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Sample records for acetyl xylan esterase

  1. Characterization and mode of action of two acetyl xylan esterases from Chrysosporium lucknowense C1 active towards acetylated xylans

    NARCIS (Netherlands)

    Pouvreau, L.A.M.; Jonathan, M.C.; Kabel, M.A.; Hinz, S.W.A.; Gruppen, H.; Schols, H.A.

    2011-01-01

    Two novel acetyl xylan esterases, Axe2 and Axe3, from Chrysosporium lucknowense (C1), belonging to the carbohydrate esterase families 5 and 1, respectively, were purified and biochemically characterized. Axe2 and Axe3 are able to hydrolyze acetyl groups both from simple acetylated

  2. Hydrolysis of Wheat Arabinoxylan by Two Acetyl Xylan Esterases from Chaetomium thermophilum

    DEFF Research Database (Denmark)

    Tong, Xiaoxue; Lange, Lene; Grell, Morten Nedergaard

    2015-01-01

    The thermophilic filamentous ascomycete Chaetomium thermophilum produces functionally diverse hemicellulases when grown on hemicellulose as carbon source. Acetyl xylan esterase (EC 3.1.1.72) is an important accessory enzyme in hemicellulose biodegradation. Although the genome of C. thermophilum has...

  3. Crystal Structure and Substrate Specificity Modification of Acetyl Xylan Esterase from Aspergillus luchuensis.

    Science.gov (United States)

    Komiya, Dai; Hori, Akane; Ishida, Takuya; Igarashi, Kiyohiko; Samejima, Masahiro; Koseki, Takuya; Fushinobu, Shinya

    2017-10-15

    Acetyl xylan esterase (AXE) catalyzes the hydrolysis of the acetyl bonds present in plant cell wall polysaccharides. Here, we determined the crystal structure of AXE from Aspergillus luchuensis ( Al AXEA), providing the three-dimensional structure of an enzyme in the Esterase_phb family. Al AXEA shares its core α/β-hydrolase fold structure with esterases in other families, but it has an extended central β-sheet at both its ends and an extra loop. Structural comparison with a ferulic acid esterase (FAE) from Aspergillus niger indicated that Al AXEA has a conserved catalytic machinery: a catalytic triad (Ser119, His259, and Asp202) and an oxyanion hole (Cys40 and Ser120). Near the catalytic triad of A lAXEA, two aromatic residues (Tyr39 and Trp160) form small pockets at both sides. Homology models of fungal FAEs in the same Esterase_phb family have wide pockets at the corresponding sites because they have residues with smaller side chains (Pro, Ser, and Gly). Mutants with site-directed mutations at Tyr39 showed a substrate specificity similar to that of the wild-type enzyme, whereas those with mutations at Trp160 acquired an expanded substrate specificity. Interestingly, the Trp160 mutants acquired weak but significant type B-like FAE activity. Moreover, the engineered enzymes exhibited ferulic acid-releasing activity from wheat arabinoxylan. IMPORTANCE Hemicelluloses in the plant cell wall are often decorated by acetyl and ferulic acid groups. Therefore, complete and efficient degradation of plant polysaccharides requires the enzymes for cleaving the side chains of the polymer. Since the Esterase_phb family contains a wide array of fungal FAEs and AXEs from fungi and bacteria, our study will provide a structural basis for the molecular mechanism of these industrially relevant enzymes in biopolymer degradation. The structure of the Esterase_phb family also provides information for bacterial polyhydroxyalkanoate depolymerases that are involved in biodegradation of

  4. An Aspergillus oryzae acetyl xylan esterase: molecular cloning and characteristics of recombinant enzyme expressed in Pichia pastoris.

    Science.gov (United States)

    Koseki, Takuya; Miwa, Yozo; Akao, Takeshi; Akita, Osamu; Hashizume, Katsumi

    2006-02-10

    We screened 20,000 clones of an expressed sequence tag (EST) library from Aspergillus oryzae (http://www.nrib.go.jp/ken/EST/db/index.html) and obtained one cDNA clone encoding a protein with similarity to fungal acetyl xylan esterase. We also cloned the corresponding gene, designated as Aoaxe, from the genomic DNA. The deduced amino acid sequence consisted of a putative signal peptide of 31-amino acids and a mature protein of 276-amino acids. We engineered Aoaxe for heterologous expression in P. pastoris. Recombinant AoAXE (rAoAXE) was secreted by the aid of fused alpha-factor secretion signal peptide and accumulated as an active enzyme in the culture medium to a final level of 190 mg/l after 5 days. Purified rAoAXEA before and after treatment with endoglycosidase H migrated by SDS-PAGE with a molecular mass of 31 and 30 kDa, respectively. Purified rAoAXE displayed the greatest hydrolytic activity toward alpha-naphthylacetate (C2), lower activity toward alpha-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. The recombinant enzyme catalyzed the release of acetic acid from birchwood xylan. No activity was detectable using methyl esters of ferulic, caffeic or sinapic acids. rAoAXE was thermolabile in comparison to other AXEs from Aspergillus.

  5. Determination of some significant batch culture conditions affecting acetyl-xylan esterase production by Penicillium notatum NRRL-1249

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    Akhtar MN

    2011-05-01

    Full Text Available Abstract Background Acetyl-xylan esterase (AXE, EC 3.1.1.72 hydrolyses acetate group from the linear chain of xylopyranose residues bound by β-1,4-linkage. The enzyme finds commercial applications in bio-bleaching of wood pulp, treating animal feed to increase digestibility, processing food to increase clarification and converting lignocellulosics to feedstock and fuel. In the present study, we report on the production of an extracellular AXE from Penicillium notatum NRRL-1249 by solid state fermentation (SSF. Results Wheat bran at a level of 10 g (with 4 cm bed height was optimized as the basal substrate for AXE production. An increase in enzyme activity was observed when 7.5 ml of mineral salt solution (MSS containing 0.1% KH2PO4, 0.05% KCl, 0.05% MgSO4.7H2O, 0.3% NaNO3, 0.001% FeSO4.2H2O and 0.1% (v/w Tween-80 as an initial moisture content was used. Various nitrogen sources including ammonium sulphate, urea, peptone and yeast extract were compared for enzyme production. Maximal enzyme activity of 760 U/g was accomplished which was found to be highly significant (p ≤ 0.05. A noticeable enhancement in enzyme activity was observed when the process parameters including incubation period (48 h, initial pH (5, 0.2% (w/w urea as nitrogen source and 0.5% (v/w Tween-80 as a stimulator were further optimized using a 2-factorial Plackett-Burman design. Conclusion From the results it is clear that an overall improvement of more than 35% in terms of net enzyme activity was achieved compared to previously reported studies. This is perhaps the first report dealing with the use of P. notatum for AXE production under batch culture SSF. The Plackett-Burman model terms were found highly significant (HS, suggesting the potential commercial utility of the culture used (df = 3, LSD = 0.126.

  6. Trichoderma reesei CE16 acetyl esterase and its role in enzymatic degradation of acetylated hemicellulose

    DEFF Research Database (Denmark)

    Biely, Peter; Cziszarava, Maria; Agger, Jane W.

    2014-01-01

    Results The combined action of GH10 xylanase and acetylxylan esterases (AcXEs) leads to formation of neutral and acidic xylooligosaccharides with a few resistant acetyl groups mainly at their non-reducing ends. We show here that these acetyl groups serve as targets for TrCE16 AcE. The most promin...

  7. Downregulation of RWA genes in hybrid aspen affects xylan acetylation and wood saccharification.

    Science.gov (United States)

    Pawar, Prashant Mohan-Anupama; Ratke, Christine; Balasubramanian, Vimal K; Chong, Sun-Li; Gandla, Madhavi Latha; Adriasola, Mathilda; Sparrman, Tobias; Hedenström, Mattias; Szwaj, Klaudia; Derba-Maceluch, Marta; Gaertner, Cyril; Mouille, Gregory; Ezcurra, Ines; Tenkanen, Maija; Jönsson, Leif J; Mellerowicz, Ewa J

    2017-06-01

    High acetylation of angiosperm wood hinders its conversion to sugars by glycoside hydrolases, subsequent ethanol fermentation and (hence) its use for biofuel production. We studied the REDUCED WALL ACETYLATION (RWA) gene family of the hardwood model Populus to evaluate its potential for improving saccharification. The family has two clades, AB and CD, containing two genes each. All four genes are expressed in developing wood but only RWA-A and -B are activated by master switches of the secondary cell wall PtNST1 and PtMYB21. Histochemical analysis of promoter::GUS lines in hybrid aspen (Populus tremula × tremuloides) showed activation of RWA-A and -B promoters in the secondary wall formation zone, while RWA-C and -D promoter activity was diffuse. Ectopic downregulation of either clade reduced wood xylan and xyloglucan acetylation. Suppressing both clades simultaneously using the wood-specific promoter reduced wood acetylation by 25% and decreased acetylation at position 2 of Xylp in the dimethyl sulfoxide-extracted xylan. This did not affect plant growth but decreased xylose and increased glucose contents in the noncellulosic monosaccharide fraction, and increased glucose and xylose yields of wood enzymatic hydrolysis without pretreatment. Both RWA clades regulate wood xylan acetylation in aspen and are promising targets to improve wood saccharification. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  8. Functional and structural characterization of a thermostable acetyl esterase from Thermotoga maritima

    NARCIS (Netherlands)

    Levisson, M.; Han, G.W.; Deller, M.C.; Hendriks, S.N.A.; Oost, van der J.; Kengen, S.W.M.

    2012-01-01

    TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100°C and pH 7.5. The positional specificity

  9. Feruloyl esterase from Aspergillus clavatus improves xylan hydrolysis of sugarcane bagasse

    Directory of Open Access Journals (Sweden)

    Dyoni M. de Oliveira

    2016-12-01

    Full Text Available Feruloyl esterase is a subclass of carboxylic acid esterases with the capacity to release ferulic acid and other cinnamic acids from plant cell walls and synthetic substrates. Feruloyl esterases act synergistically with xylanases removing ferulic acid residues esterified to arabinoxylans. Feruloyl esterase type D from Aspergillus clavatus (AcFAE was expressed in Escherichia coli, purified, and applied with a commercial xylanase consortium (Novozymes for hydrolysis of sugarcane bagasse. Feruloyl esterase plus xylanase increased 5.13-fold the releasing of ferulic acid from sugarcane bagasse. Removal of only 7.7% of ferulic acid content by AcFAE increased 97.3% the sugarcane bagasse hydrolysis by xylanase. These data support the use of AcFAE as an interesting adjuvant enzyme to improve lignocellulose digestion and biotechnological tool for biorefineries.

  10. Expression of mung bean pectin acetyl esterase in potato tubers: effect on acetylation of cell wall polymers and tuber mechanical properties.

    Science.gov (United States)

    Orfila, Caroline; Dal Degan, Florence; Jørgensen, Bodil; Scheller, Henrik Vibe; Ray, Peter M; Ulvskov, Peter

    2012-07-01

    A mung bean (Vigna radiata) pectin acetyl esterase (CAA67728) was heterologously expressed in tubers of potato (Solanum tuberosum) under the control of the granule-bound starch synthase promoter or the patatin promoter in order to probe the significance of O-acetylation on cell wall and tissue properties. The recombinant tubers showed no apparent macroscopic phenotype. The enzyme was recovered from transgenic tubers using a high ionic strength buffer and the extract was active against a range of pectic substrates. Partial in vivo de-acetylation of cell wall polysaccharides occurred in the transformants, as shown by a 39% decrease in the degree of acetylation (DA) of tuber cell wall material (CWM). Treatment of CWM using a combination of endo-polygalacturonase and pectin methyl esterase extracted more pectin polymers from the transformed tissue compared to wild type. The largest effect of the pectin acetyl esterase (68% decrease in DA) was seen in the residue from this extraction, suggesting that the enzyme is preferentially active on acetylated pectin that is tightly bound to the cell wall. The effects of acetylation on tuber mechanical properties were investigated by tests of failure under compression and by determination of viscoelastic relaxation spectra. These tests suggested that de-acetylation resulted in a stiffer tuber tissue and a stronger cell wall matrix, as a result of changes to a rapidly relaxing viscoelastic component. These results are discussed in relation to the role of pectin acetylation in primary cell walls and its implications for industrial uses of potato fibres.

  11. Trichoderma longibrachiatum acetyl xylan esterase 1 enhances hemicellulolytic preparations to degrade corn silage polysaccharides

    NARCIS (Netherlands)

    Neumüller, K.G.; Streekstra, H.; Gruppen, H.; Schols, H.A.

    2014-01-01

    Supplementation of a Trichoderma longibrachiatum preparation to an industrial Aspergillus niger/Talaromyces emersonii enzyme mixture demonstrated synergy for the saccharification of corn silage water-unextractable solids (WUS). Sub-fractions of the crude T. longibrachiatum preparation obtained after

  12. The Four Arabidopsis Reduced Wall Acetylation Genes are Expressed in Secondary Wall-Containing Cells and Required for the Acetylation of Xylan

    Science.gov (United States)

    Xylan is one of the major polysaccharides in cellulosic biomass, and understanding the mechanisms underlying xylan biosynthesis will potentially help us design strategies to produce cellulosic biomass better suited for biofuel production. Although a number of genes have been show...

  13. The Aspergillus niger faeB gene encodes a second feruloyl esterase involved in pectin and xylan degradation and is specifically induced in the presence of aromatic compounds

    NARCIS (Netherlands)

    Vries, de R.P.; vanKuyk, P.A.; Kester, H.C.M.; Visser, J.

    2002-01-01

    The faeB gene encoding a second feruloyl esterase from Aspergillus niger has been cloned and characterized. It consists of an open reading frame of 1644 bp containing one intron. The gene encodes a protein of 521 amino acids that has sequence similarity to that of an Aspergillus oryzae tannase.

  14. The Aspergillus niger faeB gene encodes a second feruloyl esterase involved in pectin and xylan degradation and is specifically induced in the presence of aromatic compounds.

    Science.gov (United States)

    de Vries, Ronald P; vanKuyk, Patricia A; Kester, Harry C M; Visser, Jaap

    2002-04-15

    The faeB gene encoding a second feruloyl esterase from Aspergillus niger has been cloned and characterized. It consists of an open reading frame of 1644 bp containing one intron. The gene encodes a protein of 521 amino acids that has sequence similarity to that of an Aspergillus oryzae tannase. However, the encoded enzyme, feruloyl esterase B (FAEB), does not have tannase activity. Comparison of the physical characteristics and substrate specificity of FAEB with those of a cinnamoyl esterase from A. niger [Kroon, Faulds and Williamson (1996) Biotechnol. Appl. Biochem. 23, 255-262] suggests that they are in fact the same enzyme. The expression of faeB is specifically induced in the presence of certain aromatic compounds, but not in the presence of other constituents present in plant-cell-wall polysaccharides such as arabinoxylan or pectin. The expression profile of faeB in the presence of aromatic compounds was compared with the expression of A. niger faeA, encoding feruloyl esterase A (FAEA), and A. niger bphA, the gene encoding a benzoate-p-hydroxylase. All three genes have different subsets of aromatic compounds that induce their expression, indicating the presence of different transcription activating systems in A. niger that respond to aromatic compounds. Comparison of the activity of FAEA and FAEB on sugar-beet pectin and wheat arabinoxylan demonstrated that they are both involved in the degradation of both polysaccharides, but have opposite preferences for these substrates. FAEA is more active than FAEB towards wheat arabinoxylan, whereas FAEB is more active than FAEA towards sugar-beet pectin.

  15. Acetylation of woody lignocellulose: significance and regulation

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    Prashant Mohan-Anupama Pawar

    2013-05-01

    Full Text Available Non-cellulosic cell wall polysaccharides constitute approximately one quarter of usable biomass for human exploitation. In contrast to cellulose, these components are usually substituted by O-acetyl groups, which affect their properties and interactions with other polymers, thus affecting their solubility and extractability. However, details of these interactions are still largely obscure. Moreover, polysaccharide hydrolysis to constituent monosaccharides, is hampered by the presence of O-acetyl groups, necessitating either enzymatic (esterase or chemical de-acetylation, increasing the costs and chemical consumption. Reduction of polysaccharide acetyl content in planta is a way to modify lignocellulose towards improved saccharification. In this review we: 1 summarize literature on lignocellulose acetylation in different tree species, 2 present data and current hypotheses concerning the role of O-acetylation in determining woody lignocellulose properties, 3 describe plant proteins involved in lignocellulose O-acetylation, 4 give examples of microbial enzymes capable to de-acetylate lignocellulose, and 5 discuss prospects for exploiting these enzymes in planta to modify xylan acetylation.

  16. Spectroscopic study of drug-binding characteristics of unmodified and pNPA-based acetylated human serum albumin: Does esterase activity affect microenvironment of drug binding sites on the protein?

    Energy Technology Data Exchange (ETDEWEB)

    Moradi, Nastaran [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Faculty of Pharmaceutical Sciences, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Ashrafi-Kooshk, Mohammad Reza [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Ghobadi, Sirous [Department of Biology, Faculty of Sciences, Razi University, Kermanshah (Iran, Islamic Republic of); Shahlaei, Mohsen [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Faculty of Pharmaceutical Sciences, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Khodarahmi, Reza, E-mail: rkhodarahmi@mbrc.ac.ir [Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of); Faculty of Pharmaceutical Sciences, Kermanshah University of Medical Sciences, Kermanshah (Iran, Islamic Republic of)

    2015-04-15

    Human serum albumin (HSA) is the most prominent extracellular protein in blood plasma. There are several binding sites on the protein which provide accommodation for structurally-unrelated endogenous and exogenous ligands and a wide variety of drugs. “Esterase-like” activity (hydrolysis of p-nitrophenyl esters) by the protein has been also reported. In the current study, we set out to investigate the interaction of indomethacin and ibuprofen with the unmodified and modified HSA (pNPA-modified HSA) using various spectroscopic techniques. Fluorescence data showed that 1:1 binding of drug to HSA is associated with quenching of the protein intrinsic fluorescence. Decrease of protein surface hydrophobicity (PSH), alteration in drug binding affinity and change of the protein stability, after esterase-like activity and permanent acetylation of HSA, were also documented. Analysis of the quenching and thermodynamic parameters indicated that forces involved in drug–HSA interactions change upon the protein modification. - Highlights: • Binding propensity of indomethacin extremely decreased upon the protein acetylation. • There is no ibuprofen binding after protein acetylation. • Protein stability changes upon drug binding as well as protein acetylation. • Drug pharmacokinetics may be influenced under co-administration of HSA-modifier drugs.

  17. Spectroscopic study of drug-binding characteristics of unmodified and pNPA-based acetylated human serum albumin: Does esterase activity affect microenvironment of drug binding sites on the protein?

    International Nuclear Information System (INIS)

    Moradi, Nastaran; Ashrafi-Kooshk, Mohammad Reza; Ghobadi, Sirous; Shahlaei, Mohsen; Khodarahmi, Reza

    2015-01-01

    Human serum albumin (HSA) is the most prominent extracellular protein in blood plasma. There are several binding sites on the protein which provide accommodation for structurally-unrelated endogenous and exogenous ligands and a wide variety of drugs. “Esterase-like” activity (hydrolysis of p-nitrophenyl esters) by the protein has been also reported. In the current study, we set out to investigate the interaction of indomethacin and ibuprofen with the unmodified and modified HSA (pNPA-modified HSA) using various spectroscopic techniques. Fluorescence data showed that 1:1 binding of drug to HSA is associated with quenching of the protein intrinsic fluorescence. Decrease of protein surface hydrophobicity (PSH), alteration in drug binding affinity and change of the protein stability, after esterase-like activity and permanent acetylation of HSA, were also documented. Analysis of the quenching and thermodynamic parameters indicated that forces involved in drug–HSA interactions change upon the protein modification. - Highlights: • Binding propensity of indomethacin extremely decreased upon the protein acetylation. • There is no ibuprofen binding after protein acetylation. • Protein stability changes upon drug binding as well as protein acetylation. • Drug pharmacokinetics may be influenced under co-administration of HSA-modifier drugs

  18. Biochemical Characterization and Relative Expression Levels of Multiple Carbohydrate Esterases of the Xylanolytic Rumen Bacterium Prevotella ruminicola 23 Grown on an Ester-Enriched Substrate ▿ †

    Science.gov (United States)

    Kabel, Mirjam A.; Yeoman, Carl J.; Han, Yejun; Dodd, Dylan; Abbas, Charles A.; de Bont, Jan A. M.; Morrison, Mark; Cann, Isaac K. O.; Mackie, Roderick I.

    2011-01-01

    We measured expression and used biochemical characterization of multiple carbohydrate esterases by the xylanolytic rumen bacterium Prevotella ruminicola 23 grown on an ester-enriched substrate to gain insight into the carbohydrate esterase activities of this hemicellulolytic rumen bacterium. The P. ruminicola 23 genome contains 16 genes predicted to encode carbohydrate esterase activity, and based on microarray data, four of these were upregulated >2-fold at the transcriptional level during growth on an ester-enriched oligosaccharide (XOSFA,Ac) from corn relative to a nonesterified fraction of corn oligosaccharides (AXOS). Four of the 16 esterases (Xyn10D-Fae1A, Axe1-6A, AxeA1, and Axe7A), including the two most highly induced esterases (Xyn10D-Fae1A and Axe1-6A), were heterologously expressed in Escherichia coli, purified, and biochemically characterized. All four enzymes showed the highest activity at physiologically relevant pH (6 to 7) and temperature (30 to 40°C) ranges. The P. ruminicola 23 Xyn10D-Fae1A (a carbohydrate esterase [CE] family 1 enzyme) released ferulic acid from methylferulate, wheat bran, corn fiber, and XOSFA,Ac, a corn fiber-derived substrate enriched in O-acetyl and ferulic acid esters, but exhibited negligible activity on sugar acetates. As expected, the P. ruminicola Axe1-6A enzyme, which was predicted to possess two distinct esterase family domains (CE1 and CE6), released ferulic acid from the same substrates as Xyn10D-Fae1 and was also able to cleave O-acetyl ester bonds from various acetylated oligosaccharides (AcXOS). The P. ruminicola 23 AxeA1, which is not assigned to a CE family, and Axe7A (CE7) were found to be acetyl esterases that had activity toward a broad range of mostly nonpolymeric acetylated substrates along with AcXOS. All enzymes were inhibited by the proximal location of other side groups like 4-O-methylglucuronic acid, ferulic acid, or acetyl groups. The unique diversity of carbohydrate esterases in P. ruminicola 23

  19. Crystallization and preliminary crystallographic analysis of Axe2, an acetylxylan esterase from Geobacillus stearothermophilus

    International Nuclear Information System (INIS)

    Lansky, Shifra; Alalouf, Onit; Solomon, Vered; Alhassid, Anat; Govada, Lata; Chayan, Naomi E.; Belrhali, Hassan; Shoham, Yuval; Shoham, Gil

    2013-01-01

    The serine acetylxylan esterase from G. stearothermophilus (Axe2) has been crystallized in the tetragonal space group I422. Complete diffraction data sets have been measured for the selenomethionine derivative (SAD data, 1.70 Å resolution) and the wild-type enzyme (1.85 Å resolution) to be used for a full three-dimensional structural analysis of the Axe2 protein. Acetylxylan esterases are part of the hemi-cellulolytic system of many microorganisms which utilize plant biomass for growth. Xylans, which are polymeric sugars that constitute a significant part of the plant biomass, are usually substituted with acetyl side groups attached at position 2 or 3 of the xylose backbone units. Acetylxylan esterases hydrolyse the ester linkages of the xylan acetyl groups and thus improve the ability of main-chain hydrolysing enzymes to break down the sugar backbone units. As such, these enzymes play an important part in the hemi-cellulolytic utilization system of many microorganisms that use plant biomass for growth. Interest in the biochemical characterization and structural analysis of these enzymes stems from their numerous potential biotechnological applications. An acetylxylan esterase (Axe2) of this type from Geobacillus stearothermophilus T-6 has recently been cloned, overexpressed, purified, biochemically characterized and crystallized. One of the crystal forms obtained (RB1) belonged to the tetragonal space group I422, with unit-cell parameters a = b = 110.2, c = 213.1 Å. A full diffraction data set was collected to 1.85 Å resolution from flash-cooled crystals of the wild-type enzyme at 100 K using synchrotron radiation. A selenomethionine derivative of Axe2 has also been prepared and crystallized for single-wavelength anomalous diffraction experiments. The crystals of the selenomethionine-derivatized Axe2 appeared to be isomorphous to those of the wild-type enzyme and enabled the measurement of a full 1.85 Å resolution diffraction data set at the selenium

  20. Structural and Enzymatic Characterization of NanS (YjhS) a 9-O-Acetyl N-acetylneuraminic Acid Esterase from Escherichia coli O157:H7

    Energy Technology Data Exchange (ETDEWEB)

    E Rangarajan; K Ruane; A Proteau; J Schrag; R Valladares; C Gonzalez; M Gilbert; A Yakunin; M Cygler

    2011-12-31

    There is a high prevalence of sialic acid in a number of different organisms, resulting in there being a myriad of different enzymes that can exploit it as a fermentable carbon source. One such enzyme is NanS, a carbohydrate esterase that we show here deacetylates the 9 position of 9-O-sialic acid so that it can be readily transported into the cell for catabolism. Through structural studies, we show that NanS adopts a SGNH hydrolase fold. Although the backbone of the structure is similar to previously characterized family members, sequence comparisons indicate that this family can be further subdivided into two subfamilies with somewhat different fingerprints. NanS is the founding member of group II. Its catalytic center contains Ser19 and His301 but no Asp/Glu is present to form the classical catalytic triad. The contribution of Ser19 and His301 to catalysis was confirmed by mutagenesis. In addition to structural characterization, we have mapped the specificity of NanS using a battery of substrates.

  1. Growth advantage of Escherichia coli O104:H4 strains on 5-N-acetyl-9-O-acetyl neuraminic acid as a carbon source is dependent on heterogeneous phage-Borne nanS-p esterases.

    Science.gov (United States)

    Saile, Nadja; Schwarz, Lisa; Eißenberger, Kristina; Klumpp, Jochen; Fricke, Florian W; Schmidt, Herbert

    2018-06-01

    Enterohemorrhagic E. coli (EHEC) are serious bacterial pathogens which are able to cause a hemorrhagic colitis or the life-threatening hemolytic-uremic syndrome (HUS) in humans. EHEC strains can carry different numbers of phage-borne nanS-p alleles that are responsible for acetic acid release from mucin from bovine submaxillary gland and 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac 2 ), a carbohydrate present in mucin. Thus, Neu5,9Ac 2 can be transformed to 5-N-acetyl neuraminic acid, an energy source used by E. coli strains. We hypothesize that these NanS-p proteins are involved in competitive growth of EHEC in the gastrointestinal tract of humans and animals. The aim of the current study was to demonstrate and characterize the nanS-p alleles of the 2011 E. coli O104:H4 outbreak strain LB226692 and analyze whether the presence of multiple nanS-p alleles in the LB226692 genome causes a competitive growth advantage over a commensal E. coli strain. We detected and characterized five heterogeneous phage-borne nanS-p alleles in the genome of E. coli O104:H4 outbreak strain LB226692 by in silico analysis of its genome. Furthermore, successive deletion of all nanS-p alleles, subsequent complementation with recombinant NanS-p13-His, and in vitro co-culturing experiments with the commensal E. coli strain AMC 198 were conducted. We could show that nanS-p genes of E. coli O104:H4 are responsible for growth inhibition of strain AMC 198, when Neu5,9Ac 2 was used as sole carbon source in co-culture. The results of this study let us suggest that multiple nanS-p alleles may confer a growth advantage by outcompeting other E. coli strains in Neu5,9Ac 2 rich environments, such as mucus in animal and human gut. Copyright © 2018 Elsevier GmbH. All rights reserved.

  2. High-temperature crystallization of the secondary alcohol dehydrogenase from the extreme thermophilic bacteria Thermoanaerobacter ethanolicus, a bifunctional alcohol dehydrogenase-acetyl-CoA thio esterase

    International Nuclear Information System (INIS)

    Watanabe, L.; Arni, R.K.

    1996-01-01

    Full text. Ethanol fermentations from Saccharomyces sp. are used in industrial ethanol production and are performed at mesophilic temperatures where final ethanol concentrations must exceed 4% (v/v) to make the process industrially economic. In addition, distillation is required to recover ethanol. Thermophilic fermentations are very attractive since they enable separation of ethanol from continuous cultures at process temperature and reduced pressure. Two different ethanol-production pathways have been identified for thermophilic bacteria; type I from Clostridium thermocellum, which contains only NADH-linked primary-alcohol dehydrogeneases, and type II from Thermoanaerobacter brockii which in addition include NADPH-linked secondary-alcohol dehydrogenases. The thermophilic anaerobic bacterium T ethanolicus 39E produces ethanol as the major end product from starch, pentose and herose substrates. The 2 Adh has a lower catalytic efficiency for the oxidation of 1 alcohols, including ethanol, than for the oxidation of secondary (2) alcohols or the reduction of ketones or aldehydes and possesses a significant acetyl-CoA reductive thioesterase activity. Large single crystals (0.7 x 0.3 x 0.3 mn) of this enzyme have been obtained at 40 0 C and diffraction data to 2.7 A resolution has been collected (R merge = 10.44%). Attempts are currently underway to obtain higher resolution data and a search for heavy atom derivatives is currently underway. The crystals belong to the space group P2 1 2 1 2 with cell constants of a a= 170.0 A, b=125.7 A and c=80.5 A. The asymmetric unit contains a tetramer as in the case of the crystals of the secondary alcohol dehydrogenase from Thermoanaerobacter brockii with a V M of 2.85 A 3 /Da. (author)

  3. Human Coronavirus HKU1 Spike Protein Uses O-Acetylated Sialic Acid as an Attachment Receptor Determinant and Employs Hemagglutinin-Esterase Protein as a Receptor-Destroying Enzyme.

