed to have a merit of effectively releasing TCs from protein matrices, but required treatment time as long as 16 h. The adoption of ultrasound irradiation was proposed to shorten the enzymatic hydrolysis time. After investigating the effects of ultrasound power and irradiation time, the conditions of the ultrasound-enhanced enzymatic hydrolysis were optimized as ultrasound power of 100 W and irradiation time of 6 min. The ultrasound-enhanced enzymatic hydrolysis treatment of 6 min yielded recovery of TCs (from protein-containing matrices) as high as that obtained by the normal enzymatic hydrolysis treatment of 16 h. The acceleration effect of ultrasound irradiation was attributed to ultrasound-induced cavitation, which increased exposure of both the functional groups of trypsin and the C-terminal amino acid in the protein that was a cleavage site for trypsin attack. When the ultrasound-enhanced enzymatic hydrolysis method was used to determine TCs in complex matrices, it was found that this new method achieved recoveries of 89.5, 117.7, 110.4 and 100.0% for oxytetracycline, tetracycline, chlortetracycline and doxycycline, being much higher than those (29.6-39.4%) obtained using the common acid deproteination process. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  3. Enzymatic hydrolysis of poly(ethyleneterephthalate) used for and analysed by pore modification of track-etched membranes.

    Science.gov (United States)

    Gamerith, Caroline; Gajda, Martyna; Ortner, Andreas; Acero, Enrique Herrero; Guebitz, Georg M; Ulbricht, Mathias

    2017-10-25

    The potential of limited enzymatic poly(ethylene terephthalate) (PET) surface hydrolysis for the modification of track-etched (TE) membranes was investigated. Cutinases 1 and 2 from Thermobifida cellulosilytica as well as a fusion protein of cutinase 1 with the polymer binding module from the polyhydroxyalkanoate depolymerase of Alcaligenes faecalis (Thc_Cut1_PBM) were shown to hydrolyse highly crystalline PET TE membranes with a pore diameter of ∼120nm at very narrow size distribution. Furthermore the effects of surface chemistry were investigated by comparison of enzymatic hydrolysis by Thc_Cut1_PBM of "as received" PET TE membranes with two surface functionalized versions towards a "hydrophilic" and a more "hydrophobic" surface. The effects of adsorbed protein and the efficacy of cleaning steps after enzymatic treatment were elucidated by complementary methods for surface analysis and membrane characterization. With the optimized cleaning protocol, all adsorbed protein could be removed from the enzyme-treated membranes and effects of chemical surface functionalization of the PET TE membranes were demonstrated. The highest efficiency of enzymatic surface hydrolysis was observed for the original PET TE membranes, leading to an 0.36% weight loss corresponding to a removal of ∼3nm PET from the entire surface of the porous membrane. This correlates very well with the measured increase of barrier pore diameter by 4nm (a radius reduction? of 2nm), leading to about a two-fold increased water permeability. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. The enzymatic nature of an anonymous protein sequence cannot reliably be inferred from superfamily level structural information alone.

    Science.gov (United States)

    Roche, Daniel Barry; Brüls, Thomas

    2015-05-01

    As the largest fraction of any proteome does not carry out enzymatic functions, and in order to leverage 3D structural data for the annotation of increasingly higher volumes of sequence data, we wanted to assess the strength of the link between coarse grained structural data (i.e., homologous superfamily level) and the enzymatic versus non-enzymatic nature of protein sequences. To probe this relationship, we took advantage of 41 phylogenetically diverse (encompassing 11 distinct phyla) genomes recently sequenced within the GEBA initiative, for which we integrated structural information, as defined by CATH, with enzyme level information, as defined by Enzyme Commission (EC) numbers. This analysis revealed that only a very small fraction (about 1%) of domain sequences occurring in the analyzed genomes was found to be associated with homologous superfamilies strongly indicative of enzymatic function. Resorting to less stringent criteria to define enzyme versus non-enzyme biased structural classes or excluding highly prevalent folds from the analysis had only modest effect on this proportion. Thus, the low genomic coverage by structurally anchored protein domains strongly associated to catalytic activities indicates that, on its own, the power of coarse grained structural information to infer the general property of being an enzyme is rather limited. © 2015 The Protein Society.

  5. Bubble electrodeposition of gold porous nanocorals for the enzymatic and non-enzymatic detection of glucose.

    Science.gov (United States)

    Sanzó, Gabriella; Taurino, Irene; Antiochia, Riccarda; Gorton, Lo; Favero, Gabriele; Mazzei, Franco; De Micheli, Giovanni; Carrara, Sandro

    2016-12-01

    Au nanocorals are grown on gold screen-printed electrodes (SPEs) by using a novel and simple one-step electrodeposition process. Scanning electron microscopy was used for the morphological characterization. The devices were assembled on a three-electrode SPE system, which is flexible and mass producible. The electroactive surface area, determined by cyclic voltammetry in sulphuric acid, was found to be 0.07±0.01cm(2) and 35.3±2.7cm(2) for bare Au and nanocoral Au, respectively. The nanocoral modified SPEs were used to develop an enzymatic glucose biosensor based on H2O2 detection. Au nanocoral electrodes showed a higher sensitivity of 48.3±0.9μA/(mMcm(2)) at +0.45V vs Ag|AgCl compared to a value of 24.6±1.3μA/(mMcm(2)) at +0.70V vs Ag|AgCl obtained with bare Au electrodes. However, the modified electrodes have indeed proven to be extremely powerful for the direct detection of glucose with a non-enzymatic approach. The results confirmed a clear peak observed by using nanocoral Au electrode even in the presence of chloride ions at physiological concentration. Amperometric study carried out at +0.15V vs Ag|AgCl in the presence of 0.12M NaCl showed a linear range for glucose between 0.1 and 13mM. Copyright © 2016. Published by Elsevier B.V.

  6. Effects of organic carbon sequestration strategies on soil enzymatic activities

    Science.gov (United States)

    Puglisi, E.; Suciu, N.; Botteri, L.; Ferrari, T.; Coppolecchia, D.; Trevisan, M.; Piccolo, A.

    2009-04-01

    Greenhouse gases emissions can be counterbalanced with proper agronomical strategies aimed at sequestering carbon in soils. These strategies must be tested not only for their ability in reducing carbon dioxide emissions, but also for their impact on soil quality: enzymatic activities are related to main soil ecological quality, and can be used as early and sensitive indicators of alteration events. Three different strategies for soil carbon sequestration were studied: minimum tillage, protection of biodegradable organic fraction by compost amendment and oxidative polimerization of soil organic matter catalyzed by biometic porfirins. All strategies were compared with a traditional agricultural management based on tillage and mineral fertilization. Experiments were carried out in three Italian soils from different pedo-climatic regions located respectively in Piacenza, Turin and Naples and cultivated with maize or wheat. Soil samples were taken for three consecutive years after harvest and analyzed for their content in phosphates, ß-glucosidase, urease and invertase. An alteration index based on these enzymatic activities levels was applied as well. The biomimetic porfirin application didn't cause changes in enzymatic activities compared to the control at any treatment or location. Enzymatic activities were generally higher in the minimum tillage and compost treatment, while differences between location and date of samplings were limited. Application of the soil alteration index based on enzymatic activities showed that soils treated with compost or subjected to minimum tillage generally have a higher biological quality. The work confirms the environmental sustainability of the carbon sequestering agronomical practices studied.

  7. Sulfate radicals enable a non-enzymatic Krebs cycle precursor.

    Science.gov (United States)

    Keller, Markus A; Kampjut, Domen; Harrison, Stuart A; Ralser, Markus

    2017-03-13

    The evolutionary origins of the Krebs cycle (tricarboxylic acid cycle) are not currently clear. Despite the existence of a simple non-enzymatic Krebs cycle catalyst being dismissed only a few years ago as 'an appeal to magic', citrate and other intermediates have since been discovered on a carbonaceous meteorite and do interconvert non-enzymatically. To identify a metabolism-like non-enzymatic Krebs cycle catalyst, we used combinatorial, quantitative high-throughput metabolomics to systematically screen iron and sulfate compounds in a reaction mixture that orients on the typical components of Archaean sediment. Krebs cycle intermediates were found to be stable in water and in the presence of most molecule species, including simple iron sulfate minerals. However, in the presence of sulfate radicals generated from peroxydisulfate, the intermediates underwent 24 interconversion reactions. These non-enzymatic reactions covered the critical topology of the oxidative Krebs cycle, the glyoxylate shunt and the succinic-semialdehyde pathway. Assembled in a chemical network, the reactions achieved over 90% carbon recovery. Our results show that a non-enzymatic precursor of the Krebs cycle is biologically sensible, efficient, and forms spontaneously in the presence of sulfate radicals.

  8. Protein S-Nitrosylation: Determinants of Specificity and Enzymatic Regulation of S-Nitrosothiol-Based Signaling.

    Science.gov (United States)

    Stomberski, Colin T; Hess, Douglas T; Stamler, Jonathan S

    2018-01-10

    Protein S-nitrosylation, the oxidative modification of cysteine by nitric oxide (NO) to form protein S-nitrosothiols (SNOs), mediates redox-based signaling that conveys, in large part, the ubiquitous influence of NO on cellular function. S-nitrosylation regulates protein activity, stability, localization, and protein-protein interactions across myriad physiological processes, and aberrant S-nitrosylation is associated with diverse pathophysiologies. Recent Advances: It is recently recognized that S-nitrosylation endows S-nitroso-protein (SNO-proteins) with S-nitrosylase activity, that is, the potential to trans-S-nitrosylate additional proteins, thereby propagating SNO-based signals, analogous to kinase-mediated signaling cascades. In addition, it is increasingly appreciated that cellular S-nitrosylation is governed by dynamically coupled equilibria between SNO-proteins and low-molecular-weight SNOs, which are controlled by a growing set of enzymatic denitrosylases comprising two main classes (high and low molecular weight). S-nitrosylases and denitrosylases, which together control steady-state SNO levels, may be identified with distinct physiology and pathophysiology ranging from cardiovascular and respiratory disorders to neurodegeneration and cancer. The target specificity of protein S-nitrosylation and the stability and reactivity of protein SNOs are determined substantially by enzymatic machinery comprising highly conserved transnitrosylases and denitrosylases. Understanding the differential functionality of SNO-regulatory enzymes is essential, and is amenable to genetic and pharmacological analyses, read out as perturbation of specific equilibria within the SNO circuitry. The emerging picture of NO biology entails equilibria among potentially thousands of different SNOs, governed by denitrosylases and nitrosylases. Thus, to elucidate the operation and consequences of S-nitrosylation in cellular contexts, studies should consider the roles of SNO-proteins as

  9. Enzymatic activities and protein profile of latex from Calotropis procera.

    Science.gov (United States)

    Freitas, Cleverson Diniz T; Oliveira, Jefferson Soares; Miranda, Maria Raquel A; Macedo, Nívea Maria R; Sales, Maurício Pereira; Villas-Boas, Laurival A; Ramos, Márcio Viana

    2007-01-01

    The laticifer fluid of Calotropis procera is rich in proteins and there is evidence that they are involved in the pharmacological properties of the latex. However, not much is known about how the latex-containing proteins are produced or their functions. In this study, laticifer proteins of C. procera were pooled and examined by 1D and 2D electrophoresis, masses spectrometry (MALDI-TOF) and characterized in respect of proteolytic activity and oxidative enzymes. Soluble laticifer proteins were predominantly composed of basic proteins (PI>6.0) with molecular masses varying between 5 and 95 kDa. Proteins with a molecular mass of approximately 26,000 Da were more evident. Strong anti-oxidative activity of superoxide dismutase (EC 1.15.1.1) (1007.74+/-91.89 Ug(-1)DM) and, to a lesser extent ascorbate peroxidase (EC 1.11.1.1) (0.117(d)+/-0.013 microMol H(2)O(2)g(-1)min(-1)), were detected. However, catalase (EC 1.11.1.6) was absent. The strong proteolytic activities of laticifer proteins from C. procera were shown to be shared by at least four distinct cysteine proteinases (EC 3.4.22.16) that were isolated by gel filtration chromatography. Serine and metaloproteinases were not detected and aspartic proteinase activities were barely visible. Chitinases (EC 3.2.1.14) were also isolated in a chitin column and their activities quantified. The presence of these enzymatic activities in latex from C. procera may confirm their involvement in resistance to phytopathogens and insects, mainly in its leaves where the latex circulates abundantly.

  10. Dynamic modeling and validation of a lignocellulosic enzymatic hydrolysis process

    DEFF Research Database (Denmark)

    Prunescu, Remus Mihail; Sin, Gürkan

    2013-01-01

    The enzymatic hydrolysis process is one of the key steps in second generation biofuel production. After being thermally pretreated, the lignocellulosic material is liquefied by enzymes prior to fermentation. The scope of this paper is to evaluate a dynamic model of the hydrolysis process on a dem......The enzymatic hydrolysis process is one of the key steps in second generation biofuel production. After being thermally pretreated, the lignocellulosic material is liquefied by enzymes prior to fermentation. The scope of this paper is to evaluate a dynamic model of the hydrolysis process...... on a demonstration scale reactor. The following novel features are included: the application of the Convection–Diffusion–Reaction equation to a hydrolysis reactor to assess transport and mixing effects; the extension of a competitive kinetic model with enzymatic pH dependency and hemicellulose hydrolysis...

  11. Bio-based alkyds by direct enzymatic bulk polymerization

    DEFF Research Database (Denmark)

    Nguyen, Hiep Dinh

    of control over the polymerization reaction. The process was used to prepare new and 100% bio-based resins. The developed enzymatic method is simple to perform, robust and allows the preparation of alkyds with much higher control over the chemical structure compared with the corresponding traditional method....... Bio-based alkyds prepared from a combination of glycerol, and tall oil fatty acids, and azelaic acid by enzymatic polymerization show improved hydrophobicity and lower glass transition temperatures compared to an alkyd prepared from the same raw materials by a classical boiling method. The enzymatic...... are considered as a good option for binders with improved curing properties. In the project various aspects of preparing a bio-based alkyd formulation have also been investigated. In particular, a reaction setup for production of larger amounts of traditional alkyds was designed to allow the production of up...

  12. Enzymatic saccharification of brown seaweed for production of fermentable sugars.

    Science.gov (United States)

    Sharma, Sandeep; Horn, Svein Jarle

    2016-08-01

    This study shows that high drying temperatures negatively affect the enzymatic saccharification yield of the brown seaweed Saccharina latissima. The optimal drying temperature of the seaweed in terms of enzymatic sugar release was found to be 30°C. The enzymatic saccharification process was optimized by investigating factors such as kinetics of sugar release, enzyme dose, solid loading and different blend ratios of cellulases and an alginate lyase. It was found that the seaweed biomass could be efficiently hydrolysed to fermentable sugars using a commercial cellulase cocktail. The inclusion of a mono-component alginate lyase was shown to improve the performance of the enzyme blend, in particular at high solid loadings. At 25% dry matter loading a combined glucose and mannitol concentration of 74g/L was achieved. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Production of ethanol from lignocellulosics: an enzymatic venture

    Science.gov (United States)

    Kuila, Arindam; Mukhopadhyay, Mainak; Tuli, D.K.; Banerjee, Rintu

    2011-01-01

    The major objective of the present investigation was to evaluate the effect of enzymatic pretreatment on Lantana camara for improved yield of reducing sugar and bioethanol production. An optimum enzymatic delignification (88.79 %) was achieved after 8 h of incubation. After delignification the substrate was further treated with the mixture of carbohydratases for appropriate saccharification. The enzyme treated substrate yielded maximum reducing sugar (713.33 mg/g dry substrate) after 9 h of saccharification. Monosaccharide content in the saccharified samples were quantified using high performance liquid chromatography (HPLC) system. Using conventional yeast strain, 9.63 g/L bioethanol was produced from saccharified samples of Lantana camara. Structural changes of Lantana camara before and after enzymatic pretreatment were further investigated through Fourier transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD) and Scanning electron microscopy (SEM). PMID:27857667

  14. 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.

  15. Enantioselective synthesis of both (-)-(R)-and (+)-(S)-angustureine controlled by enzymatic resolution

    Energy Technology Data Exchange (ETDEWEB)

    Diaz, Gaspar, E-mail: gaspardm@qui.ufmg.br [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Inst. de Ciencias Exatas. Dept. de Qumica; Diaz, Marisa A.N. [Universidade Federal de Vicosa, MG (Brazil). Dept. de Bioquimica e Biologia Molecular; Reis, Marco A. [Centro Federal de Educacao Tecnologica (CEFET), Belo Horizonte, MG (Brazil). Dept. de Quimica

    2013-09-15

    The present study describes a new synthesis of (-)-(R)- and (+)-(S)-angustureine enantiomers, as well as of racemate ({+-})-angustureine, from a racemic {beta}-amino ester controlled by kinetic enzymatic resolution. This strategy allowed to incorporate the basic skeleton, as well as to control the single stereocenter at carbon 2 in both enantiomers. The sequence of five steps starting from the chiral {beta}-amino ester and sodium carboxylate for the synthesis of both alkaloids achieved overall yields of 80 and 44%, respectively, and produced excellent enantiomeric excesses (95 and 96%, respectively) with no protection of functional groups in any of the steps. (author)

  16. Intracellular Microreactors as Artificial Organelles to Conduct Multiple Enzymatic Reactions Simultaneously

    DEFF Research Database (Denmark)

    Gallardo, Maria Godoy; Labay, Cédric Pierre; Jansman, Michelle M. T.

    2017-01-01

    The creation of artificial organelles is a new paradigm in medical therapy that aims to substitute for missing cellular function by replenishing a specific cellular task. Artificial organelles tackle the challenge of mimicking metabolism, which is the set of chemical reactions that occur within...... and Amplex Red substrates, which are specific for TRP and HRP, respectively. Conversion of the substrates into the respective fluorescent products is observed. This report on the first microreactor conducting multiple enzymatic reactions simultaneously inside a cell is a considerable step in the field...

  17. MALDI imaging of enzymatic degradation of glycerides by lipase on textile surface.

    Science.gov (United States)

    Hall-Andersen, Jonatan; Kaasgaard, Svend G; Janfelt, Christian

    2017-11-06

    Most modern laundry detergents contain enzymes such as proteases, amylases, and lipases for more efficient removal of stains containing proteins, carbohydrates, and lipids during wash at low temperature. The function of the lipases is to hydrolyse the hydrophobic triglycerides from fats and oils to the more hydrophilic lipids diglycerides, monoglycerides and free fatty acids. Here, we use MALDI imaging to study the effect of enzymatic degradation of triglycerides by lipases directly on the textile surface. Textile samples were created by using swatches of different textile blends, adding a lipid stain and simulating washing cycles using well-defined detergents with lipase concentrations ranging between 0 and 0.5ppm. After washing, the textile swatches as well as cryo-sections of the swatches were imaged using MALDI imaging in positive ion mode at pixel sizes of 15-75μm. Similar samples were imaged by DESI-MSI for comparison. Despite the rough surface and non-conductive nature of textile, MALDI imaging of glycerides on textile was readily possible. The results show extensive enzymatic degradation of triglycerides into diglycerides, and images suggest that this degradation takes place in a quite heterogeneous manner as also observed in images of cross-sections. DESI-imaging reveals the same kind of enzymatic degradation, but with a more homogeneous appearance. While the enzymatic degradation is exemplified in a few images, the overall degradations process was monitored by extraction of ion intensities from 298 individual ion masses of mono-, di- and triglycerides and free fatty acids. MALDI imaging of glycerides was possible directly from a textile surface, allowing visualization of the enzymatic degradation of fatty stains on textile during the laundry process. The images showed an inhomogeneous presence of diglycerides after lipase treatment both in planar images of the textile surface as well as in cross-sections suggesting a non-uniform enzyme effect or

  18. Moving towards a Competitive Fully Enzymatic Biodiesel Process

    Directory of Open Access Journals (Sweden)

    Silvia Cesarini

    2015-06-01

    Full Text Available Enzymatic biodiesel synthesis can solve several problems posed by the alkaline-catalyzed transesterification but it has the drawback of being too expensive to be considered competitive. Costs can be reduced by lipase improvement, use of unrefined oils, evaluation of soluble/immobilized lipase preparations, and by combination of phospholipases with a soluble lipase for biodiesel production in a single step. As shown here, convenient natural tools have been developed that allow synthesis of high quality FAMEs (EN14214 from unrefined oils in a completely enzymatic single-step process, making it fully competitive.

  19. Enzymatic network for production of ether amines from alcohols

    DEFF Research Database (Denmark)

    Palacio, Cyntia M.; Crismaru, Ciprian G.; Bartsch, Sebastian

    2016-01-01

    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 desired ether amines from the corresponding ether alcohols with inorganic ammonium as the only additional substrate. To examine conversion, individual and overall reaction equilibria were established. Using these data, it was found that the experimentally observed conversions of up to 60% observed...

  20. Enzymatic oxidation of substituted tryptamines catalysed by monoamine oxidase

    Directory of Open Access Journals (Sweden)

    Dragulska Sylwia

    2014-08-01

    Full Text Available The enzymatic deamination of 5-fl uorotryptamine and 5-hydroxytryptamine, 5-HT, catalysed by enzyme monoamine oxidase A (MAO-A, EC 1.4.3.4 was investigated using the kinetic (KIE and solvent (SIE isotope effects methods. The numerical values of deuterium isotope effects in the (1R positions of 5-F-tryptamine were determined using non-competitive spectrophotomeric method. Isotopologue 5-F-[(1R- -2H]-tryptamine, needed for kinetic studies was obtained by enzymatic decarboxylation of 5´-fl uoro-L-tryptophan, 5´-F-L-Trp, in fully deuteriated medium.

  1. 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

    The antifouling (AF) potential of hydrogen peroxide (H2O2) produced enzymatically in a coating containing starch, glucoamylase, and hexose oxidase was evaluated in a series of laboratory tests and in-sea field trials. Dissolved H2O2 inhibited bacterial biofilm formation by eight of nine marine...... 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...

  2. 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...

  3. Study on beta-galactosidase enzymatic activity of herbal yogurt.

    Science.gov (United States)

    Chowdhury, Banani Ray; Chakraborty, Runu; Raychaudhuri, Utpal

    2008-03-01

    Different types of herbal yogurts were developed by mixing standardized milk with pretreated herbs, namely tulsi leaf (Ocimum sanctum), pudina leaf (Mentha arvensis) and coriander leaf (Coriandrum sativum), with leaves separately and a 1:1 (v/v) mixture of the strains of lactic starter cultures---Lactobacillus acidophilus (NCIM 2903) and Lactobacillus plantarum (NCIM 2083)-followed by incubation at 40 degrees C for 6 h. The beta-galactosidase enzymatic activity of the abovementioned herbal yogurts was determined and interestingly noted to exhibit higher enzymatic activity compared with the control yogurt (without any herbs). Among all herbal yogurts, tulsi yogurt had the maximum beta-galactosidase activity.