    Science.gov (United States)

    Huang, Xingchuan; Dong, Wenjuan; Milewska, Aleksandra; Golda, Anna; Qi, Yonghe; Zhu, Quan K; Marasco, Wayne A; Baric, Ralph S; Sims, Amy C; Pyrc, Krzysztof; Li, Wenhui; Sui, Jianhua

    2015-07-01

    Human coronavirus (hCoV) HKU1 is one of six hCoVs identified to date and the only one with an unidentified cellular receptor. hCoV-HKU1 encodes a hemagglutinin-esterase (HE) protein that is unique to the group a betacoronaviruses (group 2a). The function of HKU1-HE remains largely undetermined. In this study, we examined binding of the S1 domain of hCoV-HKU1 spike to a panel of cells and found that the S1 could specifically bind on the cell surface of a human rhabdomyosarcoma cell line, RD. Pretreatment of RD cells with neuraminidase (NA) and trypsin greatly reduced the binding, suggesting that the binding was mediated by sialic acids on glycoproteins. However, unlike other group 2a CoVs, e.g., hCoV-OC43, for which 9-O-acetylated sialic acid (9-O-Ac-Sia) serves as a receptor determinant, HKU1-S1 bound with neither 9-O-Ac-Sia-containing glycoprotein(s) nor rat and mouse erythrocytes. Nonetheless, the HKU1-HE was similar to OC43-HE, also possessed sialate-O-acetylesterase activity, and acted as a receptor-destroying enzyme (RDE) capable of eliminating the binding of HKU1-S1 to RD cells, whereas the O-acetylesterase-inactive HKU1-HE mutant lost this capacity. Using primary human ciliated airway epithelial (HAE) cell cultures, the only in vitro replication model for hCoV-HKU1 infection, we confirmed that pretreatment of HAE cells with HE but not the enzymatically inactive mutant blocked hCoV-HKU1 infection. These results demonstrate that hCoV-HKU1 exploits O-Ac-Sia as a cellular attachment receptor determinant to initiate the infection of host cells and that its HE protein possesses the corresponding sialate-O-acetylesterase RDE activity. Human coronaviruses (hCoV) are important human respiratory pathogens. Among the six hCoVs identified to date, only hCoV-HKU1 has no defined cellular receptor. It is also unclear whether hemagglutinin-esterase (HE) protein plays a role in viral entry. In this study, we found that, similarly to other members of the group 2a CoVs, sialic

  4. Characterization of O-acetil-(4-O-methylglucurono)xylans from Eucalyptus urograndis

    International Nuclear Information System (INIS)

    Magaton, Andreia da Silva; Pilo-Veloso, Dorila; Colodette, Jorge Luiz

    2008-01-01

    The O-acetyl- 4-O-methyl-(glucurono)xylans were isolated from E. urograndis by extraction with dimethyl sulfoxide, analysed for monosaccharide composition and structurally characterized by NMR spectroscopy. These xylans contained one 4-O-methyl-glucuronic acid substituent and 5.5 acetyl groups for approximately 10 xylose residues. About 10% of 4-O-methyl-glucuronic acid (MeGlcA) units were branched at O-2. The O-acetyl-4-O-methyl-(glucurono)-xylans were composed of the following (1→4)-linked β-D-xylopyranosyl structural elements: unsubstituted (51 mol%), 2-O-acetylated (12 mol%), 3-O-acetylated (20 mol%), 2,3-di-Oacetylated (6 mol%) and [MeGlcA α-(1→2)] [3-O-acetylated] (11 mol%). The weight-average molar mass and polydispersity of this xylan were 34.9 kDa and 1.16, respectively, as measured by size-exclusion chromatography. (author)

  5. Reducing the heterogeneity of xylan through processing.

    Science.gov (United States)

    Zhang, Wei; Johnson, Amanda M; Barone, Justin R; Renneckar, Scott

    2016-10-05

    Glycerol thermal processing (GTP) of hardwood biomass at temperatures between 200 and 240°C facilitated stepwise biopolymer fractionation, while limiting significant degradation of the major hemicellulose, glucuronoxylan, into water-extractable oligosaccharides. After GTP pretreatment and sequential water and organic solvent extraction, up to 80% of the initial xylan remained in the pretreated biomass. The majority of the xylan from GTP pretreated and water/solvent extracted biomass was removed using a mild alkali extraction and the composition was compared to xylan directly isolated from untreated hardwood. The precipitated xylan from the neutralized alkaline filtrate was isolated as a water insoluble xylan portion (WIX). The residual xylan dissolved in the neutralized filtrate was precipitated in cold methanol and recovered as the water soluble xylan portion (WSX). Results showed that xylan in WIX was in a polymeric form with a number average degree of polymerization (DP) over 100, whereas the WSX had a much lower average DP of 27 (ca) and contained more substitution. As the processing severity increased during GTP pretreatment, the proportion of WIX increased and the purity of the xylan within the WIX sample reached 84% based on compositional analysis. FT-IR analysis of WIX revealed that xylan isolated after GTP contained peaks related to a reduced carbonyl signal compared to the control. Furthermore, crude WSX contained less xylan with more lignin contamination at severe GTP conditions. The recovery of the xylan in two portions facilitated a preferential purification strategy resulting in WIX with an extremely narrow polydispersity index between 1.1 and 1.25, dependent upon the GTP severity. This study provided insight into fractionating higher molecular weight xylan that may serve value-added applications such as healthcare materials and advanced packaging. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Mapping sugar beet pectin acetylation pattern.

    Science.gov (United States)

    Ralet, Marie-Christine; Cabrera, Juan Carlos; Bonnin, Estelle; Quéméner, Bernard; Hellìn, Pilar; Thibault, Jean-François

    2005-08-01

    Homogalacturonan-derived partly methylated and/or acetylated oligogalacturonates were recovered after enzymatic hydrolysis (endo-polygalacturonase+pectin methyl esterase+side-chain degrading enzymes) of sugar beet pectin followed by anion-exchange and size exclusion chromatography. Around 90% of the GalA and 75% of the acetyl groups present in the initial sugar beet pectin were recovered as homogalacturonan-derived oligogalacturonates, the remaining GalA and acetyl belonging to rhamnogalacturonic regions. Around 50% of the acetyl groups present in sugar beet homogalacturonans were recovered as partly methylated and/or acetylated oligogalacturonates of degree of polymerisation 5 whose structures were determined by electrospray ionization ion trap mass spectrometry (ESI-IT-MSn). 2-O-acetyl- and 3-O-acetyl-GalA were detected in roughly similar amounts but 2,3-di-O-acetylation was absent. Methyl-esterified GalA residues occurred mainly upstream 2-O-acetyl GalA. Oligogalacturonates containing GalA residues that are at once methyl- and acetyl-esterified were recovered in very limited amounts. A tentative mapping of the distribution of acetyl and methyl esters within sugar beet homogalacturonans is proposed. Unsubstituted GalA residues are likely to be present in limited amounts (approximately 10% of total GalA residues), due to the fact that methyl and acetyl groups are assumed to be most often not carried by the same residues.

  7. Probing Xylan-Specific Raman Bands for Label-Free Imaging Xylan in Plant Cell Wall

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Yining; Yarbrough, John M.; Mittal, Ashutosh; Tucker, Melvin P.; Vinzant, Todd; Himmel, Michael E.

    2015-06-15

    Xylan constitutes a significant portion of biomass (e.g. 22% in corn stover used in this study). Xylan is also an important source of carbohydrates, besides cellulose, for renewable and sustainable energy applications. Currently used method for the localization of xylan in biomass is to use fluorescence confocal microscope to image the fluorescent dye labeled monoclonal antibody that specifically binds to xylan. With the rapid adoption of the Raman-based label-free chemical imaging techniques in biology, identifying Raman bands that are unique to xylan would be critical for the implementation of the above label-free techniques for in situ xylan imaging. Unlike lignin and cellulose that have long be assigned fingerprint Raman bands, specific Raman bands for xylan remain unclear. The major challenge is the cellulose in plant cell wall, which has chemical units highly similar to that of xylan. Here we report using xylanase to specifically remove xylan from feedstock. Under various degree of xylan removal, with minimum impact to other major cell wall components, i.e. lignin and cellulose, we have identified Raman bands that could be further tested for chemical imaging of xylan in biomass in situ.

  8. Esterase screening using whole cells of Brazilian soil microorganisms

    Energy Technology Data Exchange (ETDEWEB)

    Mantovani, Simone M.; Oliveira, Luciana G. de; Marsaioli, Anita J., E-mail: anita@iqm.unicamp.b [Universidade Estadual de Campinas (IQ/UNICAMP), SP (Brazil). Inst. de Quimica

    2010-07-01

    A miniaturized enzymatic assay using fluorescent probes to reveal esterase producing microorganisms was optimized and applied to screen 64 soil bacterial strains. The best results were validated using traditional non-fluorogenic assays with acetyl and propanoyl phenylethanol to confirm the miniaturized results. The most active microorganisms belong to the genus Bacillus showing esterase activity and good enantiomeric ratios for the resolution of phenylethanol derivatives (E > 30). Part of the microorganisms are kept in our laboratory in glycerol or freezedried and the best microorganisms will be deposited in the CBMAI/CPQBA/UNICAMP culture collection. (author)

  9. Glucuronoyl esterase--novel carbohydrate esterase produced by Schizophyllum commune.

    Science.gov (United States)

    Spániková, Silvia; Biely, Peter

    2006-08-21

    The cellulolytic system of the wood-rotting fungus Schizophyllum commune contains an esterase that hydrolyzes methyl ester of 4-O-methyl-d-glucuronic acid. The enzyme, called glucuronoyl esterase, was purified to electrophoretic homogeneity from a cellulose-spent culture fluid. Its substrate specificity was examined on a number of substrates of other carbohydrate esterases such as acetylxylan esterase, feruloyl esterase and pectin methylesterase. The glucuronoyl esterase attacks exclusively the esters of MeGlcA. The methyl ester of free or glycosidically linked MeGlcA was not hydrolysed by other carbohydrate esterases. The results suggest that we have discovered a new type of carbohydrate esterase that might be involved in disruption of ester linkages connecting hemicellulose and lignin in plant cell walls.

  10. Carboxymethylated-, hydroxypropylsulfonated- and quaternized xylan derivative films

    Czech Academy of Sciences Publication Activity Database

    Šimkovic, I.; Kelnar, Ivan; Uhliariková, I.; Mendichi, R.; Mandalika, A.; Elder, T.

    2014-01-01

    Roč. 110, 22 September (2014), s. 464-471 ISSN 0144-8617 Institutional support: RVO:61389013 Keywords : xylan * films * properties Subject RIV: CD - Macromolecular Chemistry Impact factor: 4.074, year: 2014

  11. The natural catalytic function of CuGE glucuronoyl esterase in hydrolysis of genuine lignin-carbohydrate complexes from birch

    DEFF Research Database (Denmark)

    Mosbech, Caroline; Holck, Jesper; Meyer, Anne S.

    2018-01-01

    Glucuronoyl esterases belong to carbohydrate esterase family 15 and catalyze de-esterification. Their natural function is presumed to be cleavage of ester linkages in lignin-carbohydrate complexes particularly those linking lignin and glucuronoyl residues in xylans in hardwood. Here, we show...... for the first time a detailed product profile of aldouronic acids released from birchwood lignin by a glucuronoyl esterase from the white-rot fungus Cerrena unicolor (CuGE). CuGE releases substrate for GH10 endo-xylanase which results in significantly increased product release compared to the action of endo......-xylanase alone. CuGE also releases neutral xylo-oligosaccharides that can be ascribed to the enzymes feruloyl esterase side activity as demonstrated by release of ferulic acid from insoluble wheat arabinoxylan. The data verify the enzyme's unique ability to catalyze removal of all glucuronoxylan associated...

  12. Reduced Wall Acetylation Proteins Play Vital and Distinct Roles in Cell Wall O-Acetylation in Arabidopsis

    DEFF Research Database (Denmark)

    Manabe, Yuzuki; Verhertbruggen, Yves; Gille, Sascha

    2013-01-01

    The Reduced Wall Acetylation (RWA) proteins are involved in cell wall acetylation in plants. Previously, we described a single mutant, rwa2, which has about 20% lower level of O-acetylation in leaf cell walls and no obvious growth or developmental phenotype. In this study, we generated double....... The quadruple rwa mutant can be completely complemented with the RWA2 protein expressed under 35S promoter, indicating the functional redundancy of the RWA proteins. Nevertheless, the degree of acetylation of xylan, (gluco) mannan, and xyloglucan as well as overall cell wall acetylation is affected differently...... in different combinations of triple mutants, suggesting their diversity in substrate preference. The overall degree of wall acetylation in the rwa quadruple mutant was reduced by 63% compared with the wild type, and histochemical analysis of the rwa quadruple mutant stem indicates defects in cell...

  13. Three feruloyl esterases in Cellulosilyticum ruminicola H1 act synergistically to hydrolyze esterified polysaccharides.

    Science.gov (United States)

    Li, Jiabao; Cai, Shichun; Luo, Yuanming; Dong, Xiuzhu

    2011-09-01

    Feruloyl esterases (Faes) constitute a subclass of carboxyl esterases that specifically hydrolyze the ester linkages between ferulate and polysaccharides in plant cell walls. Until now, the described microbial Faes were mainly from fungi. In this study, we report that Cellulosilyticum ruminicola H1, a previously described fibrolytic rumen bacterium, possesses three different active feruloyl esterases, FaeI, FaeII, and FaeIII. Phylogenetic analysis classified the described bacterial Faes into two types, FaeI and FaeII in type I and FaeIII in type II. Substrate specificity assays indicated that FaeI is more active against the ester bonds in natural hemicelluloses and FaeIII preferentially attacks the ferulate esters with a small moiety, such as methyl groups, while FaeII is active on both types of substrates. Among the three feruloyl esterase genes, faeI was the only one induced significantly by xylose and xylan, while pectin appeared to moderately induce the three genes during the late log phase to stationary phase. Western blot analysis determined that FaeI and FaeIII were secreted and cytoplasmic proteins, respectively, whereas FaeII seemed to be cell associated. The addition of FaeI and FaeII but not FaeIII enhanced the activity of a xylanase on maize cob, suggesting a synergy of the former two with xylanase. Hence, we propose that the three feruloyl esterases work in concert to hydrolyze ferulate esters in natural hemicelluloses.

  14. Structural differences of xylans affect their interaction with cellulose

    NARCIS (Netherlands)

    Kabel, M.A.; Borne, van den H.; Vincken, J.P.; Voragen, A.G.J.; Schols, H.A.

    2007-01-01

    The affinity of xylan to cellulose is an important aspect of many industrial processes, e.g. production of cellulose, paper making and bio-ethanol production. However, little is known about the adsorption of structurally different xylans to cellulose. Therefore, the adsorption of various xylans to

  15. Distribution and substrate specificity of esterases in the housefly, Musca domestica L.

    NARCIS (Netherlands)

    Asperen, K. van

    1959-01-01

    Housefly homogenates perform high cholinesterase and ali-esterase activity. Warburg-manometric studies show that acetylcholine, acetyl-β-methylcholine, butyrylcholine, and benzoylcholine are exclusively hydrolysed by a cholinesterase, the properties of which are more or less comparable to those of

  16. Non-specific esterases and esterproteases in masticatory muscles from the muscular dystrophic mouse

    DEFF Research Database (Denmark)

    Kirkeby, S; Moe, D; Vilmann, H

    1989-01-01

    With the aid of histochemical and electrophoretic techniques activities for esterase and esterprotease were investigated in the digastric and masseter muscles from normal and dystrophic mice. The substrates used were alpha-naphthyl acetate and N-acetyl-L-alanine alpha-naphthyl ester. According...

  17. Enzymatic deconstruction of xylan for biofuel production

    Science.gov (United States)

    DODD, DYLAN; CANN, ISAAC K. O.

    2010-01-01

    The combustion of fossil-derived fuels has a significant impact on atmospheric carbon dioxide (CO2) levels and correspondingly is an important contributor to anthropogenic global climate change. Plants have evolved photosynthetic mechanisms in which solar energy is used to fix CO2 into carbohydrates. Thus, combustion of biofuels, derived from plant biomass, can be considered a potentially carbon neutral process. One of the major limitations for efficient conversion of plant biomass to biofuels is the recalcitrant nature of the plant cell wall, which is composed mostly of lignocellulosic materials (lignin, cellulose, and hemicellulose). The heteropolymer xylan represents the most abundant hemicellulosic polysaccharide and is composed primarily of xylose, arabinose, and glucuronic acid. Microbes have evolved a plethora of enzymatic strategies for hydrolyzing xylan into its constituent sugars for subsequent fermentation to biofuels. Therefore, microorganisms are considered an important source of biocatalysts in the emerging biofuel industry. To produce an optimized enzymatic cocktail for xylan deconstruction, it will be valuable to gain insight at the molecular level of the chemical linkages and the mechanisms by which these enzymes recognize their substrates and catalyze their reactions. Recent advances in genomics, proteomics, and structural biology have revolutionized our understanding of the microbial xylanolytic enzymes. This review focuses on current understanding of the molecular basis for substrate specificity and catalysis by enzymes involved in xylan deconstruction. PMID:20431716

  18. Carboxymethylated-, hydroxypropylsulfonated- and quaternized xylan derivative films

    Science.gov (United States)

    Ivan Simkovic; Ivan Kelnar; Iveta Uhliarikova; Raniero Mdndichi; Anurag Mandalika; Thomas Elder

    2014-01-01

    Under alkaline/water conditions carboxymethyl, 2-hydroxypropylsulfonate and trimethylammonium-2-hydroxypropyl groups were introduced into xylan in one step with the goal to prepare film specimens. The materials were characterized by NMR, SEC-MALS, TG/DTG/DTA, AFM and mechanical testing. The properties of triple, double and mono-substituted materials were compared. The...

  19. Maillard reaction products from chitosan-xylan ionic liquid solution.

    Science.gov (United States)

    Luo, Yuqiong; Ling, Yunzhi; Wang, Xiaoying; Han, Yang; Zeng, Xianjie; Sun, Runcang

    2013-10-15

    A facile method is reported to prepare Maillard reaction products (MRPs) from chitosan and xylan in co-solvent ionic liquid. UV absorbance and fluorescence changes were regarded as indicators of the occurrence of Maillard reaction. FT-IR, NMR, XRD and TG were used to investigate the structure of chitosan-xylan conjugate. The results revealed that when chitosan reacted with xylan in ionic liquid, the hydrogen bonds in chitosan were destroyed, the facts resulted in the formation of chitosan-xylan MRPs. Moreover, when the mass ratio of chitosan to xylan was 1:1, the Maillard reaction proceeded easily. In addition, relatively high antioxidant property was also noted for the chitosan-xylan conjugate with mass ratio 1:1. So the obtained chitosan-xylan MRP is a promising antioxidant agent for food industry. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Esterase Isoenzyme Variants in Barley

    DEFF Research Database (Denmark)

    Hvid, S.; Nielsen, G.

    1977-01-01

    Gene symbols are proposed for 27 esterase isoenzyme alleles representing 10 loci in barley. Two new esterase loci, Est 9 and Est 10, each with an active and a silent allele, and three new alleles in previously described loci were found. A few chemical and physical characteristics of the different...... esterase isoenzyme systems were studied. The heat inactivation temperature differed for the isoenzymes coded by most of the loci, whereas the substrate and inhibitor specificity of the isoenzymes was less distinct. A possible relationship between some of the systems is discussed....

  1. A Bacillus licheniformis pectin acetylesterase is specific for homogalacturonans acetylated at O-3

    NARCIS (Netherlands)

    Remoroza, C.A.; Wagenknecht, M.; Buchholt, H.C.; Moerschbacher, B.M.; Schols, H.A.; Gruppen, H.

    2014-01-01

    A recombinant acetylesterase from Bacillus licheniformis DSM13, belonging to carbohydrate esterase family 12, was purified and biochemically characterized. The purified enzyme, termed BliPAE, was capable of deacetylating acetylated pectins, e.g. sugar beet pectin (SBP). Contrary to its provisional

  2. Esterase profile of human masseter muscle

    DEFF Research Database (Denmark)

    Kirkeby, S; Moe, D; Vilmann, H

    1988-01-01

    The esterase profile of fresh human masseter muscle was investigated by use of histochemistry and electrophoresis. The histochemical methods included reactions for alpha-naphthyl esterase, myofibrillar ATPase, reverse myofibrillar ATPase and succinic dehydrogenase. In frozen sections of the muscle...... the coloured reaction product for esterases was present both as a diffuse sarcoplasmic coloration and as distinct granules. The intensity of diffuse reaction was used to classify the muscle fibres as strongly, moderately and weakly reacting. The fibres with strong esterase activity belonged to Type I and ii......C. iM and Type II A fibres showed a moderate esterase reaction and Type II B fibres had a low activity. The electrophoretic gels stained for esterase activity showed that the human masseter muscle possesses a slow migrating double band with high enzyme activity and a cascade of faster migrating...

  3. Effect of halogenated benzenes on acetanilide esterase, acetanilide hydroxylase and procaine esterase in rats.

    Science.gov (United States)

    Carlson, G P; Dziezak, J D; Johnson, K M

    1979-07-01

    1,2,4-Trichlorobenzene, 1,3,5-trichlorobenzene, hexachlorobenzene, 1,2,4-tribromobenzene, 1,3,5-tribromobenzene and hexabromobenzene were compared for their abilities to induce acetanilide esterase, acentailide hydroxylase and procaine esterase. Except for hexabromobenzene all induced acetanilide esterase whereas the hydroxylation of acetanilide was seen only with the fully halogenated benzenes and with 1,3,5-tribromobenzene. Hepatic procaine esterase activity was increased by the three chlorinated benzenes and 1,2,4-tribromobenzene.

  4. Acetylation/deacetylation reactions of T-2, acetyl T-2, HT-2, and acetyl HT-2 toxins in bovine rumen fluid in vitro

    International Nuclear Information System (INIS)

    Munger, C.E.; Ivie, G.W.; Christopher, R.J.; Hammock, B.D.; Phillips, T.D.

    1987-01-01

    A tritiated preparation of the trichothecene mycotoxin, T-2 toxin, underwent both acetylation and deacetylation reactions when incubated with bovine rumen fluid in vitro. Products from incubations of T-2 in rumen fluid included acetyl T-2, HT-2, and acetyl HT-2. Direct studies with tritiated samples of each of these metabolites confirmed their relatively facile interconversion in the rumen. Studies with [ 3 H]HT-2 under conditions of inhibited esterase activity (added diisopropyl fluorophosphate) showed that acetylation is preferred at C-3 vs. C-4. Studies with [ 3 H]acetyl T-2 indicated that deacetylation similarly occurs with greater rapidity at C-3. There were no indications that ester hydrolysis of these trichothecenes occurred at C-8 or C-15 or that they were subjected to epoxide reduction reactions. These data suggest that acetylation of T-2 and other trichothecenes in the rumen in situ may ultimately result in the absorption of more lipophilic metabolites whose toxicological and residual properties are at present unknown

  5. Acetylation/deacetylation reactions of T-2, acetyl T-2, HT-2, and acetyl HT-2 toxins in bovine rumen fluid in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Munger, C.E.; Ivie, G.W.; Christopher, R.J.; Hammock, B.D.; Phillips, T.D.

    A tritiated preparation of the trichothecene mycotoxin, T-2 toxin, underwent both acetylation and deacetylation reactions when incubated with bovine rumen fluid in vitro. Products from incubations of T-2 in rumen fluid included acetyl T-2, HT-2, and acetyl HT-2. Direct studies with tritiated samples of each of these metabolites confirmed their relatively facile interconversion in the rumen. Studies with (/sup 3/H)HT-2 under conditions of inhibited esterase activity (added diisopropyl fluorophosphate) showed that acetylation is preferred at C-3 vs. C-4. Studies with (/sup 3/H)acetyl T-2 indicated that deacetylation similarly occurs with greater rapidity at C-3. There were no indications that ester hydrolysis of these trichothecenes occurred at C-8 or C-15 or that they were subjected to epoxide reduction reactions. These data suggest that acetylation of T-2 and other trichothecenes in the rumen in situ may ultimately result in the absorption of more lipophilic metabolites whose toxicological and residual properties are at present unknown.

  6. Heterologous expression, purification and characterization of three novel esterases secreted by the lignocellulolytic fungus Penicillium purpurogenum when grown on sugar beet pulp.

    Science.gov (United States)

    Oleas, Gabriela; Callegari, Eduardo; Sepúlveda, Romina; Eyzaguirre, Jaime

    2017-04-18

    The lignocellulolytic fungus, Penicillium purpurogenum, grows on a variety of natural carbon sources, among them sugar beet pulp. Culture supernatants of P. purpurogenum grown on sugar beet pulp were partially purified and the fractions obtained analyzed for esterase activity by zymograms. The bands with activity on methyl umbelliferyl acetate were subjected to mass spectrometry to identify peptides. The peptides obtained were probed against the proteins deduced from the genome sequence of P. purpurogenum. Eight putative esterases thus identified were chosen for future work. Their cDNAs were expressed in Pichia pastoris. The supernatants of the recombinant clones were assayed for esterase activity, and five of the proteins were active against one or more substrates: methyl umbelliferyl acetate, indoxyl acetate, methyl esterified pectin and fluorescein diacetate. Three of those enzymes were purified, further characterized and subjected to a BLAST search. Based on their amino acid sequence and properties, they were identified as follows: RAE1, pectin acetyl esterase (CAZy family CE 12); FAEA, feruloyl esterase (could not be assigned to a CAZy family) and EAN, acetyl esterase (former CAZy family CE 10). Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. aes, the gene encoding the esterase B in Escherichia coli, is a powerful phylogenetic marker of the species

    Directory of Open Access Journals (Sweden)

    Tuffery Pierre

    2009-12-01

    Full Text Available Abstract Background Previous studies have established a correlation between electrophoretic polymorphism of esterase B, and virulence and phylogeny of Escherichia coli. Strains belonging to the phylogenetic group B2 are more frequently implicated in extraintestinal infections and include esterase B2 variants, whereas phylogenetic groups A, B1 and D contain less virulent strains and include esterase B1 variants. We investigated esterase B as a marker of phylogeny and/or virulence, in a thorough analysis of the esterase B-encoding gene. Results We identified the gene encoding esterase B as the acetyl-esterase gene (aes using gene disruption. The analysis of aes nucleotide sequences in a panel of 78 reference strains, including the E. coli reference (ECOR strains, demonstrated that the gene is under purifying selection. The phylogenetic tree reconstructed from aes sequences showed a strong correlation with the species phylogenetic history, based on multi-locus sequence typing using six housekeeping genes. The unambiguous distinction between variants B1 and B2 by electrophoresis was consistent with Aes amino-acid sequence analysis and protein modelling, which showed that substituted amino acids in the two esterase B variants occurred mostly at different sites on the protein surface. Studies in an experimental mouse model of septicaemia using mutant strains did not reveal a direct link between aes and extraintestinal virulence. Moreover, we did not find any genes in the chromosomal region of aes to be associated with virulence. Conclusion Our findings suggest that aes does not play a direct role in the virulence of E. coli extraintestinal infection. However, this gene acts as a powerful marker of phylogeny, illustrating the extensive divergence of B2 phylogenetic group strains from the rest of the species.

  8. Xylanolytic enzyme systems in Arthrobacter sp MTCC 5214 and Lactobacillus sp.

    Digital Repository Service at National Institute of Oceanography (India)

    Khandeparker, R.; Jalal, T.

    The production of extracellular xylanolytic enzymes such as xylanase, alfa-L-arabinofuranosidase (alfa-l-AFase), and acetyl xylan esterase (Axe) by marine Arthrobacter sp and Lactobacillus sp was investigated using different carbon sources Induction...

  9. Cellulose- and xylan-degrading thermophilic anaerobic bacteria from biocompost.

    Science.gov (United States)

    Sizova, M V; Izquierdo, J A; Panikov, N S; Lynd, L R

    2011-04-01

    Nine thermophilic cellulolytic clostridial isolates and four other noncellulolytic bacterial isolates were isolated from self-heated biocompost via preliminary enrichment culture on microcrystalline cellulose. All cellulolytic isolates grew vigorously on cellulose, with the formation of either ethanol and acetate or acetate and formate as principal fermentation products as well as lactate and glycerol as minor products. In addition, two out of nine cellulolytic strains were able to utilize xylan and pretreated wood with roughly the same efficiency as for cellulose. The major products of xylan fermentation were acetate and formate, with minor contributions of lactate and ethanol. Phylogenetic analyses of 16S rRNA and glycosyl hydrolase family 48 (GH48) gene sequences revealed that two xylan-utilizing isolates were related to a Clostridium clariflavum strain and represent a distinct novel branch within the GH48 family. Both isolates possessed high cellulase and xylanase activity induced independently by either cellulose or xylan. Enzymatic activity decayed after growth cessation, with more-rapid disappearance of cellulase activity than of xylanase activity. A mixture of xylan and cellulose was utilized simultaneously, with a significant synergistic effect observed as a reduction of lag phase in cellulose degradation.

  10. Hydrolysis kinetics of tulip tree xylan in hot compressed water.

    Science.gov (United States)

    Yoon, Junho; Lee, Hun Wook; Sim, Seungjae; Myint, Aye Aye; Park, Hee Jeong; Lee, Youn-Woo

    2016-08-01

    Lignocellulosic biomass, a promising renewable resource, can be converted into numerous valuable chemicals post enzymatic saccharification. However, the efficacy of enzymatic saccharification of lignocellulosic biomass is low; therefore, pretreatment is necessary to improve the efficiency. Here, a kinetic analysis was carried out on xylan hydrolysis, after hot compressed water pretreatment of the lignocellulosic biomass conducted at 180-220°C for 5-30min, and on subsequent xylooligosaccharide hydrolysis. The weight ratio of fast-reacting xylan to slow-reacting xylan was 5.25 in tulip tree. Our kinetic results were applied to three different reaction systems to improve the pretreatment efficiency. We found that semi-continuous reactor is promising. Lower reaction temperatures and shorter space times in semi-continuous reactor are recommended for improving xylan conversion and xylooligosaccharide yield. In the theoretical calculation, 95% of xylooligosaccharide yield and xylan conversion were achieved simultaneously with high selectivity (desired product/undesired product) of 100 or more. Copyright © 2016. Published by Elsevier Ltd.

  11. Esterase reactions in acute myelomonocytic leukemia.

    Science.gov (United States)

    Kass, L

    1977-05-01

    Specific and nonspecific esterase reactions of bone marrow cells from 14 patients with untreated acute myelomonocytic leukemia and six patients with acute histiomonocytic leukemia were examined. The technic for esterase determination permitted simultaneous visualization of both esterases on the same glass coverslip containing the marrow cells. In cases of acute histiomonocytic leukemia, monocytes, monocytoid hemohistioblasts and undifferentiated blasts stained intensely positive for nonspecific esterase, using alpha-naphthyl acetate as the substrate. No evidence of specific esterase activity using naphthol ASD-chloroacetate as the substrate and fast blue BBN as the dye coupler was apparent in these cells. In all of the cases of acute myelomonocytic leukemia, both specific and nonspecific esterases were visualized within monocytes, monocytoid cells, and granulocytic cells that had monocytoid-type nuclei. Nonspecific esterase activity was not observed in polymorphonuclear leukocytes in cases of myelomonocytic leukemia. The results support a current viewpoint that acute myelomonocytic leukemia may be a variant of acute myeloblastic leukemia, and that cytochemically, many of the leukemic cells in myelomonocytic leukemia share properties of both granulocytes and monocytes.

  12. Xylan polysaccharides fabricated into nanofibrous substrate for myocardial infarction

    International Nuclear Information System (INIS)

    Venugopal, J.; Rajeswari, R.; Shayanti, M.; Sridhar, R.; Sundarrajan, S.; Balamurugan, R.; Ramakrishna, S.

    2013-01-01

    Myocardial infarction, a main cause of heart failure, leads to loss of cardiac tissue impairment of left ventricular function. Repair of diseased myocardium with in vitro engineered cardiac muscle patch/injectable biopolymers with cells may become a viable option for myocardial infarction. We attempted to solve these problems by in vitro study by selecting a plant based polysaccharides beech wood Xylan for the normal functioning of infarcted myocardium. The present study fabricated Xylan based nanofibrous scaffolds cross-linked with glutaraldehyde (Glu) vapors for 24 h, 48 h and 1% Glu blended fibers for the culture of neonatal rat cardiac cells for myocardial infarction. These nanofibers were characterized by SEM, FT-IR, tensile testing and cell culture studies for the normal expression of cardiac proteins. The observed results showed that the Xylan/polyvinyl alcohol (PVA) 24 h Glu vapor cross-linked nanofibers (427 nm) having mechanical strength of 2.43 MPa and Young modulus of 3.74 MPa are suitable for the culture of cardiac cells. Cardiac cells proliferation increased only by 11% in Xylan/PVA 24 h Glu cross-linked nanofibers compared to control tissue culture plate (TCP). The normal cardiac cell morphology was observed in 24 h cross-linked Xylan/PVA nanofibers but 48 h cross-linked fibers cell morphology was changed to flattened and elongated on the fibrous surfaces. Confocal analysis for cardiac expression proteins actinin, connexin 43 was observed normally in 24 h Glu cross-linked nanofibers compared to all other nanofibrous scaffolds. The fabricated Xylan/PVA nanofibrous scaffold may have good potential for the normal functioning of infarcted myocardium. - Highlights: ► Fabrication of polysaccharides Xylan/PVA nanofibers for cardiac tissue engineering ► Nanofibers characterized by SEM, FT-IR, tensile testing and cell culture studies ► Isolation of cardiac cells and cultured on Xylan/PVA nanofibrous scaffolds ► Cultured cells on 24 h Glu cross

  13. Xylan polysaccharides fabricated into nanofibrous substrate for myocardial infarction

    Energy Technology Data Exchange (ETDEWEB)

    Venugopal, J., E-mail: nnijrv@nus.edu.sg; Rajeswari, R.; Shayanti, M.; Sridhar, R.; Sundarrajan, S.; Balamurugan, R.; Ramakrishna, S.

    2013-04-01

    Myocardial infarction, a main cause of heart failure, leads to loss of cardiac tissue impairment of left ventricular function. Repair of diseased myocardium with in vitro engineered cardiac muscle patch/injectable biopolymers with cells may become a viable option for myocardial infarction. We attempted to solve these problems by in vitro study by selecting a plant based polysaccharides beech wood Xylan for the normal functioning of infarcted myocardium. The present study fabricated Xylan based nanofibrous scaffolds cross-linked with glutaraldehyde (Glu) vapors for 24 h, 48 h and 1% Glu blended fibers for the culture of neonatal rat cardiac cells for myocardial infarction. These nanofibers were characterized by SEM, FT-IR, tensile testing and cell culture studies for the normal expression of cardiac proteins. The observed results showed that the Xylan/polyvinyl alcohol (PVA) 24 h Glu vapor cross-linked nanofibers (427 nm) having mechanical strength of 2.43 MPa and Young modulus of 3.74 MPa are suitable for the culture of cardiac cells. Cardiac cells proliferation increased only by 11% in Xylan/PVA 24 h Glu cross-linked nanofibers compared to control tissue culture plate (TCP). The normal cardiac cell morphology was observed in 24 h cross-linked Xylan/PVA nanofibers but 48 h cross-linked fibers cell morphology was changed to flattened and elongated on the fibrous surfaces. Confocal analysis for cardiac expression proteins actinin, connexin 43 was observed normally in 24 h Glu cross-linked nanofibers compared to all other nanofibrous scaffolds. The fabricated Xylan/PVA nanofibrous scaffold may have good potential for the normal functioning of infarcted myocardium. - Highlights: ► Fabrication of polysaccharides Xylan/PVA nanofibers for cardiac tissue engineering ► Nanofibers characterized by SEM, FT-IR, tensile testing and cell culture studies ► Isolation of cardiac cells and cultured on Xylan/PVA nanofibrous scaffolds ► Cultured cells on 24 h Glu cross

  14. Ferulic acid: an antioxidant found naturally in plant cell walls and feruloyl esterases involved in its release and their applications.

    Science.gov (United States)

    Mathew, Sindhu; Abraham, T Emilia

    2004-01-01

    Ferulic acid is the most abundant hydroxycinnamic acid in the plant world and maize bran with 3.1% (w/w) ferulic acid is one of the most promising sources of this antioxidant. The dehydrodimers of ferulic acid are important structural components in the plant cell wall and serve to enhance its rigidity and strength. Feruloyl esterases are a subclass of the carboxylic acid esterases that hydrolyze the ester bond between hydroxycinnamic acids and sugars present in plant cell walls and they have been isolated from a wide range of microorganisms, when grown on complex substrates such as cereal brans, sugar beet pulp, pectin and xylan. These enzymes perform a function similar to alkali in the deesterification of plant cell wall and differ in their specificities towards the methyl esters of cinnamic acids and ferulolylated oligosaccharides. They act synergistically with xylanases and pectinases and facilitate the access of hydrolases to the backbone of cell wall polymers. The applications of ferulic acid and feruloyl esterase enzymes are many and varied. Ferulic acid obtained from agricultural byproducts is a potential precursor for the production of natural vanillin, due to the lower production cost.

  15. Esterase resistant to inactivation by heavy metals

    KAUST Repository

    El, Dorry Hamza

    2014-09-25

    EstATII is an esterase that a halotolerant, thermophilic and resistant to a spectrum of heavy metals including toxic concentration of metals. It was isolated from the lowest convective layer of the Atlantis II Red Sea brine pool. The Atlantis II brine pool is an extreme environment that possesses multiple harsh conditions such as; high temperature, salinity, pH and high concentration of metals, including toxic heavy metals. A fosmid metagenomic library using DNA isolated from the lowest convective layer this pool was used to identify EstATII. Polynucleotides encoding EstATII and similar esterases are disclosed and can be used to make EstATII. EstATII or compositions or apparatuses that contain it may be used in various processes employing lipases/esterases especially when these processes are performed under harsh conditions that inactivate other kinds of lipases or esterases.

  16. Nanocomposite film prepared by depositing xylan on cellulose nanowhiskers matrix

    Science.gov (United States)

    Qining Sun; Anurag Mandalika; Thomas Elder; Sandeep S. Nair; Xianzhi Meng; Fang Huang; Art J. Ragauskas

    2014-01-01

    Novel bionanocomposite films have been prepared by depositing xylan onto cellulose nanowhiskers through a pH adjustment. Analysis of strength properties, water vapour transmission, transparency, surface morphology and thermal decomposition showed the enhancement of film performance. This provides a new green route to the utilization of biomass for sustainable...

  17. Esterase variation in Turkish white-toothed shrews (Crocidura: Record of a trimeric esterase

    Directory of Open Access Journals (Sweden)

    Tez C.

    2009-01-01

    Full Text Available This study focuses on esterase variation of the genus Crocidura in Turkey. A total of 248 white-toothed shrews were analyzed by means of cellulose acetate gel electrophoresis. Liver tissue and alfa naphthyl acetate were used to investigate esterase variation in Turkish white-toothed shrews. A different esterase banding pattern was found in one Crocidura individual. This phenotype had four anodally migrated bands on cellulose acetate gel. The Crocidura individual displaying the given phenotype was identified as Crocidura suaveolens. The different esterase banding pattern observed in this study is considered to be a result of the trimeric structure of esterase in the lesser white-toothed shrew (Crocidura suaveolens.

  18. [3H]Indole-3-acetyl-myo-inositol hydrolysis by extracts of Zea mays L. vegetative tissue

    Science.gov (United States)

    Hall, P. J.; Bandurski, R. S.

    1986-01-01

    [3H]Indole-3-acetyl-myo-inositol was hydrolyzed by buffered extracts of acetone powders prepared from 4 day shoots of dark grown Zea mays L. seedlings. The hydrolytic activity was proportional to the amount of extract added and was linear for up to 6 hours at 37 degrees C. Boiled or alcohol denatured extracts were inactive. Analysis of reaction mixtures by high performance liquid chromatography demonstrated that not all isomers of indole-3-acetyl-myo-inositol were hydrolyzed at the same rate. Buffered extracts of acetone powders were prepared from coleoptiles and mesocotyls. The rates of hydrolysis observed with coleoptile extracts were greater than those observed with mesocotyl extracts. Active extracts also catalyzed the hydrolysis of esterase substrates such as alpha-naphthyl acetate and the methyl esters of indoleacetic acid and naphthyleneacetic acid. Attempts to purify the indole-3-acetyl-myo-inositol hydrolyzing activity by chromatographic procedures resulted in only slight purification with large losses of activity. Chromatography over hydroxylapatite allowed separation of two enzymically active fractions, one of which catalyzed the hydrolysis of both indole-3-acetyl-myo-inositol and esterase substrates. With the other enzymic hydrolysis of esterase substrates was readily demonstrated, but no hydrolysis of indole-3-acetyl-myo-inositol was ever detected.

  19. [3H]Indole-3-acetyl-myo-inositol hydrolysis by extracts of Zea mays L. vegetative tissue

    International Nuclear Information System (INIS)

    Hall, P.J.; Bandurski, R.S.

    1986-01-01

    [ 3 H]Indole-3-acetyl-myo-inositol was hydrolyzed by buffered extracts of acetone powders prepared from 4 day shoots of dark grown Zea mays L. seedlings. The hydrolytic activity was proportional to the amount of extract added and was linear for up to 6 hours at 37 0 C. Boiled or alcohol denatured extracts were inactive. Analysis of reaction mixtures by high performance liquid chromatography demonstrated that not all isomers of indole-3-acetyl-myo-inositol were hydrolyzed at the same rate. Buffered extracts of acetone powders were prepared from coleoptiles and mesocotyls. The rates of hydrolysis observed with coleoptile extracts were greater than those observed with mesocotyl extracts. Active extracts also catalyzed the hydrolysis of esterase substrates such as α-naphthyl acetate and the methyl esters of indoleacetic acid and naphthyleneacetic acid. Attempts to purify the indole-3-acetyl-myo-inositol hydrolyzing activity by chromatographic procedures resulted in only slight purification with large losses of activity. Chromatography over hydroxylapatite allowed separation of two enzymically active fractions, one of which catalyzed the hydrolysis of both indole-3-acetyl-myo-inositol and esterase substrates. With the other fraction enzymic hydrolysis of esterase substrates was readily demonstrated, but no hydrolysis of indole-3-acetyl-myo-inositol was ever detected

  20. Reconstitution of a thermostable xylan-degrading enzyme mixture from the bacterium Caldicellulosiruptor bescii.

    Science.gov (United States)

    Su, Xiaoyun; Han, Yejun; Dodd, Dylan; Moon, Young Hwan; Yoshida, Shosuke; Mackie, Roderick I; Cann, Isaac K O

    2013-03-01

    Xylose, the major constituent of xylans, as well as the side chain sugars, such as arabinose, can be metabolized by engineered yeasts into ethanol. Therefore, xylan-degrading enzymes that efficiently hydrolyze xylans will add value to cellulases used in hydrolysis of plant cell wall polysaccharides for conversion to biofuels. Heterogeneous xylan is a complex substrate, and it requires multiple enzymes to release its constituent sugars. However, the components of xylan-degrading enzymes are often individually characterized, leading to a dearth of research that analyzes synergistic actions of the components of xylan-degrading enzymes. In the present report, six genes predicted to encode components of the xylan-degrading enzymes of the thermophilic bacterium Caldicellulosiruptor bescii were expressed in Escherichia coli, and the recombinant proteins were investigated as individual enzymes and also as a xylan-degrading enzyme cocktail. Most of the component enzymes of the xylan-degrading enzyme mixture had similar optimal pH (5.5 to ∼6.5) and temperature (75 to ∼90°C), and this facilitated their investigation as an enzyme cocktail for deconstruction of xylans. The core enzymes (two endoxylanases and a β-xylosidase) exhibited high turnover numbers during catalysis, with the two endoxylanases yielding estimated k(cat) values of ∼8,000 and ∼4,500 s(-1), respectively, on soluble wheat arabinoxylan. Addition of side chain-cleaving enzymes to the core enzymes increased depolymerization of a more complex model substrate, oat spelt xylan. The C. bescii xylan-degrading enzyme mixture effectively hydrolyzes xylan at 65 to 80°C and can serve as a basal mixture for deconstruction of xylans in bioenergy feedstock at high temperatures.

  1. Metagenomic mining of feruloyl esterases from termite enteric flora

    CSIR Research Space (South Africa)

    Rashamuse, K

    2014-01-01

    Full Text Available A metagenome expression library was created from Trinervitermes trinervoides termite hindgut symbionts and subsequently screened for feruloyl esterase (FAE) activities, resulting in seven recombinant fosmids conferring feruloyl esterase phenotypes...

  2. Selection and production of insoluble xylan hydrolyzing enzyme by ...

    African Journals Online (AJOL)

    Jane

    2011-03-07

    Mar 7, 2011 ... The effect of pH and temperature on the enzyme activity and stability of crude enzyme produced by T. lanuginosus THKU 56 were investigated. To study the effect of pH on activity, the reaction mixture of 0.5 ml of enzyme and 0.5 ml of 1% insoluble oat spelt xylan in 50 mM buffers with various pH values ...

  3. Modelling substrate specificity and enantioselectivity for lipases and esterases by substrate-imprinted docking

    Directory of Open Access Journals (Sweden)

    Tyagi Sadhna

    2009-06-01

    Full Text Available Abstract Background Previously, ways to adapt docking programs that were developed for modelling inhibitor-receptor interaction have been explored. Two main issues were discussed. First, when trying to model catalysis a reaction intermediate of the substrate is expected to provide more valid information than the ground state of the substrate. Second, the incorporation of protein flexibility is essential for reliable predictions. Results Here we present a predictive and robust method to model substrate specificity and enantioselectivity of lipases and esterases that uses reaction intermediates and incorporates protein flexibility. Substrate-imprinted docking starts with covalent docking of reaction intermediates, followed by geometry optimisation of the resulting enzyme-substrate complex. After a second round of docking the same substrate into the geometry-optimised structures, productive poses are identified by geometric filter criteria and ranked by their docking scores. Substrate-imprinted docking was applied in order to model (i enantioselectivity of Candida antarctica lipase B and a W104A mutant, (ii enantioselectivity and substrate specificity of Candida rugosa lipase and Burkholderia cepacia lipase, and (iii substrate specificity of an acetyl- and a butyrylcholine esterase toward the substrates acetyl- and butyrylcholine. Conclusion The experimentally observed differences in selectivity and specificity of the enzymes were reproduced with an accuracy of 81%. The method was robust toward small differences in initial structures (different crystallisation conditions or a co-crystallised ligand, although large displacements of catalytic residues often resulted in substrate poses that did not pass the geometric filter criteria.

  4. Study on the Modification of Bleached Eucalyptus Kraft Pulp Using Birch Xylan

    Science.gov (United States)

    Wenjia Han; Chuanshan Zhao; Thomas Elder; Rendang Yang; Dongho Kim; Yunqiao Pu; Jeffery Hsieh; Arthur J. Ragauskas

    2012-01-01

    In this study, birch xylan was deposited onto elementally chlorine free (ECF) bleached eucalyptus kraft pulp, and the corresponding changes in physical properties were determined. An aqueous 5% birch xylan solution at pH 9 was added to 5 wt% slurry of bleached kraft eucalyptus fibers, with stirring at 70 C for 15 min after which the pH was adjusted to 5–6. The xylan...

  5. Production of xylan-degrading enzymes by a Trichoderma harzianum strain

    Directory of Open Access Journals (Sweden)

    Cacais André O.Guerreiro

    2001-01-01

    Full Text Available Trichoderma harzianum strain 4 produced extracellular xylan-degrading enzymes, namely beta-xylanase, beta-xylosidase and alpha-arabinofuranosidase, when grown in liquid medium cultures containing oat spelt xylan as inducer. Cellulase activity was not detected. The pattern of xylan-degrading enzymes induction was influenced by the form of xylan present in the medium. They were detected in different incubation periods. Electrophoretic separation of the proteins from liquid culture filtrates by SDS-PAGE showed a variety of bands with high and low molecular weights.

  6. The effect of fixation on esterases

    DEFF Research Database (Denmark)

    Kirkeby, S; Moe, D

    1984-01-01

    The localization of reaction product for non-specific esterase from fresh and aldehyde treated glandular tissue was examined. The electrophoretical studies showed a selective inhibition of certain isoenzymes and a change in mobility of some bands caused by aldehyde fixation. In sections a granular...

  7. Esterase resistant to inactivation by heavy metals

    KAUST Repository

    El, Dorry Hamza; Siam, Rania; Mohamed, Yasmine M.

    2014-01-01

    EstATII is an esterase that a halotolerant, thermophilic and resistant to a spectrum of heavy metals including toxic concentration of metals. It was isolated from the lowest convective layer of the Atlantis II Red Sea brine pool. The Atlantis II

  8. Biochemical and Structural Analyses of Two Cryptic Esterases in Bacteroides intestinalis and their Synergistic Activities with Cognate Xylanases.

    Science.gov (United States)

    Wefers, Daniel; Cavalcante, Janaina J V; Schendel, Rachel R; Deveryshetty, Jaigeeth; Wang, Kui; Wawrzak, Zdzislaw; Mackie, Roderick I; Koropatkin, Nicole M; Cann, Isaac

    2017-08-04

    Arabinoxylans are constituents of the human diet. Although not utilizable by the human host, they can be fermented by colonic bacteria. The arabinoxylan backbone is decorated with arabinose side chains that may be substituted with ferulic acid, thus limiting depolymerization to fermentable sugars. We investigated the polypeptides encoded by two genes upregulated during growth of the colonic bacterium Bacteroides intestinalis on wheat arabinoxylan. The recombinant proteins, designated BiFae1A and BiFae1B, were functionally assigned esterase activities. Both enzymes were active on acetylated substrates, although each showed a higher ferulic acid esterase activity on methyl-ferulate. BiFae1A showed a catalytic efficiency of 12mM s -1 on para-nitrophenyl-acetate, and on methyl-ferulate, the value was 27 times higher. BiFae1B showed low catalytic efficiencies for both substrates. Furthermore, the two enzymes released ferulic acid from various structural elements, and NMR spectroscopy indicated complete de-esterification of arabinoxylan oligosaccharides from wheat bran. BiFae1A is a tetramer based on the crystal structure, whereas BiFae1B is a dimer in solution based on size exclusion chromatography. The structure of BiFae1A was solved to 1.98Å resolution, and two tetramers were observed in the asymmetric unit. A flexible loop that may act as a hinge over the active site and likely coordinates critical interactions with the substrate was prominent in BiFae1A. Sequence alignments of the esterase domains in BiFae1B with the feruloyl esterase from Clostridium thermocellum suggest that both domains lack the flexible hinge in BiFae1A, an observation that may partly provide a molecular basis for the differences in activities in the two esterases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Engineering the Xylan Utilization System in Bacillus subtilis for Production of Acidic Xylooligosaccharides

    Science.gov (United States)

    Mun Su Rhee; Lusha Wei; Neha Sawhney; John D. Rice; Franz J. St. John; Jason C. Hurlbert; James F. Preston

    2014-01-01

    Xylans are the predominant polysaccharides in hemicelluloses and an important potential source of biofuels and chemicals. The ability of Bacillus subtilis subsp. subtilis strain 168 to utilize xylans has been ascribed to secreted glycoside hydrolase family 11 (GH11) and GH30 endoxylanases, encoded by the xynA and...

  10. Diversity in Production of Xylan-Degrading Enzymes Among Species Belonging to the Trichoderma Section Longibrachiatum

    NARCIS (Netherlands)

    Toth, K.; Gool, van M.P.; Schols, H.A.; Samuels, G.J.; Gruppen, H.; Szakacs, G.

    2013-01-01

    Xylan is an important part of plant biomass and represents a renewable raw material for biorefineries. Contrary to cellulose, the structure of hemicellulose is quite complex. Therefore, the biodegradation of xylan needs the cooperation of many enzymes. For industrial production of xylanase

  11. Gorse (Ulex europaeus) as a possible source of xylans by hydrothermal treatment

    NARCIS (Netherlands)

    Ligero, P.; Vega, de A.; Kolk, van der J.C.; Dam, van J.E.G.

    2011-01-01

    Autocatalytic hydrothermal process conditions were used to study Ulex europæus (Gorse) as a source of xylan compounds. The aim was to study the possibilities for using this unutilised biomass material to produce xylans. Ulex is an evergreen shrub that grows in the northwest of Spain and has no

  12. Deep Eutectic Solvent Aqueous Solutions as Efficient Media for the Solubilization of Hardwood Xylans.

    Science.gov (United States)

    Morais, Eduarda S; Mendonça, Patrícia V; Coelho, Jorge F J; Freire, Mara G; Freire, Carmen S R; Coutinho, João A P; Silvestre, Armando J D

    2018-02-22

    This work contributes to the development of integrated lignocellulosic-based biorefineries by the pioneering exploitation of hardwood xylans by solubilization and extraction in deep eutectic solvents (DES). DES formed by choline chloride and urea or acetic acid were initially evaluated as solvents for commercial xylan as a model compound. The effects of temperature, molar ratio, and concentration of the DES aqueous solutions were evaluated and optimized by using a response surface methodology. The results obtained demonstrated the potential of these solvents, with 328.23 g L -1 of xylan solubilization using 66.7 wt % DES in water at 80 °C. Furthermore, xylans could be recovered by precipitation from the DES aqueous media in yields above 90 %. The detailed characterization of the xylans recovered after solubilization in aqueous DES demonstrated that 4-O-methyl groups were eliminated from the 4-O-methylglucuronic acids moieties and uronic acids (15 %) were cleaved from the xylan backbone during this process. The similar M w values of both pristine and recovered xylans confirmed the success of the reported procedure. DES recovery in four additional extraction cycles was also demonstrated. Finally, the successful extraction of xylans from Eucalyptus globulus wood by using aqueous solutions of DES was demonstrated. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Synthesis and characterization of birch wood xylan succinoylated in 1-n-butyl-3-methylimidazolium chloride

    DEFF Research Database (Denmark)

    Hansen, Natanya Majbritt Louie; Plackett, David

    2011-01-01

    analysis of the modified and native xylans showed a slight lowering of thermal stability with functionalization. Contact angle measurements on spin-coated surfaces of modified xylan films showed a significant increase in hydrophobicity with the introduction of the alkenyl-functionalized succinic anhydride...

  14. High oxygen nanocomposite barrier films based on xylan and nanocrystalline cellulose

    Science.gov (United States)

    Amit Saxena; Thomas J. Elder; Jeffrey Kenvin; Arthur J. Ragauskas

    2010-01-01

    The goal of this work is to produce nanocomposite film with low oxygen permeability by casting an aqueous solution containing xylan, sorbitol and nanocrystalline cellulose. The morphology of the resulting nanocomposite films was examined by scanning electron microscopy and atomic force microscopy which showed that control films containing xylan and sorbitol had a more...

  15. Synthesis and thermal characterization of xylan-graft-polyacrylonitrile.

    Science.gov (United States)

    Ünlü, Cüneyt H; Öztekin, N Simge; Atıcı, Oya Galioğlu

    2012-10-01

    In this study emulsion polymerization of acrylonitrile using xylan from agricultural waste material (corn cob) and cerium ammonium nitrate was investigated in terms of catalyst acid. Stock ceric solutions were prepared using either nitric or perchloric acid as catalyst. Optimum conditions were determined using different parameters such as reaction time, temperature, and component concentrations. Nitric acid catalyzed reactions resulted in maximum conversion ratio (96%) at 50°C, 1 h where ceric ion, acrylonitrile, xylan, and catalyst concentrations were 21.7 mmol l(-1), 0.5 mol l(-1), 0.2% (w/v), and 0.1 mol l(-1), respectively. However, 83% conversion was obtained with perchloric acid catalysis at 27 °C, 1 h where concentrations were 5.4 mmol l(-1), 0.8 mol l(-1), 0.5% (w/v), and 0.2 mol l(-1), respectively. Copolymer synthesis using perchloric acid was realized at milder conditions than using nitric acid. Thermal analyses of obtained polymers were conducted to characterize copolymers. Results showed that calculated activation energy, maximum degradation temperature, and heat of thermal decomposition changed relying mainly on molecular weight. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Cholesterol esterase activity of human intestinal mucosa

    International Nuclear Information System (INIS)

    Ponz de Leon, M.; Carubbi, F.; Di Donato, P.; Carulli, N.

    1985-01-01

    It has been suggested that cholesterol absorption in humans is dependent on bile acid pool composition and that expansion of the cholic acid pool size is followed by an increase of the absorption values. Similar observations were reported in rats. In the present study, therefore, the authors investigated some general properties of human intestinal cholesterol esterase, with particular emphasis on the effect of bile acids on this enzymatic activity. Twenty-nine segments of small intestine were taken during operations; the enzymatic activity was studied by using mucosal homogenate as a source of enzyme and oleic acid, cholesterol, and 14 C-labeled cholesterol as substrates. The time-activity relationship was linear within the first two hours; optimal pH for esterification ranged between 5 and 6.2. There was little difference between the esterifying activity of the jejunal and ileal mucosa. Esterification of cholesterol was observed with all the investigated fatty acids but was maximal with oleic acid. Bile acids did not affect cholesterol esterase activity when present in the incubation mixture at 0.1 and 1.0 mM; the enzymatic activity, however, was significantly inhibited when bile acids were added at 20 mM. In conclusion, this study has shown that the human intestinal mucosa possesses a cholesterol esterase activity; at variance with the rat, however, the human enzyme does not seem to be stimulated by trihydroxy bile acids

  17. Non-specific esterases in partly mineralized bovine enamel

    DEFF Research Database (Denmark)

    Moe, D; Kirkeby, S

    1990-01-01

    Activity for non-specific esterase was demonstrated in the matrix of developing bovine enamel with alpha-naphthyl acetate and 5-bromoindoxyl acetate as the esterase substrates. By use of high-performance liquid chromatography gel filtration, ion-exchange chromatography, and electrophoresis three...... esterases were shown to be present in the enamel matrix. The enzymes showed highest activity at pH 6.5-7.5. In sections a strong reaction was observed in the secretory ameloblasts. The esterases may be proteolytic enzymes that participate in the degradation of the matrix proteins....