  4. Particle size distribution of rice flour affecting the starch enzymatic hydrolysis and hydration properties.

    Science.gov (United States)

    de la Hera, Esther; Gomez, Manuel; Rosell, Cristina M

    2013-10-15

    Rice flour is becoming very attractive as raw material, but there is lack of information about the influence of particle size on its functional properties and starch digestibility. This study evaluates the degree of dependence of the rice flour functional properties, mainly derived from starch behavior, with the particle size distribution. Hydration properties of flours and gels and starch enzymatic hydrolysis of individual fractions were assessed. Particle size heterogeneity on rice flour significantly affected functional properties and starch features, at room temperature and also after gelatinization; and the extent of that effect was grain type dependent. Particle size heterogeneity on rice flour induces different pattern in starch enzymatic hydrolysis, with the long grain having slower hydrolysis as indicated the rate constant (k). No correlation between starch digestibility and hydration properties or the protein content was observed. It seems that in intact granules interactions with other grain components must be taken into account. Overall, particle size fractionation of rice flour might be advisable for selecting specific physico-chemical properties. Copyright © 2013. Published by Elsevier Ltd.

  5. Enzymatic Ligation of Large Biomolecules to DNA

    DEFF Research Database (Denmark)

    Sørensen, Rasmus Schøler; Okholm, Anders Hauge; Schaffert, David Henning

    2013-01-01

    The ability to synthesize, characterize, and manipulate DNA forms the foundation of a range of advanced disciplines including genomics, molecular biology, and biomolecular engineering. In particular for the latter field, DNA has proven useful as a structural or functional component in nanoscale s...

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

    African Journals Online (AJOL)

    sunny t

    2015-09-18

    Sep 18, 2015 ... bacterial and 14 fungi strains that presented positive lipolytic activity were obtained by detection through Rhodamine B Agar .... nutritive broth and 10 g of bacteriological agar in 450 ml of distilled water. The lipoidal emulsion .... amino acids (Alnahdi, 2012), were tested in accordance with their function in ...

  7. Research trends in radiobiology since 40 years. a new approach: the enzymatic repair function of DNA, internal factor in evolution of biological systems under irradiation; Etude des tendances des recherches en radiologie depuis 40 ans. Une nouvelle voie de recherche: la fonction de reparation enzymatique de l'ADN, facteur interne d'evolution des systemes biologiques sous rayonnement

    Energy Technology Data Exchange (ETDEWEB)

    Mouton, R. [Commissariat a l' Energie Atomique, Saclay (France). Centre d' Etudes Nucleaires

    1968-07-01

    In the first part of the report, the author attempts to draw an historical scheme of successive research working hypotheses in radiobiology since 1924. Less than a generation ago the effect of radiation exposure were viewed as being direct, immediate, irreparable and unmodifiable. Now it is generally accepted that radiation lesion can also be indirect, delayed, reparable and often modified with appropriate chemical or biochemical treatment. It was however in 1962-1964 that came the decisive breakthrough in radiobiology with the discovery that the cell possesses a natural active self-defense mechanism against whatever stress would affect the integrity of the genetic message contained in the DNA structure itself. The existence of what could be considered as a fourth DNA function i.e. self-repair by enzymatic action under genetic control-brings at least to radiobiology the missing molecular biology basis it needed to get out of its 'phenomenological night' after abandon of the generalization of Lea's theory through lack of experimental evidence. In the second part, which is a prospective one, the author tries to set an enlarged synthesis considering the possible role of DNA repair system not only in cell survival - in presence or absence of dose modifiers or mutagens - but also in the artificial and natural evolution of biological system exposed to sub-lethal doses of radiation. Most recent data from the literature fit well with what must be still considered as a general working hypothesis. Studies dealing with phenotypic and genotypic characters linked with the acquisition of gamma and UV radiation resistance in 'Escherichia coli K12' has been started by the author, in collaboration with O. Tremeau, in order to bring a new experimental contribution in this respect. (author) [French] Dans la premiere partie, l'auteur tente de retracer l'historique des hypotheses successives qui ont jalonne les avances de la radiobiologie depuis 1924

  8. Enzymatic Modification of Antioxidants Towards Omega-3 Oil Protection

    DEFF Research Database (Denmark)

    Yang, Zhiyong

    This PhD dissertation entitled “Enzymatic Modification of Antioxidants Towards Omega-3 Oil Protection” is primarily focused on synthesize of novel antioxidant from natural sources for better protection of oxidation-prone omega 3 oil. Selected phenolic acids were conjugated with fatty alcohols...

  9. Graphene-glucose oxidase bioanodes for enzymatic biofuel cells

    DEFF Research Database (Denmark)

    Tang, Jing; Werchmeister, Rebecka Maria Larsen; Engelbrekt, Christian

    2017-01-01

    Enzymatic biofuel cells (EBFCs) are electrochemical devices, that produce electricity from energy stored in fuel molecules under catalysis of enzymes. An EBFC contains a bioanode and/or a biocathode, in which enzymes are used to catalyse oxidation of fuel molecules such as sugars, and dioxygen...

  10. 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...

  11. Recombinant EXLX1 from Bacillus subtilis for enhancing enzymatic ...

    African Journals Online (AJOL)

    Recombinant EXLX1 from Bacillus subtilis for enhancing enzymatic hydrolysis of corn stover with low cellulase loadings. ... These results provided a feasible way for the potential application of BsEXLX1 in the efficient saccharification of cellulose materials for bioethanol production. Key word: Bacillus subtilis, BsEXLX1, ...

  12. Physiological and enzymatic changes in rice seeds stored at low ...

    African Journals Online (AJOL)

    magnolia lopes

    2015-08-05

    Aug 5, 2015 ... This study aimed to evaluate the effect of low temperatures on the physiological and enzymatic changes of rice seeds. The seeds were packed in airtight chambers and maintained at temperatures of 8 and -. 50°C for periods of 15, 30 , 45, 60, 75 and 90 days. The same procedure was adopted for the ...

  13. Non-Enzymatic Antioxidants and Nutritional Profiles in Newly ...

    African Journals Online (AJOL)

    Dr Femi Olaleye

    Full Length Research Article. Non-Enzymatic Antioxidants and. Nutritional Profiles in Newly Diagnosed. Pulmonary Tuberculosis Patients in Nigeria. Akiibinu M.O. 1. , Arinola O.G. 2*. , Ogunlewe J.O. 1. , and Onih E.A. 3. 1 Department of Chemical Pathology and Immunology, Obafemi Awolowo College of. Health Sciences.

  14. Wet explosion pretreatment of sugarcane bagasse for enhanced enzymatic hydrolysis

    DEFF Research Database (Denmark)

    Biswas, Rajib; Uellendahl, Hinrich; Ahring, Birgitte Kiær

    2014-01-01

    .7% of the theoretical maximum value. Pretreatment at 200 C with oxygen exhibited enhanced enzymatic efficiency but lower xylose recovery and formation of the degradation products such as acetate, furfural and HMF of 7.6, 3.3 and 1.0 g/L, respectively. In the hydrolysis, the total sugars (glucose + xylose) yielded...

  15. An efficient enzymatic synthesis of 5-aminovaleric acid

    NARCIS (Netherlands)

    Pukin, A.; Boeriu, C.G.; Scott, E.L.; Sanders, J.P.M.; Franssen, M.C.R.

    2010-01-01

    The title compound was prepared enzymatically from l-lysine in an excellent yield and under buffer-free conditions. l-Lysine was oxidized by the action of l-lysine a-oxidase from Trichoderma viride followed by spontaneous oxidative decarboxylation of the intermediate 6-amino-2-oxocaproic acid in the

  16. Influence of water availability on the enzymatic hydrolysis of proteins

    NARCIS (Netherlands)

    Butré, C.I.; Wierenga, P.A.; Gruppen, H.

    2014-01-01

    The overall rate of enzymatic protein hydrolysis decreases with increasing protein concentration (0.1–30% (w/v)) at constant enzyme/substrate ratio. To understand the role of water, the amount of available water was expressed as the ratio between free and bound water and experimentally determined

  17. Enzymatic activities of Dermatophilus congolensis measured by API ZYM.

    Science.gov (United States)

    Hermoso de Mendoza, J; Arenas, A; Alonso, J M; Rey, J M; Gil, M C; Anton, J M; Hermoso de Mendoza, M

    1993-10-01

    API ZYM kit was used to test enzymatic activities on eighteen strains of Dermatophilus congolensis. All strains produced lipase and acid phosphatase, which act on lipids, and leucine arylamidase which act on proteins. Another 10 exoenzymes were present in at least one of the strains.

  18. Physiological and enzymatic changes in rice seeds stored at low ...

    African Journals Online (AJOL)

    This study aimed to evaluate the effect of low temperatures on the physiological and enzymatic changes of rice seeds. The seeds were packed in airtight chambers and maintained at temperatures of 8 and -50°C for periods of 15, 30 , 45, 60, 75 and 90 days. The same procedure was adopted for the control treatment with ...

  19. Factors Impeding Enzymatic Wheat Gluten Hydrolysis at High Solid Concentrations

    NARCIS (Netherlands)

    Hardt, N.A.; Janssen, A.E.M.; Boom, R.M.; Goot, van der A.J.

    2014-01-01

    Enzymatic wheat gluten hydrolysis at high solid concentrations is advantageous from an environmental and economic point of view. However, increased wheat gluten concentrations result in a concentration effect with a decreased hydrolysis rate at constant enzyme-to-substrate ratios and a decreased

  20. A comparison of acidic and enzymatic hydrolysis of rutin | Wang ...

    African Journals Online (AJOL)

    Rutin and its hydrolysis products (isoquercitrin and quercetin) are widely used as important materials in food and pharmaceutical industry. In this study, the effects of various acids and enzymes as catalysts on the hydrolysis reaction of rutin were studied. In comparison with acidic and enzymatic catalysis of rutin, the research ...

  1. Enzymatic Browning in Sugar Beet Leaves (Beta vulgaris L.)

    NARCIS (Netherlands)

    Vissers, Anne; Kiskini, Alexandra; Hilgers, Roelant; Marinea, Marina; Wierenga, Peter Alexander; Gruppen, Harry; Vincken, Jean Paul

    2017-01-01

    Sugar beet (Beta vulgaris L.) leaves of 8 month (8m) plants showed more enzymatic browning than those of 3 month (3m). Total phenolic content increased from 4.6 to 9.4 mg/g FW in 3m and 8m, respectively, quantitated by

  2. Validation of Orchestia gammarellus enzymatic activities in several ...

    African Journals Online (AJOL)

    Validation of Orchestia gammarellus enzymatic activities in several sites of Tangier's bay (Morocco. ... Abstract. Marine biodiversity is increasingly at risk because of coastal contamination. Biomarkers of pollutant exposure can be very useful for marine biodiversity conservation. The aim of this research was to validate the ...

  3. Optimization of peptide production by enzymatic hydrolysis of tuna ...

    African Journals Online (AJOL)

    Optimization of peptide production by enzymatic hydrolysis of tuna dark muscle by-product using commercial proteases. ... The PDF file you selected should load here if your Web browser has a PDF reader plug-in installed (for example, a recent version of Adobe Acrobat Reader). If you would like more information about ...

  4. Non-enzymatic Polymerization of Nucleic Acids from Monomers

    DEFF Research Database (Denmark)

    Dörr, Mark; Löffler, Philipp M. G.; Monnard, Pierre-Alain

    2012-01-01

    This review deals with the state-of-the-art techniques in non-enzymatic nucleic acid condensation from monomers. In particular, the procedures called \\emph{monomer self-condensation} and \\emph{template-directed monomer condensation} are described, which have been developed to achieve efficient...

  5. Diversity and enzymatic characterization of Bacillus species isolated ...

    African Journals Online (AJOL)

    species. Enzymatic activity of different species identified was carried out with API ZYM system. Based on the. 16S r DNA sequence analysis, seven species of Bacillus (Bacillus subtilis, Bacillus cereus, Bacillus pumilus,. Bacillus amyloliquefaciens, Bacillus methylotrophicus, Bacillus vallismortis and Bacillus toyonensis) were.

  6. The Preparation and Enzymatic Hydrolysis of a Library of Esters

    Science.gov (United States)

    Sanford, Elizabeth M.; Smith, Traci L.

    2008-01-01

    An investigative case study involving the preparation of a library of esters using Fischer esterification and alcoholysis of acid chlorides and their subsequent enzymatic hydrolysis by pig liver esterase and orange peel esterase is described. Students work collaboratively to prepare and characterize the library of esters and complete and evaluate…

  7. Enzymatic Profiles of Activated Sludge from a Wastewater Treatment ...

    African Journals Online (AJOL)

    Activated sludge samples collected from a treatment plant, with foaming experience in the month of July, was characterized enzymatically. Hexokinase, Glyceraldehyde-3-phosphate dehydrogenase and Adenylate kinase activity assays were conducted before, during and after the foaming episode. The spectrum of enzyme ...

  8. Enzymatic saccharification of some agro-industrial cellulosic wastes ...

    African Journals Online (AJOL)

    Enzymatic saccharification of some agro-industrial cellulosic wastes by cellulose produced from a mixed culture of Aspergillus Niger and Saccharomyces ... All the sorghum pomace media recorded significantly (P<0.05) higher level of cellulase enzyme (2.06-4.06 units/ml) than that of carboxymethyl-cellulose medium (1.72 ...

  9. 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 ...

  10. Enzymatic Breakdown of Type II Collagen in the Human Vitreous

    NARCIS (Netherlands)

    van Deemter, Marielle; Pas, Hendri H.; Kuijer, Roel; van der Worp, Roelofje J.; Hooymans, Johanna M. M.; Los, Leonoor I.

    2009-01-01

    PURPOSE. To investigate whether enzymatic collagen breakdown is an active process in the human vitreous. METHODS. Human donor eyes were used for immunohistochemistry to detect the possible presence of the matrix metalloproteinase (MMP)-induced type II collagen breakdown product col2-3/4C-short in

  11. 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 ...

  12. Short-time ultrasonication treatment in enzymatic hydrolysis of biomass

    Science.gov (United States)

    Zengqian Shi; Zhiyong Cai; Siqun Wang; Qixin Zhong; Joseph J. Bozell

    2013-01-01

    To improve the conversion of enzymatic hydrolysis of biomass in an energy-efficient manner, two shorttime ultrasonication strategies were applied on six types of biomass with different structures and components. The strategies include pre-sonication before the hydrolysis and intermittent sonication during the ongoing hydrolysis. The microstructures of each type of...

  13. A Multiplex Enzymatic Machinery for Cellular Protein S-nitrosylation.

    Science.gov (United States)

    Seth, Divya; Hess, Douglas T; Hausladen, Alfred; Wang, Liwen; Wang, Ya-Juan; Stamler, Jonathan S

    2018-02-01

    S-nitrosylation, the oxidative modification of Cys residues by nitric oxide (NO) to form S-nitrosothiols (SNOs), modifies all main classes of proteins and provides a fundamental redox-based cellular signaling mechanism. However, in contrast to other post-translational protein modifications, S-nitrosylation is generally considered to be non-enzymatic, involving multiple chemical routes. We report here that endogenous protein S-nitrosylation in the model organism E. coli depends principally upon the enzymatic activity of the hybrid cluster protein Hcp, employing NO produced by nitrate reductase. Anaerobiosis on nitrate induces both Hcp and nitrate reductase, thereby resulting in the S-nitrosylation-dependent assembly of a large interactome including enzymes that generate NO (NO synthase), synthesize SNO-proteins (SNO synthase), and propagate SNO-based signaling (trans-nitrosylases) to regulate cell motility and metabolism. Thus, protein S-nitrosylation by NO in E. coli is essentially enzymatic, and the potential generality of the multiplex enzymatic mechanism that we describe may support a re-conceptualization of NO-based cellular signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Enzymatic conversion of lignocellulose into fermentable sugars

    DEFF Research Database (Denmark)

    Jørgensen, Henning; Kristensen, Jan Bach; Felby, Claus

    2007-01-01

    The economic dependency on fossil fuels and the resulting effects on climate and environment have put tremendous focus on utilizing fermentable sugars from lignocellulose, the largest known renewable carbohydrate source. The fermentable sugars in lignocellulose are derived from cellulose...... into fermentable sugars requires a number of different cellulases and hemicellulases. The hydrolysis of cellulose is a sequential breakdown of the linear glucose chains, whereas hemicellulases must be capable of hydrolysing branched chains containing different sugars and functional groups. The technology...

  15. Structural basis and enzymatic mechanism of the biosynthesis of C9- from C10-monoterpenoid indole alkaloids.

    Science.gov (United States)

    Yang, Liuqing; Hill, Marco; Wang, Meitian; Panjikar, Santosh; Stöckigt, Joachim

    2009-01-01

    Cutting carbons: The three-dimensional structure of polyneuridine aldehyde esterase (PNAE) gives insight into the enzymatic mechanism of the biosynthesis of C(9)- from C(10)-monoterpenoid indole alkaloids (see scheme). PNAE is a very substrate-specific serine esterase. It harbors the catalytic triad S87-D216-H244, and is a new member of the alpha/beta-fold hydrolase superfamily. Its novel function leads to the diversification of alkaloid structures.

  16. Enzymatic reaction paths as determined by transition path sampling

    Science.gov (United States)

    Masterson, Jean Emily

    Enzymes are biological catalysts capable of enhancing the rates of chemical reactions by many orders of magnitude as compared to solution chemistry. Since the catalytic power of enzymes routinely exceeds that of the best artificial catalysts available, there is much interest in understanding the complete nature of chemical barrier crossing in enzymatic reactions. Two specific questions pertaining to the source of enzymatic rate enhancements are investigated in this work. The first is the issue of how fast protein motions of an enzyme contribute to chemical barrier crossing. Our group has previously identified sub-picosecond protein motions, termed promoting vibrations (PVs), that dynamically modulate chemical transformation in several enzymes. In the case of human heart lactate dehydrogenase (hhLDH), prior studies have shown that a specific axis of residues undergoes a compressional fluctuation towards the active site, decreasing a hydride and a proton donor--acceptor distance on a sub-picosecond timescale to promote particle transfer. To more thoroughly understand the contribution of this dynamic motion to the enzymatic reaction coordinate of hhLDH, we conducted transition path sampling (TPS) using four versions of the enzymatic system: a wild type enzyme with natural isotopic abundance; a heavy enzyme where all the carbons, nitrogens, and non-exchangeable hydrogens were replaced with heavy isotopes; and two versions of the enzyme with mutations in the axis of PV residues. We generated four separate ensembles of reaction paths and analyzed each in terms of the reaction mechanism, time of barrier crossing, dynamics of the PV, and residues involved in the enzymatic reaction coordinate. We found that heavy isotopic substitution of hhLDH altered the sub-picosecond dynamics of the PV, changed the favored reaction mechanism, dramatically increased the time of barrier crossing, but did not have an effect on the specific residues involved in the PV. In the mutant systems

  17. Prediction and characterization of enzymatic activities guided by sequence similarity and genome neighborhood networks.

    Science.gov (United States)

    Zhao, Suwen; Sakai, Ayano; Zhang, Xinshuai; Vetting, Matthew W; Kumar, Ritesh; Hillerich, Brandan; San Francisco, Brian; Solbiati, Jose; Steves, Adam; Brown, Shoshana; Akiva, Eyal; Barber, Alan; Seidel, Ronald D; Babbitt, Patricia C; Almo, Steven C; Gerlt, John A; Jacobson, Matthew P

    2014-06-30

    Metabolic pathways in eubacteria and archaea often are encoded by operons and/or gene clusters (genome neighborhoods) that provide important clues for assignment of both enzyme functions and metabolic pathways. We describe a bioinformatic approach (genome neighborhood network; GNN) that enables large scale prediction of the in vitro enzymatic activities and in vivo physiological functions (metabolic pathways) of uncharacterized enzymes in protein families. We demonstrate the utility of the GNN approach by predicting in vitro activities and in vivo functions in the proline racemase superfamily (PRS; InterPro IPR008794). The predictions were verified by measuring in vitro activities for 51 proteins in 12 families in the PRS that represent ∼85% of the sequences; in vitro activities of pathway enzymes, carbon/nitrogen source phenotypes, and/or transcriptomic studies confirmed the predicted pathways. The synergistic use of sequence similarity networks3 and GNNs will facilitate the discovery of the components of novel, uncharacterized metabolic pathways in sequenced genomes.

  18. Dynamic Simulation, Sensitivity and Uncertainty Analysis of a Demonstration Scale Lignocellulosic Enzymatic Hydrolysis Process

    DEFF Research Database (Denmark)

    Prunescu, Remus Mihail; Sin, Gürkan

    2014-01-01

    This study presents the uncertainty and sensitivity analysis of a lignocellulosic enzymatic hydrolysis model considering both model and feed parameters as sources of uncertainty. The dynamic model is parametrized for accommodating various types of biomass, and different enzymatic complexes...

  19. Comparison of dilute mineral and organic acid pretreatment for enzymatic hydrolysis of wheat straw

    NARCIS (Netherlands)

    Kootstra, A.M.J.; Beeftink, H.H.; Scott, E.L.; Sanders, J.P.M.

    2009-01-01

    The efficiencies of fumaric, maleic, and sulfuric acid in wheat straw pretreatment were compared. As a measure for pretreatment efficiency, enzymatic digestibility of the lignocellulose was determined. Monomeric glucose and xylose concentrations were measured after subsequent enzymatic hydrolysis,

  20. Effect of enzymatic hydrolysis on native starch granule structure.

    Science.gov (United States)

    Blazek, Jaroslav; Gilbert, Elliot Paul

    2010-12-13

    Enzymatic digestion of six starches of different botanical origin was studied in real time by in situ time-resolved small-angle neutron scattering (SANS) and complemented by the analysis of native and digested material by X-ray diffraction, differential scanning calorimetry, small-angle X-ray scattering, and scanning electron microscopy with the aim of following changes in starch granule nanostructure during enzymatic digestion. This range of techniques enables coverage over five orders of length-scale, as is necessary for this hierarchically structured material. Starches studied varied in their digestibility and displayed structural differences in the course of enzymatic digestion. The use of time-resolved SANS showed that solvent-drying of digested residues does not induce any structural artifacts on the length scale followed by small-angle scattering. In the course of digestion, the lamellar peak intensity gradually decreased and low-q scattering increased. These trends were more substantial for A-type than for B-type starches. These observations were explained by preferential digestion of the amorphous growth rings. Hydrolysis of the semicrystalline growth rings was explained on the basis of a liquid-crystalline model for starch considering differences between A-type and B-type starches in the length and rigidity of amylopectin spacers and branches. As evidenced by differing morphologies of enzymatic attack among varieties, the existence of granular pores and channels and physical penetrability of the amorphous growth ring affect the accessibility of the enzyme to the substrate. The combined effects of the granule microstructure and the nanostructure of the growth rings influence the opportunity of the enzyme to access its substrate; as a consequence, these structures determine the enzymatic digestibility of granular starches more than the absolute physical densities of the amorphous growth rings and amorphous and crystalline regions of the semicrystalline

  1. Molecular dynamics study of enhanced Man5B enzymatic activity.

    Science.gov (United States)

    Bernardi, Rafael C; Cann, Isaac; Schulten, Klaus

    2014-01-01

    Biofuels are a well-known alternative to the largely used fossil-derived fuels, however the competition with food production is an ethical dilemma. Fortunately a solution is offered by second-generation biofuels which can be produced from agricultural waste or, more specifically, from plant cell wall polysaccharides. The conversion process involves typically enzymatic hydrolysis of lignocellulosic biomass and then separation of its constituent sugars that are further fermented to produce ethanol. Over the years several technologies have been developed that allow this conversion process to occur and the objective is now to make this process cost-competitive in today's markets. We observe that reduction of enzymatic efficiency in the presence of gluco-oligosaccharides is associated with a loss of the enzyme's flexibility, the latter being required to bind new substrate, while the presence of manno-oligosaccharides does not pose this problem. Molecular dynamics simulations identify key contacts between substrates and the enzyme catalytic pocket that might be modified through site-directed mutagenesis to prevent loss of enzymatic efficiency. Based on previous experimental studies and the new molecular dynamics data, we suggest that cellohexaose in the active site pocket slows down or even inhibits Man5B enzymatic activity. The assumption of such a mechanism is reasonable since when the gluco-oligosaccharide substrate is attached to the catalytic pocket it takes much longer to leave the pocket and thus prevents other substrates from reaching the active site. The insight is of crucial importance since the inhibition of enzymes by the enzymatic product or by an unsuitable substrate is a major technological problem in reducing the competitiveness of second-generation biofuel production.