  18. A p-coumaroyl esterase from Rhizoctonia solani with a pronounced chlorogenic acid esterase activity.

    Science.gov (United States)

    Nieter, Annabel; Kelle, Sebastian; Linke, Diana; Berger, Ralf G

    2017-07-25

    Extracellular esterase activity was detected in submerged cultures of Rhizoctonia solani grown in the presence of sugar beet pectin or Tween 80. Putative type B feruloyl esterase (FAE) coding sequences found in the genome data of the basidiomycete were heterologously expressed in Pichia pastoris. Recombinant enzyme production on the 5-L bioreactor scale (Rs pCAE: 3245UL -1 ) exceeded the productivity of the wild type strain by a factor of 800. Based on substrate specificity profiling, the purified recombinant Rs pCAE was classified as a p-coumaroyl esterase (pCAE) with a pronounced chlorogenic acid esterase side activity. The Rs pCAE was also active on methyl cinnamate, caffeate and ferulate and on feruloylated saccharides. The unprecedented substrate profile of Rs pCAE together with the lack of sequence similarity to known FAEs or pCAEs suggested that the Rs pCAE represents a new type of enzyme. Hydroxycinnamic acids were released from agro-industrial side-streams, such as destarched wheat bran (DSWB), sugar beet pectin (SBP) and coffee pulp (CP). Overnight incubation of coffee pulp with the Rs pCAE resulted in the efficient release of p-coumaric (100%), caffeic (100%) and ferulic acid (85%) indicating possible applications for the valorization of food processing wastes and for the enhanced degradation of lignified biomass. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Weinert, Brian Tate; Iesmantavicius, Vytautas; Moustafa, Tarek

    2014-01-01

    Lysine acetylation is a frequently occurring posttranslational modification; however, little is known about the origin and regulation of most sites. Here we used quantitative mass spectrometry to analyze acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. We found that acetylation...

  20. Study on the Hydrolysis Kinetics of Xylan on Different Acid Catalysts

    Energy Technology Data Exchange (ETDEWEB)

    Na, Byeong-Il; Lee, Jae-Won [Chonnam National University, Gwangju (Korea, Republic of)

    2014-04-15

    In this study, we investigated kinetic model for the acid-catalyzed xylan hydrolysis at temperature 120-150 .deg. C. Also, we analyzed the kinetic parameters for xylose production and furfural decomposition. The hydrolysis of xylan and the degradation of xylose were promoted by high reaction temperature and acid concentration. The optimal hydrolysis condition for the highest reaction rate constants (k{sub 1}) was different depending on the acid catalysts. Among sulfuric, oxalic and maleic acid, the xylan reaction rate constants (k{sub 1}) to xylose had the highest value of 0.0241 min{sup -1} when 100 mM sulfuric acid was used at 120 .deg. C. However, sulfuric acid induced more xylose degradation compared to oxalic and maleic acid hydrolysis. The activation energy for xylan degradation was the highest when sulfuric acid was used.

  1. Xylella fastidiosa esterase rather than hydroxynitrile lyase.

    Science.gov (United States)

    Torrelo, Guzman; Ribeiro de Souza, Fayene Zeferino; Carrilho, Emanuel; Hanefeld, Ulf

    2015-03-02

    In 2009, we reported that the product of the gene SCJ21.16 (XFa0032) from Xylella fastidiosa, a xylem-restricted plant pathogen that causes a range of diseases in several important crops, encodes a protein (XfHNL) with putative hydroxynitrile lyase activity. Sequence analysis and activity tests indicated that XfHNL exhibits an α/β-hydrolase fold and could be classified as a member of the family of FAD-independent HNLs. Here we provide a more detailed sequence analysis and new experimental data. Using pure heterologously expressed XfHNL we show that this enzyme cannot catalyse the cleavage/synthesis of mandelonitrile and that this protein is in fact a non-enantioselective esterase. Homology modelling and ligand docking simulations were used to study the active site and support these results. This finding could help elucidate the common ancestor of esterases and hydroxynitrile lyases with an α/β -hydrolase fold. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Synthesis of cell wall xylans and glucans by golgi membranes

    International Nuclear Information System (INIS)

    Gibeaut, D.M.; Carpita, N.C.

    1989-01-01

    We investigated the biosynthesis of mixed-linkage β-D-glucan and glucuronoarabinoxylans which make up the hemicellulosic matrix of the primary cell walls of maize and other cereal grasses. The Golgi apparatus was enriched from plasma membrane and other organelles by flotation density gradient centrifugation. Glucan synthase I and II, which are established markers for Golgi and plasma membrane, respectively, displayed considerable overlap in conventional separations with sucrose density gradients. Flotation gradients improved separation of the membranes substantially, but the different synthases themselves also incorporated radioactivity from either 10 μM or 1 mM UDP-[ 14 C]-glucose into polymer. Relative incorporation of radioactivity into polymers from UDP-[ 14 C]-xylose by the various membrane fractions was nearly identical to relative IDPase activities, indicating that combined xylosyl transferase-xylan synthase represents a new, unequivocal marker for the Golgi apparatus. We also have developed techniques of gas-liquid chromatography and radiogas proportional counting to achieve capillary quality separation of partially methylated alditol acetates with simultaneous determination of radioactivity in the derivatives. Digestion of polymeric products by specific endo-glycanohydrolases to diagnostic oligosaccharides also reveal specific kinds of polysaccharides synthesized by the Golgi membranes. A combination of these techniques provides unequivocal determination of the linkage structure of specific polymers synthesized by the purified Golgi apparatus

  3. Modification of Bacterial Cellulose Biofilms with Xylan Polyelectrolytes.

    Science.gov (United States)

    Santos, Sara M; Carbajo, José M; Gómez, Nuria; Ladero, Miguel; Villar, Juan C

    2017-11-28

    The effect of the addition of two [4-butyltrimethylammonium]-xylan chloride polyelectrolytes (BTMAXs) on bacterial cellulose (BC) was evaluated. The first strategy was to add the polyelectrolytes to the culture medium together with a cell suspension of the bacterium. After one week of cultivation, the films were collected and purified. The second approach consisted of obtaining a purified and homogenized BC, to which the polyelectrolytes were added subsequently. The films were characterized in terms of tear and burst indexes, optical properties, surface free energy, static contact angle, Gurley porosity, SEM, X-ray diffraction and AFM. Although there are small differences in mechanical and optical properties between the nanocomposites and control films, the films obtained by BC synthesis in the presence of BTMAXs were remarkably less opaque, rougher, and had a much lower specular gloss. The surface free energy depends on the BTMAXs addition method. The crystallinity of the composites is lower than that of the control material, with a higher reduction of this parameter in the composites obtained by adding the BTMAXs to the culture medium. In view of these results, it can be concluded that BC-BTMAX composites are a promising new material, for example, for paper restoration.

  4. Modification of Bacterial Cellulose Biofilms with Xylan Polyelectrolytes

    Directory of Open Access Journals (Sweden)

    Sara M. Santos

    2017-11-01

    Full Text Available The effect of the addition of two [4-butyltrimethylammonium]-xylan chloride polyelectrolytes (BTMAXs on bacterial cellulose (BC was evaluated. The first strategy was to add the polyelectrolytes to the culture medium together with a cell suspension of the bacterium. After one week of cultivation, the films were collected and purified. The second approach consisted of obtaining a purified and homogenized BC, to which the polyelectrolytes were added subsequently. The films were characterized in terms of tear and burst indexes, optical properties, surface free energy, static contact angle, Gurley porosity, SEM, X-ray diffraction and AFM. Although there are small differences in mechanical and optical properties between the nanocomposites and control films, the films obtained by BC synthesis in the presence of BTMAXs were remarkably less opaque, rougher, and had a much lower specular gloss. The surface free energy depends on the BTMAXs addition method. The crystallinity of the composites is lower than that of the control material, with a higher reduction of this parameter in the composites obtained by adding the BTMAXs to the culture medium. In view of these results, it can be concluded that BC–BTMAX composites are a promising new material, for example, for paper restoration.

  5. Characterization and distribution of esterase activity in activated sludge

    NARCIS (Netherlands)

    Boczar, BA; Forney, LJ; Begley, WM; Larson, RJ; Federle, TW

    2001-01-01

    The location and activity of esterase enzymes in activated Sludge from three Municipal wastewater treatment plants were characterized using model Substrate, and denaturing and nondenaturing polyacrylamide gel electrophoresis (PAGE) Of particulate, freeze thaw (primarily periplasmic enzymes and those

  6. Novel recombinant ethyl ferulate esterase from Burkholderia multivorans

    CSIR Research Space (South Africa)

    Rashamuse, KJ

    2007-11-01

    Full Text Available Isolation and identification of bacterial isolates with specific ferulic acid (FA) esterase activity and cloning of a gene encoding activity. A micro-organism with ethyl ferulate hydrolysing (EFH) activity was isolated by culture enrichment...

  7. Pectin methyl esterase activity in apple and orange pulps

    International Nuclear Information System (INIS)

    Abdullaev, A.; Djumaev, B.B.; Djumaev, N.B.; Mukhidinov, Z.K.

    2008-01-01

    The results of pectin methyl esterase activity from apple, orange pulp and orange peel depending of ph and temperature are discussed. It's shown that the methyl esterase activity form apple and orange pulps higher in range of temperatures from +37...+60 d ig C . The analysis of dependence of its activity from ph has shown that in both case the enzyme activity increase with increase of ph

  8. Esterase activity as a novel parameter of spore germination in Bacillus anthracis

    International Nuclear Information System (INIS)

    Ferencko, Linda; Cote, Mindy A.; Rotman, Boris

    2004-01-01

    Spores of Bacillus anthracis were shown to produce esterase activity about 4 min after exposure to conventional germinants such as combinations of amino acids and purine ribosides. Neither amino acids nor ribosides alone induce germination and esterase activity. Expression of esterase activity was chloramphenicol resistant, and correlated with loss of spore refractivity, a traditional parameter of early germination. Based on these observations, we hypothesized that esterase activity could be used as a novel parameter for quantifying early events during spore germination. To test this hypothesis, we measured expression of esterase activity under a variety of germinating conditions. Using diacetyl fluorescein as fluorogenic substrate of esterases, we demonstrated that esterase activity was invariably induced whenever spores were triggered by known germinants. Moreover, D-alanine, an inhibitor of L-alanine-mediated germination, was found to significantly inhibit expression of esterase activity. In terms of molecular mechanisms, esterase expression could represent activation of proteases at the onset of spore germination

  9. Impact of the fouling mechanism on enzymatic depolymerization of xylan in different configurations of membrane reactors

    DEFF Research Database (Denmark)

    Mohd Sueb, Mohd Shafiq Bin; Luo, Jianquan; Meyer, Anne S.

    2017-01-01

    In order to maximize enzymatic xylan depolymerization while simultaneously purifying the resulting monosaccharide (xylose), different ultrafiltration (UF) membrane reactor configurations were evaluated. Initial results showed that the two hydrolytic enzymes required for complete depolymerization...... which hindered enzymatic attack in addition to fouling. Reaction with both enzymes followed by UF was found to be the optimal configuration, providing at least 40% higher xylan hydrolysis than the cascade configuration (involving sequential reaction with each of the enzymes separately......) and the simultaneous reaction-filtration with both enzymes, respectively. This study thus confirmed that the reactor configuration has a crucial impact on the performance of both the reaction and the separation process of xylose during enzymatic xylan degradation, and that the type of fouling mechanism varies...

  10. Designer biomass for next-generation biorefineries: leveraging recent insights into xylan structure and biosynthesis.

    Science.gov (United States)

    Smith, Peter J; Wang, Hsin-Tzu; York, William S; Peña, Maria J; Urbanowicz, Breeanna R

    2017-01-01

    Xylans are the most abundant noncellulosic polysaccharides in lignified secondary cell walls of woody dicots and in both primary and secondary cell walls of grasses. These polysaccharides, which comprise 20-35% of terrestrial biomass, present major challenges for the efficient microbial bioconversion of lignocellulosic feedstocks to fuels and other value-added products. Xylans play a significant role in the recalcitrance of biomass to degradation, and their bioconversion requires metabolic pathways that are distinct from those used to metabolize cellulose. In this review, we discuss the key differences in the structural features of xylans across diverse plant species, how these features affect their interactions with cellulose and lignin, and recent developments in understanding their biosynthesis. In particular, we focus on how the combined structural and biosynthetic knowledge can be used as a basis for biomass engineering aimed at developing crops that are better suited as feedstocks for the bioconversion industry.

  11. Efficient extraction of xylan from delignified corn stover using dimethyl sulfoxide

    Energy Technology Data Exchange (ETDEWEB)

    Rowley, John; Decker, Stephen R.; Michener, William; Black, Stuart

    2013-09-13

    Xylan can be extracted from biomass using either alkali (KOH or NaOH) or dimethyl sulfoxide (DMSO); however, DMSO extraction is the only method that produces a water-soluble xylan. In this study, DMSO extraction of corn stover was studied at different temperatures with the objective of finding a faster, more efficient extraction method. The temperature and time of extraction were compared followed by a basic structural analysis to ensure that no significant structural changes occurred under different temperatures. The resulting data showed that heating to 70 degrees C during extraction can give a yield comparable to room temperature extraction while reducing the extraction time by ~90 %. This method of heating was shown to be the most efficient method currently available and was shown to retain the important structural characteristics of xylan extracted with DMSO at room temperature.

  12. Properties of plasticized composite films prepared from nanofibrillated cellulose and birch wood xylan

    DEFF Research Database (Denmark)

    Hansen, Natanya Majbritt Louie; Blomfeldt, Thomas O. J.; Hedenqvist, Mikael S.

    2012-01-01

    was combined with nanofibrillated cellulose (NFC) and films were cast with and without glycerol, sorbitol or methoxypolyethylene glycol (MPEG) as plasticizers. Microscopy revealed some NFC agglomeration in the composite films as well as a layered nanocellulose structure. Equilibrium moisture content...... in plasticized films increased with glycerol content but was independent of xylan:NFC ratio in unplasticized films. Sorbitol- and MPEG-plasticized films showed equilibrium moisture contents of approximately 10 wt% independent of plasticizer content. Tensile testing revealed increases in tensile strength...... with increased NFC content in the xylan:NFC composition range from 50:50 to 80:20 and plasticizer addition generally provided less brittle films. The oxygen permeability of unplasticized xylan-NFC films fell into a range which was similar to that for previously measured pure NFC films and was statistically...

  13. In Vitro Antioxidant, Anticoagulant and Antimicrobial Activity and in Inhibition of Cancer Cell Proliferation by Xylan Extracted from Corn Cobs

    Science.gov (United States)

    Melo-Silveira, Raniere Fagundes; Fidelis, Gabriel Pereira; Costa, Mariana Santana Santos Pereira; Telles, Cinthia Beatrice Silva; Dantas-Santos, Nednaldo; de Oliveira Elias, Susana; Ribeiro, Vanessa Bley; Barth, Afonso Luis; Macedo, Alexandre José; Leite, Edda Lisboa; Rocha, Hugo Alexandre Oliveira

    2012-01-01

    Xylan is one of most abundant polymer after cellulose. However, its potential has yet to be completely recognized. Corn cobs contain a considerable reservoir of xylan. The aim of this work was to study some of the biological activities of xylan obtained from corn cobs after alkaline extraction enhanced by ultrasonication. Physical chemistry and infrared analyses showed 130 kDa heteroxylan containing mainly xylose:arabinose: galactose:glucose (5.0:1.5:2.0:1.2). Xylan obtained exhibited total antioxidant activity corresponding to 48.5 mg of ascorbic acid equivalent/g of xylan. Furthermore, xylan displayed high ferric chelating activity (70%) at 2 mg/mL. Xylan also showed anticoagulant activity in aPTT test. In antimicrobial assay, the polysaccharide significantly inhibited bacterial growth of Klebsiella pneumoniae. In a test with normal and tumor human cells, after 72 h, only HeLa tumor cell proliferation was inhibited (p < 0.05) in a dose-dependent manner by xylan, reaching saturation at around 2 mg/mL, whereas 3T3 normal cell proliferation was not affected. The results suggest that it has potential clinical applications as antioxidant, anticoagulant, antimicrobial and antiproliferative compounds. PMID:22312261

  14. Biomimetic Inks Based on Cellulose Nanofibrils and Cross-Linkable Xylans for 3D Printing.

    Science.gov (United States)

    Markstedt, Kajsa; Escalante, Alfredo; Toriz, Guillermo; Gatenholm, Paul

    2017-11-22

    This paper presents a sustainable all-wood-based ink which can be used for 3D printing of constructs for a large variety of applications such as clothes, furniture, electronics, and health care products with a customized design and versatile gel properties. The 3D printing technologies where the material is dispensed in the form of liquids, so called inks, have proven suitable for 3D printing dispersions of cellulose nanofibrils (CNFs) because of their unique shear thinning properties. In this study, novel inks were developed with a biomimetic approach where the structural properties of cellulose and the cross-linking function of hemicelluloses that are found in the plant cell wall were utilized. The CNF was mixed with xylan, a hemicellulose extracted from spruce, to introduce cross-linking properties which are essential for the final stability of the printed ink. For xylan to be cross-linkable, it was functionalized with tyramine at different degrees. Evaluation of different ink compositions by rheology measurements and 3D printing tests showed that the degree of tyramine substitution and the ratio of CNFs to xylan-tyramine in the prepared inks influenced the printability and cross-linking density. Both two-layered gridded structures and more complex 3D constructs were printed. Similarly to conventional composites, the interactions between the components and their miscibility are important for the stability of the printed and cross-linked ink. Thus, the influence of tyramine on the adsorption of xylan to cellulose was studied with a quartz crystal microbalance to verify that the functionalization had little influence on xylan's adsorption to cellulose. Utilizing xylan-tyramine in the CNF dispersions resulted in all-wood-based inks which after 3D printing can be cross-linked to form freestanding gels while at the same time, the excellent printing properties of CNFs remain intact.

  15. Imidazole, a New Tunable Reagent for Producing Nanocellulose, Part I: Xylan-Coated CNCs and CNFs

    Directory of Open Access Journals (Sweden)

    Jia Mao

    2017-09-01

    Full Text Available Imidazole is reported to be an effective reactant for the production of nanocellulose from hardwood pulp. The morphologies and surface properties of the nanocellulose can be simply tailored according to the water content in the imidazole system: with pure imidazole, cellulose nanofibrils (CNFs in a yield of 10 wt % can be produced. With 25 wt % of water in imidazole, cellulose nanocrystals (CNCs are obtained in 20 wt % yield. Both nanocelluloses exhibit crystallinity indices in the order of 70%. Interestingly, they retain the original xylan from the pulp with ca. 9–10 wt % of residual xylan content.

  16. Arabidopsis GUX Proteins Are Glucuronyltransferases Responsible for the Addition of Glucuronic Acid Side Chains onto Xylan

    Science.gov (United States)

    Xylan, the second most abundant cell wall polysaccharide, is composed of a linear backbone of β-(1,4)-linked xylosyl residues that are often substituted with sugar side chains, such as glucuronic acid (GlcA) and methylglucuronic acid (MeGlcA). It has recently been shown that muta...

  17. Properties of polyvinyl alcohol/xylan composite films with citric acid.

    Science.gov (United States)

    Wang, Shuaiyang; Ren, Junli; Li, Weiying; Sun, Runcang; Liu, Shijie

    2014-03-15

    Composite films of xylan and polyvinyl alcohol were produced with citric acid as a new plasticizer or a cross-linking agent. The effects of citric acid content and polyvinyl alcohol/xylan weight ratio on the mechanical properties, thermal stability, solubility, degree of swelling and water vapor permeability of the composite films were investigated. The intermolecular interactions and morphology of composite films were characterized by FTIR spectroscopy and SEM. The results indicated that polyvinyl alcohol/xylan composite films had good compatibility. With an increase in citric acid content from 10% to 50%, the tensile strength reduced from 35.1 to 11.6 MPa. However, the elongation at break increased sharply from 15.1% to 249.5%. The values of water vapor permeability ranged from 2.35 to 2.95 × 10(-7)g/(mm(2)h). Interactions between xylan and polyvinyl alcohol in the presence of citric acid become stronger, which were caused by hydrogen bond and ester bond formation among the components during film forming. Copyright © 2013. Published by Elsevier Ltd.

  18. In situ enzyme aided adsorption of soluble xylan biopolymers onto cellulosic material.

    Science.gov (United States)

    Chimphango, Annie F A; Görgens, J F; van Zyl, W H

    2016-06-05

    The functional properties of cellulose fibers can be modified by adsorption of xylan biopolymers. The adsorption is improved when the degree of biopolymers substitution with arabinose and 4-O-methyl-glucuronic acid (MeGlcA) side groups, is reduced. α-l-Arabinofuranosidase (AbfB) and α-d-glucuronidase (AguA) enzymes were applied for side group removal, to increase adsorption of xylan from sugarcane (Saccharum officinarum L) bagasse (BH), bamboo (Bambusa balcooa) (BM), Pinus patula (PP) and Eucalyptus grandis (EH) onto cotton lint. The AguA treatment increased the adsorption of all xylans by up to 334%, whereas, the AbfB increased the adsorption of the BM and PP by 31% and 44%, respectively. A combination of AguA and AbfB treatment increased the adsorption, but to a lesser extent than achieved with AguA treatment. This indicated that the removal of the glucuronic acid side groups provided the most significant increase in xylan adsorption to cellulose, in particular through enzymatic treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Xylan hydrolysis in Populus trichocarpa × P. deltoides and model substrates during hydrothermal pretreatment.

    Science.gov (United States)

    Trajano, Heather L; Pattathil, Sivakumar; Tomkins, Bruce A; Tschaplinski, Timothy J; Hahn, Michael G; Van Berkel, Gary J; Wyman, Charles E

    2015-03-01

    Previous studies defined easy and difficult to hydrolyze fractions of hemicellulose that may result from bonds among cellulose, hemicellulose, and lignin. To understand how such bonds affect hydrolysis, Populus trichocarpa × Populus deltoides, holocellulose isolated from P. trichocarpa × P. deltoides and birchwood xylan were subjected to hydrothermal flow-through pretreatment. Samples were characterized by glycome profiling, HPLC, and UPLC-MS. Glycome profiling revealed steady fragmentation and removal of glycans from solids during hydrolysis. The extent of polysaccharide fragmentation, hydrolysis rate, and total xylose yield were lowest for P. trichocarpa × P. deltoides and greatest for birchwood xylan. Comparison of results from P. trichocarpa × P. deltoides and holocellulose suggested that lignin-carbohydrate complexes reduce hydrolysis rates and limit release of large xylooligomers. Smaller differences between results with holocellulose and birchwood xylan suggest xylan-cellulose hydrogen bonds limited hydrolysis, but to a lesser extent. These findings imply cell wall structure strongly influences hydrolysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Analysis of acetylation stoichiometry suggests that SIRT3 repairs nonenzymatic acetylation lesions

    DEFF Research Database (Denmark)

    Weinert, Brian T; Moustafa, Tarek; Iesmantavicius, Vytautas

    2015-01-01

    or suppresses acetylation. Using quantitative mass spectrometry, we measured acetylation stoichiometry in mouse liver tissue and found that SIRT3 suppressed acetylation to a very low stoichiometry at its target sites. By examining acetylation changes in the liver, heart, brain, and brown adipose tissue...... of fasted mice, we found that SIRT3-targeted sites were mostly unaffected by fasting, a dietary manipulation that is thought to regulate metabolism through SIRT3-dependent deacetylation. Globally increased mitochondrial acetylation in fasted liver tissue, higher stoichiometry at mitochondrial acetylation...... functions as a protein repair factor that removes acetylation lesions from lysine residues....

  1. Acetyl-Phosphate Is a Critical Determinant of Lysine Acetylation in E. coli

    DEFF Research Database (Denmark)

    Weinert, Brian T; Iesmantavicius, Vytautas; Wagner, Sebastian A

    2013-01-01

    Lysine acetylation is a frequently occurring posttranslational modification in bacteria; however, little is known about its origin and regulation. Using the model bacterium Escherichia coli (E. coli), we found that most acetylation occurred at a low level and accumulated in growth-arrested cells...... acetylate lysine residues in vitro and that AcP levels are correlated with acetylation levels in vivo, suggesting that AcP may acetylate proteins nonenzymatically in cells. These results uncover a critical role for AcP in bacterial acetylation and indicate that most acetylation in E. coli occurs at a low...

  2. 21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Esterase-lipase derived from Mucor miehei. 173.140... HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme derived from Mucor miehei var. Cooney et Emerson by...

  3. Insight into glycoside hydrolases for debranched xylan degradation from extremely thermophilic bacterium Caldicellulosiruptor lactoaceticus.

    Directory of Open Access Journals (Sweden)

    Xiaojing Jia

    Full Text Available Caldicellulosiruptor lactoaceticus 6A, an anaerobic and extremely thermophilic bacterium, uses natural xylan as carbon source. The encoded genes of C. lactoaceticus 6A for glycoside hydrolase (GH provide a platform for xylan degradation. The GH family 10 xylanase (Xyn10A and GH67 α-glucuronidase (Agu67A from C. lactoaceticus 6A were heterologously expressed, purified and characterized. Both Xyn10A and Agu67A are predicted as intracellular enzymes as no signal peptides identified. Xyn10A and Agu67A had molecular weight of 47.0 kDa and 80.0 kDa respectively as determined by SDS-PAGE, while both appeared as homodimer when analyzed by gel filtration. Xyn10A displayed the highest activity at 80 °C and pH 6.5, as 75 °C and pH 6.5 for Agu67A. Xyn10A had good stability at 75 °C, 80 °C, and pH 4.5-8.5, respectively, and was sensitive to various metal ions and reagents. Xyn10A possessed hydrolytic activity towards xylo-oligosaccharides (XOs and beechwood xylan. At optimum conditions, the specific activity of Xyn10A was 44.6 IU/mg with beechwood xylan as substrate, and liberated branched XOs, xylobiose, and xylose. Agu67A was active on branched XOs with methyl-glucuronic acids (MeGlcA sub-chains, and primarily generated XOs equivalents and MeGlcA. The specific activity of Agu67A was 1.3 IU/mg with aldobiouronic acid as substrate. The synergistic action of Xyn10A and Agu67A was observed with MeGlcA branched XOs and xylan as substrates, both backbone and branched chain of substrates were degraded, and liberated xylose, xylobiose, and MeGlcA. The synergism of Xyn10A and Agu67A provided not only a thermophilic method for natural xylan degradation, but also insight into the mechanisms for xylan utilization of C. lactoaceticus.

  4. A Highly Efficient Xylan-Utilization System in Aspergillus niger An76: A Functional-Proteomics Study

    Directory of Open Access Journals (Sweden)

    Weili Gong

    2018-03-01

    Full Text Available Xylan constituted with β-1,4-D-xylose linked backbone and diverse substituted side-chains is the most abundant hemicellulose component of biomass, which can be completely and rapidly degraded into fermentable sugars by Aspergillus niger. This is of great value for obtaining renewable biofuels and biochemicals. To clarify the underlying mechanisms associated with highly efficient xylan degradation, assimilation, and metabolism by A. niger, we utilized functional proteomics to analyze the secreted proteins, sugar transporters, and intracellular proteins of A. niger An76 grown on xylan-based substrates. Results demonstrated that the complete xylanolytic enzyme system required for xylan degradation and composed of diverse isozymes was secreted in a sequential order. Xylan-backbone-degrading enzymes were preferentially induced by xylose or other soluble sugars, which efficiently produced large amounts of xylooligosaccharides (XOS and xylose; however, XOS was more efficient than xylose in triggering the expression of the key transcription activator XlnR, resulting in higher xylanase activity and shortening xylanase-production time. Moreover, the substituted XOS was responsible for improving the abundance of side-chain-degrading enzymes, specific transporters, and key reductases and dehydrogenases in the pentose catabolic pathway. Our findings indicated that industries might be able to improve the species and concentrations of xylan-degrading enzymes and shorten fermentation time by adding abundant intermediate products of natural xylan (XOS to cultures of filamentous fungi.

  5. A Highly Efficient Xylan-Utilization System in Aspergillus niger An76: A Functional-Proteomics Study

    Science.gov (United States)

    Gong, Weili; Dai, Lin; Zhang, Huaiqiang; Zhang, Lili; Wang, Lushan

    2018-01-01

    Xylan constituted with β-1,4-D-xylose linked backbone and diverse substituted side-chains is the most abundant hemicellulose component of biomass, which can be completely and rapidly degraded into fermentable sugars by Aspergillus niger. This is of great value for obtaining renewable biofuels and biochemicals. To clarify the underlying mechanisms associated with highly efficient xylan degradation, assimilation, and metabolism by A. niger, we utilized functional proteomics to analyze the secreted proteins, sugar transporters, and intracellular proteins of A. niger An76 grown on xylan-based substrates. Results demonstrated that the complete xylanolytic enzyme system required for xylan degradation and composed of diverse isozymes was secreted in a sequential order. Xylan-backbone-degrading enzymes were preferentially induced by xylose or other soluble sugars, which efficiently produced large amounts of xylooligosaccharides (XOS) and xylose; however, XOS was more efficient than xylose in triggering the expression of the key transcription activator XlnR, resulting in higher xylanase activity and shortening xylanase-production time. Moreover, the substituted XOS was responsible for improving the abundance of side-chain-degrading enzymes, specific transporters, and key reductases and dehydrogenases in the pentose catabolic pathway. Our findings indicated that industries might be able to improve the species and concentrations of xylan-degrading enzymes and shorten fermentation time by adding abundant intermediate products of natural xylan (XOS) to cultures of filamentous fungi. PMID:29623069

  6. A Highly Efficient Xylan-Utilization System in Aspergillus niger An76: A Functional-Proteomics Study.

    Science.gov (United States)

    Gong, Weili; Dai, Lin; Zhang, Huaiqiang; Zhang, Lili; Wang, Lushan

    2018-01-01

    Xylan constituted with β-1,4-D-xylose linked backbone and diverse substituted side-chains is the most abundant hemicellulose component of biomass, which can be completely and rapidly degraded into fermentable sugars by Aspergillus niger . This is of great value for obtaining renewable biofuels and biochemicals. To clarify the underlying mechanisms associated with highly efficient xylan degradation, assimilation, and metabolism by A. niger , we utilized functional proteomics to analyze the secreted proteins, sugar transporters, and intracellular proteins of A. niger An76 grown on xylan-based substrates. Results demonstrated that the complete xylanolytic enzyme system required for xylan degradation and composed of diverse isozymes was secreted in a sequential order. Xylan-backbone-degrading enzymes were preferentially induced by xylose or other soluble sugars, which efficiently produced large amounts of xylooligosaccharides (XOS) and xylose; however, XOS was more efficient than xylose in triggering the expression of the key transcription activator XlnR, resulting in higher xylanase activity and shortening xylanase-production time. Moreover, the substituted XOS was responsible for improving the abundance of side-chain-degrading enzymes, specific transporters, and key reductases and dehydrogenases in the pentose catabolic pathway. Our findings indicated that industries might be able to improve the species and concentrations of xylan-degrading enzymes and shorten fermentation time by adding abundant intermediate products of natural xylan (XOS) to cultures of filamentous fungi.

  7. Identification of an algal xylan synthase indicates that there is functional orthology between algal and plant cell wall biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Jensen, Jacob Kruger [Michigan State Univ., East Lansing, MI (United States). Dept. of Plant Biology; Michigan State Univ., East Lansing, MI (United States). DOE Great Lakes Bioenergy Research Center; Busse-Wicher, Marta [Univ. of Cambridge (United Kingdom). Dept. of Biochemistry; Poulsen, Christian Peter [Carlsberg Research Lab., Copenhagen (Denmark); Fangel, Jonatan Ulrik [Carlsberg Research Lab., Copenhagen (Denmark); Smith, Peter James [Univ. of Georgia, Athens, GA (United States). Complex Carbohydrate Research Center; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC); Yang, Jeong-Yeh [Univ. of Georgia, Athens, GA (United States). Complex Carbohydrate Research Center; Peña, Maria-Jesus [Univ. of Georgia, Athens, GA (United States). Complex Carbohydrate Research Center; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC); Dinesen, Malene Hessellund [Carlsberg Research Lab., Copenhagen (Denmark); Martens, Helle Juel [Univ. of Copenhagen (Denmark). Dept. of Plant and Environmental Sciences; Melkonian, Michael [Univ. zu Koln (Germany). Botanical Inst., Dept. of Biological Sciences; Wong, Gane Ka-Shu [BGI-Shenzhen, Shenzhen, Guangdong (China); Moremen, Kelley W. [Univ. of Georgia, Athens, GA (United States). Complex Carbohydrate Research Center; Wilkerson, Curtis Gene [Michigan State Univ., East Lansing, MI (United States). Dept. of Plant Biology; Michigan State Univ., East Lansing, MI (United States). DOE Great Lakes Bioenergy Research Center; Michigan State Univ., East Lansing, MI (United States). Dept. of Biochemistry and Molecular Biology; Scheller, Henrik Vibe [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Environmental Genomics and Systems Biology Division; Dupree, Paul [Univ. of Cambridge (United Kingdom). Dept. of Biochemistry; Ulvskov, Peter [Univ. of Georgia, Athens, GA (United States). Complex Carbohydrate Research Center; Urbanowicz, Breeanna Rae [Univ. of Georgia, Athens, GA (United States). Complex Carbohydrate Research Center; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC); Harholt, Jesper [Carlsberg Research Lab., Copenhagen (Denmark)

    2018-02-20

    Insights into the evolution of plant cell walls have important implications for comprehending these diverse and abundant biological structures. In order to understand the evolving structure-function relationships of the plant cell wall, it is imperative to trace the origin of its different components. The present study is focused on plant 1,4-β-xylan, tracing its evolutionary origin by genome and transcriptome mining followed by phylogenetic analysis, utilizing a large selection of plants and algae. It substantiates the findings by heterologous expression and biochemical characterization of a charophyte alga xylan synthase. Of the 12 known gene classes involved in 1,4-β-xylan formation, XYS1/IRX10 in plants, IRX7, IRX8, IRX9, IRX14 and GUX occurred for the first time in charophyte algae. An XYS1/IRX10 ortholog from Klebsormidium flaccidum, designated K. flaccidumXYLAN SYNTHASE-1 (KfXYS1), possesses 1,4-β-xylan synthase activity, and 1,4-β-xylan occurs in the K. flaccidum cell wall. Finally, these data suggest that plant 1,4-β-xylan originated in charophytes and shed light on the origin of one of the key cell wall innovations to occur in charophyte algae, facilitating terrestrialization and emergence of polysaccharide-based plant cell walls.

  8. Evidence for a blockwise distribution of acetyl groups onto homogalacturonans from a commercial sugar beet (Beta vulgaris) pectin.

    Science.gov (United States)

    Ralet, Marie-Christine; Crépeau, Marie-Jeanne; Bonnin, Estelle

    2008-06-01

    Commercial acid-extracted sugar beet pectin was extensively hydrolysed using an endo-polygalacturonase (AnPGI from Aspergillus niger or AnPGII from A. niger or FmPG from Fusarium moniliforme) in combination with Aspergillus aculeatus pectin methyl-esterase (AaPME). The homogalacturonan-derived oligogalacturonates released were quantified by high-performance anion-exchange chromatography and their structure determined by mass spectrometry. The different endo-polygalacturonases exhibited variable tolerance towards acetyl groups. AnPGI was the most active and FmPG the less. A hypothetical homogalacturonan was constructed using the AnPGI-recovered oligogalacturonates as building blocks and the validity of the model was checked taking into account FmPG observed requirements and hydrolysis products. A blockwise repartition of the acetyl groups onto sugar beet pectin homogalacturonan is proposed.

  9. Activity of pectin methyl esterase during blanching of peaches

    NARCIS (Netherlands)

    Tijskens, L.M.M.; Rodis, P.S.; Hertog, M.L.A.T.M.; Proxenia, N.; Dijk, van C.

    1999-01-01

    The activity of pectin methyl esterase (PE) in peaches during blanching treatments was modelled and analyzed. It was postulated that the enzyme exists in two configurations, one bound and one soluble. The bound configuration can be converted into the soluble configuration. These two configurations

  10. A feruloyl esterase derived from a leachate metagenome library

    CSIR Research Space (South Africa)

    Rashamuse, K

    2012-01-01

    Full Text Available A feruloyl esterase encoding gene (designated fae6), derived from a leachate metagenomic library, was cloned and the nucleotide sequence of the insert DNA determined. Translational analysis revealed that fae6 consists of a 515 amino acid polypeptide...

  11. New cholesterol esterase inhibitors based on rhodanine and thiazolidinedione scaffolds

    DEFF Research Database (Denmark)

    Heng, Sabrina; Tieu, William; Hautmann, Stephanie

    2011-01-01

    We present a new class of inhibitors of pancreatic cholesterol esterase (CEase) based on 'priviledged' 5-benzylidenerhodanine and 5-benzylidene-2,4-thiazolidinedione structural scaffolds. The lead structures (5-benzylidenerhodanine 4a and 5-benzylidene-2,4-thiazolidinedione 4b) were identified in...

  12. Esterase polymorphism marking cultivars of Manihot esculenta, Crantz

    Directory of Open Access Journals (Sweden)

    Adriana Gazoli Resende

    2004-07-01

    Full Text Available Esterase isozymes were used to detected substrate-preference polymorphism in twenty cultivars of Manihot esculenta, and to show cultivar-specific variation of this species. A relatively complex extraction solution of proteins from leaves was needed to show a larger number of esterase isozymes. Similarity between cultivars from six groups ranged from 51 to 96%. The cultivars identified by the same name seemed to be biochemically different regarding esterase isozymes. Esterase isozyme electrophoretic patterns could, therefore, be used to discriminate the cultivars identified by the same name, and to monitor the vegetative propagation of cultivars maintained in the germplasm collection. In breeding strategies, isoesterase analysis could be used to avoid intercrossing between the similar genotypes.Isoenzimas esterases foram usadas no presente estudo, para detectar polimorfismos específicos para diferentes substratos em vinte cultivares de Manihot esculenta, e para mostrar variações específicas de cultivares nesta espécie. Os diferentes cultivares de M. esculenta tem sido mantidos na coleção de germoplasma do Departamento de Agronomia da Universidade Estadual de Maringá (Maringá, PR, e foram provenientes de cultivares tradicionais coletados nas regiões sudoeste e noroeste do Estado. Foi necessário a utilização de uma solução de extração de proteínas relativamente mais complexa, para evidenciar um maior número de isoenzimas esterases. A similaridade entre os cultivares variou de 51 a 96%. Cultivares identificados pelo mesmo nome parecem ser bioquimicamente diferentes para as isoenzimas esterases. Os padrões eletroforéticos das isoesterases podem, portanto, serem usados para discriminar os cultivares que são identificados pelo mesmo nome, e para monitorar a propagação vegetativa dos cultivares mantidos na coleção de germoplasma. A análise das isoesterases pode também ser usada para evitar cruzamentos entre genótipos mais

  13. Esterase activity able to hydrolyze dietary antioxidant hydroxycinnamates is distributed along the intestine of mammals

    DEFF Research Database (Denmark)

    Andreasen, Mette Findal; Kroon, P A; Williamson, G

    2001-01-01

    and may contribute to the beneficial effects derived from consumption of cereal bran. However, these compounds are ester linked to the main polymers in the plant cell wall and cannot be absorbed in this complex form. The present work shows that esterases with activity toward esters of the major dietary...... hydroxycinnamates are distributed throughout the intestinal tract of mammals. In rats, the cinnamoyl esterase activity in the small intestine is derived mainly from the mucosa, whereas in the large intestine the esterase activity was found predominantly in the luminal microflora. Mucosa cell-free extracts obtained...... from human duodenum, jejunum, and ileum efficiently hydrolyzed various hydroxycinnamoyl esters, providing the first evidence of human cinnamoyl esterase(s). This study first demonstrates the release by human colonic esterase(s) (mostly of microbial origin) of sinapic acid and p-coumaric acid from rye...

  14. Conversion of xylan by recyclable spores of Bacillus subtilis displaying thermophilic enzymes.

    Science.gov (United States)

    Mattossovich, Rosanna; Iacono, Roberta; Cangiano, Giuseppina; Cobucci-Ponzano, Beatrice; Isticato, Rachele; Moracci, Marco; Ricca, Ezio

    2017-11-28

    The Bacillus subtilis spore has long been used to display antigens and enzymes. Spore display can be accomplished by a recombinant and a non-recombinant approach, with the latter proved more efficient than the recombinant one. We used the non-recombinant approach to independently adsorb two thermophilic enzymes, GH10-XA, an endo-1,4-β-xylanase (EC 3.2.1.8) from Alicyclobacillus acidocaldarius, and GH3-XT, a β-xylosidase (EC 3.2.1.37) from Thermotoga thermarum. These enzymes catalyze, respectively, the endohydrolysis of (1-4)-β-D-xylosidic linkages of xylans and the hydrolysis of (1-4)-β-D-xylans to remove successive D-xylose residues from the non-reducing termini. We report that both purified enzymes were independently adsorbed on purified spores of B. subtilis. The adsorption was tight and both enzymes retained part of their specific activity. When spores displaying either GH10-XA or GH3-XT were mixed together, xylan was hydrolysed more efficiently than by a mixture of the two free, not spore-adsorbed, enzymes. The high total activity of the spore-bound enzymes is most likely due to a stabilization of the enzymes that, upon adsorption on the spore, remained active at the reaction conditions for longer than the free enzymes. Spore-adsorbed enzymes, collected after the two-step reaction and incubated with fresh substrate, were still active and able to continue xylan degradation. The recycling of the mixed spore-bound enzymes allowed a strong increase of xylan degradation. Our results indicate that the two-step degradation of xylans can be accomplished by mixing spores displaying either one of two required enzymes. The two-step process occurs more efficiently than with the two un-adsorbed, free enzymes and adsorbed spores can be reused for at least one other reaction round. The efficiency of the process, the reusability of the adsorbed enzymes, and the well documented robustness of spores of B. subtilis indicate the spore as a suitable platform to display enzymes

  15. In vitro comparison of rat and chicken brain neurotoxic esterase

    International Nuclear Information System (INIS)

    Novak, R.; Padilla, S.

    1986-01-01

    A systematic comparison was undertaken to characterize neurotoxic esterase (NTE) from rat and chicken brain in terms of inhibitor sensitivities, pH optima, and molecular weights. Paraoxon titration of phenyl valerate (PV)-hydrolyzing carboxylesterases showed that rat esterases were more sensitive than chicken to paraoxon inhibition at concentrations less than or equal to microM and superimposable with chicken esterases at concentrations of 2.5-1000 microM. Mipafox titration of the paraoxon-resistant esterases at a fixed paraoxon concentration of 100 microM (mipafox concentration: 0-1000 microM) resulted in a mipafox I50 of 7.3 microM for chicken brain NTE and 11.6 microM for rat brain NTE. NTE (i.e., paraoxon-resistant, mipafox-sensitive esterase activity) comprised 80% of chicken and 60% of rat brain paraoxon-resistant activity with the specific activity of chicken brain NTE approximately twice that of rat brain NTE. The pH maxima for NTE from both species was similar showing broad, slightly alkaline optima from pH 7.9 to 8.6. [ 3 H]Diisopropyl phosphorofluoridate (DFP)-labeled NTE from the brains of both species had an apparent mol wt of 160,000 measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In conclusion, NTE from both species was very similar, with the mipafox I50 for rat NTE within the range of reported values for chicken and human NTE, and the inhibitor parameters of the chicken NTE assay were applicable for the rat NTE assay

  16. Simultaneous catalytic conversion of cellulose and corncob xylan under temperature programming for enhanced sorbitol and xylitol production.

    Science.gov (United States)

    Ribeiro, Lucília Sousa; Órfão, José J de Melo; Pereira, Manuel Fernando Ribeiro

    2017-11-01

    Sorbitol and xylitol yields can be improved by converting cellulose and xylan simultaneously, due to a synergetic effect between both substrates. Furthermore, both yields can be greatly enhanced by simply adjusting the reaction conditions regarding the optimum for the production of each product, since xylitol (from xylan) and sorbitol (from cellulose) yields are maximized when the reaction is carried out at 170 and 205°C, respectively. Therefore, the combination of a simultaneous conversion of cellulose and xylan with a two-step temperature approach, which consists in the variation of the reaction temperature from 170 to 205°C after 2h, showed to be a good strategy for maximizing the production of sorbitol and xylitol directly from mixture of cellulose and xylan. Using this new and environmentally friendly approach, yields of sorbitol and xylitol of 75 and 77%, respectively, were obtained after 6h of reaction. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. A Highly Efficient Xylan-Utilization System in Aspergillus niger An76: A Functional-Proteomics Study

    OpenAIRE

    Weili Gong; Lin Dai; Huaiqiang Zhang; Lili Zhang; Lushan Wang; Lushan Wang

    2018-01-01

    Xylan constituted with β-1,4-D-xylose linked backbone and diverse substituted side-chains is the most abundant hemicellulose component of biomass, which can be completely and rapidly degraded into fermentable sugars by Aspergillus niger. This is of great value for obtaining renewable biofuels and biochemicals. To clarify the underlying mechanisms associated with highly efficient xylan degradation, assimilation, and metabolism by A. niger, we utilized functional proteomics to analyze the secre...

  18. Biochemical and structural insights into xylan utilization by the thermophilic bacterium Caldanaerobius polysaccharolyticus.

    Science.gov (United States)

    Han, Yejun; Agarwal, Vinayak; Dodd, Dylan; Kim, Jason; Bae, Brian; Mackie, Roderick I; Nair, Satish K; Cann, Isaac K O

    2012-10-12

    Hemicellulose is the next most abundant plant cell wall component after cellulose. The abundance of hemicellulose such as xylan suggests that their hydrolysis and conversion to biofuels can improve the economics of bioenergy production. In an effort to understand xylan hydrolysis at high temperatures, we sequenced the genome of the thermophilic bacterium Caldanaerobius polysaccharolyticus. Analysis of the partial genome sequence revealed a gene cluster that contained both hydrolytic enzymes and also enzymes key to the pentose-phosphate pathway. The hydrolytic enzymes in the gene cluster were demonstrated to convert products from a large endoxylanase (Xyn10A) predicted to anchor to the surface of the bacterium. We further use structural and calorimetric studies to demonstrate that the end products of Xyn10A hydrolysis of xylan are recognized and bound by XBP1, a putative solute-binding protein, likely for transport into the cell. The XBP1 protein showed preference for xylo-oligosaccharides as follows: xylotriose > xylobiose > xylotetraose. To elucidate the structural basis for the oligosaccharide preference, we solved the co-crystal structure of XBP1 complexed with xylotriose to a 1.8-Å resolution. Analysis of the biochemical data in the context of the co-crystal structure reveals the molecular underpinnings of oligosaccharide length specificity.

  19. A molecular modeling approach to understand the structure and conformation relationship of (GlcpA)Xylan.

    Science.gov (United States)

    Guo, Qingbin; Kang, Ji; Wu, Yan; Cui, Steve W; Hu, Xinzhong; Yada, Rickey Y

    2015-12-10

    The structure and conformation relationships of a heteropolysaccharide (GlcpA)Xylan in terms of various molecular weights, Xylp/GlcpA ratio and the distribution of GlcpA along xylan chain were investigated using computer modeling. The adiabatic contour maps of xylobiose, XylpXylp(GlcpA) and (GlcpA)XylpXylp(GlcpA) indicated that the insertion of the side group (GlcpA) influenced the accessible conformational space of xylobiose molecule. RIS-Metropolis Monte Carlo method indicated that insertion of GlcpA side chain induced a lowering effect of the calculated chain extension at low GlcpA:Xylp ratio (GlcpA:Xylp = 1:3). The chain, however, became extended when the ratio of GlcpA:Xylp above 2/3. It was also shown that the spatial extension of the polymer chains was dependent on the distribution of side chain: the random distribution demonstrated the most flexible structure compared to block and alternative distribution. The present studies provide a unique insight into the dependence of both side chain ratio and distribution on the stiffness and flexibility of various (GlcpA)Xylan molecules. Copyright © 2015. Published by Elsevier Ltd.

  20. Accumulation of Peptidoglycan O-Acetylation Leads to Altered Cell Wall Biochemistry and Negatively Impacts Pathogenesis Factors of Campylobacter jejuni.

    Science.gov (United States)

    Ha, Reuben; Frirdich, Emilisa; Sychantha, David; Biboy, Jacob; Taveirne, Michael E; Johnson, Jeremiah G; DiRita, Victor J; Vollmer, Waldemar; Clarke, Anthony J; Gaynor, Erin C

    2016-10-21

    Campylobacter jejuni is a leading cause of bacterial gastroenteritis in the developed world. Despite its prevalence, its mechanisms of pathogenesis are poorly understood. Peptidoglycan (PG) is important for helical shape, colonization, and host-pathogen interactions in C. jejuni Therefore, changes in PG greatly impact the physiology of this organism. O-acetylation of peptidoglycan (OAP) is a bacterial phenomenon proposed to be important for proper cell growth, characterized by acetylation of the C6 hydroxyl group of N-acetylmuramic acid in the PG glycan backbone. The OAP gene cluster consists of a PG O-acetyltransferase A (patA) for translocation of acetate into the periplasm, a PG O-acetyltransferase B (patB) for O-acetylation, and an O-acetylpeptidoglycan esterase (ape1) for de-O-acetylation. In this study, reduced OAP in ΔpatA and ΔpatB had minimal impact on C. jejuni growth and fitness under the conditions tested. However, accumulation of OAP in Δape1 resulted in marked differences in PG biochemistry, including O-acetylation, anhydromuropeptide levels, and changes not expected to result directly from Ape1 activity. This suggests that OAP may be a form of substrate level regulation in PG biosynthesis. Ape1 acetylesterase activity was confirmed in vitro using p-nitrophenyl acetate and O-acetylated PG as substrates. In addition, Δape1 exhibited defects in pathogenesis-associated phenotypes, including cell shape, motility, biofilm formation, cell surface hydrophobicity, and sodium deoxycholate sensitivity. Δape1 was also impaired for chick colonization and adhesion, invasion, intracellular survival, and induction of IL-8 production in INT407 cells in vitro The importance of Ape1 in C. jejuni biology makes it a good candidate as an antimicrobial target. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Engineering of plants with improved properties as biofuels feedstocks by vessel-specific complementation of xylan biosynthesis mutants

    Directory of Open Access Journals (Sweden)

    Petersen Pia Damm

    2012-11-01

    Full Text Available Abstract Background Cost-efficient generation of second-generation biofuels requires plant biomass that can easily be degraded into sugars and further fermented into fuels. However, lignocellulosic biomass is inherently recalcitrant toward deconstruction technologies due to the abundant lignin and cross-linked hemicelluloses. Furthermore, lignocellulosic biomass has a high content of pentoses, which are more difficult to ferment into fuels than hexoses. Engineered plants with decreased amounts of xylan in their secondary walls have the potential to render plant biomass a more desirable feedstock for biofuel production. Results Xylan is the major non-cellulosic polysaccharide in secondary cell walls, and the xylan deficient irregular xylem (irx mutants irx7, irx8 and irx9 exhibit severe dwarf growth phenotypes. The main reason for the growth phenotype appears to be xylem vessel collapse and the resulting impaired transport of water and nutrients. We developed a xylan-engineering approach to reintroduce xylan biosynthesis specifically into the xylem vessels in the Arabidopsis irx7, irx8 and irx9 mutant backgrounds by driving the expression of the respective glycosyltransferases with the vessel-specific promoters of the VND6 and VND7 transcription factor genes. The growth phenotype, stem breaking strength, and irx morphology was recovered to varying degrees. Some of the plants even exhibited increased stem strength compared to the wild type. We obtained Arabidopsis plants with up to 23% reduction in xylose levels and 18% reduction in lignin content compared to wild-type plants, while exhibiting wild-type growth patterns and morphology, as well as normal xylem vessels. These plants showed a 42% increase in saccharification yield after hot water pretreatment. The VND7 promoter yielded a more complete complementation of the irx phenotype than the VND6 promoter. Conclusions Spatial and temporal deposition of xylan in the secondary cell wall of

  2. Overexpression of esterase D in kidney from trisomy 13 fetuses

    Energy Technology Data Exchange (ETDEWEB)

    Loughna, S.; Moore, G. (Institute of Obstetrics and Gynaecology, London (United Kingdom)); Gau, G.; Blunt, S. (Cytogenetics Lab., London (United Kingdom)); Nicolaides, K. (King' s College School of Medicine and Dentistry, London (United Kingdom))

    1993-10-01

    Human trisomy 13 (Patau syndrome) occurs in approximately 1 in 5,000 live births. It is compatible with life, but prolonged survival is rare. Anomalies often involve the urogenital, cardiac, craniofacial, and central nervous systems. It is possible that these abnormalities may be due to the overexpression of developmentally important genes on chromosome 13. The expression of esterase D (localized to chromosome 13q14.11) has been investigated in both muscle and kidney from trisomy 13 fetuses and has been compared with normal age- and sex-matched fetal tissues, by using northern analysis. More than a twofold increase in expression of esterase D was found in the kidney of two trisomy 13 fetuses, with normal levels in a third. Overexpression was not seen in the muscle tissues from these fetuses. 34 refs., 3 figs., 2 tabs.

  3. Leucocyte esterase in the rapid diagnosis of paediatric septic arthritis.

    LENUS (Irish Health Repository)

    Kelly, E G

    2013-02-01

    Septic arthritis may affect any age group but is more common in the paediatric population. Infection is generally bacterial in nature. Prompt diagnosis is crucial, as delayed treatment is associated with lifelong joint dysfunction. A clinical history and application of Kocher\\'s criteria may indicate that there is a septic arthritis. However, definitive diagnosis is made on culture of septic synovial fluid. The culture process can take over 24h for the initial culture to yield bacterial colonies. Leucocyte esterase is released by leucocytes at the site of an infection. We hypothesise that leucocyte esterase can be utilized in the rapid diagnosis of septic arthritis and shorten the time to decisive treatment whilst simultaneously decreasing unnecessary treatment of non-septic joints.

  4. Non-enzymatic N-acetylation of Lysine Residues by AcetylCoA Often Occurs via a Proximal S-acetylated Thiol Intermediate Sensitive to Glyoxalase II

    Directory of Open Access Journals (Sweden)

    Andrew M. James

    2017-02-01

    Full Text Available Summary: Acetyl coenzyme A (AcCoA, a key intermediate in mitochondrial metabolism, N-acetylates lysine residues, disrupting and, in some cases, regulating protein function. The mitochondrial lysine deacetylase Sirtuin 3 (Sirt3 reverses this modification with benefits reported in diabetes, obesity, and aging. We show that non-enzymatic lysine N-acetylation by AcCoA is greatly enhanced by initial acetylation of a cysteine residue, followed by SN-transfer of the acetyl moiety to a nearby lysine on mitochondrial proteins and synthetic peptides. The frequent occurrence of an S-acetyl intermediate before lysine N-acetylation suggests that proximity to a thioester is a key determinant of lysine susceptibility to acetylation. The thioesterase glyoxalase II (Glo2 can limit protein S-acetylation, thereby preventing subsequent lysine N-acetylation. This suggests that the hitherto obscure role of Glo2 in mitochondria is to act upstream of Sirt3 in minimizing protein N-acetylation, thus limiting protein dysfunction when AcCoA accumulates. : James et al. show that the non-enzymatic N-acetylation of lysine residues in mitochondrial proteins frequently occurs via a proximal S-acetylated thiol intermediate. Glutathione equilibrates with this intermediate, allowing the thioesterase glyoxalase II to limit protein lysine N-acetylation. These findings expand our understanding of how protein acetylation arises. Keywords: AcetylCoA, lysine acetylation, glyoxalase

  5. Inhibition of Pectin Methyl Esterase Activity By Green Tea Catechins

    OpenAIRE

    Sagi, Irit; Lewis, Kristin; Tworowski, Dmitry; Shahar, Chen; Selzer, Tzvia

    2008-01-01

    Pectin methyl esterases (PMEs) and their endogenous inhibitors are involved in the regulation of many processes in plant physiology, ranging from tissue growth and fruit ripening to parasitic plant haustorial formation and host invasion. Thus, control of PME activity is critical for enhancing our understanding of plant physiological processes and regulation. Here we report on the identification of epigallocatechin gallate (EGCG), a green tea component, as a natural inhibitor for pectin ...

  6. Acetyl coenzyme A: alpha-glucosaminide N-acetyltransferase. Evidence for a transmembrane acetylation mechanism

    International Nuclear Information System (INIS)

    Bame, K.J.; Rome, L.H.

    1985-01-01

    The lysosomal membrane enzyme acetyl-CoA: alpha-glucosaminide N-acetyltransferase catalyzes the transfer of an acetyl group from acetyl-CoA to terminal alpha-linked glucosamine residues of heparan sulfate. The reaction mechanism was examined using highly purified lysosomal membranes from rat liver. The reaction was followed by measuring the acetylation of a monosaccharide acetyl acceptor, glucosamine. The enzyme reaction was optimal above pH 5.5, and a 2-3-fold stimulation of activity was observed when the membranes were assayed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicated that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. Further evidence to support this mechanism was provided by characterization of the enzyme half-reactions. Membranes incubated with acetyl-CoA and [ 3 H]CoA were found to produce acetyl-[ 3 H]CoA. This exchange was optimal at pH values above 7.0. Treating membranes with [ 3 H] acetyl-CoA resulted in the formation of an acetyl-enzyme intermediate. The acetyl group could then be transferred to glucosamine, forming [ 3 H]N-acetylglucosamine. The transfer of the acetyl group from the enzyme to glucosamine was optimal between pH 4 and 5. The results suggest that acetyl-CoA does not cross the lysosomal membrane. Instead, the enzyme is acetylated on the cytoplasmic side of the lysosome and the acetyl group is then transferred to the inside where it is used to acetylate heparan sulfate

  7. 3 Benzyl-6-chloropyrone: a suicide inhibitor of cholesterol esterase

    International Nuclear Information System (INIS)

    Saint, C.; Gallo, I.; Kantorow, M.; Bailey, J.M.

    1986-01-01

    Cholesterol, absorbed from the intestine, appears in lymph as the ester. Cholesterol esterase is essential for this process, since depletion of the enzyme blocks and repletion restores, absorption. Selective inhibitors of cholesterol esterase may thus prove useful in reducing cholesterol uptake. A series of potential suicide substrates were synthesized which, following cleavage by the enzyme, would attack the putative nucleophile in the active site. One of these, 3-benzyl-6-chloropyrone (3BCP), inhibited both synthesis and hydrolysis of 14 C-cholesteryl oleate with an I 50 of approximately 150 μM. The inactivation was time-dependent and characteristic of a suicide mechanism. The α pyrone structure (lactone analog) is cleaved by a serine-hydroxyl in the active site. This generates an enoyl chloride which inactivates the imidazole believed to play a part in the catalytic function of the enzyme. Inhibition by 3BCP is selective for cholesterol esterase. The activity of pancreatic lipase as not affected by concentrations up to 1 mM

  8. Inhibition of pectin methyl esterase activity by green tea catechins.

    Science.gov (United States)

    Lewis, Kristin C; Selzer, Tzvia; Shahar, Chen; Udi, Yael; Tworowski, Dmitry; Sagi, Irit

    2008-10-01

    Pectin methyl esterases (PMEs) and their endogenous inhibitors are involved in the regulation of many processes in plant physiology, ranging from tissue growth and fruit ripening to parasitic plant haustorial formation and host invasion. Thus, control of PME activity is critical for enhancing our understanding of plant physiological processes and regulation. Here, we report on the identification of epigallocatechin gallate (EGCG), a green tea component, as a natural inhibitor for pectin methyl esterases. In a gel assay for PME activity, EGCG blocked esterase activity of pure PME as well as PME extracts from citrus and from parasitic plants. Fluorometric tests were used to determine the IC50 for a synthetic substrate. Molecular docking analysis of PME and EGCG suggests close interaction of EGCG with the catalytic cleft of PME. Inhibition of PME by the green tea compound, EGCG, provides the means to study the diverse roles of PMEs in cell wall metabolism and plant development. In addition, this study introduces the use of EGCG as natural product to be used in the food industry and agriculture.