  2. Integration of molecular and enzymatic catalysts on graphene for biomimetic generation of antithrombotic species

    Science.gov (United States)

    Xue, Teng; Peng, Bo; Xue, Min; Zhong, Xing; Chiu, Chin-Yi; Yang, Si; Qu, Yongquan; Ruan, Lingyan; Jiang, Shan; Dubin, Sergey; Kaner, Richard B.; Zink, Jeffrey I.; Meyerhoff, Mark E.; Duan, Xiangfeng; Huang, Yu

    2014-02-01

    The integration of multiple synergistic catalytic systems can enable the creation of biocompatible enzymatic mimics for cascading reactions under physiologically relevant conditions. Here we report the design of a graphene-haemin-glucose oxidase conjugate as a tandem catalyst, in which graphene functions as a unique support to integrate molecular catalyst haemin and enzymatic catalyst glucose oxidase for biomimetic generation of antithrombotic species. Monomeric haemin can be conjugated with graphene through π-π interactions to function as an effective catalyst for the oxidation of endogenous L-arginine by hydrogen peroxide. Furthermore, glucose oxidase can be covalently linked onto graphene for local generation of hydrogen peroxide through the oxidation of blood glucose. Thus, the integrated graphene-haemin-glucose oxidase catalysts can readily enable the continuous generation of nitroxyl, an antithrombotic species, from physiologically abundant glucose and L-arginine. Finally, we demonstrate that the conjugates can be embedded within polyurethane to create a long-lasting antithrombotic coating for blood-contacting biomedical devices.

  3. Relationship between hyperglycemia, antioxidant capacity and some enzymatic and non-enzymatic antioxidants in African patients with type 2 diabetes.

    Science.gov (United States)

    Pieme, Constant Anatole; Tatangmo, Jérôme Antony; Simo, Gustave; Biapa Nya, Prosper Cabral; Ama Moor, Vicky Jocelyne; Moukette Moukette, Bruno; Tankeu Nzufo, Francine; Njinkio Nono, Borgia Legrand; Sobngwi, Eugene

    2017-03-29

    Studies demonstrate that free radicals are involved in the pathogenesis of diabetic complications. The aim of this study was to determine the implication of total antioxidant capacity (TAC) and some enzymatic and non-enzymatic antioxidants as suitable biomarkers of diabetic complications risk factors. A total of 90 patients (70 patients with or without diabetic complications +20 normal healthy) were examined by evaluating the level of lipid peroxidation, nitrogen monoxide (NO), fasting blood glucose, glycated haemoglobin (HbA1c), enzymatic and non-enzymatic antioxidants using standard spectrophotometric methods. The fasting blood glucose and HbA1c levels were respectively 2.05 and 2.32 times higher in the group of patients with diabetes and complications (DPWC) compared to those of healthy persons. A statistically higher level of malondialdehyde (MDA), NO and TAC was observed in a group of patients with diabetes and complications compared to those without complications (DPNC). A significant positive correlation was found between catalase (CAT) and fasting blood glucose while a significant and negative correlation was noted between reduced glutathione (GSH) and fasting blood glucose. Also was noted a significant relationship between HbA1c and other markers of oxidative stress. The results suggest that the plasma levels of CAT, TAC and reduced glutathione could give information on the risk of developing complications of diabetes, considering that the modification of these biomarkers levels were associated with oxidative stress.

  4. Stimulation and inhibition of enzymatic hydrolysis by organosolv lignins as determined by zeta potential and hydrophobicity

    Science.gov (United States)

    Yang Huang; Shaolong Sun; Chen Huang; Qiang Yong; Thomas Elder; Maobing Tu

    2017-01-01

    Background: Lignin typically inhibits enzymatic hydrolysis of cellulosic biomass, but certain organosolv lignins or lignosulfonates enhance enzymatic hydrolysis. The hydrophobic and electrostatic interactions between lignin and cellulases play critical roles in the enzymatic hydrolysis process. However, how to incorporate these two...

  5. Characterization of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties.

    Science.gov (United States)

    Mertsalov, Ilya B; Novikov, Boris N; Scott, Hilary; Dangott, Lawrence; Panin, Vladislav M

    2016-07-01

    CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CSASs that normally localize in the nucleus, Drosophila melanogaster CSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterized its activity in vitro Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn(2+), Fe(2+), Co(2+) and Mn(2+), whereas the activity with Mg(2+) was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in co-ordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulate Drosophila sialylation, thus modulating neural transmission. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

  6. Characterisation of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties

    Science.gov (United States)

    Mertsalov, Ilya B.; Novikov, Boris N.; Scott, Hilary; Dangott, Lawrence; Panin, Vladislav M.

    2016-01-01

    CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CMP-Sia synthetases that normally localize in the nucleus, Drosophila melanogaster CSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterised its activity in vitro. Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn2+, Fe2+, Co2+ and Mn2+, while the activity with Mg2+ was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in coordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulate Drosophila sialylation, thus modulating neural transmission. PMID:27114558

  7. The evolution of photosynthetic capacity and the antioxidant enzymatic system during acclimatization of micropropagated Calathea plants.

    Science.gov (United States)

    Van Huylenbroeck JM; Piqueras; Debergh

    2000-06-12

    The effects of an increased PPFD on photosynthesis, the functioning of the photosynthetic apparatus and the response of the antioxidant enzymatic system were studied during the ex vitro establishment of micropropagated Calathea 'Maui Queen' plantlets. Measured chlorophyll and carotenoids contents in ex vitro formed leaves were almost three times higher compared to the in vitro formed ones. At the end of the acclimatization, an inverse relation between PPFD and the chlorophyll (a+b)/carotenoids ratio was observed. During the first days after transplantation Calathea plants are not photosynthetically active, as is illustrated by the photosynthetic light response curves. With the appearance of new leaves, higher photosynthetic capacities were observed and light saturation point increased (days 17 and 25). Also the maximal photosynthetic efficiency enlarged as shown by the increased initial slope of the curves. F(v)/F(m) decreased directly after transplantation of the micropropagated plantlets, afterwards a recovery was observed, but highest F(v)/F(m) values were observed in low light (LL) plants. The photochemical quenching coefficient increased gradually during the first two weeks of the acclimatization. In high light (HL) plants, q(P) decreased directly after transfer, while this was not observed in LL and medium light (ML). During the acclimatization period to increasing light intensities significant changes in the activity of the antioxidant enzymatic system were observed. A decrease in superoxide dismutase (SOD) activity was measured during the first half of the acclimatization period followed by a recovery in ML and HL plants by day 35. Dehydroascorbate reductase (DHAR) activity decreased during acclimatization. At the end of the experimental period the lowest levels were measured in ML plants. Catalase (CAT) activity increased significantly during the first two weeks after transfer, a clear inverse relationship to PPFD was detected. The relation between the

  8. Aza‐Michael addition reaction: Post‐polymerization modification and preparation of PEI/PEG‐based polyester hydrogels from enzymatically synthesized reactive polymers

    DEFF Research Database (Denmark)

    Hoffmann, Christian; Stuparu, Mihaiela C.; Daugaard, Anders Egede

    2015-01-01

    The utility of aza‐Michael addition chemistry for post‐polymerization functionalization of enzymatically prepared polyesters is established. For this, itaconate ester and oligoethylene glycol are selected as monomers. A Candida Antarctica lipase B catalyzed polycondensation reaction between the t...

  9. Enzymatic Cellulose Palmitate Synthesis Using Immobilized Lipase

    Directory of Open Access Journals (Sweden)

    Anna Roosdiana

    2017-06-01

    Full Text Available Bacterial cellulose can be modified by esterification using palmitic acid and Mucor miehei  lipase  as catalyst. The purpose of this research was to determine the optimum conditions of esterification reaction of cellulose and palmitic acid . The esterification reaction was carried out at the time variation  of  6, 12, 18, 24 and 30 hours and the mass ratio of cellulose: palmitic acid (1: 11: 2, 1: 3, 1: 4, 1: 5,1:6 at 50 °C. The   cellulose palmitate  was examined  its  physical and chemical properties by using FTIR spectrophotometer, XRD, bubble point test and saponification  apparatus. The results showed that the optimum reaction time of esterification reaction of cellulose and palmitic acid occurred within 24 hours and the mass ratio of cellulose: palmitic acid was 1: 3 resulting in DS of  0.376 with  swelling index of 187 %, crystallinity index of 61.95%,  and Φ porous of 2.40 μm. Identification of functional groups using FTIR spectrophotometer showed that C=O ester group  was observed at 1737.74 cm-1 and strengthened  by  the appearance of C-O ester peak at 1280 cm-1. The conclusion of this study is reaction time and reactant ratio influence significantly the DS of cellulose ester.

  10. Solvent viscosity dependence for enzymatic reactions

    CERN Document Server

    Sitnitsky, A E

    2008-01-01

    A mechanism for relationship of solvent viscosity with reaction rate constant at enzyme action is suggested. It is based on fluctuations of electric field in enzyme active site produced by thermally equilibrium rocking (cranckshaft motion) of the rigid plane (in which the dipole moment $\\approx 3.6 D$ lies) of a favourably located and oriented peptide group (or may be a few of them). Thus the rocking of the plane leads to fluctuations of the electric field of the dipole moment. These fluctuations can interact with the reaction coordinate because the latter in its turn has transition dipole moment due to separation of charges at movement of the reacting system along it. The rocking of the plane of the peptide group is sensitive to the microviscosity of its environment in protein interior and the latter is a function of the solvent viscosity. Thus we obtain an additional factor of interrelationship for these characteristics with the reaction rate constant. We argue that due to the properties of the cranckshaft ...

  11. Amyloglucosidase enzymatic reactivity inside lipid vesicles

    Directory of Open Access Journals (Sweden)

    Kim Jin-Woo

    2007-10-01

    Full Text Available Abstract Efficient functioning of enzymes inside liposomes would open new avenues for applications in biocatalysis and bioanalytical tools. In this study, the entrapment of amyloglucosidase (AMG (EC 3.2.1.3 from Aspergillus niger into dipalmitoylphosphatidylcholine (DPPC multilamellar vesicles (MLVs and large unilamellar vesicles (LUVs was investigated. Negative-stain, freeze-fracture, and cryo-transmission electron microscopy images verified vesicle formation in the presence of AMG. Vesicles with entrapped AMG were isolated from the solution by centrifugation, and vesicle lamellarity was identified using fluorescence laser confocal microscopy. The kinetics of starch hydrolysis by AMG was modeled for two different systems, free enzyme in aqueous solution and entrapped enzyme within vesicles in aqueous suspension. For the free enzyme system, intrinsic kinetics were described by a Michaelis-Menten kinetic model with product inhibition. The kinetic constants, Vmax and Km, were determined by initial velocity measurements, and Ki was obtained by fitting the model to experimental data of glucose concentration-time curves. Predicted concentration-time curves using these kinetic constants were in good agreement with experimental measurements. In the case of the vesicles, the time-dependence of product (glucose formation was experimentally determined and simulated by considering the kinetic behavior of the enzyme and the permeation of substrate into the vesicle. Experimental results demonstrated that entrapped enzymes were much more stable than free enyzme. The entrapped enzyme could be recycled with retention of 60% activity after 3 cycles. These methodologies can be useful in evaluating other liposomal catalysis operations.

  12. Aiming for the complete utilization of sugar-beet pulp: Examination of the effects of mild acid and hydrothermal pretreatment followed by enzymatic digestion

    Directory of Open Access Journals (Sweden)

    Gruppen Harry

    2011-05-01

    Full Text Available Abstract Background Biomass use for the production of bioethanol or platform chemicals requires efficient breakdown of biomass to fermentable monosaccharides. Lignocellulosic feedstocks often require physicochemical pretreatment before enzymatic hydrolysis can begin. The optimal pretreatment can be different for different feedstocks, and should not lead to biomass destruction or formation of toxic products. Methods We examined the influence of six mild sulfuric acid or water pretreatments at different temperatures on the enzymatic degradability of sugar-beet pulp (SBP. Results We found that optimal pretreatment at 140°C of 15 minutes in water was able to solubilize 60% w/w of the total carbohydrates present, mainly pectins. More severe treatments led to the destruction of the solubilized sugars, and the subsequent production of the sugar-degradation products furfural, hydroxymethylfurfural, acetic acid and formic acid. The pretreated samples were successfully degraded enzymatically with an experimental cellulase preparation. Conclusions In this study, we found that pretreatment of SBP greatly facilitated the subsequent enzymatic degradation within economically feasible time ranges and enzyme levels. In addition, pretreatment of SBP can be useful to fractionate functional ingredients such as arabinans and pectins from cellulose. We found that the optimal combined severity factor to enhance the enzymatic degradation of SBP was between log R'0 = -2.0 and log R'0 = -1.5. The optimal pretreatment and enzyme treatment solubilized up to 80% of all sugars present in the SBP, including ≥90% of the cellulose.

  13. Chemicals effect on the enzymatic digestibility of rape straw over the thermo-mechanical pretreatment using a continuous twin screw-driven reactor (CTSR).

    Science.gov (United States)

    Um, Byung-Hwan; Choi, Chang Ho; Oh, Kyeong Keun

    2013-02-01

    Rape straw pretreated by a continuous twin screw-driven reactor (CTSR) with hot water presented a distinctive particle-size distribution profile as a function of the operating temperature. The relative amount of finer particle size dramatically increased as the ratio of solid to liquid was increased. Size reduction through physical CTSR process effectively promoted the enzymatic hydrolysis of pretreated rape straw. Meanwhile, the crystallinity of the physically pretreated straw was not a greater factor affecting the enzyme digestibility. The glucose conversion from the enzymatic hydrolysis of the straw pretreated by CTSR with hot water was maximized at 52%. Using the chemicals as catalyst have affected considerably for increasing the digestibility at same condition with hot water pretreatment. The enzymatic digestibilities of the straw pretreated by CTSR with sodium hydroxide and sulfuric acid were 60% and 77%, respectively. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. Activity profiling of ectomycorrhiza communities in two forest soils using multiple enzymatic tests.

    Science.gov (United States)

    Courty, Pierre-Emmanuel; Pritsch, Karin; Schloter, Michael; Hartmann, Anton; Garbaye, Jean

    2005-07-01

    Data on the diversity and distribution of enzyme activities in native ectomycorrhizal (ECM) communities are inadequate. A microplate multiple enzymatic test was developed which makes it possible to measure eight enzyme activities on 14 individual, excised ECM root tips. Hydrolytic and oxidative enzymes are involved in the decomposition of lignocellulose, chitin and phosphorus-containing organic compounds. This test system was used to describe the functional diversity of ECM communities in two forest sites. This set of tests proved to be accurate and sensitive enough to reveal a high diversity of activity profiles, depending on the fungal symbiont and the soil horizon. Ectomycorrhizas can be classified into specialists and generalists, and appear to complement each other in the same horizon to collectively perform all eight activities studied. By including a higher number of different assays for more detailed analyses, ECM activity profiling will provide a valuable tool for studying the functional diversity of ECM communities.

  15. Enzymatic formation of carbohydrate rings catalyzed by single-walled carbon nanotubes.

    Science.gov (United States)

    Hyun, Moon Seop; Park, Jong Pil; Seo, Dongkyun; Chang, Sung-Jin; Lee, Seok Jae; Lee, Sang Yup; Kwak, Kyungwon; Park, Tae Jung

    2016-05-01

    Macrocyclic carbohydrate rings were formed via enzymatic reactions around single-walled carbon nanotubes (SWNTs) as a catalyst. Cyclodextrin glucanotransferase, starch substrate and SWNTs were reacted in buffer solution to yield cyclodextrin (CD) rings wrapped around individual SWNTs. Atomic force microscopy showed the resulting complexes to be rings of 12-50 nm in diameter, which were highly soluble and dispersed in aqueous solution. They were further characterized by Raman and Fourier transform infrared spectroscopy and molecular simulation using density functional theory calculation. In the absence of SWNT, hydrogen bonding between glucose units determines the structure of maltose (the precursor of CD) and produces the curvature along the glucose chain. Wrapping SWNT along the short axis was preferred with curvature in the presence of SWNTs and with the hydrophobic interactions between the SWNTs and CD molecules. This synthetic approach may be useful for the functionalization of carbon nanotubes for development of nanostructures.

  16. Impacts of silver nanoparticles on performance and microbial community and enzymatic activity of a sequencing batch reactor.

    Science.gov (United States)

    Xu, Qiaoyan; Li, Shanshan; Wan, Yiping; Wang, Sen; Ma, Bingrui; She, Zonglian; Guo, Liang; Gao, Mengchun; Zhao, Yangguo; Jin, Chunji; Dong, Junwei; Li, Zhiwei

    2017-12-15

    The performance, microbial community and enzymatic activity of a sequencing batch reactor (SBR) were evaluated under silver nanoparticles (Ag NPs) stress. Over 5 mg/L Ag NPs inhibited the COD and phosphorus removals, whereas the NH4(+) removal kept stable during the whole operational period. The organic matter, nitrogen and phosphorus removal rates were obviously inhibited under Ag NPs stress, which showed similar varying trends with the corresponding microbial enzymatic activities. The change of Ag content in the activated sludge indicated that some Ag NPs were absorbed by the sludge. The presence of Ag NPs promoted the increase of reactive oxygen species (ROS) and lactate dehydrogenase (LDH) of microorganism due to the microbial response to the Ag NPs toxicity, which could impact on the microbial morphology and physiological functions. The presence of Ag NPs could produce some evident changes in the microbial community. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Novel investigation of enzymatic biodiesel reaction by isothermal calorimetry

    DEFF Research Database (Denmark)

    Søtoft, Lene Fjerbaek; Westh, Peter; Christensen, Knud V.

    2010-01-01

    Isothermal calorimetry (ITC) was used to investigate solvent-free enzymatic biodiesel production. The transesterification of rapeseed oil with methanol and ethanol was catalyzed by immobilized lipase Novozym 435 at 40 °C. The aim of the study was to determine reaction enthalpy for the enzymatic...... transesterification and to elucidate the mass transfer and energetic processes taking place. Based on the measured enthalpy and composition change in the system, the heat of reaction at 40 °C for the two systems was determined as −9.8 ± 0.9 kJ/mole biodiesel formed from rapeseed oil and methanol, and −9.3 ± 0.7 k...

  18. On-chip enzymatic microbiofuel cell-powered integrated circuits.

    Science.gov (United States)

    Mark, Andrew G; Suraniti, Emmanuel; Roche, Jérôme; Richter, Harald; Kuhn, Alexander; Mano, Nicolas; Fischer, Peer

    2017-05-16

    A variety of diagnostic and therapeutic medical technologies rely on long term implantation of an electronic device to monitor or regulate a patient's condition. One proposed approach to powering these devices is to use a biofuel cell to convert the chemical energy from blood nutrients into electrical current to supply the electronics. We present here an enzymatic microbiofuel cell whose electrodes are directly integrated into a digital electronic circuit. Glucose oxidizing and oxygen reducing enzymes are immobilized on microelectrodes of an application specific integrated circuit (ASIC) using redox hydrogels to produce an enzymatic biofuel cell, capable of harvesting electrical power from just a single droplet of 5 mM glucose solution. Optimisation of the fuel cell voltage and power to match the requirements of the electronics allow self-powered operation of the on-board digital circuitry. This study represents a step towards implantable self-powered electronic devices that gather their energy from physiological fluids.

  19. Biological activity of camel milk casein following enzymatic digestion.

    Science.gov (United States)

    Salami, Maryam; Moosavi-Movahedi, Ali Akbar; Moosavi-Movahedi, Faezeh; Ehsani, Mohammad Reza; Yousefi, Reza; Farhadi, Mohammad; Niasari-Naslaji, Amir; Saboury, Ali Akbar; Chobert, Jean-Marc; Haertlé, Thomas

    2011-11-01

    The aim of this study was to investigate the effects of enzymatic hydrolysis with digestive enzymes of camel whole casein and beta-casein (β-CN) on their antioxidant and Angiotensin Converting Enzyme (ACE)-inhibitory properties. Peptides in each hydrolysate were fractionated with ultra-filtration membranes. The antioxidant activity was determined using a Trolox equivalent antioxidant capacity (TEAC) scale. After enzymatic hydrolysis, both antioxidant and ACE-inhibitory activities of camel whole casein and camel β-CN were enhanced. Camel whole casein and β-CN showed significant ACE-inhibitory activities after hydrolysis with pepsin alone and after pepsinolysis followed by trypsinolysis and chymotrypsinolysis. Camel β-CN showed high antioxidant activity after hydrolysis with chymotrypsin. The results of this study suggest that when camel milk is consumed and digested, the produced peptides start to act as natural antioxidants and ACE-inhibitors.

  20. Quantifying the limits of transition state theory in enzymatic catalysis.

    Science.gov (United States)

    Zinovjev, Kirill; Tuñón, Iñaki

    2017-11-21

    While being one of the most popular reaction rate theories, the applicability of transition state theory to the study of enzymatic reactions has been often challenged. The complex dynamic nature of the protein environment raised the question about the validity of the nonrecrossing hypothesis, a cornerstone in this theory. We present a computational strategy to quantify the error associated to transition state theory from the number of recrossings observed at the equicommittor, which is the best possible dividing surface. Application of a direct multidimensional transition state optimization to the hydride transfer step in human dihydrofolate reductase shows that both the participation of the protein degrees of freedom in the reaction coordinate and the error associated to the nonrecrossing hypothesis are small. Thus, the use of transition state theory, even with simplified reaction coordinates, provides a good theoretical framework for the study of enzymatic catalysis. Copyright © 2017 the Author(s). Published by PNAS.

  1. Fed-Batch Feeding Strategies for Enzymatic Biodiesel Production

    DEFF Research Database (Denmark)

    Price, Jason Anthony; Nordblad, Mathias; Woodley, John

    2014-01-01

    of the differences in the interfacial and bulk concentrations of the enzyme. The model is then used to evaluate various feeding strategies to improve the enzymatic biodiesel production. The feeding strategies investigated, gave insight into how the methanol should be fed to potentially mitigate enzyme deactivation......In this work a kinetic model for the enzymatic transesterification of rapeseed oil using a solubilised lipase (Callera Trans L-Thermomyces lanuginos us) was developed from first principles. The model is based on a Ping-Pong Bi-Bi mechanism, with methanol inhibition, along with consideration...... while improving the biodiesel yield. The best experimental results gave a yield of 703 .76 g FAME L-1 and a reactor productivity of 28.12 g FAME L-1 h-1. In comparison, to reach the same yield, the optimised two step feeding strategy took 6.25 hours less, which equates to an increase the reactor...