  9. Investigation of acetyl migrations in furanosides

    Directory of Open Access Journals (Sweden)

    Migaud ME

    2006-07-01

    Full Text Available Abstract Standard reaction conditions for the desilylation of acetylated furanoside (riboside, arabinoside and xyloside derivatives facilitate acyl migration. Conditions which favour intramolecular and intermolecular mechanisms have been identified with intermolecular transesterifications taking place under mild basic conditions when intramolecular orthoester formations are disfavoured. In acetyl ribosides, acyl migration could be prevented when desilylation was catalysed by cerium ammonium nitrate.

  10. Histone Acetylation in Fungal Pathogens of Plants

    Directory of Open Access Journals (Sweden)

    Junhyun Jeon

    2014-03-01

    Full Text Available Acetylation of histone lysine residues occurs in different organisms ranging from yeast to plants and mammals for the regulation of diverse cellular processes. With the identification of enzymes that create or reverse this modification, our understanding on histone acetylation has expanded at an amazing pace during the last two decades. In fungal pathogens of plants, however, the importance of such modification has only just begun to be appreciated in the recent years and there is a dearth of information on how histone acetylation is implicated in fungal pathogenesis. This review covers the current status of research related to histone acetylation in plant pathogenic fungi and considers relevant findings in the interaction between fungal pathogens and host plants. We first describe the families of histone acetyltransferases and deacetylases. Then we provide the cases where histone acetylation was investigated in the context of fungal pathogenesis. Finally, future directions and perspectives in epigenetics of fungal pathogenesis are discussed.

  11. Mode of action of Fusarium moniliforme endopolygalacturonase towards acetylated pectin.

    NARCIS (Netherlands)

    Bonnin, E.; Alebeek, van G.J.W.M.; Voragen, A.G.J.; Thibault, J.F.

    2003-01-01

    Endopolygalacturonase from Fusarium moniliforme was used to degrade acetylated homogalacturonan previously prepared from sugar beet pulp. The initial velocity and the final percentage of hydrolysis decreased-very rapidly with increasing degree of acetylation, showing that acetyl substitution

  12. A comparison between activities for non-specific esterases and esterproteases

    DEFF Research Database (Denmark)

    Kirkeby, S; Moe, D

    1988-01-01

    Electrophoretic separation of non-specific esterases and esterproteases from kidney, lung, and liver have been carried out in polyacrylamide gels. By use of zone electrophoresis, isoelectric focusing, and 2-dimensional electrophoresis it was found that most of the esterprotease bands had the same...... localization in the gels as non-specific esterase bands. A number of esterase bands showed no activity towards the esterprotease substrates and a single kidney band possessed esterprotease activity only. Isozymes of the ES-6 and ES-9 zones showed sex dependent esterprotease reactions. In sections esterase...

  13. Characterisation of a New Family of Carboxyl Esterases with an OsmC Domain.

    Directory of Open Access Journals (Sweden)

    Mai-Britt V Jensen

    Full Text Available Proteins in the serine esterase family are widely distributed in bacterial phyla and display activity against a range of biologically produced and chemically synthesized esters. A serine esterase from the psychrophilic bacterium Pseudoalteromonas arctica with a C-terminal OsmC-like domain was recently characterized; here we report on the identification and characterization of further putative esterases with OsmC-like domains constituting a new esterase family that is found in a variety of bacterial species from different environmental niches. All of these proteins contained the Ser-Asp-His motif common to serine esterases and a highly conserved pentapeptide nucleophilic elbow motif. We produced these proteins heterologously in Escherichia coli and demonstrated their activity against a range of esterase substrates. Two of the esterases characterized have activity of over two orders of magnitude higher than other members of the family, and are active over a wide temperature range. We determined the crystal structure of the esterase domain of the protein from Rhodothermus marinus and show that it conforms to the classical α/β hydrolase fold with an extended 'lid' region, which occludes the active site of the protein in the crystal. The expansion of characterized members of the esterase family and demonstration of activity over a wide-range of temperatures could be of use in biotechnological applications such as the pharmaceutical, detergent, bioremediation and dairy industries.

  14. Acetylation-mediated suppression of transcription-independent memory: bidirectional modulation of memory by acetylation.

    Directory of Open Access Journals (Sweden)

    Katja Merschbaecher

    Full Text Available Learning induced changes in protein acetylation, mediated by histone acetyl transferases (HATs, and the antagonistic histone deacetylases (HDACs play a critical role in memory formation. The status of histone acetylation affects the interaction between the transcription-complex and DNA and thus regulates transcription-dependent processes required for long-term memory (LTM. While the majority of studies report on the role of elevated acetylation in memory facilitation, we address the impact of both, increased and decreased acetylation on formation of appetitive olfactory memory in honeybees. We show that learning-induced changes in the acetylation of histone H3 at aminoacid-positions H3K9 and H3K18 exhibit distinct and different dynamics depending on the training strength. A strong training that induces LTM leads to an immediate increase in acetylation at H3K18 that stays elevated for hours. A weak training, not sufficient to trigger LTM, causes an initial increase in acetylation at H3K18, followed by a strong reduction in acetylation at H3K18 below the control group level. Acetylation at position H3K9 is not affected by associative conditioning, indicating specific learning-induced actions on the acetylation machinery. Elevating acetylation levels by blocking HDACs after conditioning leads to an improved memory. While memory after strong training is enhanced for at least 2 days, the enhancement after weak training is restricted to 1 day. Reducing acetylation levels by blocking HAT activity after strong training leads to a suppression of transcription-dependent LTM. The memory suppression is also observed in case of weak training, which does not require transcription processes. Thus, our findings demonstrate that acetylation-mediated processes act as bidirectional regulators of memory formation that facilitate or suppress memory independent of its transcription-requirement.

  15. A new microplate screening method for the simultaneous activity quantification of feruloyl esterases, tannases, and chlorogenate esterases.

    Science.gov (United States)

    Ramírez, L; Arrizon, J; Sandoval, G; Cardador, A; Bello-Mendoza, R; Lappe, P; Mateos-Díaz, J C

    2008-12-01

    Feruloyl, chlorogenate esterases, and tannases are enzymes useful in phenolic modifications of pharmaceutical relevance as protectors against several degenerative human diseases. Therefore, there is a growing interest in discovering new sources of these enzymes. However, traditional methods for their activity measurements are time-consuming and poorly adapted for high-throughput screening. In this study, a successful new microplate high-throughput screening method for the simultaneous quantification of all mentioned activities is demonstrated. This method allows the detection of activities as low as 1.7 mU ml(-1). Furthermore, reaction rates increased proportionally with the amount of enzyme added, and no interferences with the other commercial hydrolases tested were found. The utility of the method was demonstrated after simultaneously screening feruloyl, chlorogenate esterase, and tannase activities in solid state fermentation extracts obtained during the kinetics of production of 20 fungal strains. Among these, seven strains were positive for at least one of the esterase activities tested. This result shows the potential for the rapid routine screening assays for multiple samples of moderate low to high enzymatic levels.

  16. Towards the industrialization of new biosurfactants: Biotechnological opportunities for the lactone esterase gene from Starmerella bombicola.

    Science.gov (United States)

    Roelants, Sophie L K W; Ciesielska, Katarzyna; De Maeseneire, Sofie L; Moens, Helena; Everaert, Bernd; Verweire, Stijn; Denon, Quenten; Vanlerberghe, Brecht; Van Bogaert, Inge N A; Van der Meeren, Paul; Devreese, Bart; Soetaert, Wim

    2016-03-01

    Although sophorolipids (SLs) produced by S. bombicola are a real showcase for the industrialization of microbial biosurfactants, some important drawbacks are associated with this efficient biological process, e.g., the simultaneous production of acidic and lactonic SLs. Depending on the application, there is a requirement for the naturally produced mixture to be manipulated to give defined ratios of the components. Recently, the enzyme responsible for the lactonization of SLs was discovered. The discovery of the gene encoding this lactone esterase (sble) enabled the development of promising S. bombicola strains producing either solely lactonic (using a sble overexpression strain described in this paper: oe sble) or solely acidic SLs (using a sble deletion strain, which was recently described, but not characterized yet: Δsble). The new S. bombicola strains were used to investigate the production processes (fermentation and purification) of either lactonic or acidic SLs. The strains maintain the high inherent productivities of the wild-type or even perform slightly better and thus represent a realistic industrial opportunity. 100% acidic SLs with a mixed acetylation pattern were obtained for the Δsble strain, while the inherent capacity to selectively produce lactonic SLs was significantly increased (+42%) for the oe sble strain (99% lactonic SLs). Moreover, the regulatory effect of citrate on lactone SL formation for the wild-type was absent in this new strain, which indicates that it is more robust and better suited for the industrial production of lactonic SLs. Basic parameters were determined for the purified SLs, which confirm that the two new strains produce molecules with distinctive properties of which the application potential can now easily be investigated independently. © 2015 Wiley Periodicals, Inc.

  17. Effect of pH on production of xylanase by Trichoderma reesei on xylan- and cellulose-based media

    Energy Technology Data Exchange (ETDEWEB)

    Bailey, M.J. (VTT, Biotechnical Lab., Espoo (Finland)); Buchert, J. (VTT, Biotechnical Lab., Espoo (Finland)); Viikari, L. (VTT, Biotechnical Lab., Espoo (Finland))

    1993-11-01

    Trichoderma reesei VTT-D-86271 (Rut C-30) was cultivated on media based on cellulose and xylan as the main carbon source in fermentors with different pH minimum controls. Production of xylanase was favoured by a rather high pH minimum control between 6.0 and 7.0 on both cellulose- and xylan-based media. Although xylanase was produced efficiently on cellulose as well as on xylan as the carbon source, significant production of cellulase was observed only on the cellulose-based medium and best production was at lower pH (4.0 minimum). Production of xylanase at pH 7.0 was shown to be dependent on the nature of the xylan in the cultivation medium but was independent of other organic components. Best production of xylanase was observed on insoluble, unsubstituted beech xylan at pH 7.0. Similar results were obtained in laboratory and pilot (200-1) fermentors. Downstream processing of the xylanase-rich, low-cellulase culture filtrate presented no technical problems despite apparent autolysis of the fungus at the high pH. Enzyme produced in the 200-1 pilot fermentor was shown to be suitable for use in enzyme-aided bleaching of kraft pulp. Due to the high xylanase/cellulase ratio of enzyme activities in the culture filtrate, pretreatment for removal of cellulase activity prior to pulp bleaching was unnecessary. (orig.)

  18. Effect of xylan and lignin removal by batch and flowthrough pretreatment on the enzymatic digestibility of corn stover cellulose.

    Science.gov (United States)

    Yang, Bin; Wyman, Charles E

    2004-04-05

    Compared with batch systems, flowthrough and countercurrent reactors have important potential advantages for pretreating cellulosic biomass, including higher hemicellulose sugar yields, enhanced cellulose digestibility, and reduced chemical additions. Unfortunately, they suffer from high water and energy use. To better understand these trade-offs, comparative data are reported on xylan and lignin removal and enzymatic digestibility of cellulose for corn stover pretreated in batch and flowthrough reactors over a range of flow rates between 160 degrees and 220 degrees C, with water only and also with 0.1 wt% sulfuric acid. Increasing flow with just water enhanced the xylan dissolution rate, more than doubled total lignin removal, and increased cellulose digestibility. Furthermore, adding dilute sulfuric acid increased the rate of xylan removal for both batch and flowthrough systems. Interestingly, adding acid also increased the lignin removal rate with flow, but less lignin was left in solution when acid was added in batch. Although the enzymatic hydrolysis of pretreated cellulose was related to xylan removal, as others have shown, the digestibility was much better for flowthrough compared with batch systems, for the same degree of xylan removal. Cellulose digestibility for flowthrough reactors was related to lignin removal as well. These results suggest that altering lignin also affects the enzymatic digestibility of corn stover. Copyright 2004 Wiley Periodicals, Inc.

  19. Characterization of xylan in the early stages of secondary cell wall formation in tobacco bright yellow-2 cells.

    Science.gov (United States)

    Ishii, Tadashi; Matsuoka, Keita; Ono, Hiroshi; Ohnishi-Kameyama, Mayumi; Yaoi, Katsuro; Nakano, Yoshimi; Ohtani, Misato; Demura, Taku; Iwai, Hiroaki; Satoh, Shinobu

    2017-11-15

    The major polysaccharides present in the primary and secondary walls surrounding plant cells have been well characterized. However, our knowledge of the early stages of secondary wall formation is limited. To address this, cell walls were isolated from differentiating xylem vessel elements of tobacco bright yellow-2 (BY-2) cells induced by VASCULAR-RELATED NAC-DOMAIN7 (VND7). The walls of induced VND7-VP16-GR BY-2 cells consisted of cellulose, pectic polysaccharides, hemicelluloses, and lignin, and contained more xylan and cellulose compared with non-transformed BY-2 and uninduced VND7-VP16-GR BY-2 cells. A reducing end sequence of xylan containing rhamnose and galaturonic acid- residues is present in the walls of induced, uninduced, and non-transformed BY-2 cells. Glucuronic acid residues in xylan from walls of induced cells are O-methylated, while those of xylan in non-transformed BY-2 and uninduced cells are not. Our results show that xylan changes in chemical structure and amounts during the early stages of xylem differentiation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Acetyl Fentanyl Toxicity: Two Case Reports.

    Science.gov (United States)

    Fort, Chelsea; Curtis, Byron; Nichols, Clay; Niblo, Cheryl

    2016-11-01

    Acetyl fentanyl is an illicit fentanyl analog recently appearing in forensic casework. A quantitative method was created for measuring acetyl fentanyl in various biological matrices acquired post-mortem due to recent positive screening results in casework. Initial detection by immunoassay and standard gas chromatography mass spectrometry (GC/MS) methods have been previously reported for acetyl fentanyl and are examined further here. A Selective Ion Monitoring (SIM) method was created using a GC/MS for quantitation. In two separate cases, acetyl fentanyl was found to be in similar concentrations to those previously reported and ruled to be the cause of death. Acetyl fentanyl concentrations were determined in blood samples, liver, brain, vitreous humor, and urine. Individual 1 had acetyl fentanyl concentrations as follows: heart blood-285 ng/mL, femoral blood-192 ng/mL, liver-1,100 ng/g, brain-620 ng/g, and urine-3,420 ng/mL. Individual 2 had acetyl fentanyl concentrations as follows: heart blood-210 ng/mL, femoral blood-255 ng/mL, urine-2,720 ng/mL and vitreous humor-140 ng/mL. Experimental conditions for screening and quantitation are provided, using immunoassay and GC/MS methods. Due to the recent emergence of acetyl fentanyl, more data will need to be generated to fully differentiate recreational and fatal concentrations of acetyl fentanyl to assist toxicologists accurately understanding its physiological impact. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  1. Xylan oligosaccharides and cellobiohydrolase I (TrCel7A interaction and effect on activity

    Directory of Open Access Journals (Sweden)

    Baumann Martin J

    2011-10-01

    Full Text Available Abstract Background The well-studied cellulase mixture secreted by Trichoderma reesei (anamorph to Hypocrea jecorina contains two cellobiohydolases (CBHs, cellobiohydrolase I (TrCel7A and cellobiohydrolase II (TrCeI6A, that are core enzymes for the solubilisation of cellulose. This has attracted significant research interest because of the role of the CBHs in the conversion of biomass to fermentable sugars. However, the CHBs are notoriously slow and susceptible to inhibition, which presents a challenge for the commercial utilisation of biomass. The xylans and xylan fragments that are also present in the biomass have been suggested repeatedly as one cause of the reduced activity of CHBs. Yet, the extent and mechanisms of this inhibition remain poorly elucidated. Therefore, we studied xylan oligosaccharides (XOSs of variable lengths with respect to their binding and inhibition of both TrCel7A and an enzyme variant without the cellulose-binding domain (CBM. Results We studied the binding of XOSs to TrCel7A by isothermal titration calorimetry. We found that XOSs bind to TrCel7A and that the affinity increases commensurate with XOS length. The CBM, on the other hand, did not affect the affinity significantly, which suggests that XOSs may bind to the active site. Activity assays of TrCel7A clearly demonstrated the negative effect of the presence of XOSs on the turnover number. Conclusions On the basis of these binding data and a comparison of XOS inhibition of the activity of the two enzyme variants towards, respectively, soluble and insoluble substrates, we propose a competitive mechanism for XOS inhibition of TrCel7A with phosphoric swollen cellulose as a substrate.

  2. Direct hydrodeoxygenation of cellulose and xylan to lower alkanes on ruthenium catalysts in subcritical water

    International Nuclear Information System (INIS)

    Osaka, Yuriko; Ikeda, Yoichi; Hashizume, Daisuke; Iwamoto, Masakazu

    2013-01-01

    Nano particles of Ru, Rh, Pd, Ir, Pt, and Au, protected by polyvinyl pyrrolidone (PVP), were applied to the hydrodeoxygenation of cellulose and xylan in water and 5 MPa H 2 at 543 K. The distributions of products generated from cellulose and xylan were roughly similar to each other under the present reaction conditions, and therefore, the former was intensively studied. The Ru-PVP catalyst afforded mainly methane and lower alkanes, rather than producing water soluble organic compounds, such as diols and alcohols, that were formed with the use of the other catalysts. The changes in the product distributions with reaction temperature and time indicated that the reaction consisted of two consecutive reactions: cellulose or xylan → water soluble compounds → hydrogenolysis. The first transformation was promoted in subcritical water, and the second step was catalyzed by the Ru catalyst. The Ru catalyst that was supported on CeO 2 , γ-Al 2 O 3 , or activated carbon yielded a similar product distribution to that on Ru-PVP; however, the loading of Ru on TiO 2 , ZrO 2 , SiO 2 –Al 2 O 3 , or SiO 2 resulted in the increment of diols. After the reaction a small portion of the CeO 2 and most of the SiO 2 –Al 2 O 3 and SiO 2 were dissolved in water, and a portion of the Al 2 O 3 was transformed to boehmite AlO(OH) from the γ-alumina. Little change in the catalytic activity however was observed upon the reuse of Ru/Al 2 O 3 in the second run. Highlights: •One-path hydrodeoxygenation of cellulose and xylan to methane and lower alkanes was studied. •Ru-PVP catalysts gave the best yields among Ru-, Rh-, Pd-, Ir-, Pt-, and Au-PVP. •The reaction pathways were cellulose → water soluble compounds → hydrogenolysis. •The catalytic activity of Ru was greatly dependent on the supports

  3. Engineering of plants with improved properties as biofuels feedstocks by vessel-specific complementation of xylan biosynthesis mutants

    DEFF Research Database (Denmark)

    Petersen, Pia; Lau, Jane; Ebert, Berit

    2012-01-01

    Background: Cost-efficient generation of second-generation biofuels requires plant biomass that can easily be degraded into sugars and further fermented into fuels. However, lignocellulosic biomass is inherently recalcitrant toward deconstruction technologies due to the abundant lignin and cross......-linked hemicelluloses. Furthermore, lignocellulosic biomass has a high content of pentoses, which are more difficult to ferment into fuels than hexoses. Engineered plants with decreased amounts of xylan in their secondary walls have the potential to render plant biomass a more desirable feedstock for biofuel production...... in the xylem vessels is sufficient to complement the irx phenotype of xylan deficient mutants, while maintaining low overall amounts of xylan and lignin in the cell wall. This engineering approach has the potential to yield bioenergy crop plants that are more easily deconstructed and fermented into biofuels....

  4. Green synthesis of silver nanoparticles in xylan solution via Tollens reaction and their detection for Hg(2+).

    Science.gov (United States)

    Luo, Yuqiong; Shen, Suqin; Luo, Jiwen; Wang, Xiaoying; Sun, Runcang

    2015-01-14

    This work reported a facile and green method to prepare highly stable and uniformly distributed Ag nanoparticles (AgNPs), in which a biopolymer xylan was used as the stabilizing and reducing agent via the Tollens reaction under microwave irradiation. Different variables were evaluated to optimize the reaction conditions. Complete characterization was performed using UV-Vis, XRD, TEM, size distribution analysis and XPS. The results revealed that AgNPs were well dispersed with diameters of 20-35 nm due to the packing of xylan. The optimal conditions were as follows: microwave irradiation temperature was 60-70 °C, microwave power was 800 W, microwave time was 30 min, the ratio of xylan to AgNO3 was 50 mg: 0.13 mmol, and ammonia concentration was 2%. In addition, the AgNPs were collected via high-speed centrifugal separation, and the supernatant was tested by HPAEC, GPC, FT-IR, and NMR. By comparing the structure of xylan before and after the reaction, the reaction mechanism was discussed. It was noted that the xylan-AgNPs composites showed high selectivity and sensitivity for Hg(2+) detection. The other 15 metal ions used had no obvious effect on the detection of Hg(2+), and the limit of detection (LOD) was 4.6 nM, which is lower than the allowed maximum level of 30 nM for drinking water by WHO. In addition, the xylan-AgNPs composites can be applied for Hg(2+) detection in real water samples. This study provides a novel way for the high-value utilization of a rich biomass resource, and a green method for the synthesis of AgNPs for the selective and sensitive detection of harmful heavy metals.

  5. Esterases of laboratory-reared and field-collected cotton boll weevils, Anthonomus grandis Boh.: polymorphism of adult esterases and formal genetics of esterase II.

    Science.gov (United States)

    Biggers, C J; Bancroft, H R

    1977-04-01

    The esterases of the cotton boll weevil were separated by polyacrylamide gel electrophoresis into four major regions. These were named Est I-IV in order of migration from anode to origin. Polymorphism was observed in all regions. The Est II region was shown to consist of no more than two bands (fast and slow). The inheritance of the fast and slow bands of Est II was demonstrated to be controlled by codominant autosomal alleles. Analysis of the gene frequency of the Est II region showed that one field population was consistent with the Hardy-Weinberg law (P = 0.995), while a second field population was not at equilibrium (P less than 0.001).

  6. Alicyclobacillus acidocaldarius Thermophilic Esterase EST2's Activity in Milk and Cheese Models

    NARCIS (Netherlands)

    Mandrich, L.; Manco, M.; Rossie, M.; Floris, E.; Jansen-van den Bosch, T.; Smit, G.; Wouters, J.A.

    2006-01-01

    The aim of this work was to investigate the behavior of thermophilic esterase EST2 from Alicyclobacillus acidocaldarius in milk and cheese models. The pure enzyme was used to compare the EST2 hydrolytic activity to the activity of endogenous esterase EstA from Lactococcus lactis. The results

  7. Esterases in striated muscle from mice with the Chediak-Higashi syndrome

    DEFF Research Database (Denmark)

    Kirkeby, S; Moe, D

    1981-01-01

    In this paper a localized strong reaction for non-specific esterase forming cylindric structures is described within skeletal muscle fibres from the beige mouse. It seems from zymograms and protein electrophoresis that this esterase is membrane bound, highly reactive and present in rather small...

  8. Esterase Isoenzyme Profiles in Acute and Chronic Leukemias.

    Science.gov (United States)

    Drexler, H G; Gignac, S M; Hoffbrand, A V; Minowada, J

    1991-01-01

    Using isoelectric focusing (IEF) a number of carboxylic esterase isoenzymes (EC 3.1.1.1) with isoelectric points between pH 4.5-8.0 can be separated. One particular isoenzyme with an isoelectric point at about pH 6.0, the Mono-band, can be selectively and completely inhibited by sodium fluoride; this isoenzyme comprises a number of closely related subcomponents and may appear in more than one band on the gel. We analyzed the expression of typical esterase isoenzyme patterns in cells from a large panel of leukemias which were tested under identical conditions by IEF on horizontal thin-layer polyacrylamide gels with an ampholyte of pH 2-11. The 442 cases of acute and chronic myeloid and lymphoid leukemia (AML/AMMoL, CML/CMML, ALL, CLL) were classified according to clinical, morpho-cytochemical and immunophenotyping criteria. While bands between pH 4.5-5.5 appeared not to be specific for lineage or stage of differentiation, isoenzymes between pH 6.6-7.7 provided information on the type of leukemia involved. Seven typical isoenzyme patterns termed Mono1/Mono2 (fo monocyte-associated), My1/My2 (myeloid), Lym1/Lym2 (lymphoid) and Und (undifferentiated) could be discerned. Lym and Und patterns are characterized by fewer bands with a weaker staining intensity than Mono and My patterns. Nearly all cases of lymphoid leukemias (acute and chronic) expressed only Lym or Und esterase isoenzyme patterns, but no Mono or My patterns. Cases of acute or chronic myeloid and (myelo)monocytic leukemia showed strong isoenzyme staining displaying predominantly Mono or My isoenzyme patterns. The isoenzyme patterns found in CML in lymphoid or myeloid blast crisis corresponded to those seen in the respective acute leukemias, ALL or AML. The Mono-band was found in most cases of leukemias with monocytic elements (AMMoL 80%, CML 44%, CMML 100%), in the occasional case of CML-myeloid blast crisis or AML, but in none of the cases of ALL or CLL. This isoenzyme is a distinctive, specific marker for

  9. Organophosphate acetylcholine esterase inhibitor poisoning from a home-made shampoo.

    Science.gov (United States)

    Sadaka, Yair; Broides, Arnon; Tzion, Raffi Lev; Lifshitz, Matitiahu

    2011-07-01

    Organophosphate acetylcholine esterase inhibitor poisoning is a major health problem in children. We report an unusual cause of organophosphate acetylcholine esterase inhibitor poisoning. Two children were admitted to the pediatric intensive care unit due to organophosphate acetylcholine esterase inhibitor poisoning after exposure from a home-made shampoo that was used for the treatment of head lice. Owing to no obvious source of poisoning, the diagnosis of organophosphate acetylcholine esterase inhibitor poisoning in one of these patients was delayed. Both patients had an uneventful recovery. Organophosphate acetylcholine esterase inhibitor poisoning from home-made shampoo is possible. In cases where the mode of poisoning is unclear, direct questioning about the use of home-made shampoo is warranted, in these cases the skin and particularly the scalp should be rinsed thoroughly as soon as possible.

  10. Studies on the oxidizing system in Holt's medium for histochemical demonstration of esterase activity

    DEFF Research Database (Denmark)

    Kirkeby, S; Blecher, S R

    1978-01-01

    Esterase activity in guinea-pig thyroid and mouse epididymis epithelial cells has been studied using 5-bromoindoxyl acetate as substrate. The pattern of esterase activity in the thyroid of the guinea-pig is constant, irrespective of whether ferri-ferrocyanide (FFC) or certain copper compounds...... cells contain an esterase activity which is not inhibited by conventional SH blocking agents, nor by high concentrations of FFC. From these results it appears that the mode of action of FFC in Holt's medium is as follows. At low concentrations FFC appears to act primarily as a catalytic agent...... in oxidation of indoxyl to indigoid. At high concentration FFC acts as an inhibitor of guinea-pig thyroid esterase, by oxidation of SH groups in the active centre. The esterase of mouse epididymis cell type EH 1 is not subject to this inhibition by FFC, presumably because it does not contain accessible SH...

  11. p53 Acetylation: Regulation and Consequences

    Energy Technology Data Exchange (ETDEWEB)

    Reed, Sara M. [Department of Pharmacology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Medical Scientist Training Program, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Quelle, Dawn E., E-mail: dawn-quelle@uiowa.edu [Department of Pharmacology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Medical Scientist Training Program, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States); Department of Pathology, The University of Iowa Carver College of Medicine, Iowa City, IA 52242 (United States)

    2014-12-23

    Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo evidence from mouse models questions the importance of p53 acetylation (at least at certain sites) as well as canonical p53 functions (cell cycle arrest, senescence and apoptosis) to tumor suppression. This review discusses the cumulative findings regarding p53 acetylation, with a focus on the acetyltransferases that modify p53 and the mechanisms regulating their activity. We also evaluate what is known regarding the influence of other post-translational modifications of p53 on its acetylation, and conclude with the current outlook on how p53 acetylation affects tumor suppression. Due to redundancies in p53 control and growing understanding that individual modifications largely fine-tune p53 activity rather than switch it on or off, many questions still remain about the physiological importance of p53 acetylation to its role in preventing cancer.

  12. p53 Acetylation: Regulation and Consequences

    International Nuclear Information System (INIS)

    Reed, Sara M.; Quelle, Dawn E.

    2014-01-01

    Post-translational modifications of p53 are critical in modulating its tumor suppressive functions. Ubiquitylation, for example, plays a major role in dictating p53 stability, subcellular localization and transcriptional vs. non-transcriptional activities. Less is known about p53 acetylation. It has been shown to govern p53 transcriptional activity, selection of growth inhibitory vs. apoptotic gene targets, and biological outcomes in response to diverse cellular insults. Yet recent in vivo evidence from mouse models questions the importance of p53 acetylation (at least at certain sites) as well as canonical p53 functions (cell cycle arrest, senescence and apoptosis) to tumor suppression. This review discusses the cumulative findings regarding p53 acetylation, with a focus on the acetyltransferases that modify p53 and the mechanisms regulating their activity. We also evaluate what is known regarding the influence of other post-translational modifications of p53 on its acetylation, and conclude with the current outlook on how p53 acetylation affects tumor suppression. Due to redundancies in p53 control and growing understanding that individual modifications largely fine-tune p53 activity rather than switch it on or off, many questions still remain about the physiological importance of p53 acetylation to its role in preventing cancer

  13. Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination.

    Science.gov (United States)

    Rejón, Juan D; Zienkiewicz, Agnieszka; Rodríguez-García, María Isabel; Castro, Antonio J

    2012-10-01

    A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized. The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods. Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles. In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma.