  2. Optimization of Substrate Feeding for Enzymatic Biodiesel Production

    DEFF Research Database (Denmark)

    Price, Jason Anthony; Huusom, Jakob Kjøbsted; Nordblad, Mathias

    Many traditional bio-processes are operated in semi-batch mode, in which, a feed stream containing substrate and or nutrients is fed into the reactor during the course of the reaction. One key advantage of a semi-batch operation is that regulation of the substrate concentration has been found...... (both the vegetable oil and alcohol) feed rate/concentration is manipulated simultaneously. The results of the simulation were tested in the laboratory and are sufficiently positive to suggest the implementation of a feeding strategy for large scale enzymatic biodiesel production....... to be effective in mitigating the effects of substrate inhibition. Using enzymatic biodiesel production as a case study, the volumetric productivity of the reactor is increased while minimizing inactivation of the enzyme due to the alcohol. This is done by using a simple optimization routine where the substrate...

  3. Optimization of Substrate Feeding for Enzymatic Biodiesel Production

    DEFF Research Database (Denmark)

    Price, Jason Anthony; Huusom, Jakob Kjøbsted; Nordblad, Mathias

    2013-01-01

    Many traditional bio-processes are operated in semi-batch mode, in which, a feed stream containing substrate and or nutrients is fed into the reactor during the course of the reaction. One key advantage of a semi-batch operation is that regulation of the substrate concentration has been found...... (both the vegetable oil and alcohol) feed rate/concentration is manipulated simultaneously. The results of the simulation were tested in the laboratory and are sufficiently positive to suggest the implementation of a feeding strategy for large scale enzymatic biodiesel production...... to be effective in mitigating the effects of substrate inhibition. Using enzymatic biodiesel production as a case study, the volumetric productivity of the reactor is increased while minimizing inactivation of the enzyme due to the alcohol. This is done by using a simple optimization routine where the substrate...

  4. Enzymatic network for production of ether amines from alcohols.

    Science.gov (United States)

    Palacio, Cyntia M; Crismaru, Ciprian G; Bartsch, Sebastian; Navickas, Vaidotas; Ditrich, Klaus; Breuer, Michael; Abu, Rohana; Woodley, John M; Baldenius, Kai; Wu, Bian; Janssen, Dick B

    2016-09-01

    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 desired ether amines from the corresponding ether alcohols with inorganic ammonium as the only additional substrate. To examine conversion, individual and overall reaction equilibria were established. Using these data, it was found that the experimentally observed conversions of up to 60% observed for reactions containing 10 mM alcohol and up to 280 mM ammonia corresponded well to predicted conversions. The results indicate that efficient amination can be driven by high concentrations of ammonia and may require improving enzyme robustness for scale-up. Biotechnol. Bioeng. 2016;113: 1853-1861. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  5. Influence of magnetic field on enzymatic ONOO- production

    Science.gov (United States)

    Dranova, T.; Petrovskii, D.; Ershov, N.; Slepneva, I.; Stass, D.

    2017-08-01

    Enzymatic oxidation of L-arginine catalyzed by inducible nitric oxide synthase gives nitric oxide as the main product and superoxide anion as a side reaction product. Recombination of these radicals gives a very reactive species - peroxynitrite, which is involved in many biochemical processes. In the current work it was shown that such a system can be a usable model system for investigating the influence of magnetic field on enzymatic peroxynitrite formation. Using a selective fluorescent probe for peroxynitrite - coumarin boronic acid and an adopted for the experimental purpose incubation mixture, magnetic field experiments have been done at 11.7T. The averaged magnetic field effect is equal to 2.8±0.9%.

  6. 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...... achievable by intermittent product removal during cellulose hydrolysis....

  7. Enzymatic synthesis of a CCK-4 tripeptide fragment.

    Science.gov (United States)

    Guo, Li; Lu, Zi-min; Eckstein, Heiner

    2003-04-01

    To synthesize a tripeptide derivative Phac-Met-Asp(OMe)-Phe -NH2, which is a fragment of the gastrin C-terminal tetrapeptide CCK-4, by enzymatic reaction. Three free enzymes, alpha-chymotrypsin, papain and thermolysin from acyl donor Phac-Met-OCam was involved in three steps. The choice of appropriate enzymes and solvents was selected. All enzymatic reactions were obtained in reasonable yields(63%-92%). FAB-MS and FD-MS verified the correct molecular mass of the peptides. Studies on the alpha-chymotrypsin catalyzed coupling reaction between Phac-Met-OCam and H-Asp(OMe)2 have focused on the low water content media. By papain catalyzed saponification of Phac-Met-Asp(OMe)2, alpha-methyl ester of aspartic acid is selectively hydrolyzed to retain beta-methyl ester, and Phac-Met-Asp(OMe)-OH and H-Phe-NH2 can be coupled efficiently by thermolysin.

  8. Alginate oligosaccharides: enzymatic preparation and antioxidant property evaluation.

    Science.gov (United States)

    Falkeborg, Mia; Cheong, Ling-Zhi; Gianfico, Carlo; Sztukiel, Katarzyna Magdalena; Kristensen, Kasper; Glasius, Marianne; Xu, Xuebing; Guo, Zheng

    2014-12-01

    Alginate oligosaccharides (AOs) prepared from alginate, by alginate lyase-mediated depolymerization, were structurally characterized by mass spectrometry, infrared spectrometry and thin layer chromatography. Studies of their antioxidant activities revealed that AOs were able to completely (100%) inhibit lipid oxidation in emulsions, superiorly to ascorbic acid (89% inhibition). AOs showed radical scavenging activity towards ABTṠ, hydroxyl, and superoxide radicals, which might explain their excellent antioxidant activity. The radical scavenging activity is suggested to originate mainly from the presence of the conjugated alkene acid structure formed during enzymatic depolymerization. According to the resonance hybrid theory, the parent radicals of AOs are delocalized through allylic rearrangement, and as a consequence, the reactive intermediates are stabilized. AOs were weak ferrous ion chelators. This work demonstrated that AOs obtained from a facile enzymatic treatment of abundant alginate is an excellent natural antioxidant, which may find applications in the food industry. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. 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....

  10. Coulometric titration of D(+)-glucose using its enzymatic oxidation.

    Science.gov (United States)

    Tanaka, T; Shutto, E; Mizoguchi, T; Fukushima, K

    2001-02-01

    A definitive method is described for the indirect assay of milligram quantities of D(+)-glucose by coulometric titration. D(+)-Glucose was aerobically oxidized by glucose oxidase in an acetate buffer solution (pH 5.1). Subsequently, the enzymatically formed hydrogen peroxide was titrated coulometrically with electrogenerated hypobromite in sodium bromide-sodium tetraborate medium of pH 8.6, with biamperometric end-point detection. Parameters affecting the enzymatically catalyzed oxidation and coulometric titration were evaluated. The optimized conditions for the oxidation of up to 20 mg of D(+)-glucose include the addition of 4500 U of glucose oxidase and stirring over a 10-min interval at 25 degrees C. Under proposed conditions, the assay values of several commercial D(+)-glucose reagents were somewhat lower than the guaranteed minimum values, with RSDs (n = 5) of 0.071 - 0.106%.

  11. Identification and functional analysis of the gene cluster for L-arabinose utilization in Corynebacterium glutamicum.

    Science.gov (United States)

    Kawaguchi, Hideo; Sasaki, Miho; Vertès, Alain A; Inui, Masayuki; Yukawa, Hideaki

    2009-06-01

    Corynebacterium glutamicum ATCC 31831 grew on l-arabinose as the sole carbon source at a specific growth rate that was twice that on d-glucose. The gene cluster responsible for l-arabinose utilization comprised a six-cistron transcriptional unit with a total length of 7.8 kb. Three l-arabinose-catabolizing genes, araA (encoding l-arabinose isomerase), araB (l-ribulokinase), and araD (l-ribulose-5-phosphate 4-epimerase), comprised the araBDA operon, upstream of which three other genes, araR (LacI-type transcriptional regulator), araE (l-arabinose transporter), and galM (putative aldose 1-epimerase), were present in the opposite direction. Inactivation of the araA, araB, or araD gene eliminated growth on l-arabinose, and each of the gene products was functionally homologous to its Escherichia coli counterpart. Moreover, compared to the wild-type strain, an araE disruptant exhibited a >80% decrease in the growth rate at a lower concentration of l-arabinose (3.6 g liter(-1)) but not at a higher concentration of l-arabinose (40 g liter(-1)). The expression of the araBDA operon and the araE gene was l-arabinose inducible and negatively regulated by the transcriptional regulator AraR. Disruption of araR eliminated the repression in the absence of l-arabinose. Expression of the regulon was not repressed by d-glucose, and simultaneous utilization of l-arabinose and d-glucose was observed in aerobically growing wild-type and araR deletion mutant cells. The regulatory mechanism of the l-arabinose regulon is, therefore, distinct from the carbon catabolite repression mechanism in other bacteria.

  12. The integrated enzymatic production and downstream processing of glucosides

    OpenAIRE

    Roode, de, B.M.

    2001-01-01

    Glucosides are of commercial interest for the industry in general and for the pharmaceutical and food industry in particular. Chemical preparation of glycosides is not applicable in the food industry, and therefore an enzyme-catalyzed reaction would be an alternative. However, until now the low yield in the enzymatic reaction prevents the production of glycosides on a commercial scale. Therefore, high yields should be established by a combination of optimum reaction conditions and a ...

  13. Nanosilver: A Catalyst in Enzymatic Hydrolysis of Starch

    Directory of Open Access Journals (Sweden)

    Falkowska Marta

    2014-09-01

    Full Text Available Silver nanoparticles are widely used, because of their antimicrobial properties. In this paper, the rate of starch digestion in the presence of nanocatalyst was compared with the rate of reaction without nanosilver. The rate of enzymatic degradation of starch was found to be increased in the presence of silver nanoparticles. It is considered that α-amylase was immobilized onto the surface of nanoparticles.

  14. High volumetric power density, non-enzymatic, glucose fuel cells

    OpenAIRE

    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...

  15. Biodiesel production by chemical or enzymatic esterification of sunflower oil

    Energy Technology Data Exchange (ETDEWEB)

    Passarinho, Paula C.; Rosa, M. Fernanda; Oliveira, A.C.; Pingarilho, M.S.; Beirao, S.G.; Vieira, Ana Maria Soares

    1998-07-01

    In this work, two processes of sunflower oil transesterification, with methanol or ethanol, were studied for biodiesel production: chemical (catalyst- NaOH) and enzymatic (catalyst - rhizomucor miehei lipase). The chemical catalysis proved to be more efficient, having been obtained higher conversion yields and a better quality biodiesel, mainly in the case where methanol was used. The transesterification product had, in all cases, to be purified in order to be used as a diesel substitute.

  16. Improvement of Soybean Oil Solvent Extraction through Enzymatic Pretreatment

    OpenAIRE

    Grasso, F. V.; P. A. Montoya; Camusso, C. C.; Maroto, B. G.

    2012-01-01

    The purpose of this study is to evaluate multienzyme hydrolysis as a pretreatment option to improve soybean oil solvent extraction and its eventual adaptation to conventional processes. Enzymatic action causes the degradation of the cell structures that contain oil. Improvements in terms of extraction, yield, and extraction rate are expected to be achieved. Soybean flakes and collets were used as materials and hexane was used as a solvent. Temperature, pH, and incubation time were optimized a...

  17. Monitoring of enzymatic reactions using capillary electrophoresis with conductivity detection

    OpenAIRE

    Schuchert-Shi, Aiping

    2009-01-01

    Capillary electrophoresis combined with contactless conductivity detection allows to separate and detect the ionic species, which are neither UV absorbing nor fluorescent. This thesis focuses on the applications of this method on enzymatic reactions in different analytical tasks. First, the non-ionic species ethanol, glucose, ethyl acetate and ethyl butyrate were made accessible for analysis by capillary electrophoresis via charged products or byproducts obtained in enzymati...

  18. Apple phenolics and their contribution to enzymatic browning reactions

    Directory of Open Access Journals (Sweden)

    Wiesław Oleszek

    2014-01-01

    Full Text Available Chlorogenic acid, epicatechin, procyanidin B2 and C1 were isolated from apple skin. These compounds as well as quercetine and phloretine glycosides isolated from apples were studied individually and as mixtures for their participation in the enzymatic browning reactions. The importance of quercetine glycosides and the synergistic effect of phloridzin and phloretine xyloglucoside with chlorogenic acid and flavans in the browning reaction are reported.

  19. Study on enzymatic browning in suspension cultures of licorice cells

    OpenAIRE

    Yali Li; Tingting Meng; Yuxi Wang; Xiaoli Zhang

    2016-01-01

    Enzymatic browning is one of the main obstacles encountered in the establishment of suspension systems of licorice cells. Browning of cells may result in decreased viability, poor growth and even death. The present study investigated the mechanism of browning reactions and the effective controlling methods. The results showed that the cell viability and membrane permeabilization obviously changed when the cells were transferred to liquid medium. The transformation caused rapid increase in the...

  20. 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.

  1. 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.

  2. Enzymatic Hydrolysis Optimization to Ethanol Production by Simultaneous Saccharification and Fermentation

    Science.gov (United States)

    Vásquez, Mariana Peñuela; da Silva, Juliana Nascimento C.; de Souza, Maurício Bezerra; Pereira, Nei

    There is tremendous interest in using agro-industrial wastes, such as cellulignin, as starting materials for the production of fuels and chemicals. Cellulignin are the solids, which result from the acid hydrolysis of the sugarcane bagasse. The objective of this work was to optimize the enzymatic hydrolysis of the cellulose fraction of cellulignin, and to study its fermentation to ethanol using Saccharomyces cerevisiae. Cellulose conversion was optimized using response surface methods with pH, enzyme loading, solid percentage, and temperature as factor variables. The optimum conditions that maximized the conversion of cellulose to glucose, calculated from the initial dried weight of pretreated cellulignin, (43°C, 2%, and 24.4 FPU/g of pretreated cellulignin) such as the glucose concentration (47°C, 10%, and 25.6 FPU/g of pretreated cellulignin) were found. The desirability function was used to find conditions that optimize both, conversion to glucose and glucose concentration (47°C, 10%, and 25.9 FPU/g of pretreated cellulignin). The resulting enzymatic hydrolyzate was fermented yielding a final ethanol concentration of 30.0 g/L, in only 10 h, and reaching a volumetric productivity of 3.0 g/L·h, which is close to the values obtained in the conventional ethanol fermentation of sugar cane juice (5.0-8.0 g/L·h) in Brazil.

  3. Kinetics of enzymatic transesterification and thermal deactivation using immobilized Burkholderia lipase as catalyst.

    Science.gov (United States)

    Tran, Dang-Thuan; Chang, Jo-Shu

    2014-03-01

    The most effective way of enzymatic synthesis of biodiesel is through lipase-catalyzed transesterification, while its performance and economic feasibility should still be improved. In this study, lipase produced by an isolated Burkholderia sp. was immobilized on microsize Celite materials functionally modified with long alkyl groups. The specific activity of the immobilized lipase was 1,154 U/g. The methanolysis of olive oil catalyzed by the immobilized lipase obeyed Ping Pong Bi Bi model with an estimated V max, K m,TG, K m,M and K i,M value of 0.61 mol/(L min), 7.93 mol/L, 1.01 mol/L, and 0.24 mol/L, respectively. The activation energy of the enzymatic reaction is estimated as 15.51 kJ/mol. The immobilized lipase exhibits high thermal stability with thermal deactivation energy of 83 kJ/mol and a long half-life. The enthalpy, Gibb's free energy, and entropy of the immobilized lipase were in the range of 80.02-80.35 kJ/mol, 88.35-90.13 kJ/mol, and -28.22 to -25.11 J/(mol K), respectively.

  4. Enzymatic transesterification of palm stearin and olein blends to produce zero-trans margarine fat

    Science.gov (United States)

    2012-01-01

    Background Food industries aim to replace trans fat in their products by formulations having equivalent functionality and economic viability. Enzymatic transesterification can be a technological option to produce trans free fats targeting commercial applications. Results Palm stearin and palm olein blends in different ratios were enzymatically transesterified in a solvent free system using a Rhizopus oryzae lipase immobilised onto CaCO3 to produce a suitable fat for margarine formulation. Slip melting points and triacylglycerols profiles were evaluated upon transesterification. Results indicated that all transesterified blends had lower slip melting points than their non transesterified counterparts. Furthermore, the triacylglycerols profile showed a decrease in the concentration of the high melting point triacylglycerols. The rheological analysis showed that margarine prepared with the transesterified blend showed a better spreadability than that of a control margarine prepared with non transesterified fat. Adding powder of dry bark orange to margarine preparation improved its colour and fairly affected its spreadability and rheological behaviour. The margarine prepared with transesterified fat displayed a rheological behaviour that was comparable to that of commercial sample. Conclusions This study is an ecofriendly approach to the utilization of relatively low value bioresources like palm stearin and palm olein for making margarine free of trans fatty acids that are now implicated as risk factor for heart diseases. PMID:22889174

  5. Enzymatic collapse of artificial polymer composite material containing double-stranded DNA.

    Science.gov (United States)

    Yamada, Masanori; Amoo, Mariko

    2008-06-01

    Large amounts of DNA-enriched biomaterials, such as salmon milts and shellfish gonads, are discarded as industrial waste around the world. Therefore, the utilizations of DNA with the specific function are important for the biomaterial science and the curce technology. We could convert the discarded DNA to an enzymatic collapsible material by the addition of DNA to the artificial polymer material, such as nylon. Although these DNA-artificial polymer composite materials were stable in water, these materials indicated the collapsibility at the DNA-hydrolyzed enzyme, such as Micrococcal nuclease, condition. Additionally, these collapsibilities under enzyme condition were controlled by the number of imino groups in the components of the artificial polymer. Furthermore, these composite materials could create the fiber form with a highly ordered molecular orientation by the reaction at the liquid/liquid interface. The DNA-artificial polymer composite materials may have the potential utility as a novel bio-, medical-, and environmental materials with the enzymatic collapsibility and degradability.

  6. Enzymatic transesterification of palm stearin and olein blends to produce zero-trans margarine fat

    Directory of Open Access Journals (Sweden)

    Sellami Mohamed

    2012-08-01

    Full Text Available Abstract Background Food industries aim to replace trans fat in their products by formulations having equivalent functionality and economic viability. Enzymatic transesterification can be a technological option to produce trans free fats targeting commercial applications. Results Palm stearin and palm olein blends in different ratios were enzymatically transesterified in a solvent free system using a Rhizopus oryzae lipase immobilised onto CaCO3 to produce a suitable fat for margarine formulation. Slip melting points and triacylglycerols profiles were evaluated upon transesterification. Results indicated that all transesterified blends had lower slip melting points than their non transesterified counterparts. Furthermore, the triacylglycerols profile showed a decrease in the concentration of the high melting point triacylglycerols. The rheological analysis showed that margarine prepared with the transesterified blend showed a better spreadability than that of a control margarine prepared with non transesterified fat. Adding powder of dry bark orange to margarine preparation improved its colour and fairly affected its spreadability and rheological behaviour. The margarine prepared with transesterified fat displayed a rheological behaviour that was comparable to that of commercial sample. Conclusions This study is an ecofriendly approach to the utilization of relatively low value bioresources like palm stearin and palm olein for making margarine free of trans fatty acids that are now implicated as risk factor for heart diseases.

  7. Structural insights into the enzymatic activity and potential substrate promiscuity of human 3-phosphoglycerate dehydrogenase (PHGDH).

    Science.gov (United States)

    Unterlass, Judith E; Wood, Robert J; Baslé, Arnaud; Tucker, Julie; Cano, Céline; Noble, Martin M E; Curtin, Nicola J

    2017-11-28

    Cancer cells reprogram their metabolism and energy production to sustain increased growth, enable metastasis and overcome resistance to cancer treatments. Although primary roles for many metabolic proteins have been identified, some are promiscuous in regards to the reaction they catalyze. To efficiently target these enzymes, a good understanding of their enzymatic function and structure, as well as knowledge regarding any substrate or catalytic promiscuity is required. Here we focus on the characterization of human 3-phosphoglycerate dehydrogenase (PHGDH). PHGDH catalyzes the NAD + -dependent conversion of 3-phosphoglycerate to phosphohydroxypyruvate, which is the first step in the de novo synthesis pathway of serine, a critical amino acid for protein and nucleic acid biosynthesis. We have investigated substrate analogues to assess whether PHGDH might possess other enzymatic roles that could explain its occasional over-expression in cancer, as well as to help with the design of specific inhibitors. We also report the crystal structure of the catalytic subunit of human PHGDH, a dimer, solved with bound cofactor in one monomer and both cofactor and L -tartrate in the second monomer. In vitro enzyme activity measurements show that the catalytic subunit of PHGDH is still active and that PHGDH activity could be significantly inhibited with adenosine 5'-diphosphoribose.

  8. 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.

  9. ENZYMATIC RESOLUTION OF ANTIDEPRESSANT DRUG PRECURSORS IN AN UNDERGRADUATE LABORATORY

    Directory of Open Access Journals (Sweden)

    Luís M. R. Solano

    2015-02-01

    Full Text Available The use of biocatalysts in synthetic chemistry is a conventional methodology for preparing enantiomerically enriched compounds. Despite this fact, the number of experiments in chemical teaching laboratories that demonstrate the potential of enzymes in synthetic organic chemistry is limited. We describe a laboratory experiment in which students synthesized a chiral secondary alcohol that can be used in the preparation of antidepressant drugs. This experiment was conducted by individual students as part of a Drug Synthesis course held at the Pharmacy Faculty, Lisbon University. This laboratory experiment requires six laboratory periods, each lasting four hours. During the first four laboratory periods, students synthesized and characterized a racemic ester using nuclear magnetic resonance spectroscopy and gas chromatography. During the last two laboratory periods, they performed enzymatic hydrolysis resolution of the racemic ester using Candida antarctica lipase B to yield enantiomerically enriched secondary alcohol. Students successfully prepared the racemic ester with a 70%-81% overall yield in three steps. The enzymatic hydrolysis afforded (R- secondary alcohol with good enantioselectivity (90%-95% and reasonable yields (10%-19%. In these experiments, students were exposed to theoretical and practical concepts of aromatic acylation, ketone reduction, esterification, and enzymatic hydrolysis.

  10. Process Evaluation Tools for Enzymatic Cascades Welcome Message

    DEFF Research Database (Denmark)

    Abu, Rohana

    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 to synthes......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...... to synthesize valuable compounds (e.g. chiral molecules) especially as pharmaceutical intermediates and to assist complex reactions that otherwise has problems as single step system. Despite this interest, process evaluation of many enzymatic cascades has only rarely been reported in the search for process...... the equilibrium positions in the main syntheses. In principle, this strategy could successfully achieve high conversion, using ammonia as the sole reagent used in excess to drive the conversion. The findings herein indicate that quantitatively the possibilities for improving the conversion of thermodynamically...