  14. Branched nanotrees with immobilized acetylcholine esterase for nanobiosensor applications

    Energy Technology Data Exchange (ETDEWEB)

    Risveden, Klas; Bhand, Sunil; Danielsson, Bengt [Department of Pure and Applied Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, PO Box 124, SE-22100 Lund (Sweden); Dick, Kimberly A; Samuelson, Lars [Solid State Physics, Lund University, Box 118, S-22100 Lund (Sweden); Rydberg, Patrik, E-mail: Kimberly.Dick@ftf.lth.se [Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen (Denmark)

    2010-02-05

    A novel lab-on-a-chip nanotree enzyme reactor is demonstrated for the detection of acetylcholine. The reactors are intended for use in the RISFET (regional ion sensitive field effect transistor) nanosensor, and are constructed from gold-tipped branched nanorod structures grown on SiN{sub x}-covered wafers. Two different reactors are shown: one with simple, one-dimensional nanorods and one with branched nanorod structures (nanotrees). Significantly higher enzymatic activity is found for the nanotree reactors than for the nanorod reactors, most likely due to the increased gold surface area and thereby higher enzyme binding capacity. A theoretical calculation is included to show how the enzyme kinetics and hence the sensitivity can be influenced and increased by the control of electrical fields in relation to the active sites of enzymes in an electronic biosensor. The possible effects of electrical fields employed in the RISFET on the function of acetylcholine esterase is investigated using quantum chemical methods, which show that the small electric field strengths used are unlikely to affect enzyme kinetics. Acetylcholine esterase activity is determined using choline oxidase and peroxidase by measuring the amount of choline formed using the chemiluminescent luminol reaction.

  15. Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes

    KAUST Repository

    Zhang, Meiling; Chekan, Jonathan R.; Dodd, Dylan; Hong, Pei-Ying; Radlinsk, Lauren; Revindran, Vanessa; Nair, Satish K.; Mackie, Roderick Ian; Cann, Isaac Ko O

    2014-01-01

    highly induced carbohydrate active genes encode a unique glycoside hydrolase (GH) family 10 endoxylanase (BiXyn10A or BACINT-04215 and BACOVA-04390) that is highly conserved in the Bacteroidetes xylan utilization system. The BiXyn10A modular architecture

  16. A bacterial cocaine esterase protects against cocaine-induced epileptogenic activity and lethality.

    Science.gov (United States)

    Jutkiewicz, Emily M; Baladi, Michelle G; Cooper, Ziva D; Narasimhan, Diwahar; Sunahara, Roger K; Woods, James H

    2009-09-01

    Cocaine toxicity results in cardiovascular complications, seizures, and death and accounts for approximately 20% of drug-related emergency department visits every year. Presently, there are no treatments to eliminate the toxic effects of cocaine. The present study hypothesizes that a bacterial cocaine esterase with high catalytic efficiency would provide rapid and robust protection from cocaine-induced convulsions, epileptogenic activity, and lethality. Cocaine-induced paroxysmal activity and convulsions were evaluated in rats surgically implanted with radiotelemetry devices (N=6 per treatment group). Cocaine esterase was administered 1 minute after a lethal dose of cocaine or after cocaine-induced convulsions to determine the ability of the enzyme to prevent or reverse, respectively, the effects of cocaine. The cocaine esterase prevented all cocaine-induced electroencephalographic changes and lethality. This effect was specific for cocaine because the esterase did not prevent convulsions and death induced by a cocaine analog, (-)-2beta-carbomethoxy-3beta-phenyltropane. The esterase prevented lethality even after cocaine-induced convulsions occurred. In contrast, the short-acting benzodiazepine, midazolam, prevented cocaine-induced convulsions but not the lethal effects of cocaine. The data showed that cocaine esterase successfully degraded circulating cocaine to prevent lethality and that cocaine-induced convulsions alone are not responsible for the lethal effects of cocaine in this model. Therefore, further investigation into the use of cocaine esterase for treating cocaine overdose and its toxic effects is warranted.

  17. Functional classification of esterases from leaves of Aspidosperma polyneuron M. Arg. (Apocynaceae

    Directory of Open Access Journals (Sweden)

    Carvalho Vanda Marilza de

    2003-01-01

    Full Text Available Polyacrylamide gel electrophoresis system (PAGE and inhibition tests for biochemical characterization of alpha- and beta-esterases were used to obtain a functional classification of esterases fromAspidosperma polyneuron. The characterization of alpha- and beta-esterases from young leaves of A. polyneuron by the PAGE system showed fourteen esterase isozymes. The differential staining pattern showed that Est-2 isozyme hydrolyzes beta-naphthyl acetate; Est-6, Est-7 and Est-8 isozymes hydrolyze alpha-naphthyl acetate, and Est-1, Est-3, Est-4, Est-5, Est-9, Est-10, Est-11, Est-12, Est-13, and Est-14 isozymes hydrolyze both alpha- and b-naphthyl acetate. Inhibition pattern of a- and beta-esterases showed that Folidol is a more potent inhibitor that Malathion, while Thiamethoxan (an insecticide with organophosphorus-like action acts as an Est-4 and Est-6 inhibitor and induces the appearance of Est-5 and Est-7 isozymes as more intensely stained bands. Inhibition tests showed that OPC insecticides inhibit or activate plant esterases. Thus, plant esterases may be used as bioindicators to detect the presence and toxicity of residues of topically applied insecticides in agriculture and may be valuable for monitoring pollutants in the environment.

  18. A Novel Cold Active Esterase from a Deep Sea Sponge Stelletta normani Metagenomic Library

    Directory of Open Access Journals (Sweden)

    Erik Borchert

    2017-09-01

    Full Text Available Esterases catalyze the hydrolysis of ester bonds in fatty acid esters with short-chain acyl groups. Due to the widespread applications of lipolytic enzymes in various industrial applications, there continues to be an interest in novel esterases with unique properties. Marine ecosystems have long been acknowledged as a significant reservoir of microbial biodiversity and in particular of bacterial enzymes with desirable characteristics for industrial use, such as for example cold adaptation and activity in the alkaline pH range. We employed a functional metagenomic approach to exploit the enzymatic potential of one particular marine ecosystem, namely the microbiome of the deep sea sponge Stelletta normani. Screening of a metagenomics library from this sponge resulted in the identification of a number of lipolytic active clones. One of these encoded a highly, cold-active esterase 7N9, and the recombinant esterase was subsequently heterologously expressed in Escherichia coli. The esterase was classified as a type IV lipolytic enzyme, belonging to the GDSAG subfamily of hormone sensitive lipases. Furthermore, the recombinant 7N9 esterase was biochemically characterized and was found to be most active at alkaline pH (8.0 and displays salt tolerance over a wide range of concentrations. In silico docking studies confirmed the enzyme's activity toward short-chain fatty acids while also highlighting the specificity toward certain inhibitors. Furthermore, structural differences to a closely related mesophilic E40 esterase isolated from a marine sediment metagenomics library are discussed.

  19. Structural, kinetic and proteomic characterization of acetyl phosphate-dependent bacterial protein acetylation.

    Directory of Open Access Journals (Sweden)

    Misty L Kuhn

    Full Text Available The emerging view of Nε-lysine acetylation in eukaryotes is of a relatively abundant post-translational modification (PTM that has a major impact on the function, structure, stability and/or location of thousands of proteins involved in diverse cellular processes. This PTM is typically considered to arise by the donation of the acetyl group from acetyl-coenzyme A (acCoA to the ε-amino group of a lysine residue that is reversibly catalyzed by lysine acetyltransferases and deacetylases. Here, we provide genetic, mass spectrometric, biochemical and structural evidence that Nε-lysine acetylation is an equally abundant and important PTM in bacteria. Applying a recently developed, label-free and global mass spectrometric approach to an isogenic set of mutants, we detected acetylation of thousands of lysine residues on hundreds of Escherichia coli proteins that participate in diverse and often essential cellular processes, including translation, transcription and central metabolism. Many of these acetylations were regulated in an acetyl phosphate (acP-dependent manner, providing compelling evidence for a recently reported mechanism of bacterial Nε-lysine acetylation. These mass spectrometric data, coupled with observations made by crystallography, biochemistry, and additional mass spectrometry showed that this acP-dependent acetylation is both non-enzymatic and specific, with specificity determined by the accessibility, reactivity and three-dimensional microenvironment of the target lysine. Crystallographic evidence shows acP can bind to proteins in active sites and cofactor binding sites, but also potentially anywhere molecules with a phosphate moiety could bind. Finally, we provide evidence that acP-dependent acetylation can impact the function of critical enzymes, including glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, and RNA polymerase.

  20. Studies on esterase isozymes and mycelium growth speed of ganoderma lucidum carried by Shenzhou spaceship

    International Nuclear Information System (INIS)

    Qi Jianjun; Chen Xiangdong; Lan Jin

    2002-01-01

    The esterase isozymes and mycelium growth speed of four Ganoderma lucidum strains carried by Shenzhou spaceship were studied. The results showed that different effects occurred to esterase and mycelium growth speed. The SX, S3 esterase band had changed compared with their control CX, C3, respectively, but there were no differences between SH and CH, S4 and C4. The growth speed of S4 strain was faster than its control C4, SX strain lower than its control CX, and there were no difference between SH and CH, S3 and C3

  1. Plasma B-esterase activities in European raptors.

    Science.gov (United States)

    Roy, Claudie; Grolleau, Gérard; Chamoulaud, Serge; Rivière, Jean-Louis

    2005-01-01

    B-esterases are serine hydrolases composed of cholinesterases, including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and carboxylesterase (CbE). These esterases, found in blood plasma, are inhibited by organophosphorus (OP) and carbamate (CB) insecticides and can be used as nondestructive biomarkers of exposure to anticholinesterase insecticides. Furthermore, B-esterases are involved in detoxification of these insecticides. In order to establish the level of these enzymes and to have reference values for their normal activities, total plasma cholinesterase (ChE), AChE and BChE activities, and plasma CbE activity were determined in 729 European raptors representing 20 species, four families, and two orders. The diurnal families of the Falconiforme order were represented by Accipitridae and Falconidae and the nocturnal families of the Strigiforme order by Tytonidae and Strigidae. Intraspecies differences in cholinesterase activities according to sex and/or age were investigated in buzzards (Buteo buteo), sparrowhawks (Accipiter nisus), kestrels (Falco tinnunculus), barn owls (Tyto alba), and tawny owls (Strix aluco). Sex-related differences affecting ChE and AChE activities were observed in young kestrels (2-3-mo-old) and age-related differences in kestrels (ChE and AChE), sparrowhawks (AChE), and tawny owls (ChE, AChE, and BChE). The interspecies analysis yielded a negative correlation between ChE activity and body mass taking into account the relative contribution of AChE and BChE to ChE activity, with the exception of the honey buzzard (Pernis apivorus). The lowest ChE activities were found in the two largest species, Bonelli's eagle (Hieraaetus fasciatus) and Egyptian vulture (Neophron percnopterus) belonging to the Accipitridae family. The highest ChE activities were found in the relatively small species belonging to the Tytonidae and Strigidae families and in honey buzzard of the Accipitridae family. Species of the Accipitridae, Tytonidae, and

  2. Functional Characterization of ATM Kinase Using Acetylation-Specific Antibodies.

    Science.gov (United States)

    Sun, Yingli; Du, Fengxia

    2017-01-01

    The activation of ATM is critical in the DNA double strand breaks repair pathway. Acetylation of ATM by Tip60 histone acetyltransferase (HAT) plays a key role in the activation of ATM kinase activity in response to DNA damage. ATM forms a stable complex with Tip60 through the FATC domain of ATM. Tip60 acetylates lysine3016 of ATM, and this acetylation induces the activation of ATM. Several techniques are included in the study of ATM acetylation by Tip60, such as in vitro kinase assay, systematic mutagenesis, western blots. Here, we describe how to study the acetylation of ATM using acetylation-specific antibodies.

  3. Production of Nα-acetyl Tα1-HSA through in vitro acetylation by RimJ.

    Science.gov (United States)

    Chen, Jing; Li, Haibin; Wang, Tao; Sun, Shuyang; Liu, Jia; Chen, Jianhua

    2017-11-10

    Thymosin alpha 1 (Tα1) is an important immunomodulating agent with various clinical applications. The natural form of Tα1 is N α -acetylated, which was supposed to be related to in vivo stability of the hormone. In this study, fusion protein Tα1-HSA was constructed and expressed in Pichia pastoris . RimJ, a N α -acetyltransferase from E.coli , was also overexpressed and purified to homogeneity. In vitro acetylation of Tα1-HSA in the presence of RimJ and acetyl coenzyme A resulted in N α -acetyl Tα1-HSA. The N α -acetylation was determined by LC-MS/MS. Kinetic assay indicated that RimJ had a higher affinity to desacetyl Tα1 than to Tα1-HSA. Bioactivity assay revealed fully retained activity of Tα1 when the hormone was connected to the N-terminus of the fusion protein, while the activity was compromised in our previously constructed HSA-Tα1. With fully retained activity and N-terminal acetylation, N α -acetyl Tα1-HSA was expected to be a more promising pharmaceutical agent than Tα1.

  4. Non-enzymatic N -acetylation of Lysine Residues by AcetylCoA Often Occurs via a Proximal S -acetylated Thiol Intermediate Sensitive to Glyoxalase II

    OpenAIRE

    James, Andrew M.; Hoogewijs, Kurt; Logan, Angela; Hall, Andrew R.; Ding, Shujing; Fearnley, Ian M.; Murphy, Michael P.

    2017-01-01

    Summary: Acetyl coenzyme A (AcCoA), a key intermediate in mitochondrial metabolism, N-acetylates lysine residues, disrupting and, in some cases, regulating protein function. The mitochondrial lysine deacetylase Sirtuin 3 (Sirt3) reverses this modification with benefits reported in diabetes, obesity, and aging. We show that non-enzymatic lysine N-acetylation by AcCoA is greatly enhanced by initial acetylation of a cysteine residue, followed by SN-transfer of the acetyl moiety to a nearby lysin...

  5. Non-enzymatic N-acetylation of Lysine Residues by AcetylCoA Often Occurs via a Proximal S-acetylated Thiol Intermediate Sensitive to Glyoxalase II.

    Science.gov (United States)

    James, Andrew M; Hoogewijs, Kurt; Logan, Angela; Hall, Andrew R; Ding, Shujing; Fearnley, Ian M; Murphy, Michael P

    2017-02-28

    Acetyl coenzyme A (AcCoA), a key intermediate in mitochondrial metabolism, N-acetylates lysine residues, disrupting and, in some cases, regulating protein function. The mitochondrial lysine deacetylase Sirtuin 3 (Sirt3) reverses this modification with benefits reported in diabetes, obesity, and aging. We show that non-enzymatic lysine N-acetylation by AcCoA is greatly enhanced by initial acetylation of a cysteine residue, followed by SN-transfer of the acetyl moiety to a nearby lysine on mitochondrial proteins and synthetic peptides. The frequent occurrence of an S-acetyl intermediate before lysine N-acetylation suggests that proximity to a thioester is a key determinant of lysine susceptibility to acetylation. The thioesterase glyoxalase II (Glo2) can limit protein S-acetylation, thereby preventing subsequent lysine N-acetylation. This suggests that the hitherto obscure role of Glo2 in mitochondria is to act upstream of Sirt3 in minimizing protein N-acetylation, thus limiting protein dysfunction when AcCoA accumulates. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  6. Fitness differences due to allelic variation at Esterase-4 locus in ...

    Indian Academy of Sciences (India)

    KAVITA KRISHNAMOORTI

    2017-08-31

    Aug 31, 2017 ... Keywords. esterases; null allele; reproductive fitness; natural selection; Drosophila ananassae. .... cific substrate (1-naphthylacetate AR) and stain (fast blue. RR). On the ... transferred to fresh food vials and eggs were counted.

  7. Common and Distant Structural Characteristics of Feruloyl Esterase Families from Aspergillus oryzae

    DEFF Research Database (Denmark)

    Udatha, D. B. R. K. Gupta; Mapelli, Valeria; Panagiotou, Gianni

    2012-01-01

    Background: Feruloyl esterases (FAEs) are important biomass degrading accessory enzymes due to their capability of cleaving the ester links between hemicellulose and pectin to aromatic compounds of lignin, thus enhancing the accessibility of plant tissues to cellulolytic and hemicellulolytic...

  8. Multiple nucleophilic elbows leading to multiple active sites in a single module esterase from Sorangium cellulosum

    DEFF Research Database (Denmark)

    Udatha, D.B.R.K. Gupta; Madsen, Karina Marie; Panagiotou, Gianni

    2015-01-01

    The catalytic residues in carbohydrate esterase enzyme families constitute a highly conserved triad: serine, histidine and aspartic acid. This catalytic triad is generally located in a very sharp turn of the protein backbone structure, called the nucleophilic elbow and identified by the consensus...... sequence GXSXG. An esterase from Sorangium cellulosum Soce56 that contains five nucleophilic elbows was cloned and expressed in Escherichia coli and the function of each nucleophilic elbowed site was characterized. In order to elucidate the function of each nucleophilic elbow, site directed mutagenesis....... To our knowledge, this is the first report presenting the role of multiple nucleophilic elbows in the catalytic promiscuity of an esterase. Further structural analysis at protein unit level indicates the new evolutionary trajectories in emerging promiscuous esterases....

  9. Esterase-sensitive sulfur dioxide prodrugs inspired by modified Julia olefination.

    Science.gov (United States)

    Wang, Wenyi; Wang, Binghe

    2017-09-12

    Sulfur dioxide (SO 2 ) is an endogenously produced gaseous molecule, and is emerging as a potential gasotransmitter. Herein, we describe the first series of esterase-sensitive prodrugs inspired by modified Julia olefination as SO 2 donors.

  10. Esterase-D and chromosome patterns in Central Amazon piranha (Serrasalmus rhombeus Linnaeus, 1766 from Lake Catalão

    Directory of Open Access Journals (Sweden)

    Aylton Saturnino Teixeira

    2006-01-01

    Full Text Available This study presents additional genetic data on piranha (Serrasalmus rhombeus Linnaeus, 1766 complex previously diagnosed due to the presence of distinct cytotypes 2n = 58 and 2n = 60. Three esterase-D enzyme loci (Est-D1, Est-D2 and Est-D3 were examined and complemented with chromosomal data from 66 piranha specimens collected from Lake Catalão. For all specimens the Est-D1 and Est-D2 loci were monomorphic. In contrast, the Est-D3 locus was polymorphic with genotypes and alleles being differentially distributed in the previously described cytotypes and served as the basis for detecting a new cytotype (2n = 60 B. In cytotype 2n = 58 the Est-D3 locus was also polymorphic and presented Mendelian allelic segregation with four genotypes (Est-D3(11, Est-D3(12, Est-D3(22 and Est-D3(33 out of six theoretically possible genotypes, presumably encoded by alleles Est-D3¹ (frequency = 0.237, EsT-D3² (0.710 and Est-D3³ (0.053. A Chi-squared (chi2 test for Hardy-Weinberg equilibrium was applied to the Est-D3 locus and revealed a genetic unbalance in cytotype 2n = 58, indicating the probable existence in the surveyed area of different stocks for that karyotypic structure. A silent null allele (Est-D3(0 with a high frequency (0.959 occurred exclusively in the 2n = 60 cytotype. On the other hand, the new cytotype 2n = 60 B described here for the first time was monomorphic for the presumably fixed Est-D3³ allele. The data as a whole should contribute to the better understanding the rhombeus complex taxonomic status definition in the Central Amazon.

  11. Serine esterase and hemolytic activity in human cloned cytotoxic T lymphocytes

    OpenAIRE

    1988-01-01

    Target cell lysis by most murine cytotoxic T lymphocytes appears to be mediated by a complement (C9)-like protein called perforin, contained in high-density cytoplasmic granules. These granules also contain high levels of serine esterase activity, which may also play a role in cytolysis. Analysis of 17 cloned human cytotoxic T lymphocytes revealed the presence of serine esterase that is very similar to its murine counterpart in substrate and inhibitor specificities, pH optimum, and molecular ...

  12. The search of the target of promotion: Phenylbenzoate esterase activities in hen peripheral nerve

    International Nuclear Information System (INIS)

    Moretto, A.; Nicolli, A.; Lotti, M.

    2007-01-01

    Certain esterase inhibitors, such as carbamates, phosphinates and sulfonyl halides, do not cause neuropathy as some organophosphates, but they may exacerbate chemical or traumatic insults to axons. This phenomenon is called promotion of axonopathies. Given the biochemical and toxicological characteristics of these compounds, the hypothesis was made that the target of promotion is a phenyl valerate (PV) esterase similar to neuropathy target esterase (NTE), the target of organophosphate induced delayed polyneuropathy. However, attempts to identify a PV esterase in hen peripheral nerve have been, so far, unsuccessful. We tested several esters, other than PV, as substrates of esterases from crude homogenate of the hen peripheral nerve. The ideal substrate should be poorly hydrolysed by NTE but extensively by enzyme(s) that are insensitive to non-promoters, such as mipafox, and sensitive to promoters, such as phenyl methane sulfonyl fluoride (PMSF). When phenyl benzoate (PB) was used as substrate, about 65% of total activity was resistant to the non-promoter mipafox (up to 0.5 mM, 20 min, pH 8.0), that inhibits NTE and other esterases. More than 90% of this resistant activity was sensitive to the classical promoter PMSF (1 mM, 20 min, pH 8.0) with an IC 50 of about 0.08 mM (20 min, pH 8.0). On the contrary, the non-promoter p-toluene sulfonyl fluoride caused only about 10% inhibition at 0.5 mM. Several esterase inhibitors including, paraoxon, phenyl benzyl carbamate, di-n-butyl dichlorovinyl phosphate and di-isopropyl fluorophosphate, were tested both in vitro and in vivo for inhibition of this PB activity. Mipafox-resistant PMSF-sensitive PB esterase activity(ies) was inhibited by promoters but not by non promoters and neuropathic compounds

  13. Esterase inhibition by synergists in the western flower thrips Frankliniella occidentalis.

    Science.gov (United States)

    López-Soler, Neus; Cervera, Amelia; Quinto, Vicente; Abellán, Jaime; Bielza, Pablo; Martínez-Pardo, Rafael; Garcerá, Maria Dolores

    2011-12-01

    Western flower thrips (WFT), Frankliniella occidentalis (Pergande), is among the most important crop pests in the south-eastern region of Spain. Its increasing resistance to insecticides constitutes a serious problem, and understanding the mechanisms involved is therefore of great interest. Use of synergists to inhibit the enzymes involved in insecticide detoxification is widely used to determine their responsibility for insecticide resistance. However, they do not always act as intended or expected, and caution must be exercised when interpreting synergist results. Laboratory-selected strains of WFT were used to analyse the effects of the synergists piperonyl butoxide (PBO), S,S,S-tributyl phosphorotrithioate (DEF) and methiocarb on total esterase activity. Significant differences were found, indicating esterase activity inhibition by DEF, a lower effect for methiocarb and a small inhibition of the activity by PBO. Esterase isoenzyme inhibition by these compounds showed a similar result; this assay revealed an extreme sensitivity of Triplet A (resistance-associated esterases) to DEF. In an in vivo assay carried out with these compounds at different incubation times, only DEF caused posterior in vitro esterase activity inhibition, with a maximum effect 1 h after treatment. In this work, only DEF shows true synergistic inhibition of WFT esterases. Copyright © 2011 Society of Chemical Industry.

  14. Three novel rice genes closely related to the Arabidopsis IRX9, IRX9L, and IRX14 genes and their roles in xylan biosynthesis

    Directory of Open Access Journals (Sweden)

    Dawn eChiniquy

    2013-04-01

    Full Text Available Xylan is the second most abundant polysaccharide on Earth, and represents a major component of both dicot wood and the cell walls of grasses. Much knowledge has been gained from studies of xylan biosynthesis in the model plant, Arabidopsis. In particular, the irregular xylem (irx mutants, named for their collapsed xylem cells, have been essential in gaining a greater understanding of the genes involved in xylan biosynthesis. In contrast, xylan biosynthesis in grass cell walls is poorly understood. We identified three rice genes Os07g49370 (OsIRX9, Os01g48440 (OsIRX9L, and Os06g47340 (OsIRX14, from glycosyltransferase family 43 as putative orthologs to the putative β-1,4-xylan backbone elongating Arabidopsis IRX9, IRX9L, and IRX14 genes, respectively. We demonstrate that the overexpression of the closely related rice genes, in full or partly complement the two well-characterized Arabidopsis irregular xylem (irx mutants: irx9 and irx14. Complementation was assessed by measuring dwarfed phenotypes, irregular xylem cells in stem cross sections, xylose content of stems, xylosyltransferase activity of stems, and stem strength. The expression of OsIRX9 in the irx9 mutant resulted in xylosyltransferase activity of stems that was over double that of wild type plants, and the stem strength of this line increased to 124% above that of wild type. Taken together, our results suggest that OsIRX9/OsIRX9L, and OsIRX14, have similar functions to the Arabidopsis IRX9 and IRX14 genes, respectively. Furthermore, our expression data indicate that OsIRX9 and OsIRX9L may function in building the xylan backbone in the secondary and primary cell walls, respectively. Our results provide insight into xylan biosynthesis in rice and how expression of a xylan synthesis gene may be modified to increase stem strength.

  15. Adhesives for Achieving Durable Bonds with Acetylated Wood

    Science.gov (United States)

    Charles Frihart; Rishawn Brandon; James Beecher; Rebecca Ibach

    2017-01-01

    Acetylation of wood imparts moisture durability, decay resistance, and dimensional stability to wood; however, making durable adhesive bonds with acetylated wood can be more difficult than with unmodified wood. The usual explanation is that the acetylated surface has fewer hydroxyl groups, resulting in a harder-to-wet surface and in fewer hydrogen bonds between wood...

  16. Effect of Drying Pretreatment on the Acetylation of Nanofibrillated Cellulose

    Directory of Open Access Journals (Sweden)

    Vesna Zepič

    2015-10-01

    Full Text Available The aim of this study was to evaluate the effect of different morphologies of solvent-exchanged (NFCSE, spray-dried (NFCSD, and freeze-dried (NFCFD nano-fibrillated cellulose on the susceptibility to surface modification with the acetic anhydride/pyridine system. The degree of substitution (DS, morphology, degree of crystallinity (Icr, hydrophobicity, and thermal stability of acetylated products were examined. Acetylated NFCSD and NFCFD had higher DS than acetylated NFCSE, suggesting that drying pre-treatment increased the susceptibility of NFC for acetylation. The morphology of acetylated NFCFD and NFCSD with higher DS was different from unmodified samples, while that of NFCSE was not affected by acetylation. Microspheres of acetylated NFCSD started to dissolve when the highest DS was reached. As opposed to unmodified NFCFD, the nanofibrillar units of acetylated NFCFD became individualised at lower DS. Acetylated samples had lower Icr than the unmodified samples. A significant increase in the contact angle was observed at higher DS of acetylated NFC samples. Acetylation markedly elevated the thermal stability of the acetylated NFC samples.

  17. The growing landscape of lysine acetylation links metabolism and cell signalling

    DEFF Research Database (Denmark)

    Choudhary, Chuna Ram; Weinert, Brian Tate; Nishida, Yuya

    2014-01-01

    Lysine acetylation is a conserved protein post-translational modification that links acetyl-coenzyme A metabolism and cellular signalling. Recent advances in the identification and quantification of lysine acetylation by mass spectrometry have increased our understanding of lysine acetylation...

  18. CHEMICAL VALORIZATION OF AGRICULTURAL BY-PRODUCTS: ISOLATION AND CHARACTERIZATION OF XYLAN-BASED ANTIOXIDANTS FROM ALMOND SHELL BIOMASS

    Directory of Open Access Journals (Sweden)

    Anna Ebringerová

    2008-02-01

    Full Text Available The isolation of non-cellulosic polysaccharides from both almond shells and their solid residue after autohydrolysis using a two-step alkaline extraction without and in combination with short ultrasonic treatment was investigated. The obtained polysaccharide preparations were characterized by yield, chemical composition and structural features, and the antioxidant activity of the water-soluble preparations was discussed in relation to the content of phenolics. The results suggested that, depending on the extraction conditions used, xylan associated to various extent with pectic polysaccharides and phenolics can be prepared, and the reaction time significantly shortened by application of ultrasound. The xylan polymers might serve as biopolymer sources in native form or after targeted modification for production of value-added substances and polysaccharide-based antioxidants, applicable in food, cosmetics and other areas.

  19. Switching catalysis from hydrolysis to perhydrolysis in P. fluorescens esterase

    Science.gov (United States)

    Yin, De Lu (Tyler); Bernhardt, Peter; Morley, Krista L.; Jiang, Yun; Cheeseman, Jeremy D.; Purpero, Vincent; Schrag, Joseph D.; Kazlauskas, Romas J.