  11. 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. PMID:23390576

  12. Recent Research Trends on the Enzymatic Synthesis of Structured Lipids.

    Science.gov (United States)

    Kim, Byung Hee; Akoh, Casimir C

    2015-08-01

    Structured lipids (SLs) are lipids that have been chemically or enzymatically modified from their natural biosynthetic form. Because SLs are made to possess desired nutritional, physicochemical, or textural properties for various applications in the food industry, many research activities have been aimed at their commercialization. The production of SLs by enzymatic procedures has a great potential in the future market because of the specificity of lipases and phospholipases used as the biocatalysts. The aim of this review is to provide concise information on the recent research trends on the enzymatic synthesis of SLs of commercial interest, such as medium- and long-chain triacylglycerols, human milk fat substitutes, cocoa butter equivalents, trans-free or low-trans plastic fats (such as margarines and shortenings), low-calorie fats/oils, health-beneficial fatty acid-rich fats/oils, mono- or diacylglycerols, and structurally modified phospholipids. This limited review covers 108 research articles published between 2010 and 2014 which were searched in Web of Science. © 2015 Institute of Food Technologists®

  13. Architecture and regulation of negative-strand viral enzymatic machinery.

    Science.gov (United States)

    Kranzusch, Philip J; Whelan, Sean P J

    2012-07-01

    Negative-strand (NS) RNA viruses initiate infection with a unique polymerase complex that mediates both mRNA transcription and subsequent genomic RNA replication. For nearly all NS RNA viruses, distinct enzymatic domains catalyzing RNA polymerization and multiple steps of 5' mRNA cap formation are contained within a single large polymerase protein (L). While NS RNA viruses include a variety of emerging human and agricultural pathogens, the enzymatic machinery driving viral replication and gene expression remains poorly understood. Recent insights with Machupo virus and vesicular stomatitis virus have provided the first structural information of viral L proteins, and revealed how the various enzymatic domains are arranged into a conserved architecture shared by both segmented and nonsegmented NS RNA viruses. In vitro systems reconstituting RNA synthesis from purified components provide new tools to understand the viral replicative machinery, and demonstrate the arenavirus matrix protein regulates RNA synthesis by locking a polymerase-template complex. Inhibition of gene expression by the viral matrix protein is a distinctive feature also shared with influenza A virus and nonsegmented NS RNA viruses, possibly illuminating a conserved mechanism for coordination of viral transcription and polymerase packaging.

  14. Use of a cyanine dye probe to estimate the composition of the vitreous body after enzymatic treatment

    Science.gov (United States)

    Panova, Ina G.; Tatikolov, Alexander S.; Sharova, Natalia P.

    2010-02-01

    The aim of this work was to study the effect of enzymes such as proteinase K, trypsin, collagenase with hyaluronidase, as well as a mixture of all these enzymes, on albumin and collagens incorporated in the vitreous body, using a cyanine dye as a spectral-fluorescent probe. We studied the vitreous body of the eyes of 19/20-week human fetuses, in which, as we showed earlier, the concentration of albumin in the vitreous body is sufficiently high. Proteinase K steeply decreased the albumin content in the vitreous body, whereas trypsin and hyaluronidase with collagenase had no effect on the albumin content. Collagen was not subjected to proteinase K. Enzymatic digestion of collagen occurred under the action of collagenase with hyaluronidase. The content of albumin and collagen sharply decreased in the system after treatment of the vitreous body with mixture of all enzymes. Hence, the results obtained showed that, even being in the mixture, these enzymes have a selective effect on albumin and collagens. The possibility to study the dose-dependent character of enzymatic vitreolysis using a cyanine dye probe has been shown. The spectral-fluorescent probe for albumin and collagens proved to be useful for experimental approaches at screening the enzymatic mixtures possessing the selective action. The study performed is considered as a preclinical trial, and the method presented as promising for the further research in this field. The effect of the enzymes used for therapeutic purposes on the functional conditions of the vitreous body should be studied.

  15. Ultrahigh pressure-assisted enzymatic extraction maximizes the yield of longan pulp polysaccharides and their acetylcholinesterase inhibitory activity in vitro.

    Science.gov (United States)

    Bai, Yajuan; Liu, Lei; Zhang, Ruifen; Huang, Fei; Deng, Yuanyuan; Zhang, Mingwei

    2017-03-01

    An extraction method employing ultrahigh pressure-assisted enzymatic treatment was developed and optimized by response surface methodology to increase the yield of longan pulp polysaccharides (LP-UE). A maximum polysaccharides yield of 8.55% was obtained under the optimal conditions of 407MPa ultrahigh pressure maintained for 6min with an enzyme to pretreated material ratio of 1:100, an enzymolysis time of 1.7h and a water to pretreated material ratio of 42ml/g. Subsequently, the physicochemical properties and acetylcholinesterase (AChE) inhibitory activity of LP-UE were compared to those of longan pulp polysaccharides (LP) extracted by hot water (LP-H), ultrahigh pressure (LP-U) or enzymatic treatment (LP-E). Results demonstrated that the extraction yield, hexuronic acid content and AChE inhibitory activity of LP-UE was the highest among the four LP samples. LP-UE was primarily made up of arabinose, glucose, and galactose and was linked mainly by β-type glycosidic linkage. The FTIR spectrum of LP-UE was very similar to those of LP-H, LP-U, and LP-E. In summary, ultrahigh pressure-assisted enzymatic treatment is a more efficient technique for extracting LP with considerable improvement of both yield and memory enhancement function. Copyright © 2016. Published by Elsevier B.V.

  16. Enhanced enzymatic saccharification of pretreated biomass using glycerol thermal processing (GTP).

    Science.gov (United States)

    Zhang, Wei; Sathitsuksanoh, Noppadon; Barone, Justin R; Renneckar, Scott

    2016-01-01

    Biomass was heated (200-240°C) in the presence of glycerol, for 4-12 min, under shear to disrupt the native cell wall architecture. The impact of this method, named glycerol thermal processing (GTP), on saccharification efficiency of the hardwood Liquidambar styraciflua, and a control cellulose sample was studied as a function of treatment severity. Furthermore, the enzymatic conversion of samples with varying compositions was studied after extraction of the structural polymers. Interestingly, the sweet gum processed materials crystallinity index increased by 10% of the initial value. The experiments revealed that the residual lignin was not a barrier to limiting the digestibility of cellulose after pretreatment yielding up to 70% glucose based on the starting wood material. Further xylan removal greatly improved the cellulose hydrolysis rate, converting nearly 70% of the cellulose into glucose within 24h, and reaching 78% of ultimate glucan digestibility after 72 h. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Enzymatic cellulose oxidation is linked to lignin by long-range electron transfer

    Science.gov (United States)

    Westereng, Bjørge; Cannella, David; Wittrup Agger, Jane; Jørgensen, Henning; Larsen Andersen, Mogens; Eijsink, Vincent G.H.; Felby, Claus

    2015-01-01

    Enzymatic oxidation of cell wall polysaccharides by lytic polysaccharide monooxygenases (LPMOs) plays a pivotal role in the degradation of plant biomass. While experiments have shown that LPMOs are copper dependent enzymes requiring an electron donor, the mechanism and origin of the electron supply in biological systems are only partly understood. We show here that insoluble high molecular weight lignin functions as a reservoir of electrons facilitating LPMO activity. The electrons are donated to the enzyme by long-range electron transfer involving soluble low molecular weight lignins present in plant cell walls. Electron transfer was confirmed by electron paramagnetic resonance spectroscopy showing that LPMO activity on cellulose changes the level of unpaired electrons in the lignin. The discovery of a long-range electron transfer mechanism links the biodegradation of cellulose and lignin and sheds new light on how oxidative enzymes present in plant degraders may act in concert. PMID:26686263

  18. Kinetics of ethanol production from sugarcane bagasse enzymatic hydrolysate concentrated with molasses under cell recycle.

    Science.gov (United States)

    de Andrade, Rafael Ramos; Maugeri Filho, Francisco; Maciel Filho, Rubens; da Costa, Aline Carvalho

    2013-02-01

    In this work, a kinetic model for ethanol fermentation from sugarcane bagasse enzymatic hydrolysate concentrated with molasses was developed. A model previously developed for fermentation of pure molasses was modified by the inclusion of a new term for acetic acid inhibition on microorganism growth rate and the kinetic parameters were estimated as functions of temperature. The influence of the hydrolysate on the kinetic parameters is analyzed by comparing with the parameters from fermentation of pure molasses. The impact of cells recycling in the kinetic parameters is also evaluated, as well as on the ethanol yield and productivity. The model developed described accurately most of the fermentations performed in several successive batches for temperatures from 30 to 38°C. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Optimal control strategy for fed-batch enzymatic hydrolysis of lignocellulosic biomass based on epidemic modeling.

    Science.gov (United States)

    Tai, Chao; Keshwani, Deepak R; Voltan, Diego S; Kuhar, Pankaj S; Engel, Aaron J

    2015-07-01

    A mathematical optimal control strategy for feeding operation was developed for fed-batch enzymatic hydrolysis of dilute acid pretreated lignocellulosic biomass based on a modified epidemic model. Cellulose conversion was maximized and glucose concentration achieved highest possible value over a fixed hydrolysis time. Boundaries of feeding rate and lignin content were set for feasible controls. Using the optimal control feeding strategy, glucose concentration and accumulated cellulose conversion reached up to 77.31 g/L and 72.08% in 100 h, which are 108.76% and 37.50% higher than in batch hydrolysis with same amount of enzyme consumption. Solids content in feeding source has a significant interference on system mass transfer. Optimal control is a useful tool for guiding operations in fed-batch and continuous processes as it enables process optimization through clear objective functions and feasible controls. © 2015 Wiley Periodicals, Inc.

  20. Utilisation Of Spirulinasp. And Chlorellapyrenoidosa Biomass For The Productionof Enzymatic Protein Hydrolysates

    Directory of Open Access Journals (Sweden)

    Cristiane R. Lisboa

    2014-05-01

    Full Text Available This aim of this study was to assess the hydrolysis reaction of the biomass of Chlorella pyrenoidosaandSpirulinasp. LEB 18,using commercial proteases that act in different pH ranges, to obtain protein hydrolysates with promising application in food or food supplement, improving functional and nutritional food properties. Threecentral composite study designs were carried out for each microalga (Chlorella and Spirulina. The 2 3 type central composite design was utilized with three replications at the central point, varying the enzyme concentration (5 to 10 U.mL-1 , the concentrationof substrate (5 to 10 % and reaction time (60 to 240 min, for a total of 11 experiments per planning. The highestdegrees of hydrolysis (52.9% and 55.31% forSpirulinaand Chlorella,respectively, were obtained with 4 h of reaction. The results show that it is possible to obtain enzymatic protein hydrolysates with different DH from microalgae biomass.

  1. Carbohydrate microarrays for enzymatic reactions and quantification of binding affinities for glycan-protein interactions.

    Science.gov (United States)

    Lee, Myung-Ryul; Park, Sungjin; Shin, Injae

    2012-01-01

    Glycans are involved in a variety of physiological and pathological processes through interactions with proteins. Thus, the molecular basis of glycan-protein interactions provides valuable information on understanding biological phenomena and exploiting more effective carbohydrate-based therapeutic agents and diagnostic tools. Carbohydrate microarray technology has become a powerful tool for evaluating glycan-mediated biological events in a high-throughput manner. This technology is mostly applied for rapid analysis of glycans-protein interactions in the field of functional glycomics. In order to expand application areas of glycan microarrays, we have used carbohydrate microarrays for measurement of binding affinities between glycans and proteins and profiling of glycosyltransferase activities. The glycan microarrays used for these studies are constructed by immobilizing maleimide or hydrazide-conjugated glycans on the thiol or hydrazide-derivatized glass slides, respectively. This protocol describes the fabrication of carbohydrate microarrays and their applications to enzymatic reactions and determination of quantitative binding affinities.

  2. MALDI imaging of enzymatic degradation of glycerides by lipase on textile surface

    DEFF Research Database (Denmark)

    Hall-Andersen, Jonatan; Kaasgaard, Svend G; Janfelt, Christian

    2017-01-01

    Most modern laundry detergents contain enzymes such as proteases, amylases, and lipases for more efficient removal of stains containing proteins, carbohydrates, and lipids during wash at low temperature. The function of the lipases is to hydrolyse the hydrophobic triglycerides from fats and oils...... stain and simulating washing cycles using well-defined detergents with lipase concentrations ranging between 0 and 0.5ppm. After washing, the textile swatches as well as cryo-sections of the swatches were imaged using MALDI imaging in positive ion mode at pixel sizes of 15-75μm. Similar samples were...... of ion intensities from 298 individual ion masses of mono-, di- and triglycerides and free fatty acids. MALDI imaging of glycerides was possible directly from a textile surface, allowing visualization of the enzymatic degradation of fatty stains on textile during the laundry process. The images showed...

  3. Enzymatic production of highly soluble myricitrin glycosides using beta-galactosidase.

    Science.gov (United States)

    Shimizu, Ryosuke; Shimabayashi, Hiroshi; Moriwaki, Masamitsu

    2006-04-01

    Myricitrin, a botanical flavonol glycoside, could be a useful ingredient of functional foods, cosmetics, and medicines because of its high anti-oxidative activity. However, due to its insolubility in water, it has a limited range of use. To improve this solubility, we glycosylated myricitrin by an enzymatic transglycosylate reaction. Myricitrin was galactosylated by beta-galactosidase from Bacillus circulans using lactose as a sugar donor. The reaction product was 480 times more soluble than myricitrin. Four myricitrin galactosides were isolated from the reaction products by column chromatography, and their molecular structures were identified by using ESI-MS, 1H-NMR, 13C-NMR, 1H-1H COSY, 1H-13C HMQC and 1H-13C HMBC analysis. The solubility of these four myricitrin galactosides was more than 3.9 x 10(3) fold that of myricitrin, and each had similar anti-oxidative activity to that of myricitrin.

  4. Synthesis and enzymatic incorporation of α-L-threofuranosyl adenine triphosphate (tATP).

    Science.gov (United States)

    Zhang, Su; Chaput, John C

    2013-03-01

    Threose nucleic acid (TNA) is an artificial genetic polymer in which the natural ribose sugar found in RNA has been replaced with an unnatural threose sugar. TNA can be synthesized enzymatically using Therminator DNA polymerase to copy DNA templates into TNA. Here, we expand the substrate repertoire of Therminator DNA polymerase to include threofuranosyl adenine 3'-triphsophate (tATP). We chemically synthesized tATP by two different methods from the 2'-O-acetyl derivative. Enzyme-mediated polymerization reveals that tATP functions as an efficient substrate for Therminator DNA polymerase, indicating that tATP can replace the diaminopurine analogue (tDTP) in TNA transcription reactions. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Improved Functional Characteristics of Whey Protein Hydrolysates in Food Industry

    OpenAIRE

    Jeewanthi, Renda Kankanamge Chaturika; Lee, Na-Kyoung; Paik, Hyun-Dong

    2015-01-01

    This review focuses on the enhanced functional characteristics of enzymatic hydrolysates of whey proteins (WPHs) in food applications compared to intact whey proteins (WPs). WPs are applied in foods as whey protein concentrates (WPCs), whey protein isolates (WPIs), and WPHs. WPs are byproducts of cheese production, used in a wide range of food applications due to their nutritional validity, functional activities, and cost effectiveness. Enzymatic hydrolysis yields improved functional and nutr...

  6. Understanding of alkaline pretreatment parameters for corn stover enzymatic saccharification

    Directory of Open Access Journals (Sweden)

    Chen Ye

    2013-01-01

    Full Text Available Abstract Background Previous research on alkaline pretreatment has mainly focused on optimization of the process parameters to improve substrate digestibility. To achieve satisfactory sugar yield, extremely high chemical loading and enzyme dosages were typically used. Relatively little attention has been paid to reduction of chemical consumption and process waste management, which has proven to be an indispensable component of the bio-refineries. To indicate alkali strength, both alkali concentration in pretreatment solution (g alkali/g pretreatment liquor or g alkali/L pretreatment liquor and alkali loading based on biomass solids (g alkali/g dry biomass have been widely used. The dual approaches make it difficult to compare the chemical consumption in different process scenarios while evaluating the cost effectiveness of this pretreatment technology. The current work addresses these issues through pretreatment of corn stover at various combinations of pretreatment conditions. Enzymatic hydrolysis with different enzyme blends was subsequently performed to identify the effects of pretreatment parameters on substrate digestibility as well as process operational and capital costs. Results The results showed that sodium hydroxide loading is the most dominant variable for enzymatic digestibility. To reach 70% glucan conversion while avoiding extensive degradation of hemicellulose, approximately 0.08 g NaOH/g corn stover was required. It was also concluded that alkali loading based on total solids (g NaOH/g dry biomass governs the pretreatment efficiency. Supplementing cellulase with accessory enzymes such as α-arabinofuranosidase and β-xylosidase significantly improved the conversion of the hemicellulose by 6–17%. Conclusions The current work presents the impact of alkaline pretreatment parameters on the enzymatic hydrolysis of corn stover as well as the process operational and capital investment costs. The high chemical consumption for alkaline

  7. A multicentric evaluation of IDMS-traceable creatinine enzymatic assays.

    Science.gov (United States)

    Piéroni, Laurence; Delanaye, Pierre; Boutten, Anne; Bargnoux, Anne-Sophie; Rozet, Eric; Delatour, Vincent; Carlier, Marie-Christine; Hanser, Anne-Marie; Cavalier, Etienne; Froissart, Marc; Cristol, Jean-Paul

    2011-11-20

    Chronic kidney disease definition is based on glomerular filtration rate (GFR) estimations which are derived from creatinine-based equations. The accuracy of GFR estimation is thus largely dependent of those of serum creatinine assays. International recommendations highlight the need for traceable creatinine assays. The French Society of Clinical Biochemistry conducted a study for measuring accuracy of creatinine enzymatic methods. This evaluation involved 25 clinical laboratories. Creatinine was measured in serum pools ranging from 35.9±0.9 μmol/L to 174.5±3.1 μmol/L (IDMS determination) using 12 creatinine enzymatic methods. For all creatinine values greater than 74.4±1.4 μmol/L, the bias and imprecision did not exceed 5% and 5.9%, respectively. For the lowest value (35.9±0.9 μmol/L), the bias ranged from -1.8 to 9.9% (with one exception). At this level, the imprecision ranged from 1.9 to 7.8%. The true performances of the assays (couples of bias and relative standard deviation), were evaluated using Monte-Carlo simulations. Most of the assays fall within the maximum Total Error of 12% at all concentrations. This study demonstrates substantial improvements in the calibration, traceability and precision of the enzymatic methods, reaching the NKDEP recommendations. Moreover, most of these assays allowed accurate creatinine measurements for creatinine levels lower than 40 μmol/L. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Enzymatic synthesis of hydrophobic compounds integrated with membrane separation

    Directory of Open Access Journals (Sweden)

    Noworyta Andrzej

    2016-03-01

    Full Text Available The enzymatic synthesis of a highly hydrophobic product (dipeptide precursor in which the reaction is accompanied by the mass transfer of the reaction product to the organic phase and the substrates to the water phase is considered. Equations describing both continuous and batch processes are formulated. The range of variability in the operating parameters of such a bioreactor is specified, and the correlations reported in the literature to describe mass transfer in the membrane contactor are validated. The proposed process was verified experimentally, and good agreement between the determined and calculated concentrations was obtained in both phases.

  9. Enzymatic preparation and characterization of soybean lecithin-based emulsifiers

    Directory of Open Access Journals (Sweden)

    R. C. Reddy Jala

    2016-12-01

    Full Text Available Simple enzymatic methods were developed for the synthesis of lysolecithin, glycerolyzed lecithin and hydrolyzed lecithin. The products were characterized in terms of their acetone insoluble matter, hexane insoluble matter, moisture, phospholipid distribution and fatty acid composition. The HLB value ranges of different products with different acid values were detected. The efficiency of optimally hydrolyzed lecithin was examined at high calcium ion, low pH, and aqueous solutions and compared with commercially available standard lecithin-based emulsifiers. Overall, lysolecithin powder was proven to be the best emulsifier even at strong and medium acidic conditions.

  10. Sugar ester surfactants: enzymatic synthesis and applications in food industry.

    Science.gov (United States)

    Neta, Nair S; Teixeira, José A; Rodrigues, Lígia R

    2015-01-01

    Sugar esters are non-ionic surfactants that can be synthesized in a single enzymatic reaction step using lipases. The stability and efficiency of lipases under unusual conditions and using non-conventional media can be significantly improved through immobilization and protein engineering. Also, the development of de novo enzymes has seen a significant increase lately under the scope of the new field of synthetic biology. Depending on the esterification degree and the nature of fatty acid and/or sugar, a range of sugar esters can be synthesized. Due to their surface activity and emulsifying capacity, sugar esters are promising for applications in food industry.

  11. The Mechanisms of Plant Cell Wall Deconstruction during Enzymatic Hydrolysis

    DEFF Research Database (Denmark)

    Thygesen, Lisbeth Garbrecht; E. Thybring, Emil; Johansen, Katja Salomon

    2014-01-01

    Mechanical agitation during enzymatic hydrolysis of insoluble plant biomass at high dry matter contents is indispensable for the initial liquefaction step in biorefining. It is known that particle size reduction is an important part of liquefaction, but the mechanisms involved are poorly understood....... Here we put forward a simple model based on mechanical principles capable of capturing the result of the interaction between mechanical forces and cell wall weakening via hydrolysis of glucosidic bonds. This study illustrates that basic material science insights are relevant also within biochemistry...

  12. Overcoming Aminoglycoside Enzymatic Resistance: Design of Novel Antibiotics and Inhibitors

    Directory of Open Access Journals (Sweden)

    Sandra G. Zárate

    2018-01-01

    Full Text Available Resistance to aminoglycoside antibiotics has had a profound impact on clinical practice. Despite their powerful bactericidal activity, aminoglycosides were one of the first groups of antibiotics to meet the challenge of resistance. The most prevalent source of clinically relevant resistance against these therapeutics is conferred by the enzymatic modification of the antibiotic. Therefore, a deeper knowledge of the aminoglycoside-modifying enzymes and their interactions with the antibiotics and solvent is of paramount importance in order to facilitate the design of more effective and potent inhibitors and/or novel semisynthetic aminoglycosides that are not susceptible to modifying enzymes.

  13. Detecting platform for phenolic compounds-characteristic of enzymatic electrode

    Science.gov (United States)

    Cabaj, Joanna; Chyla, Antoni; Jędrychowska, Agnieszka; Olech, Kamila; Sołoducho, Jadwiga

    2012-08-01

    We report here on simple and universal method for the highly efficient, electrolytic immobilization of tyrosinase (from Agaricus bisporus), for amperometric biosensing purposes. Tyrosinase has been successfully deposited on the surface of thin, ordered films of copolymerized derivative of thiophene (3-methylthiophene/3-thiopheneacetic acid/bis(ethylenedioxythiophene)diphenylamine). The tyrosinase retains well its activity well within the fabricated copolymer matrix. Reduction peaks, observed in cyclic voltammetry at 0.125 to +0.07 V, were attributed to the reduction of enzymatically liberated quinone species. Considering the fact, that immobilization strategy showed high efficiency, obtained results suggest that the method for phenoloxidase immobilization has a great potential for fabrication of bioelectronics' devices.

  14. Modelling and operation of reactors for enzymatic biodiesel production

    DEFF Research Database (Denmark)

    Price, Jason Anthony

    to the production of high fructose corn syrup, upgrading of fats and oils and biodiesel production to name a few. Despite these examples of industrial enzymatic applications, it is still not “clear cut” how to implement biocatalyst in industry and how best to optimize the processes. This is because the processing...... aspects of the enzyme with reaction/reactor engineering is performed. This strategy is applied to a case study of biodiesel production catalysed by a liquid enzyme formulation. The use of enzymes for biodiesel production is still in its infancy with non-optimized process designs. Furthermore is it unclear...

  15. Identification and Functional Analysis of the Gene Cluster for l-Arabinose Utilization in Corynebacterium glutamicum▿ †

    Science.gov (United States)

    Kawaguchi, Hideo; Sasaki, Miho; Vertès, Alain A.; Inui, Masayuki; Yukawa, Hideaki

    2009-01-01

    Corynebacterium glutamicum ATCC 31831 grew on l-arabinose as the sole carbon source at a specific growth rate that was twice that on d-glucose. The gene cluster responsible for l-arabinose utilization comprised a six-cistron transcriptional unit with a total length of 7.8 kb. Three l-arabinose-catabolizing genes, araA (encoding l-arabinose isomerase), araB (l-ribulokinase), and araD (l-ribulose-5-phosphate 4-epimerase), comprised the araBDA operon, upstream of which three other genes, araR (LacI-type transcriptional regulator), araE (l-arabinose transporter), and galM (putative aldose 1-epimerase), were present in the opposite direction. Inactivation of the araA, araB, or araD gene eliminated growth on l-arabinose, and each of the gene products was functionally homologous to its Escherichia coli counterpart. Moreover, compared to the wild-type strain, an araE disruptant exhibited a >80% decrease in the growth rate at a lower concentration of l-arabinose (3.6 g liter−1) but not at a higher concentration of l-arabinose (40 g liter−1). The expression of the araBDA operon and the araE gene was l-arabinose inducible and negatively regulated by the transcriptional regulator AraR. Disruption of araR eliminated the repression in the absence of l-arabinose. Expression of the regulon was not repressed by d-glucose, and simultaneous utilization of l-arabinose and d-glucose was observed in aerobically growing wild-type and araR deletion mutant cells. The regulatory mechanism of the l-arabinose regulon is, therefore, distinct from the carbon catabolite repression mechanism in other bacteria. PMID:19346355

  16. Glucose obtained from rice bran by ultrasound-assisted enzymatic hydrolysis

    OpenAIRE

    Raquel Cristine Kuhn; Marcio Antonio Mazutti; Edson Luiz Foletto; Valéria Dal Prá; Eduardo Zimmermann; Matheus Souza; Vitória Segabinazzi Foletto; Tanisa Paula Silveira Maleski; Felipe Cavalheiro Lunelli; Pâmela Sfalcin

    2015-01-01

    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 p...

  17. Enzymatic and non-enzymatic antioxidant potentials of Chlorella vulgaris grown in effluent of a confectionery industry.

    Science.gov (United States)

    Kumar, R Ranjith; Rao, P Hanumantha; Subramanian, V V; Sivasubramanian, V

    2014-02-01

    Enzymatic and non-enzymatic antioxidant potentials of Chlorella vulgaris have gained considerable importance in recent decades. C. vulgaris strain highly tolerant to extreme pH variations was isolated and mass-cultivated in the wastewater from a confectionery industry. C.vulgaris showed better growth in wastewater than in improvised CFTRI medium. The microalgal biomass was then screened for the following antioxidants: peroxidase, superoxide dismutase, polyphenol oxidase, glutathione peroxidase, chlorophyll a, ascorbic acid, α-tocopherol and reduced glutathione. The total polyphenol content of the strain was also studied. The strain showed a high degree of enzymatic antioxidant activity (0.195 × 10(-5) ± 0.0072 units/cell peroxidase, 0.04125 × 10(-5) ± 0.001 units/cell superoxide dismutase, 0.2625 × 10(-5) ± 0.003 units/cell polyphenol oxidase and 0.025 × 10(-5) ± 0.003 glutathione peroxidase). The microalgal biomass also showed, per milligram weight, 0.2182 ± 0.005 μg of ascorbic acid, 0.00264 ± 0.001 μg of α-tocopherol and 0.07916 ± 0.004 μg of reduced glutathione. These results represent the possibility of using C. vulgaris grown in confectionery industry wastewater as a source of nutritious supplement, which is highly promising in terms of both economic and nutritional point of view.

  18. Medium- and short-chain dehydrogenase/reductase gene and protein families : the SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes.

    Science.gov (United States)

    Kavanagh, K L; Jörnvall, H; Persson, B; Oppermann, U

    2008-12-01

    Short-chain dehydrogenases/reductases (SDRs) constitute a large family of NAD(P)(H)-dependent oxidoreductases, sharing sequence motifs and displaying similar mechanisms. SDR enzymes have critical roles in lipid, amino acid, carbohydrate, cofactor, hormone and xenobiotic metabolism as well as in redox sensor mechanisms. Sequence identities are low, and the most conserved feature is an alpha/beta folding pattern with a central beta sheet flanked by 2 - 3 alpha-helices from each side, thus a classical Rossmannfold motif for nucleotide binding. The conservation of this element and an active site, often with an Asn-Ser-Tyr-Lys tetrad, provides a platform for enzymatic activities encompassing several EC classes, including oxidoreductases, epimerases and lyases. The common mechanism is an underlying hydride and proton transfer involving the nicotinamide and typically an active site tyrosine residue, whereas substrate specificity is determined by a variable C-terminal segment. Relationships exist with bacterial haloalcohol dehalogenases, which lack cofactor binding but have the active site architecture, emphasizing the versatility of the basic fold in also generating hydride transfer-independent lyases. The conserved fold and nucleotide binding emphasize the role of SDRs as scaffolds for an NAD(P)(H) redox sensor system, of importance to control metabolic routes, transcription and signalling.

  19. Enzymatic digestion of articular cartilage results in viscoelasticity changes that are consistent with polymer dynamics mechanisms

    National Research Council Canada - National Science Library

    June, Ronald K; Fyhrie, David P

    2009-01-01

    .... This study tested whether the predictions of polymer dynamics were consistent with changes in cartilage mechanics caused by enzymatic digestion of specific cartilage extracellular matrix molecules...

  20. Enzymatic synthesis of galacto-oligosaccharides and other lactose derivatives (hetero-oligosaccharides) from lactose

    National Research Council Canada - National Science Library

    Gänzle, Michael G

    2012-01-01

    .... Oligosaccharides derived through enzymatic synthesis from lactose, i.e., galacto-oligosaccharides, lactulose and lactosucrose, account for a major part of the annual oligosaccharide production...

  1. Novel investigation of enzymatic biodiesel reaction by isothermal calorimetry

    Energy Technology Data Exchange (ETDEWEB)

    Sotoft, Lene Fjerbaek, E-mail: lfj@kbm.sdu.dk [Institute of Chemical Engineering, Biotechnology and Environmental Technology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M (Denmark); Westh, Peter [Department of Life Science and Chemistry, Roskilde University, PO Box 260, DK-4000 Roskilde (Denmark); Christensen, Knud V.; Norddahl, Birgir [Institute of Chemical Engineering, Biotechnology and Environmental Technology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M (Denmark)

    2010-03-30

    Isothermal calorimetry (ITC) was used to investigate solvent-free enzymatic biodiesel production. The transesterification of rapeseed oil with methanol and ethanol was catalyzed by immobilized lipase Novozym 435 at 40 {sup o}C. The aim of the study was to determine reaction enthalpy for the enzymatic transesterification and to elucidate the mass transfer and energetic processes taking place. Based on the measured enthalpy and composition change in the system, the heat of reaction at 40 {sup o}C for the two systems was determined as -9.8 {+-} 0.9 kJ/mole biodiesel formed from rapeseed oil and methanol, and -9.3 {+-} 0.7 kJ/mole when rapeseed oil and ethanol was used. Simple Michaelis-Menten kinetics was not an appropriate choice for describing the kinetics of this heterogeneous system. The experiments demonstrated the possibility of investigating complex reaction mixtures using ITC. Although it is possible to determine thermodynamic properties such as reaction enthalpy and reaction rate, the difficulty in actually measuring the true non-mass-transfer-limited reaction kinetics is exposed by the high time resolution of ITC.

  2. Improvement of Soybean Oil Solvent Extraction through Enzymatic Pretreatment

    Directory of Open Access Journals (Sweden)

    F. V. Grasso

    2012-01-01

    Full Text Available The purpose of this study is to evaluate multienzyme hydrolysis as a pretreatment option to improve soybean oil solvent extraction and its eventual adaptation to conventional processes. Enzymatic action causes the degradation of the cell structures that contain oil. Improvements in terms of extraction, yield, and extraction rate are expected to be achieved. Soybean flakes and collets were used as materials and hexane was used as a solvent. Temperature, pH, and incubation time were optimized and diffusion coefficients were estimated for each solid. Extractions were carried out in a column, oil content was determined according to time, and a mathematical model was developed to describe the system. The optimum conditions obtained were pH 5.4, 38°C, and 9.7 h, and pH 5.8, 44°C, and 5.8h of treatment for flakes and collets, respectively. Hydrolyzed solids exhibited a higher yield. Diffusion coefficients were estimated between 10-11 and 10-10. The highest diffusion coefficient was obtained for hydrolyzed collets. 0.73 g oil/mL and 0.7 g oil/mL were obtained at 240 s in a column for collets and flakes, respectively. Hydrolyzed solids exhibited a higher yield. The enzymatic incubation accelerates the extraction rate and allows for higher yield. The proposed model proved to be appropriate.

  3. Queueing up for enzymatic processing: correlated signaling through coupled degradation

    Science.gov (United States)

    Cookson, Natalie A; Mather, William H; Danino, Tal; Mondragón-Palomino, Octavio; Williams, Ruth J; Tsimring, Lev S; Hasty, Jeff

    2011-01-01

    High-throughput technologies have led to the generation of complex wiring diagrams as a post-sequencing paradigm for depicting the interactions between vast and diverse cellular species. While these diagrams are useful for analyzing biological systems on a large scale, a detailed understanding of the molecular mechanisms that underlie the observed network connections is critical for the further development of systems and synthetic biology. Here, we use queueing theory to investigate how ‘waiting lines' can lead to correlations between protein ‘customers' that are coupled solely through a downstream set of enzymatic ‘servers'. Using the E. coli ClpXP degradation machine as a model processing system, we observe significant cross-talk between two networks that are indirectly coupled through a common set of processors. We further illustrate the implications of enzymatic queueing using a synthetic biology application, in which two independent synthetic networks demonstrate synchronized behavior when common ClpXP machinery is overburdened. Our results demonstrate that such post-translational processes can lead to dynamic connections in cellular networks and may provide a mechanistic understanding of existing but currently inexplicable links. PMID:22186735

  4. Highly Efficient Enzymatic Preparation of Daidzein in Deep Eutectic Solvents

    Directory of Open Access Journals (Sweden)

    Qi-Bin Cheng

    2017-01-01

    Full Text Available Daidzein, which is scarce in nature, has gained significant attention due to its superior biological activity and bioavailability compared with daidzin. So far, it has been widely used in the medicine and health care products industries. The enzymatic approach for the preparation of daidzein has prevailed, benefitted by its high efficiency and eco-friendly nature. Our present research aimed at providing a preparation method of daidzein by enzymatic hydrolysis of daidzin in a new “green” reaction medium-deep eutectic solvents (DESs. Herein, the DESs were screened via evaluating enzyme activity, enzyme stability and the substrate solubility, and the DES (ChCl/EG 2:1, 30 vol % was believed to be the most appropriate co-solvent to improve the bioconversion efficiency. Based on the yield of daidzein, response surface methodology (RSM was employed to model and optimize the reaction parameters. Under these optimum process conditions, the maximum yield of 97.53% was achieved and the purity of daidzein crude product reached more than 70%, which is more efficient than conversions in DESs-free buffer. Importantly, it has been shown that DESs medium could be reused for six batches of the process with a final conversion of above 50%. The results indicated that this procedure could be considered a mild, environmentally friendly, highly efficient approach to the economical production of daidzein, with a simple operation process and without any harmful reagents being involved.

  5. Study on enzymatic browning in suspension cultures of licorice cells

    Directory of Open Access Journals (Sweden)

    Yali Li

    2016-03-01

    Full Text Available Enzymatic browning is one of the main obstacles encountered in the establishment of suspension systems of licorice cells. Browning of cells may result in decreased viability, poor growth and even death. The present study investigated the mechanism of browning reactions and the effective controlling methods. The results showed that the cell viability and membrane permeabilization obviously changed when the cells were transferred to liquid medium. The transformation caused rapid increase in the levels of polyphenol oxidase activity and in the production of polyphenols. Osmotic and hydrodynamic stresses arising from liquid culture were regarded as the major causes of enzymatic browning. Ascorbic acid and L-cysteine were found to be the most significant anti-browning agents that could decrease the degree of browning with 55.8% and 52.2%, respectively, at the end of the suspension culture's lag phase. When cultured with a cycle of 21 days, the maximum biomass of the cells cultured with ascorbic acid and L-cysteine increased with 31.1% and 26.5%, respectively, when compared to the control. These findings may be essential for the development of licorice cell cultures devoted to browning prevention and cell viability maintaining.

  6. Structure and solution properties of enzymatically synthesized glycogen.

    Science.gov (United States)

    Kajiura, Hideki; Takata, Hiroki; Kuriki, Takashi; Kitamura, Shinichi

    2010-04-19

    Recently, a new enzymatic process for glycogen production was developed. In this process, short-chain amylose is used as a substrate for branching enzymes (BE, EC 2.4.1.18). The molecular weight of the enzymatically synthesized glycogen (ESG) depends on the size and concentration of the substrate. Structural and physicochemical properties of ESG were compared to those of natural source glycogen (NSG). The average chain length, interior chain length, and exterior chain length of ESG were 8.2-11.6, 2.0-3.3, and 4.2-7.6, respectively. These values were within the range of variation of NSG. The appearances of both ESG and NSG in solution were opalescent (milky white and slightly bluish). Furthermore, transmission electron microscopy and atomic force microscopy showed that ESG molecules formed spherical particles, and that there were no differences between ESG and NSG. Viscometric analyses also showed the spherical nature of both glycogens. When ESG and NSG were treated with pullulanase, a glucan-hydrolyzing enzyme known to degrade glycogen only on its surface portion, both glycogens were similarly degraded. These analyses revealed that ESG shares similar molecular shapes and surface properties with NSG. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  7. Enzymatic hydrolysis of fructans in the tequila production process.

    Science.gov (United States)

    Avila-Fernández, Angela; Rendón-Poujol, Xóchitl; Olvera, Clarita; González, Fernando; Capella, Santiago; Peña-Alvarez, Araceli; López-Munguía, Agustín

    2009-06-24

    In contrast to the hydrolysis of reserve carbohydrates in most plant-derived alcoholic beverage processes carried out with enzymes, agave fructans in tequila production have traditionally been transformed to fermentable sugars through acid thermal hydrolysis. Experiments at the bench scale demonstrated that the extraction and hydrolysis of agave fructans can be carried out continuously using commercial inulinases in a countercurrent extraction process with shredded agave fibers. Difficulties in the temperature control of large extraction diffusers did not allow the scaling up of this procedure. Nevertheless, batch enzymatic hydrolysis of agave extracts obtained in diffusers operating at 60 and 90 degrees C was studied at the laboratory and industrial levels. The effects of the enzymatic process on some tequila congeners were studied, demonstrating that although a short thermal treatment is essential for the development of tequila's organoleptic characteristics, the fructan hydrolysis can be performed with enzymes without major modifications in the flavor or aroma, as determined by a plant sensory panel and corroborated by the analysis of tequila congeners.

  8. Synergistic enzymatic saccharification and fermentation of agar for biohydrogen production.

    Science.gov (United States)

    Wu, Yi-Rui; Zhang, Mingming; Zhong, Mingqi; Hu, Zhong

    2017-10-01

    Nowadays, marine biomass is gradually considered as another utilizable material for the sustainable bioenergy development. In the present study, galactose, the main component of agar polysaccharide, was utilized for the biohydrogen production by Enterobacter sp. CN1. The highest hydrogen yield of 303.2mL/g was obtained in the cultivation media containing 5.87g/L of galactose, together with initial pH of 7.3 and incubation temperature of 36°C, after the response surface methodology (RSM) analysis. After the saccharification process by the agarase (AgaXa) and neoagarobiose hydrolase (NH852), the agar hydrolysate obtained was further applied to generate biohydrogen by strain CN1. Under the synergistic enzymatic saccharification and fermentation process, the production of biohydrogen was obtained to be 5047±228mL/L from 50g/L of agar, resulting in 3.86-fold higher than the control without enzymatic pretreatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Ultrasound enhanced enzymatic hydrolysis of Parthenium hysterophorus: A mechanistic investigation.

    Science.gov (United States)

    Singh, Shuchi; Agarwal, Mayank; Bhatt, Aditya; Goyal, Arun; Moholkar, Vijayanand S

    2015-09-01

    This study has attempted to establish the mechanism of the ultrasound-induced enhancement of enzymatic hydrolysis of pretreated and delignified biomass of Parthenium hysterophorus. A dual approach of statistical optimization of hydrolysis followed by application of sonication at optimum conditions has been adopted. The kinetics of hydrolysis shows a marked 6× increase with sonication, while net sugar yield shows marginal rise of ∼ 20%. The statistical experimental design reveals the hydrolysis process to be enzyme limited. Profile of sugar yield in ultrasound-assisted enzymatic hydrolysis has been analyzed using HCH-1 model coupled with Genetic Algorithm optimization. The trends in the kinetic and physiological parameters of HCH-1 model reveal that sonication enhances enzyme/substrate affinity and reaction velocity of hydrolysis. The product inhibition of enzyme in all forms (free, adsorbed, complexed) also reduces with ultrasound. These effects are attributed to intense micro-convection induced by ultrasound and cavitation in the liquid medium. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Chemical and Enzymatic Hydrolysis of Polyurethane/Polylactide Blends

    Directory of Open Access Journals (Sweden)

    Joanna Brzeska

    2015-01-01

    Full Text Available Polyether-esterurethanes containing synthetic poly[(R,S-3-hydroxybutyrate] (R,S-PHB and polyoxytetramethylenediol in soft segments and polyesterurethanes with poly(ε-caprolactone and poly[(R,S-3-hydroxybutyrate] were blended with poly([D,L]-lactide (PLA. The products were tested in terms of their oil and water absorption. Oil sorption tests of polyether-esterurethane revealed their higher response in comparison to polyesterurethanes. Blending of polyether-esterurethanes with PLA caused the increase of oil sorption. The highest water sorption was observed for blends of polyether-esterurethane, obtained with 10% of R,S-PHB in soft segments. The samples mass of polyurethanes and their blends were almost not changed after incubation in phosphate buffer and trypsin and lipase solutions. Nevertheless the molecular weight of polymers was significantly reduced after degradation. It was especially visible in case of incubation of samples in phosphate buffer what suggested the chemical hydrolysis of polymer chains. The changes of surface of polyurethanes and their blends, after incubation in both enzymatic solutions, indicated on enzymatic degradation, which had been started despite the lack of mass lost. Polyurethanes and their blends, contained more R,S-PHB in soft segments, were degraded faster.

  11. 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.

  12. Production and characterization of cowpea protein hydrolysate with optimum nitrogen solubility by enzymatic hydrolysis using pepsin.

    Science.gov (United States)

    Mune Mune, Martin Alain; Minka, Samuel René

    2017-06-01

    Cowpea is a source of low-cost and good nutritional quality protein for utilization in food formulations in replacement of animal proteins. Therefore it is necessary that cowpea protein exhibits good functionality, particularly protein solubility which affects the other functional properties. The objective of this study was to produce cowpea protein hydrolysate exhibiting optimum solubility by the adequate combination of hydrolysis parameters, namely time, solid/liquid ratio (SLR) and enzyme/substrate ratio (ESR), and to determine its functional properties and molecular characteristics. A Box-Behnken experimental design was used for the experiments, and a second-order polynomial to model the effects of hydrolysis time, SLR and ESR on the degree of hydrolysis and nitrogen solubility index. The optimum hydrolysis conditions of time 208.61 min, SLR 1/15 (w/w) and ESR 2.25% (w/w) yielded a nitrogen solubility of 75.71%. Protein breakdown and the peptide profile following enzymatic hydrolysis were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and size exclusion chromatography. Cowpea protein hydrolysate showed higher oil absorption capacity, emulsifying activity and foaming ability compared with the concentrate. The solubility of cowpea protein hydrolysate was adequately optimized by response surface methodology, and the hydrolysate showed adequate functionality for use in food. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  13. Enzymatic production of DFA III from fresh dahlia tubers as raw material

    Science.gov (United States)

    Budiwati, Thelma A.; Ratnaningrum, D.; Pudjiraharti, S.

    2017-01-01

    Dahlia is an annual ornamental plants and tubers that have not been widely used in Indonesia. Dahlia tubers contain nearly 70 per cent of the starch in the form of inulin. Inulin addition can be used as a food ingredient can also be used as a raw material for making DFA III (ie functional oligosaccharides), using inulin fructotransferase (IFTase) Nonomuraea sp. In this study conducted production of DFA III through enzymatic reactions and yeast fermentation, using inulin from fresh dahlia tubers and fresh dahlia tuber extract. Dahlia tubers which is one source of inulin, do blanching before extracted. Most dahlia tuber extract used directly for enzymatic reactions in the production of DFA III and some extracts are processed to produce inulin by precipitation using ethanol and then inulin is used for the enzymatic reaction. Syrup DFA III was measured volume and viscosity, and then do decolorization and then crystallization. The analysis was done of Thin Layer Chromatography (to see DFA III formed) and HPLC to see the purity of the product. The results showed that the average of inulin from precipitation with ethanol in the two batch of 113,5 g with an average water content of 7.41%, average whiteness degree 62.29% and an average yield 7.345% (w/w, wb dahlia tuber). From the average of DFA III liquid of 480 mL with density of 14.15%, the result of the average of DFA III crystal from enzyme reaction in the two reactor using inulin dahlia tubers as a substrate, was obtained of 55.4 g with an average whiteness degree of 93.8%, and the average of yield 3.56% w/w (wb dahlia tuber) or 48.89% w/w (db inulin). And then from the average of 475 mL with density of 16.85% was obtained an average DFA III crystals of 29 g from the enzyme reaction in the two reactor using fresh dahlia tuber extract as a substrate, with an average whiteness degree o 80.75% and the average of the yield of 1.86% w/w (wb dahlia tuber).

  14. Vertical and horizontal distributions of microbial abundances and enzymatic activities in propylene-glycol-affected soils.

    Science.gov (United States)

    Biró, Borbála; Toscano, Giuseppe; Horváth, Nikoletta; Matics, Heléna; Domonkos, Mónika; Scotti, Riccardo; Rao, Maria A; Wejden, Bente; French, Helen K

    2014-01-01

    The natural microbial activity in the unsaturated soil is vital for protecting groundwater in areas where high loads of biodegradable contaminants are supplied to the surface, which usually is the case for airports using aircraft de-icing fluids (ADF) in the cold season. Horizontal and vertical distributions of microbial abundance were assessed along the western runway of Oslo Airport (Gardermoen, Norway) to monitor the effect of ADF dispersion with special reference to the component with the highest chemical oxygen demand (COD), propylene glycol (PG). Microbial abundance was evaluated by several biondicators: colony-forming units (CFU) of some physiological groups (aerobic and anaerobic heterotrophs and microscopic fungi), most probable numbers (MPN) of PG degraders, selected catabolic enzymatic activities (fluorescein diacetate (FDA) hydrolase, dehydrogenase, and β-glucosidase). High correlations were found between the enzymatic activities and microbial counts in vertical soil profiles. All microbial abundance indicators showed a steep drop in the first meter of soil depth. The vertical distribution of microbial abundance can be correlated by a decreasing exponential function of depth. The horizontal trend of microbial abundance (evaluated as total aerobic CFU, MPN of PG-degraders, and FDA hydrolase activity) assessed in the surface soil at an increasing distance from the runway is correlated negatively with the PG and COD loads, suggesting the relevance of other chemicals in the modulation of microbial growth. The possible role of potassium formate, component of runway de-icers, has been tested in the laboratory by using mixed cultures of Pseudomonas spp., obtained by enrichment with a selective PG medium from soil samples taken at the most contaminated area near the runway. The inhibitory effect of formate on the growth of PG degraders is proven by the reduction of biomass yield on PG in the presence of formate.

  15. [Development of novel asymmetric reactions oriented to next-generation enzymatic organic syntheses].

    Science.gov (United States)

    Akai, Shuji

    2003-11-01

    Enzyme-catalyzed organic syntheses have enormous potential in the development of environmentally benign processes. In the last two decades, increased efforts have been devoted to making this a reality. However, the utility of the enzymes is generally limited. For example, although the lipases are extensively used for the kinetic resolution or the desymmetrization of alcohols and carboxylic acid derivatives, little is known about their ability to catalyze carbon-carbon bond-forming reactions. In the past several years, we have been engaged in exploiting a next-generation enzymatic synthesis by using the lipases for the construction of carbon skeletons. This review article describes the results. First, the development of a new type of acyl donor, 1-ethoxyvinyl esters (EVEs), that have significant advantages over common acyl donors (e.g., vinyl esters) for lipase-catalyzed esterification reactions. Second, the highly enantioselective desymmetrization of prochiral 1,3-propanediols and meso 1,2-diols using 1-ethoxyvinyl 2-furoate. The application of this technology for the asymmetric syntheses of fredericamycin A analogues and the oxindoles with a chiral, nonracemic quaternary carbon center has been demonstrated. Third, the first lipase-catalyzed domino reaction using EVEs possessing a suitably functionalized acyl moiety is described. The acyl moiety installed during the enzymatic kinetic resolution was used as a part of the constituent structure for the subsequent Diels--Alder reaction to product optically pure tricyclic compounds with five chiral carbon centers via a one-pot operation. The potential influence of the lipase on the Diets--Alder reaction and the domino process accompanied by the dynamic kinetic resolution are also described.

  16. 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.

  17. Microbial/enzymatic synthesis of chiral drug intermediates.

    Science.gov (United States)

    Patel, R N

    2000-01-01

    Biocatalytic processes were used to prepare chiral intermediates for pharmaceuticals. These include the following processes. Enzymatic synthesis of [4S-(4a,7a,10ab)]1-octahydro-5-oxo-4-[[(phenylmethoxy) carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid methyl ester (BMS-199541-01), a key chiral intermediate for synthesis of a new vasopeptidase inhibitor. Enzymatic oxidation of the epsilon-amino group of lysine in dipeptide dimer N2-[N[[(phenylmethoxy)carbonyl] L-homocysteinyl] L-lysine)1,1-disulfide (BMS-201391-01) to produce BMS-199541-01 using a novel L-lysine epsilon-aminotransferase from S. paucimobilis SC16113 was demonstrated. This enzyme was overexpressed in E. coli, and a process was developed using recombinant enzyme. The aminotransferase reaction required alpha-ketoglutarate as the amine acceptor. Glutamate formed during this reaction was recycled back to alpha-ketoglutarate by glutamate oxidase from S. noursei SC6007. Synthesis and enzymatic conversion of 2-keto-6-hydroxyhexanoic acid 5 to L-6-hydroxy norleucine 4 was demonstrated by reductive amination using beef liver glutamate dehydrogenase. To avoid the lengthy chemical synthesis of ketoacid 5, a second route was developed to prepare the ketoacid by treatment of racemic 6-hydroxy norleucine (readily available from hydrolysis of 5-(4-hydroxybutyl) hydantoin, 6) with D-amino acid oxidase from porcine kidney or T. variabilis followed by reductive amination to convert the mixture to L-6-hydroxynorleucine in 98% yield and 99% enantiomeric excess. Enzymatic synthesis of (S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (allysine ethylene acetal, 7), one of three building blocks used for synthesis of a vasopeptidase inhibitor, was demonstrated using phenylalanine dehydrogenase from T. intermedius. The reaction requires ammonia and NADH. NAD produced during the reaction was recycled to NADH by oxidation of formate to CO2 using formate dehydrogenase. Efficient synthesis of chiral

  18. Alkaline-sulfite pretreatment and use of surfactants during enzymatic hydrolysis to enhance ethanol production from sugarcane bagasse.

    Science.gov (United States)

    Mesquita, Jéssica Faria; Ferraz, André; Aguiar, André

    2016-03-01

    Sugarcane bagasse is a by-product from the sugar and ethanol industry which contains approximately 70 % of its dry mass composed by polysaccharides. To convert these polysaccharides into fuel ethanol it is necessary a pretreatment step to increase the enzymatic digestibility of the recalcitrant raw material. In this work, sugarcane bagasse was pretreated by an alkaline-sulfite chemithermomechanical process for increasing its enzymatic digestibility. Na2SO3 and NaOH ratios were fixed at 2:1, and three increasing chemical loads, varying from 4 to 8 % m/m Na2SO3, were used to prepare the pretreated materials. The increase in the alkaline-sulfite load decreased the lignin content in the pretreated material up to 35.5 % at the highest chemical load. The pretreated samples presented enhanced glucose yields during enzymatic hydrolysis as a function of the pretreatment severity. The maximum glucose yield (64 %) was observed for the samples pretreated with the highest chemical load. The use of 2.5 g l(-1) Tween 20 in the hydrolysis step further increased the glucose yield to 75 %. Semi-simultaneous hydrolysis and fermentation of the pretreated materials indicated that the ethanol yield was also enhanced as a function of the pretreatment severity. The maximum ethanol yield was 56 ± 2 % for the sample pretreated with the highest chemical load. For the sample pretreated with the lowest chemical load (2 % m/m NaOH and 4 % m/m Na2SO3), adding Tween 20 during the hydrolysis process increased the ethanol yield from 25 ± 3 to 39.5 ± 1 %.

  19. The enzymatic determination of starch in food, feed and raw materials of the starch industry

    NARCIS (Netherlands)

    Brunt, K.; Sanders, P.; Rozema, T.

    1998-01-01

    An enzymatic starch determination which can be used for the analysis of starch in a very broad range of different samples is evaluated, ranging from starch in plants, feed and food to industrial applications as starch in starch. The method is based on a complete enzymatic conversion of the starch

  20. Effect of Maize Biomass Composition on the Optimization of Dilute-Acid Pretreatments and Enzymatic Saccharification

    NARCIS (Netherlands)

    Torres Salvador, A.F.; Weijde, van der R.T.; Dolstra, O.; Visser, R.G.F.; Trindade, L.M.

    2013-01-01

    At the core of cellulosic ethanol research are innovations leading to reductions in the chemical and energetic stringency of thermochemical pretreatments and enzymatic saccharification. In this study, key compositional features of maize cell walls influencing the enzymatic conversion of biomass into

  1. The effect of high intensity mixing on the enzymatic hydrolysis of concentrated cellulose fiber suspensions

    Science.gov (United States)

    Joseph R. Samaniuk; C. Tim Scott; Thatcher W. Root; Daniel J. Klingenberg

    2011-01-01

    Enzymatic hydrolysis of lignocellulosic biomass in a high shear environment was examined. The conversion of cellulose to glucose in samples mixed in a torque rheometer producing shear flows similar to those found in twin screw extruders was greater than that of unmixed samples. In addition, there is a synergistic effect of mixing and enzymatic hydrolysis; mixing...

  2. Disruption of Methicillin-resistant Staphylococcus aureus Biofilms with Enzymatic Therapeutics

    Science.gov (United States)

    2015-04-29

    NAVAL MEDICAL RESEARCH UNIT SAN ANTONIO Disruption of Methicillin-resistant Staphylococcus aureus Biofilms with Enzymatic...Methicillin-resistant Staphylococcus aureus MSSA Methicillin-sensitive Staphylococcus aureus OD Optical density PBS Phosphate-buffered saline SEM... Staphylococcus aureus biofilm model that mimics wound-like conditions and employ this model to evaluate the anti-biofilm activity of four enzymatic compounds

  3. ENZYMATIC HYDROLYSIS AS AN ENVIRONMENTALLY FRIENDLY PROCESS COMPARED TO THERMAL HYDROLYSIS FOR INSTANT COFFEE PRODUCTION

    Directory of Open Access Journals (Sweden)

    I. J. Baraldi

    Full Text Available Abstract Conventional production of instant coffee is based on solubilisation of polysaccharides present in roasted coffee. Higher process temperatures increase the solubilisation yield, but also lead to carbohydrate degradation and formation of undesirable volatile compounds. Enzymatic hydrolysis of roasted coffee is an alternative to minimize carbohydrate degradation. In this work, products obtained from thermal and enzymatic processes were compared in terms of carbohydrates and volatiles composition. Roasted coffee was extracted with water at 125 °C, and spent coffee was processed by thermal (180 °C or enzymatic hydrolysis. Enzymatic hydrolysis experiments were carried out at 50 °C using the commercial enzyme preparations Powercell (Prozyn, Galactomannanase (HBI-Enzymes, and Ultraflo XL (Novozymes. These formulations were previously selected from eleven different commercial enzyme preparations, and their main enzymatic activities included cellulase, galactomannanase, galactanase, and β-glucanase. Enzymatic hydrolysis yield was 18% (dry basis, similar to the extraction yield at 125 °C (20%, but lower than the thermal hydrolysis yield at 180 °C (28%. Instant coffee produced by enzymatic hydrolysis had a low content of undesirable volatile compounds and 21% (w/w of total carbohydrates. These results point to the enzymatic process as a feasible alternative for instant coffee production, with benefits including improved quality as well as reduced energy consumption.

  4. Effects of lignin-metal complexation on enzymatic hydrolysis of cellulose

    Science.gov (United States)

    H. Liu; Junyong Zhu; S.Y. Fu

    2010-01-01

    This study investigated the inhibition of enzymatic hydrolysis by unbound lignin (soluble and insoluble) with or without the addition of metal compounds. Sulfonated, Organosolv, and Kraft lignin were added in aqueous enzyme-cellulose systems at different concentrations before hydrolysis. The measured substrate enzymatic digestibility (SED) of cellulose was decreased by...

  5. One-pot peptide and protein conjugation: a combination of enzymatic transamidation and click chemistry.

    Science.gov (United States)

    Rachel, N M; Pelletier, J N

    2016-02-11

    Enzymatic transamidation and copper-catalyzed azide-alkyne cycloaddition (CuAAC) were combined to yield covalently conjugated peptides and proteins. The addition of glutathione preserved enzymatic activity in the presence of copper. Tuning the reaction kinetics was key to success, providing up to 95% conversion. This one-pot reaction allowed for targeted fluorescent protein labeling.

  6. Understanding the effects of lignosulfonate on enzymatic saccharification of pure cellulose

    Science.gov (United States)

    Hongming Lou; Haifeng Zhou; Xiuli Li; Mengxia Wang; J.Y. Zhu; Xueqing Qiu

    2014-01-01

    The effects of lignosulfonate (LS) on enzymatic saccharification of pure cellulose were studied. Four fractions of LS with different molecular weight (MW) prepared by ultrafiltration of a commercial LS were applied at different loadings to enzymatic hydrolysis of Whatman paper under different pH. Using LS fractions with low MW and high degree of sulfonation can enhance...

  7. Transport equations in an enzymatic glucose fuel cell

    Science.gov (United States)

    Jariwala, Soham; Krishnamurthy, Balaji

    2018-01-01

    A mathematical model is developed to study the effects of convective flux and operating temperature on the performance of an enzymatic glucose fuel cell with a membrane. The model assumes isothermal operating conditions and constant feed rate of glucose. The glucose fuel cell domain is divided into five sections, with governing equations describing transport characteristics in each region, namely - anode diffusion layer, anode catalyst layer (enzyme layer), membrane, cathode catalyst layer and cathode diffusion layer. The mass transport is assumed to be one-dimensional and the governing equations are solved numerically. The effects flow rate of glucose feed on the performance of the fuel cell are studied as it contributes significantly to the convective flux. The effects of operating temperature on the performance of a glucose fuel cell are also modeled. The cell performances are compared using cell polarization curves, which were found compliant with experimental observations.

  8. Production of resistant starch by enzymatic debranching in legume flours.

    Science.gov (United States)

    Morales-Medina, Rocío; Del Mar Muñío, María; Guadix, Emilia M; Guadix, Antonio

    2014-01-30

    Resistant starch (RS) was produced by enzymatic hydrolysis of flours from five different legumes: lentil, chickpea, faba bean, kidney bean and red kidney bean. Each legume was firstly treated thermally, then hydrolyzed with pullulanase for 24h at 50°C and pH 5 and lyophilized. At the end of each hydrolysis reaction, the RS amount ranged from 4.7% for red kidney beans to 7.5% for chickpeas. With respect to the curves of RS against hydrolysis time, a linear increase was observed initially and a plateau was generally achieved by the end of reaction. These curves were successfully modeled by a kinetic equation including three parameters: initial RS, RS at long operation time and a kinetic constant (k). Furthermore, the relative increase in hydrolysis, calculated using the kinetic parameters, was successfully correlated to the percentage of amylose. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Enzymatic transesterification of waste vegetable oil to produce biodiesel.

    Science.gov (United States)

    Lopresto, C G; Naccarato, S; Albo, L; De Paola, M G; Chakraborty, S; Curcio, S; Calabrò, V

    2015-11-01

    An experimental study on enzymatic transesterification was performed to produce biodiesel from waste vegetable oils. Lipase from Pseudomonas cepacia was covalently immobilized on a epoxy-acrylic resin support. The immobilized enzyme exhibited high catalytic specific surface and allowed an easy recovery, regeneration and reutilisation of biocatalyst. Waste vegetable oils - such as frying oils, considered not competitive with food applications and wastes to be treated - were used as a source of glycerides. Ethanol was used as a short chain alcohol and was added in three steps with the aim to reduce its inhibitory effect on lipase activity. The effect of biocatalyst/substrate feed mass ratios and the waste oil quality have been investigated in order to estimate the process performances. Biocatalyst recovery and reuse have been also studied with the aim to verify the stability of the biocatalyst for its application in industrial scale. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Enzymatic production of hydrogen gas from glucose and cellulose

    Energy Technology Data Exchange (ETDEWEB)

    Mattingly, S.M.; Woodward, J. [Oak Ridge National Lab., TN (United States)

    1996-10-01

    An enzymatic process has been used to convert glucose to molecular hydrogen with the ultimate goal of converting cellulose to hydrogen. Two enzymes from the Archae, Thermoplasma acidophilium glucose dehydrogenase (GDH) and Pyrococcus furiosus hydrogenase, were used to oxidize glucose and NADPH respectively, resulting in the formation of molecular hydrogen. The stoichiometric yield of hydrogen from glucose was close to the theoretical maximum expected. Further, the molar amount of hydrogen produced was greater than the molar equivalent of NADP{sup +} present in the reaction mixture indicating that this GDH cofactor was regenerated throughout the course of the reaction. Hydrogen was also shown to be produced from cellulose if cellulase was included in the reaction mixture.

  11. 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.

  12. Enzymatic hydrolysis of pretreated barley and wheat straw

    DEFF Research Database (Denmark)

    Rosgaard, Lisa

    2007-01-01

    feeding strategy to increase the substrate loading in the hydrolysis reaction. The substrate for the enzymatic hydrolysis was primarily steam pretreated wheat and barley straw since these substrates were the primary feedstocks for the Babilafuente Bioethanol process. The initial work showed...... that there was indeed potential to boost the enzyme activities in Celluclast (arising from Trichoderma reesei) by addition of small amounts of fermentation broth from fungal sources other than T. reesei at optimal reaction conditions for Celluclast, pH 5, 50 °C. The activity(ies) related to the boosting effect were...... indicated to arise from more efficient or different endoglucanase activities than those found in Celluclast. Evaluating of the extent of hydrolysis using the 4 major enzyme activities in Celluclast, which constituted a complete set of enzymes for hydrolysis of cellulose, showed that the most efficient...

  13. Structural Characterization and Enzymatic Modification of Soybean Polysaccharides

    DEFF Research Database (Denmark)

    Pierce, Brian; Wichmann, Jesper

    investigations utilized TrCel61A, an AA9 LPMO from Trichoderma reesei. This enzyme showed no oxidative activity on native soybean polysaccharides; however, significant oxidative degradation was observed on NaOH pretreated soybean polysaccharides. The oxidation products were evaluated using HPAEC and MS......, with the results showing oxi-dation at both the C1 and C4 positions of cellulose. In addition, a synergistic effect between TrCel61A and a GH5 endo-β-1,4-glucanase was discovered, boosting the glucose release from NaOH pretreated soybean polysaccha-rides. Building upon these observations, twenty-three additional...... LPMOs from seven fungal sources were evaluated (using TrCel61A as a benchmark), with none showing oxidative activity on native soybean polysaccharides. However, NaOH pretreatment of the raw material was shown to improve the enzymatic accessibility of the soybean cellulose through the removal of non...

  14. An infrared radiation based thermal biosensor for enzymatic biochemical reactions.

    Science.gov (United States)

    Zhang, Lei; Dong, Tao; Zhao, Xinyan; Yang, Zhaochu; Pires, Nuno M M

    2012-01-01

    In this paper, a thermal biosensor based on the infrared radiation energy is proposed for calorimetric measurement of biochemical reactions. Having a good structure design combined with MEMS technology as well as employing the Si /SiGe quantum well sensing material with a high TCR and low 1/f noise, the sensor shows potentials to be high sensitive and real-time. The urea enzymatic reaction was tested to verify the performance of sensor, which demonstrates a linear detection range from 0.5mM to 150mM and a relative standard deviation less than 1%. For the sensor fabrication, wafer-level transfer bonding is a key process, which makes the integration of quantum well material and a free standing structure possible. It reduces the heat loss from the sensor to the surrounding environment.

  15. Chemo-enzymatic epoxidation of sunflower oil methyl esters

    Energy Technology Data Exchange (ETDEWEB)

    Schneider, Rosana de Cassia S.; Lara, Luciano R.S. [Universidade de Santa Cruz do Sul, RS (Brazil). Dept. de Quimica e Fisica], e-mail: rosana@unisc.br; Bitencourt, Thiago B.; Nascimento, Maria da Graca [Universidade Federal de Santa Catarina (UFSC), Florianopolis, SC (Brazil). Dept. de Quimica; Nunes, Marta R. dos Santos [Universidade de Caxias do Sul, RS (Brazil). Centro de Ciencias Exatas e Tecnologia

    2009-07-01

    The chemo-enzymatic epoxidation of the methyl esters of sunflower oil with lipase from Candida antarctica B and aqueous H{sub 2}O{sub 2} in the presence and absence of an acyl donor was investigated. The biphasic system (CH{sub C}l{sub 2}/H{sub 2}O) comprised Candida antarctica B lipase (CALB, 1000 u g{sup -1}) and 30% (v/v) aqueous hydrogen peroxide. In some cases the conversion was higher than 99%. The best results were obtained for the biphasic system after 16 h of reaction, at 30 deg C, using 10 mmol of octanoic acid in relation to 1 g of the oil, 6 mL of dichloromethane and 5 mL of water. (author)

  16. Rapid enzymatic response to compensate UV radiation in copepods.

    Directory of Open Access Journals (Sweden)

    María Sol Souza

    Full Text Available Ultraviolet radiation (UVR causes physical damage to DNA, carboxylation of proteins and peroxidation of lipids in copepod crustaceans, ubiquitous and abundant secondary producers in most aquatic ecosystems. Copepod adaptations for long duration exposures include changes in behaviour, changes in pigmentation and ultimately changes in morphology. Adaptations to short-term exposures are little studied. Here we show that short-duration exposure to UVR causes the freshwater calanoid copepod, Eudiaptomus gracilis, to rapidly activate production of enzymes that prevent widespread collateral peroxidation (glutathione S-transferase, GST, that regulate apoptosis cell death (Caspase-3, Casp-3, and that facilitate neurotransmissions (cholinesterase-ChE. None of these enzyme systems is alone sufficient, but they act in concert to reduce the stress level of the organism. The interplay among enzymatic responses provides useful information on how organisms respond to environmental stressors acting on short time scales.

  17. Global Warming Potential Of A Waste Refinery Using Enzymatic Treatment

    DEFF Research Database (Denmark)

    Tonini, Davide; Astrup, Thomas

    2010-01-01

    and fossil resources. This is especially important with respect to the residual waste (i.e. the remains after source-separation and separate collection) which is typically incinerated or landfilled. In this paper the energy and Global Warming performance of a pilot-scale waste refinery for the enzymatic......Decrease of fossil fuel dependence and resource saving has become increasingly important during the last years. In this perspective, higher recycling rates for valuable materials as well as energy recovery from waste streams could play a significant role substituting for virgin material production...... treatment of municipal solid waste (MSW) was presented. The refinery produced a liquid (liquefied organic materials and paper) and a solid fraction (non-degradable materials) from the initial waste. A number of scenarios for the utilization of the two outputs were analyzed. Co-combustion in existing power...

  18. Waste management and enzymatic treatment of Municipal Solid Waste

    DEFF Research Database (Denmark)

    Jensen, Jacob Wagner

    % of the organic and degradable material. Source sorting is another way of collecting the household waste in its respective fractions. However, this separation technique is hard to enforce and expensive. Future waste management calls for novel and efficient technologies for the separation of unsorted MSW in order......The work carried out during the Ph.D. project is part of the Danish Energy Authority funded research project called PSO REnescience and is focussed on studying the enzymatic hydrolysis and liquefaction of waste biomass. The purpose of studying the liquefaction of waste biomass is uniform slurry...... generation for subsequent biogas production. Municipal solid waste (MSW) is produced in large amounts every year in the developed part of the world. The household waste composition varies between geographical areas and between seasons. However the overall content of organic and degradable material is rather...

  19. Recent advances in Carbon Nanotube based Enzymatic Fuel Cells

    Directory of Open Access Journals (Sweden)

    Serge eCosnier

    2014-10-01

    Full Text Available This review summarizes recent trends in the field of enzymatic fuel cells. Thanks to the high specificity of enzymes, biofuel cells can generate electrical energy by oxidation of a targeted fuel (sugars, alcohols or hydrogen at the anode and reduction of oxidants (O2, H2O2 at the cathode in complex media. The combination of carbon nanotubes, enzymes and redox mediators was widely exploited to develop biofuel cells since the electrons, involved in the bio-electrocatalytic processes, can be efficiently transferred from or to an external circuit. Original approaches to construct electron transfer based CNT-bioelectrodes and impressive biofuel cell performances are reported as well as biomedical applications.

  20. Enzymatic-fluorometric quantification of cholesterol in bovine milk

    DEFF Research Database (Denmark)

    Larsen, Torben

    2012-01-01

    The present paper describes an enzymatic–fluorometric method for the determination of cholesterol in milk and other opaque matrices. The initial step of the method is to liberate chemically and physically bound cholesterol from the milk fat globule membrane by enzymatic action. The method is able...... to discriminate between esterified and free cholesterol in milk. The analysis is cost effective and is developed to work directly on whole, fresh milk thereby eliminating time consuming and tedious pre-treatment procedures of the sample. More than 1000 milk samples were analysed on the day of sampling. The total...... concentration of milk cholesterol ranged from 80 to 756 μM (n = 1068; mean 351 μM). Milk cholesterol was significantly correlated to milk fat concentration as analysed by mid-infra red spectrometry (r = 0.630; n = 853) and by an enzymatic–fluorometric method (triacylglycerol) (r = 0.611; n = 842)....

  1. Enzymatic Modification of Corn Starch Influences Human Fecal Fermentation Profiles.

    Science.gov (United States)

    Dura, Angela; Rose, Devin J; Rosell, Cristina M

    2017-06-14

    Enzymatically modified starches have been widely used in food applications to develop new products, but information regarding digestion and fecal fermentation of these products is sparse. The objective of this study was to determine the fermentation properties of corn starch modified with α-amylase, amyloglucosidase, or cyclodextrin glycosyltransferase and the possible role of hydrolysis products. Samples differed in their digestibility and availability to be fermented by the microbiota, resulting in differences in microbial metabolites produced during in vitro fermentation. The presence or absence of hydrolysis products and gelatinization affected starch composition and subsequent metabolite production by the microbiota. Amyloglucosidase-treated starch led to the greatest production of short- and branched-chain fatty acid production by the microbiota. Results from this study could be taken into consideration to confirm the possible nutritional claims and potential health benefits of these starches as raw ingredients for food development.

  2. A xylanase-aided enzymatic pretreatment facilitates cellulose nanofibrillation.

    Science.gov (United States)

    Long, Lingfeng; Tian, Dong; Hu, Jinguang; Wang, Fei; Saddler, Jack

    2017-11-01

    Although biological pretreatment of cellulosic fiber based on endoglucanases has shown some promise to facilitate cellulose nanofibrillation, its efficacy is still limited. In this study, a xylanase-aided endoglucanase pretreatment was assessed on the bleached hardwood and softwood Kraft pulps to facilitate the downstream cellulose nanofibrillation. Four commercial xylanase preparations were compared and the changes of major fiber physicochemical characteristics such as cellulose/hemicellulose content, gross fiber properties, fiber morphologies, cellulose accessibility/degree of polymerization (DP)/crystallinity were systematically evaluated before and after enzymatic pretreatment. It showed that the synergistic cooperation between endoglucanase and certain xylanase (Biobrite) could efficiently "open up" the hardwood Kraft pulp with limited carbohydrates degradation (cellulose nanofibrillation during mild sonication process (90Wh) with more uniform disintegrated nanofibril products (50-150nm, as assessed by scanning electron microscopy and UV-vis spectroscopy). Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Epidemic based modeling of enzymatic hydrolysis of lignocellulosic biomass.

    Science.gov (United States)

    Tai, Chao; Arellano, Maria G; Keshwani, Deepak R

    2014-01-01

    An epidemic based model was developed to describe the enzymatic hydrolysis of a lignocellulosic biomass, dilute sulfuric acid pretreated corn stover. The process of substrate getting adsorbed and digested by enzyme was simulated as susceptibles getting infected by viruses and becoming removed and recovered. This model simplified the dynamic enzyme "infection" process and the catalysis of cellulose into a two-parameter controlled, enzyme behavior guided mechanism. Furthermore, the model incorporates the adsorption block by lignin and inhibition effects on cellulose catalysis. The model satisfactorily predicted the enzyme adsorption and hydrolysis, negative role of lignin, and inhibition effects over hydrolysis for a broad range of substrate and enzyme loadings. Sensitivity analysis was performed to evaluate the incorporation of lignin and other inhibition effects. Our model will be a useful tool for evaluating the effects of parameters during hydrolysis and guide a design strategy for continuous hydrolysis and the associated process control. © 2014 American Institute of Chemical Engineers.

  4. Enzymatic denitrification of 2-nitropropane in uninduced mouse liver microsomes.

    Science.gov (United States)

    Marker, E K; Kulkarni, A P

    1985-08-01

    Hepatic microsomes from 5 strains of untreated mice were tested for the ability to enzymatically cleave the nitro group from 2-nitropropane (2NP). All strains showed significant NADPH-dependent nitrite release at pH 7.6 and pH 8.8. Statistical differences in nitrite-releasing activity between strains were found between BALB and PL/J and ATH strains at pH 7.6. At pH 8.8, BIO.M differed from CD-1 and BALB. These results are in contrast to a report of little or no denitrification activity in uninduced rats and suggest that the 2NP microsomal metabolism may be of greater importance than previously thought.

  5. Non-enzymatic glucose detection using magnetic nanoemulsions

    Energy Technology Data Exchange (ETDEWEB)

    Mahendran, V.; Philip, John, E-mail: philip@igcar.gov.in [SMARTS, Metallurgy and Materials Group, Indira Gandhi Centre for Atomic Research, Kalpakkam 603 102, Tamil Nadu (India)

    2014-09-22

    We probe the optical properties and intermolecular interactions in magnetically responsive nanoemulsions in the presence of glucose. The equilibrium interdroplet distance between the emulsion droplets in an one-dimensional array increases by several nanometers in the presence of glucose because of intermolecular hydrogen bonding with sodium dodecyl sulphate molecules at the oil-water interface that gives rise to stretched lamellae-like structure. The observed large red shift in the diffracted Bragg peak (∼50–100 nm) and the linear response in the glucose concentration range of 0.25–25 mM offer a simple, fast, and cost effective non-enzymatic approach for glucose detection.

  6. Enzymatic antioxidants status in patients with recurrent aphthous stomatitis.

    Science.gov (United States)

    Zhang, Zichuan; Li, Shan; Fang, Huiqing

    2017-10-01

    Oxidative stress (OS) has been thought to play a main role in the etiopathogenesis of recurrent aphthous stomatitis (RAS), which is one of the most common oral mucosal diseases characterized by recurrent and painful oral ulcers. The aim of this investigation was to evaluate the enzymatic antioxidants status in patients with RAS in the active stage and remission stage. Ninety-seven patients with idiopathic minor RAS and 102 race-, age- and gender-matched healthy individuals were recruited. All these subjects were allocated to three groups: RAS patients with active lesion (group A); the same patients in group A in the remission stage of RAS (group B); and healthy individuals without RAS (group C). Following an overnight fast, blood samples were obtained. The serum levels of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSHPx) were measured by the spectrophotometric method. Independent-samples t-test and paired t-test were performed for statistical evaluation. The serum levels of SOD, GSHPx, and CAT (83.9 ± 17.1 U/ml, 6687.2 ± 2629.2 U/ml, 1789.7 ± 593.8 U/l) were found to be significantly lower in group A as compared to those of group B (99.8 ± 11.1 U/ml, 9364.1 ± 1607.9 U/ml, 2789.1 ± 1113.4 U/l; P 0.05). Our results indicate that enzymatic antioxidant defense system is impaired in RAS patients with active lesion and seems to play a crucial role in its pathogenesis. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Optimization of the Enzymatic Saccharification Process of Milled Orange Wastes

    Directory of Open Access Journals (Sweden)

    Daniel Velasco

    2017-08-01

    Full Text Available Orange juice production generates a very high quantity of residues (Orange Peel Waste or OPW-50–60% of total weight that can be used for cattle feed as well as feedstock for the extraction or production of essential oils, pectin and nutraceutics and several monosaccharides by saccharification, inversion and enzyme-aided extraction. As in all solid wastes, simple pretreatments can enhance these processes. In this study, hydrothermal pretreatments and knife milling have been analyzed with enzyme saccharification at different dry solid contents as the selection test: simple knife milling seemed more appropriate, as no added pretreatment resulted in better final glucose yields. A Taguchi optimization study on dry solid to liquid content and the composition of the enzymatic cocktail was undertaken. The amounts of enzymatic preparations were set to reduce their impact on the economy of the process; however, as expected, the highest amounts resulted in the best yields to glucose and other monomers. Interestingly, the highest content in solid to liquid (11.5% on dry basis rendered the best yields. Additionally, in search for process economy with high yields, operational conditions were set: medium amounts of hemicellulases, polygalacturonases and β-glucosidases. Finally, a fractal kinetic modelling of results for all products from the saccharification process indicated very high activities resulting in the liberation of glucose, fructose and xylose, and very low activities to arabinose and galactose. High activity on pectin was also observed, but, for all monomers liberated initially at a fast rate, high hindrances appeared during the saccharification process.

  8. Use of bio-enzymatic preparations for enhancement biogas production

    Directory of Open Access Journals (Sweden)

    Tomáš Vítěz

    2011-01-01

    Full Text Available Biogas is a renewable energy resource with high increasing developed in last few decades. It’s big opportunity for stabilization rural areas, concretely agriculture sector. This technology can decentralize supply of energy. The number of operated biogas plants is rapidly increasing. Biogas plants require a high level of intensity and stableness of the process of anaerobic fermentation with biogas production for efficiency treatment, also for good quality of development biogas and fertilization effect of the rest of fermentation. If this is not completed the operator has problem to keep the process in optimal condition for anaerobic fermentation. Researchers have tried different techniques to enhance biogas production. In order to achieve the aforementioned state, it is essential to ensure increased activity of microorganisms that contribute to the anaerobic fermentation. The metabolic activity of microorganisms is preconditioned by availability of easily decomposable solids. Adding of bacterial and enzymatic cultures into a fermented substrate represents one of the possibilities. The enzymes contained in this preparation are responsible for better exposing methanogenic bacteria to the material. The tested bio-enzymatic preparation, APD BIO GAS, is a mixture that contains bacteria and enzymes which are essential for the efficient progress of anaerobic fermentation. The reference biogas laboratory of the Mendel University in Brno was used for the purpose of testing of APD BIOGAS in mesophilic conditions of anaerobic fermentation on a substrate consisting of a mixture of maize silage and liquid manure. The producer of this preparation declare enhancement of quality and quantity of developed biogas, elimination of smell level of the rest of fermentation its higher homogenity. For the test were used lab scale fermenters of batch type with work volume 0.12 m3. An increase of biogas production by 15% was determined in connection with addition of the

  9. Enzymatic saccharification of hemicellulose extracted from palm oil mill wastes

    Directory of Open Access Journals (Sweden)

    Poonsuk Prasertsan

    2001-11-01

    Full Text Available Various parameters affecting the extraction of hemicellulose from palm cake by alkali method and sterilizer condensate by solvent method were investigated. For extraction of hemicellulose from palm cake, the optimal ratio of palm cake to sodium hydroxide (NaOH (1.5% conc. was 1:10. However, potassium hydroxide (KOH was a better source of alkali than NaOH and the optimum ratio of palm cake to 12% KOH was 1:50 (w/v. Temperature over 100ºC (100 and 121ºC extracted significantly higher hemicellulose than at 80ºC after 20 min treatment. The addition of ethanol to the extracted solution in the ratio of 1:1 (v/v gave the highest hemicellulose yield of 8.67 g/100 g palm cake. For extraction of hemicellulose from sterilizer condensate, the optimum ratio of ethanol to the condensate was 2:1 (v/v, which gave a hemicellulose yield of 6.42 g/100 ml. The enzymatic saccharification of the hemicelllulose extracted from palm cake (HEPC and from sterilizer condensate (HESC was 3-10 times lower than that of xylan. The enzyme from Aspergillus niger ATCC 6275 and Meicellase gave higher saccharification rates than that of Sumyzyme. The contents of reducing sugars in xylan, HEPC and HESC were 96.4, 36.2 and 20.6%, respectively and 75.3, 67.9 and 97.6% of these values could be hydrolysed by the enzymes. Hence, palm cake was a better source of substrate for extraction of hemicellulose while hemicellulose extracted from sterilizer condensate gave higher percentage of enzymatic saccharification.

  10. Effect of nitrogen oxide pretreatments on enzymatic hydrolysis of cellulose

    Energy Technology Data Exchange (ETDEWEB)

    Borrevik, R.K.; Wilke, C.R.; Brink, D.L.

    1978-09-01

    This work considers the effect of nitrogen oxide pretreatments on the subsequent enzymatic hydrolysis by Trichoderma viride cellulase of the cellulose occurring in wheat straw; Triticum Aestivum-L, em. Thell. In the pretreatment scheme the straw is first reacted with nitric oxide and air, and then extracted in aqueous solution. In this way, overall sugar yields increased from 17% for the case of no pretreatment to 70%. The glucose yield increased from 20 to 60%. The yield of glucose during enzymatic hydrolysis is dependent on the reaction time of the gas phase reaction. For a 24 hour reaction the yield is 60%, but drops to 45% for a reaction time of 2 hours. Xylose, a potentially valuable side product of the pretreatment, is obtained by dilute acid hydrolysis during the extraction stage in yields of 90 to 96%. In acidic media, the kinetics of both the rate of formation and destruction of xylose were found to follow the first-order rate laws reported in the literature. These were determined to be 4.5 (liter/gmole)(hr./sup -1/) and 0.03 hr./sup -1/, respectively. However, the rate of formation is much greater (20.4 (liter/gmole) (hr./sup -1/)) when the extraction liquor is recycled. The most likely explanation for this is that the increased total acidity of the recycled liquor compensates for diffusional limitations. A preliminary design and cost analysis of the pretreatment-hydrolysis scheme indicates that glucose can be produced at 10.86 cents per pound, exclusive of straw cost. The corresponding cost per pound of total sugars produced is 5.0 cents. Sensitivity analyses indicate that 42% of the pretreatment cost (excluding hydrolysis) can be attributed to nitric oxide production, and the high yield of sugar obtained is advantageous when considering the cost of straw.

  11. Acute hormonal, immunological and enzymatic responses to a basketball game

    Directory of Open Access Journals (Sweden)

    Denis Foschini

    2008-12-01

    Full Text Available The objective of the present study was to analyze the acute hormonal, immunological and enzymatic responses of professional basketball players to a basketball game. The sample was composed of eight basketball athletes, with a minimum of 4 years’ experience in basketball. A real game was simulated with a total duration of 40 minutes, divided into two halves of 20 minutes each and an interval of 10 minutes between halves. Blood samples were collected before andimmediately after the game (20 ml, vacuum tube system. The variables analyzed were: testosterone and cortisol hormones, total leukocytes, neutrophils, lymphocytes, monocytes and the enzymes creatine kinase (CK and lactate dehydrogenase (LDH. Statistical analysis was with descriptive statistics and the Student’s t test for paired samples to p≤0.05. The pre (13.34 nmol/L and 301.97 nmol/L and post game (17.34 nmol/L and 395.91 nmol/L levels of testosterone and cortisol were statistically different, with higher levels after the game for both hormones. The immune cell counts exhibited significant differences for total leukocytes (6393.75 nmol/L and 9158.75 nmol/L and neutrophils (3532.5 nmol/L and 6392.62 nmol/L, with levels being higher after the game. No statistical differences were observed for the enzymatic variables. Therefore, based on the markers analyzed, testosterone and cortisol exhibited pronounced increases after the game and the samebehavior was observed for total leukocytes and neutrophils.

  12. Soil solid materials affect the kinetics of extracellular enzymatic reactions

    Science.gov (United States)

    Lammirato, C.; Miltner, A.; Kästner, M.

    2009-04-01

    INTRODUCTION Soil solid materials affect the degradation processes of many organic compounds by decreasing the bioavailability of substrates and by interacting with degraders. The magnitude of this effect in the environment is shown by the fact that xenobiotics which are readily metabolized in aquatic environments can have long residence times in soil. Extracellular enzymatic hydrolysis of cellobiose (enzyme: beta-glucosidase from Aspergillus niger) was chosen as model degradation process since it is easier to control and more reproducible than a whole cell processes. Furthermore extracellular enzymes play an important role in the environment since they are responsible for the first steps in the degradation of organic macromolecules; beta-glucosidase is key enzyme in the degradation of cellulose and therefore it is fundamental in the carbon cycle and for soil in general. The aims of the project are: 1) quantification of solid material effect on degradation, 2) separation of the effects of minerals on enzyme (adsorption →change in activity) and substrate (adsorption →change in bioavailability). Our hypothesis is that a rate reduction in the enzymatic reaction in the presence of a solid phase results from the sum of decreased bioavailability of the substrate and decreased activity of enzyme molecules. The relative contribution of the two terms to the overall effect can vary widely depending on the chemical nature of the substrate, the properties of the enzyme and on the surface properties of the solid materials. Furthermore we hypothesize that by immobilizing the enzyme in an appropriate carrier the adsorption of enzymes to soil materials can be eliminated and that therefore immobilization can increase the overall reaction rate (activity loss caused by immobilization standard experimental conditions: 66 mM phosphate buffer, pH 5, 25°C, 20 mg solid/ml buffer). The enzyme in an immobilized form (covalent bonding to oxirane groups on the surfaces of macroporous

  13. Monitoring of Enzymatic Proteolysis Using Self-Assembled Quantum Dot-Protein Substrate Sensors

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    Aaron R. Clapp

    2008-07-01

    Full Text Available We have previously utilized hybrid semiconductor quantum dot- (QD- peptide substrates for monitoring of enzymatic proteolysis. In this report, we expand on this sensing strategy to further monitor protein-protease interactions. We utilize QDs self-assembled with multiple copies of dye-labeled proteins as substrates for the sensing of protease activity. Detection of proteolysis is based on changes in the rate of fluorescence resonance energy transfer (FRET between the QDs and the proximal dye-labeled proteins following protein digestion by added enzyme. Our study focused on two representative proteolytic enzymes: the cysteine protease papain and the serine protease endoproteinase K. Analysis of the enzymatic digestion allowed us to estimate minimal values for the enzymatic activities of each enzyme used. Mechanisms of enzymatic inhibition were also inferred from the FRET data collected in the presence of inhibitors. Potential applications of this technology include drug discovery assays and in vivo cellular monitoring of enzymatic activity.

  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. Recent Progress and Novel Applications in Enzymatic Conversion of Carbon Dioxide

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    Nguyen Van Duc Long

    2017-04-01

    Full Text Available Turning carbon dioxide (CO2 into fuels and chemicals using chemical, photochemical, electrochemical, and enzymatic methods could be used to recycle large quantities of carbon. The enzymatic method, which is inspired by cellular CO2 metabolism, has attracted considerable attention for efficient CO2 conversion due to improved selectivity and yields under mild reaction conditions. In this review, the research progress of green and potent enzymatic conversion of CO2 into useful fuels and chemicals was discussed. Furthermore, applications of the enzymatic conversion of CO2 to assist in CO2 capture and sequestration were highlighted. A summary including the industrial applications, barriers, and some perspectives on the research and development of the enzymatic approach to convert CO2 were introduced.

  16. 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

  17. Effect of Cross-Linking and Enzymatic Hydrolysis Composite Modification on the Properties of Rice Starches

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    Gao-Qiang Liu

    2012-07-01

    Full Text Available Native rice starch lacks the versatility necessary to function adequately under rigorous industrial processing, so modified starches are needed to meet the functional properties required in food products. This work investigated the impact of enzymatic hydrolysis and cross-linking composite modification on the properties of rice starches. Rice starch was cross-linked with epichlorohydrin (EPI with different concentrations (0.5%, 0.7%, 0.9% w/w, on a dry starch basis, affording cross-linked rice starches with the three different levels of cross-linking that were named R1, R2, and R3, respectively. The cross-linked rice starches were hydrolyzed by α-amylase and native, hydrolyzed, and hydrolyzed cross-linked rice starches were comparatively studied. It was found that hydrolyzed cross-linked rice starches showed a lower the degree of amylase hydrolysis compared with hydrolyzed rice starch. The higher the degree of cross-linking, the higher the capacity to resist enzyme hydrolysis. Hydrolyzed cross-linked rice starches further increased the adsorptive capacities of starches for liquids and decreased the trend of retrogradation, and it also strengthened the capacity to resist shear compared to native and hydrolyzed rice starches.

  18. Characterization and enzymatic properties of protein kinase ACR4 from Arabidopsis thaliana.

    Science.gov (United States)

    Zhao, Yu; Liu, Xuehe; Xu, Ziyan; Yang, Hui; Li, Jixi

    2017-07-22

    Serine/threonine-protein kinase-like protein ARABIDOPSIS CRINKLY4 (ACR4), a transmembrane protein of Arabidopsis thaliana, plays important roles in cell division and differentiation. Although accumulating studies shed light on the function of ACR4, the structure and catalytic mechanism of ACR4 remain to be elucidated. Here, we report the purification and enzymatic properties of the intracellular kinase domain (residues 464-799) of ACR4 (ACR4IKD). Through Ni-affinity chromatography and gel filter chromatography methods, we successfully obtain high-purity ACR4IKD protein from Escherichia coli. Dynamic light scattering and gel-filtration methods reveal that ACR4IKD distributes with high homogeneity and exists as a monomer in solution. In addition, the ACR4IKD protein has typical kinase activity with myelin basic protein (MBP) as the substrate. Our study may lay the foundation for structure determination of ACR4IKD and further functional research, for example, screening significant substrates of ACR4 in Arabidopsis thaliana. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Chemical and Enzymatic Approaches to Carbohydrate-Derived Spiroketals: Di-D-Fructose Dianhydrides (DFAs

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    José M. García Fernández

    2008-08-01

    Full Text Available Di-D-fructose dianhydrides (DFAs comprise a unique family of stereoisomeric spiro-tricyclic disaccharides formed upon thermal and/or acidic activation of sucroseand/ or D-fructose-rich materials. The recent discovery of the presence of DFAs in food products and their remarkable nutritional features has attracted considerable interest from the food industry. DFAs behave as low-caloric sweeteners and have proven to exert beneficial prebiotic nutritional functions, favouring the growth of Bifidobacterium spp. In the era of functional foods, investigation of the beneficial properties of DFAs has become an important issue. However, the complexity of the DFA mixtures formed during caramelization or roasting of carbohydrates by traditional procedures (up to 14 diastereomeric spiroketal cores makes evaluation of their individual properties a difficult challenge. Great effort has gone into the development of efficient procedures to obtain DFAs in pure form at laboratory and industrial scale. This paper is devoted to review the recent advances in the stereoselective synthesis of DFAs by means of chemical and enzymatic approaches, their scope, limitations, and complementarities.

  20. Shear-induced unfolding and enzymatic cleavage of full-length VWF multimers

    CERN Document Server

    Lippok, Svenja; Obser, Tobias; Kleemeier, Lars; Schneppenheim, Reinhard; Budde, Ulrich; Netz, Roland R; Rädler, Joachim O

    2015-01-01

    Proteolysis of the multimeric blood coagulation protein von Willebrand Factor (VWF) by ADAMTS13 is crucial for prevention of microvascular thrombosis. ADAMTS13 cleaves VWF within the mechanosensitive A2 domain, which is believed to open under shear flow. Here, we combine Fluorescence Correlation Spectroscopy (FCS) and a microfluidic shear cell to monitor real-time kinetics of full-length VWF proteolysis as a function of shear stress. For comparison, we also measure the Michaelis-Menten kinetics of ADAMTS13 cleavage of wild-type VWF in the absence of shear but partially denaturing conditions. Under shear, ADAMTS13 activity on full-length VWF arises without denaturing agent as evidenced by FCS and gel-based multimer analysis. In agreement with Brownian hydrodynamics simulations, we find a sigmoidal increase of the enzymatic rate as a function of shear at a threshold shear rate 5522/s. The same flow-rate dependence of ADAMTS13 activity we also observe in blood plasma, which is relevant to predict hemostatic dysf...