    2010-01-01

    Many serine hydrolases catalyze perhydrolysis – the reversible formation of per-acids from carboxylic acids and hydrogen peroxide. Recently we showed that a single amino acid substitution in the alcohol binding pocket - L29P - in Pseudomonas fluorescens (SIK WI) aryl esterase (PFE) increased the specificity constant of PFE for peracetic acid formation >100-fold [Bernhardt et al. Angew. Chem. Intl. Ed. 2005, 44, 2742]. In this paper, we extend this work to address the three following questions. First, what is the molecular basis of the increase in perhydrolysis activity? We previously proposed that the L29P substitution creates a hydrogen bond between the enzyme and hydrogen peroxide in the transition state. Here we report two x-ray structures of L29P PFE that support this proposal. Both structures show a main chain carbonyl oxygen closer to the active-site serine as expected. One structure further shows acetate in the active site in an orientation consistent with reaction by an acyl-enzyme mechanism. We also detected an acyl-enzyme intermediate in the hydrolysis of ε-caprolactone by mass spectrometry. Second, can we further increase perhydrolysis activity? We discovered that the reverse reaction – hydrolysis of peracetic acid to acetic acid and hydrogen peroxide – occurs at nearly the diffusion limited rate. Since the reverse reaction cannot increase further, neither can the forward reaction. Consistent with this prediction, two variants with additional amino acid substitutions showed two fold higher kcat, but Km also increased so the specificity constant, kcat/Km, remained similar. Third, how does the L29P substitution change the esterase activity? Ester hydrolysis decreased for most esters (75-fold for ethyl acetate), but not for methyl esters. In contrast, L29P PFE catalyzed hydrolysis of ε-caprolactone five times more efficiently than wild-type PFE. Molecular modeling suggests that moving the carbonyl group closer to the active site blocks access for

  20. Switching Catalysis from Hydrolysis to Perhydrolysis in Pseudomonas fluorescens Esterase

    Energy Technology Data Exchange (ETDEWEB)

    Yin, D.; Bernhardt, P; Morley, K; Jiang, Y; Cheeseman, J; Purpero, V; Schrag, J; Kazlauskas, R

    2010-01-01

    Many serine hydrolases catalyze perhydrolysis, the reversible formation of peracids from carboxylic acids and hydrogen peroxide. Recently, we showed that a single amino acid substitution in the alcohol binding pocket, L29P, in Pseudomonas fluorescens (SIK WI) aryl esterase (PFE) increased the specificity constant of PFE for peracetic acid formation >100-fold [Bernhardt et al. (2005) Angew. Chem., Int. Ed. 44, 2742]. In this paper, we extend this work to address the three following questions. First, what is the molecular basis of the increase in perhydrolysis activity? We previously proposed that the L29P substitution creates a hydrogen bond between the enzyme and hydrogen peroxide in the transition state. Here we report two X-ray structures of L29P PFE that support this proposal. Both structures show a main chain carbonyl oxygen closer to the active site serine as expected. One structure further shows acetate in the active site in an orientation consistent with reaction by an acyl-enzyme mechanism. We also detected an acyl-enzyme intermediate in the hydrolysis of {var_epsilon}-caprolactone by mass spectrometry. Second, can we further increase perhydrolysis activity? We discovered that the reverse reaction, hydrolysis of peracetic acid to acetic acid and hydrogen peroxide, occurs at nearly the diffusion limited rate. Since the reverse reaction cannot increase further, neither can the forward reaction. Consistent with this prediction, two variants with additional amino acid substitutions showed 2-fold higher k{sub cat}, but K{sub m} also increased so the specificity constant, k{sub cat}/K{sub m}, remained similar. Third, how does the L29P substitution change the esterase activity? Ester hydrolysis decreased for most esters (75-fold for ethyl acetate) but not for methyl esters. In contrast, L29P PFE catalyzed hydrolysis of {var_epsilon}-caprolactone five times more efficiently than wild-type PFE. Molecular modeling suggests that moving the carbonyl group closer to the

  1. Enzymic hydrolysis of xylans. I. A high xylanase and beta-xylosidase producing strain of Aspergillus niger

    Energy Technology Data Exchange (ETDEWEB)

    Conrad, D.

    1981-01-01

    Aspergillus niger, strain 110.42 (CBS) was selected as a producer of high xylanolytic activities. The time course of xylanase and beta-xylosidase production as well as the effect of pH and temperature on the activity of these enzymes were studied. High-performance liquid chromatography analysis of the enzymic degradation of arabinoxylan showed a nearly complete conversion to pentose sugars. Aspects of using crude xylanase preparations for enzymic saccharification of xylans are discussed.

  2. Facilitating the enzymatic saccharification of pulped bamboo residues by degrading the remained xylan and lignin-carbohydrates complexes.

    Science.gov (United States)

    Huang, Caoxing; He, Juan; Li, Xin; Min, Douyong; Yong, Qiang

    2015-09-01

    Kraft pulping was performed on bamboo residues and its impact on the chemical compositions and the enzymatic digestibility of the samples were investigated. To improve the digestibility of sample by degrading the xylan and lignin-carbohydrates complexes (LCCs), xylanase and α-L-arabinofuranosidase (AF) were supplemented with cellulase. The results showed more carbohydrates were remained in the samples pulped with low effective alkali (EA) charge, compared to conventional kraft pulping. When 120 IU/g xylanase and 15 IU/g AF were supplemented with 20 FPU/g cellulase, the xylan degradation yield of the sample pulped with 12% EA charge increased from 68.20% to 88.35%, resulting in an increased enzymatic saccharification efficiency from 58.98% to 83.23%. The amount of LCCs in this sample decreased from 8.63/100C9 to 2.99/100C9 after saccharification with these enzymes. The results indicated that degrading the remained xylan and LCCs in the pulp could improve its enzymatic digestibility. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Direct transformation of xylan-type hemicelluloses to furfural via SnCl₄ catalysts in aqueous and biphasic systems.

    Science.gov (United States)

    Wang, Wenju; Ren, Junli; Li, Huiling; Deng, Aojie; Sun, Runcang

    2015-05-01

    Direct catalytic transformation of xylan-type hemicelluloses to furfural in the aqueous system and the biphasic system were comparatively investigated under mild conditions. Screening of several promising chlorides for conversion of beech xylan in the aqueous system revealed the Lewis acid SnCl4 was the most effective catalyst. Comparing to the single aqueous system, the bio-based 2-methyltetrahydrofuran (2-MTHF)/H2O biphasic system was more conducive to the synthesis of furfural, in which the highest furfural yield of 78.1% was achieved by using SnCl4 as catalysts under the optimized reaction conditions (150°C, 120 min). Additionally, the influences of xylan-type hemicelluloses with different chemical and structural features from beech, corncob and bagasse on the furfural production were studied. It was found that furfural yield to some extent was determined by the xylose content in hemicelluloses and also had relationships with the molecular weight of hemicelluloses and the degree of crystallization. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. NetAcet: prediction of N-terminal acetylation sites

    DEFF Research Database (Denmark)

    Kiemer, Lars; Bendtsen, Jannick Dyrløv; Blom, Nikolaj

    2005-01-01

    Summary: We present here a neural network based method for prediction of N-terminal acetylation-by far the most abundant post-translational modification in eukaryotes. The method was developed on a yeast dataset for N-acetyltransferase A (NatA) acetylation, which is the type of N-acetylation for ......Summary: We present here a neural network based method for prediction of N-terminal acetylation-by far the most abundant post-translational modification in eukaryotes. The method was developed on a yeast dataset for N-acetyltransferase A (NatA) acetylation, which is the type of N...

  5. Structural analysis of thermostabilizing mutations of cocaine esterase

    Energy Technology Data Exchange (ETDEWEB)

    Narasimhan, Diwahar; Nance, Mark R.; Gao, Daquan; Ko, Mei-Chuan; Macdonald, Joanne; Tamburi, Patricia; Yoon, Dan; Landry, Donald M.; Woods, James H.; Zhan, Chang-Guo; Tesmer, John J.G.; Sunahara, Roger K. (Michigan); (Columbia); (Kentucky)

    2010-09-03

    Cocaine is considered to be the most addictive of all substances of abuse and mediates its effects by inhibiting monoamine transporters, primarily the dopamine transporters. There are currently no small molecules that can be used to combat its toxic and addictive properties, in part because of the difficulty of developing compounds that inhibit cocaine binding without having intrinsic effects on dopamine transport. Most of the effective cocaine inhibitors also display addictive properties. We have recently reported the use of cocaine esterase (CocE) to accelerate the removal of systemic cocaine and to prevent cocaine-induced lethality. However, wild-type CocE is relatively unstable at physiological temperatures ({tau}{sub 1/2} {approx} 13 min at 37 C), presenting challenges for its development as a viable therapeutic agent. We applied computational approaches to predict mutations to stabilize CocE and showed that several of these have increased stability both in vitro and in vivo, with the most efficacious mutant (T172R/G173Q) extending half-life up to 370 min. Here we present novel X-ray crystallographic data on these mutants that provide a plausible model for the observed enhanced stability. We also more extensively characterize the previously reported variants and report on a new stabilizing mutant, L169K. The improved stability of these engineered CocE enzymes will have a profound influence on the use of this protein to combat cocaine-induced toxicity and addiction in humans.

  6. Acetylcholine esterase activity in mild cognitive impairment and Alzheimer's disease

    International Nuclear Information System (INIS)

    Herholz, Karl

    2008-01-01

    Impairment of cholinergic neurotransmission is a well-established fact in Alzheimer's disease (AD), but there is controversy about its relevance at the early stages of the disease and in mild cognitive impairment (MCI). In vivo positron emission tomography imaging of cortical acetylcholine esterase (AChE) activity as a marker of cholinergic innervation that is expressed by cholinergic axons and cholinoceptive neurons has demonstrated a reduction of this enzyme activity in manifest AD. The technique is also useful to measure the inhibition of cerebral AChE induced by cholinesterase inhibitors for treatment of dementia symptoms. A reduction of cortical AchE activity was found consistently in all studies of AD and in few cases of MCI who later concerted to AD. The in vivo findings in MCI and very mild AD are still preliminary, and studies seem to suggest that cholinergic innervation and AChE as the main degrading enzyme are both reduced, which might result in partial compensation of their effect. (orig.)

  7. Acetylation and characterization of banana (Musa paradisiaca) starch.

    Science.gov (United States)

    Bello-Pérez, L A; Contreras-Ramos, S M; Jìmenez-Aparicio, A; Paredes-López, O

    2000-01-01

    Banana native starch was acetylated and some of its functional properties were evaluated and compared to corn starch. In general, acetylated banana starch presented higher values in ash, protein and fat than corn acetylated starch. The modified starches had minor tendency to retrogradation assessed as % transmittance of starch pastes. At high temperature acetylated starches presented a water retention capacity similar to their native counterpart. The acetylation considerably increased the solubility of starches, and a similar behavior was found for swelling power. When freeze-thaw stability was studied, acetyl banana starch drained approximately 60% of water in the first and second cycles, but in the third and fourth cycles the percentage of separated water was low. However, acetyl corn starch showed lower freeze-thaw stability than the untreated sample. The modification increased the viscosity of banana starch pastes.

  8. Enhanced biosurfactant production through cloning of three genes and role of esterase in biosurfactant release

    Science.gov (United States)

    2011-01-01

    Background Biosurfactants have been reported to utilize a number of immiscible substrates and thereby facilitate the biodegradation of panoply of polyaromatic hydrocarbons. Olive oil is one such carbon source which has been explored by many researchers. However, studying the concomitant production of biosurfactant and esterase enzyme in the presence of olive oil in the Bacillus species and its recombinants is a relatively novel approach. Results Bacillus species isolated from endosulfan sprayed cashew plantation soil was cultivated on a number of hydrophobic substrates. Olive oil was found to be the best inducer of biosurfactant activity. The protein associated with the release of the biosurfactant was found to be an esterase. There was a twofold increase in the biosurfactant and esterase activities after the successful cloning of the biosurfactant genes from Bacillus subtilis SK320 into E.coli. Multiple sequence alignment showed regions of similarity and conserved sequences between biosurfactant and esterase genes, further confirming the symbiotic correlation between the two. Biosurfactants produced by Bacillus subtilis SK320 and recombinant strains BioS a, BioS b, BioS c were found to be effective emulsifiers, reducing the surface tension of water from 72 dynes/cm to as low as 30.7 dynes/cm. Conclusion The attributes of enhanced biosurfactant and esterase production by hyper-producing recombinant strains have many utilities from industrial viewpoint. This study for the first time has shown a possible association between biosurfactant production and esterase activity in any Bacillus species. Biosurfactant-esterase complex has been found to have powerful emulsification properties, which shows promising bioremediation, hydrocarbon biodegradation and pharmaceutical applications. PMID:21707984

  9. Gender differences in the activities of aspirin-esterases in rat tissues

    Directory of Open Access Journals (Sweden)

    Benedito M.A.C.

    1998-01-01

    Full Text Available The activities of aspirin (acetylsalicylic acid-esterases were measured in several tissues (liver, kidney, adrenal glands, brain and serum from adult male and female Wistar rats. In males, both aspirin-esterase I (assayed at pH 5.5 and II (assayed at pH 7.4 activities were higher in liver homogenates when compared to females (aspirin-esterase I: males 48.9 ± 4.8 (N = 8 and females 29.3 ± 4.2 (N = 8 nmol of salicylic acid formed min-1 mg protein-1; aspirin-esterase II: males 41.4 ± 4.1 (N = 8 and females 26.1 ± 4.5 (N = 8 nmol of salicylic acid formed min-1 mg protein-1, P<0.001. In serum, enzyme activity was higher in females than in males (aspirin-esterase I: males 0.85 ± 0.06 (N = 6 and females 1.18 ± 0.11 (N = 6 nmol of salicylic acid formed min-1 mg protein-1; aspirin-esterase II: males 1.03 ± 0.13 (N = 6 and females 1.34 ± 0.11 (N = 6 nmol of salicylic acid formed min-1 mg protein-1, P<0.001. In the other tissues assayed, no statistically significant difference between males and females was found. There were no statistically significant differences when the enzymes were assayed in different phases of the estrous cycle in liver and serum. These results show that the differences in aspirin-esterase activity observed between males and females are not due to the estrous cycle. The gender difference obtained in our study may indicate an involvement of gonadal hormones in the control of the hydrolysis of aspirin. This possibility is currently under investigation.

  10. Altered Expression of Genes Implicated in Xylan Biosynthesis Affects Penetration Resistance against Powdery Mildew.

    Science.gov (United States)

    Chowdhury, Jamil; Lück, Stefanie; Rajaraman, Jeyaraman; Douchkov, Dimitar; Shirley, Neil J; Schwerdt, Julian G; Schweizer, Patrick; Fincher, Geoffrey B; Burton, Rachel A; Little, Alan

    2017-01-01

    Heteroxylan has recently been identified as an important component of papillae, which are formed during powdery mildew infection of barley leaves. Deposition of heteroxylan near the sites of attempted fungal penetration in the epidermal cell wall is believed to enhance the physical resistance to the fungal penetration peg and hence to improve pre-invasion resistance. Several glycosyltransferase (GT) families are implicated in the assembly of heteroxylan in the plant cell wall, and are likely to work together in a multi-enzyme complex. Members of key GT families reported to be involved in heteroxylan biosynthesis are up-regulated in the epidermal layer of barley leaves during powdery mildew infection. Modulation of their expression leads to altered susceptibility levels, suggesting that these genes are important for penetration resistance. The highest level of resistance was achieved when a GT43 gene was co-expressed with a GT47 candidate gene, both of which have been predicted to be involved in xylan backbone biosynthesis. Altering the expression level of several candidate heteroxylan synthesis genes can significantly alter disease susceptibility. This is predicted to occur through changes in the amount and structure of heteroxylan in barley papillae.

  11. Fragrance material review on acetyl carene.

    Science.gov (United States)

    Scognamiglio, J; Letizia, C S; Api, A M

    2013-12-01

    A toxicologic and dermatologic review of acetyl carene when used as a fragrance ingredient is presented. Acetyl carene is a member of the fragrance structural group Alkyl Cyclic Ketones. These fragrances can be described as being composed of an alkyl, R1, and various substituted and bicyclic saturated or unsaturated cyclic hydrocarbons, R2, in which one of the rings may include up to 12 carbons. Alternatively, R2 may be a carbon bridge of C2-C4 carbon chain length between the ketone and cyclic hydrocarbon. This review contains a detailed summary of all available toxicology and dermatology papers that are related to this individual fragrance ingredient and is not intended as a stand-alone document. Available data for acetyl carene were evaluated then summarized and includes physical properties, acute toxicity, skin irritation, and skin sensitization data. A safety assessment of the entire Alkyl Cyclic Ketones will be published simultaneously with this document; please refer to Belsito et al. (Belsito, D., Bickers, D., Bruze, M., Calow, P., Dagli, M., Fryer, A.D., Greim, H., Miyachi, Y., Saurat, J.H., Sipes, I.G., 2013A Toxicologic and dermatologic assessment of alkyl cyclic ketones when used as fragrance ingredients. (submitted for publication).) for an overall assessment of the safe use of this material and all Alkyl Cyclic Ketones in fragrances. Copyright © 2013. Published by Elsevier Ltd.

  12. Expanding the feruloyl esterase gene family of Aspergillus niger by characterization of a feruloyl esterase, FaeC.

    Science.gov (United States)

    Dilokpimol, Adiphol; Mäkelä, Miia R; Mansouri, Sadegh; Belova, Olga; Waterstraat, Martin; Bunzel, Mirko; de Vries, Ronald P; Hildén, Kristiina S

    2017-07-25

    A feruloyl esterase (FAE) from Aspergillus niger N402, FaeC was heterologously produced in Pichia pastoris X-33 in a yield of 10mg/L. FaeC was most active at pH 7.0 and 50°C, and showed broad substrate specificity and catalyzed the hydrolysis of methyl 3,4-dimethoxycinnamate, ethyl ferulate, methyl ferulate, methyl p-coumarate, ethyl coumarate, methyl sinapate, and methyl caffeate. The enzyme released both ferulic acid and p-coumaric acid from wheat arabinoxylan and sugar beet pectin (up to 3mg/g polysaccharide), and acted synergistically with a commercial xylanase increasing the release of ferulic acid up to six-fold. The expression of faeC increased over time in the presence of feruloylated polysaccharides. Cinnamic, syringic, caffeic, vanillic and ferulic acid induced the expression of faeC. Overall expression of faeC was very low in all tested conditions, compared to two other A. niger FAE encoding genes, faeA and faeB. Our data showed that the fae genes responded differently towards the feruloylated polysaccharides and tested monomeric phenolic compounds suggesting that the corresponding FAE isoenzymes may target different substrates in a complementary manner. This may increase the efficiency of the degradation of diverse plant biomass. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome.

    Science.gov (United States)

    Privé, Florence; Newbold, C Jamie; Kaderbhai, Naheed N; Girdwood, Susan G; Golyshina, Olga V; Golyshin, Peter N; Scollan, Nigel D; Huws, Sharon A

    2015-07-01

    Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78% following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses.

  14. Solid-state fermentation as a potential technique for esterase/lipase production by halophilic archaea.

    Science.gov (United States)

    Martin del Campo, Martha; Camacho, Rosa M; Mateos-Díaz, Juan C; Müller-Santos, Marcelo; Córdova, Jesus; Rodríguez, Jorge A

    2015-11-01

    Halophilic archaea are extremophiles, adapted to high-salt environments, showing a big biotechnological potential as enzyme, lipids and pigments producers. Four inert supports (perlite, vermiculite, polyurethane foam and glass fiber) were employed for solid-state fermentation (SSF) of the halophilic archaeon Natronococcus sp. TC6 to investigate biomass and esterase production. A very low esterase activity and high water activity were observed when perlite, vermiculite and polyurethane were used as supports. When glass fiber was employed, an important moisture loss was observed (8.6%). Moreover, moisture retention was improved by mixing polyurethane and glass fiber, resulting in maximal biomass and esterase production. Three halophilic archaea: Natronococcus sp. TC6, Halobacterium sp. NRC-1 and Haloarcula marismortui were cultured by submerged fermentation (SmF) and by SSF; an improvement of 1.3- to 6.2-fold was observed in the biomass and esterase production when SSF was used. Growth was not homogeneous in the mixture, but was predominant in the glass fiber thus was probably because the glass fiber provides a holder to the cells, while the polyurethane acts as an impregnation medium reservoir. To the best of our knowledge, this work is the first report on haloarchaea cultivation by SSF aiming biomass and esterase/lipase activity production.

  15. Esterase Active in Polar Organic Solvents from the Yeast Pseudozyma sp. NII 08165

    Directory of Open Access Journals (Sweden)

    Deepthy Alex

    2014-01-01

    Full Text Available Esterases/lipases active in water miscible solvents are highly desired in biocatalysis where substrate solubility is limited and also when the solvent is desired as an acyl acceptor in transesterification reactions, as with the case of biodiesel production. We have isolated an esterase from the glycolipid producing yeast-Pseudozyma sp. NII 08165 which in its crude form was alkali active, thermo stable, halo tolerant and also capable of acting in presence of high methanol concentration. The crude enzyme which maintained 90% of its original activity after being treated at 70°C was purified and the properties were characterized. The partially purified esterase preparation had temperature and pH optima of 60°C and 8.0 respectively. The enzyme retained almost complete activity in presence of 25% methanol and 80% activity in the same strength of ethanol. Conditions of enzyme production were optimized, which lead to 9 fold increase in the esterase yield. One of the isoforms of the enzyme LIP1 was purified to homogeneity and characterized. Purified LIP1 had a Km and Vmax of 0.01 and 1.12, respectively. The purified esterase lost its thermo and halo tolerance but interestingly, retained 97% activity in methanol.

  16. Cellular function of neuropathy target esterase in lysophosphatidylcholine action

    International Nuclear Information System (INIS)

    Vose, Sarah C.; Fujioka, Kazutoshi; Gulevich, Alex G.; Lin, Amy Y.; Holland, Nina T.; Casida, John E.

    2008-01-01

    Neuropathy target esterase (NTE) plays critical roles in embryonic development and maintenance of peripheral axons. It is a secondary target of some organophosphorus toxicants including analogs of insecticides and chemical warfare agents. Although the mechanistic role of NTE in vivo is poorly defined, it is known to hydrolyze lysophosphatidylcholine (LPC) in vitro and may protect cell membranes from cytotoxic accumulation of LPC. To determine the cellular function of NTE, Neuro-2a and COS-7 cells were transfected with a full-length human NTE-containing plasmid yielding recombinant NTE (rNTE). We find the same inhibitor sensitivity and specificity profiles for rNTE assayed with LPC or phenyl valerate (a standard NTE substrate) and that this correlation extends to the LPC hydrolases of human brain, lymphocytes and erythrocytes. All of these LPC hydrolases are therefore very similar to each other in respect to a conserved inhibitor binding site conformation. NTE is expressed in brain and lymphocytes and contributes to LPC hydrolase activities in these tissues. The enzyme or enzymes responsible for erythrocyte LPC hydrolase activity remain to be identified. We also show that rNTE protects Neuro-2a and COS-7 cells from exogenous LPC cytotoxicity. Expression of rNTE in Neuro-2a cells alters their phospholipid balance (analyzed by liquid chromatography-mass spectrometry with single ion monitoring) by lowering LPC-16:0 and LPC-18:0 and elevating glycerophosphocholine without a change in phosphatidylcholine-16:0/18:1 or 16:0/18:2. NTE therefore serves an important function in LPC homeostasis and action

  17. Xylan degradation by the human gut Bacteroides xylanisolvens XB1A(T) involves two distinct gene clusters that are linked at the transcriptional level.

    Science.gov (United States)

    Despres, Jordane; Forano, Evelyne; Lepercq, Pascale; Comtet-Marre, Sophie; Jubelin, Gregory; Chambon, Christophe; Yeoman, Carl J; Berg Miller, Margaret E; Fields, Christopher J; Martens, Eric; Terrapon, Nicolas; Henrissat, Bernard; White, Bryan A; Mosoni, Pascale

    2016-05-04

    Plant cell wall (PCW) polysaccharides and especially xylans constitute an important part of human diet. Xylans are not degraded by human digestive enzymes in the upper digestive tract and therefore reach the colon where they are subjected to extensive degradation by some members of the symbiotic microbiota. Xylanolytic bacteria are the first degraders of these complex polysaccharides and they release breakdown products that can have beneficial effects on human health. In order to understand better how these bacteria metabolize xylans in the colon, this study was undertaken to investigate xylan breakdown by the prominent human gut symbiont Bacteroides xylanisolvens XB1A(T). Transcriptomic analyses of B. xylanisolvens XB1A(T) grown on insoluble oat-spelt xylan (OSX) at mid- and late-log phases highlighted genes in a polysaccharide utilization locus (PUL), hereafter called PUL 43, and genes in a fragmentary remnant of another PUL, hereafter referred to as rPUL 70, which were highly overexpressed on OSX relative to glucose. Proteomic analyses supported the up-regulation of several genes belonging to PUL 43 and showed the important over-production of a CBM4-containing GH10 endo-xylanase. We also show that PUL 43 is organized in two operons and that the knockout of the PUL 43 sensor/regulator HTCS gene blocked the growth of the mutant on insoluble OSX and soluble wheat arabinoxylan (WAX). The mutation not only repressed gene expression in the PUL 43 operons but also repressed gene expression in rPUL 70. This study shows that xylan degradation by B. xylanisolvens XB1A(T) is orchestrated by one PUL and one PUL remnant that are linked at the transcriptional level. Coupled to studies on other xylanolytic Bacteroides species, our data emphasize the importance of one peculiar CBM4-containing GH10 endo-xylanase in xylan breakdown and that this modular enzyme may be used as a functional marker of xylan degradation in the human gut. Our results also suggest that B. xylanisolvens

  18. Enzymatic degradation of lignin‐carbohydrate complexes (LCCs): Model studies using a fungal glucuronoyl esterase from Cerrena unicolor

    DEFF Research Database (Denmark)

    d'Errico, Clotilde; Jørgensen, Jonas O.; Krogh, Kristian B. R. M.

    2015-01-01

    Lignin‐carbohydrate complexes (LCCs) are believed to influence the recalcitrance of lignocellulosic plant material preventing optimal utilization of biomass in e.g. forestry, feed and biofuel applications. The recently emerged carbohydrate esterase (CE) 15 family of glucuronoyl esterases (GEs) has...

  19. Preparation of Polyvinyl Alcohol/Xylan Blending Films with 1,2,3,4-Butane Tetracarboxylic Acid as a New Plasticizer

    Directory of Open Access Journals (Sweden)

    Cun-dian Gao

    2014-01-01

    Full Text Available Miscible, biodegradable polyvinyl alcohol (PVA/xylan blending films were firstly prepared in the range of the PVA/xylan weight ratio from 1 : 2 to 3 : 1 by casting method using 1,2,3,4-butane tetracarboxylic acid (BTCA as a new plasticizer. The properties of blending films as functions of PVA/xylan weight ratio and BTCA amount were discussed. XRD and FT-IR were applied to characterize the blending films. Experimental results indicated that tensile strength (TS and elongation at break (EAB of blending films decreased along with the decrease of the PVA/xylan weight ratio. Both of TS and EAB firstly increased and then decreased as the amount of BTCA was increased. More importantly, blending films were biodegraded almost by 41% with an addition of 10% BTCA in blending films within 30 days in soil. For all hydroxyl functionalized polymers (xylan and PVA, their molecular interactions and miscibility with BTCA endowed blending films with the biocompatibility and biodegradability. Therefore, these blending films are environmentally friendly materials which could be applied as biodegradable plastics for food packaging and agricultural applications.

  20. Fragrance material review on acetyl cedrene.

    Science.gov (United States)

    Scognamiglio, J; Letizia, C S; Politano, V T; Api, A M

    2013-12-01

    A toxicologic and dermatologic review of acetyl cedrene when used as a fragrance ingredient is presented. Acetyl cedrene is a member of the fragrance structural group Alkyl Cyclic Ketones. The generic formula for this group can be represented as (R1)(R2)CO. These fragrances can be described as being composed of an alkyl, R1, and various substituted and bicyclic saturated or unsaturated cyclic hydrocarbons, R2, in which one of the rings may include up to 12 carbons. Alternatively, R2 may be a carbon bridge of C2-C4 carbon chain length between the ketone and cyclic hydrocarbon. This review contains a detailed summary of all available toxicology and dermatology papers that are related to this individual fragrance ingredient and is not intended as a stand-alone document. Available data for acetyl cedrene were evaluated then summarized and includes physical properties, acute toxicity, skin irritation, mucous membrane (eye) irritation, skin sensitization, elicitation, phototoxicity, photoallergy, toxicokinetics, repeated dose, reproductive toxicity, and genotoxicity data. A safety assessment of the entire Alkyl Cyclic Ketones will be published simultaneously with this document; please refer to Belsito et al. (2013) (Belsito, D., Bickers, D., Bruze, M., Calow, P., Dagli, M., Fryer, A.D., Greim, H., Miyachi, Y., Saurat, J.H., Sipes, I.G., 2013. A Toxicologic and Dermatologic Assessment of Alkyl Cyclic Ketones When Used as Fragrance Ingredients. Submitted with this manuscript.) for an overall assessment of the safe use of this material and all Alkyl Cyclic Ketones in fragrances. Copyright © 2013. Published by Elsevier Ltd.

  1. Contribution of soil esterase to biodegradation of aliphatic polyester agricultural mulch film in cultivated soils.

    Science.gov (United States)

    Yamamoto-Tamura, Kimiko; Hiradate, Syuntaro; Watanabe, Takashi; Koitabashi, Motoo; Sameshima-Yamashita, Yuka; Yarimizu, Tohru; Kitamoto, Hiroko

    2015-01-01

    The relationship between degradation speed of soil-buried biodegradable polyester film in a farmland and the characteristics of the predominant polyester-degrading soil microorganisms and enzymes were investigated to determine the BP-degrading ability of cultivated soils through characterization of the basal microbial activities and their transition in soils during BP film degradation. Degradation of poly(butylene succinate-co-adipate) (PBSA) film was evaluated in soil samples from different cultivated fields in Japan for 4 weeks. Both the degradation speed of the PBSA film and the esterase activity were found to be correlated with the ratio of colonies that produced clear zone on fungal minimum medium-agarose plate with emulsified PBSA to the total number colonies counted. Time-dependent change in viable counts of the PBSA-degrading fungi and esterase activities were monitored in soils where buried films showed the most and the least degree of degradation. During the degradation of PBSA film, the viable counts of the PBSA-degrading fungi and the esterase activities in soils, which adhered to the PBSA film, increased with time. The soil, where the film was degraded the fastest, recorded large PBSA-degrading fungal population and showed high esterase activity compared with the other soil samples throughout the incubation period. Meanwhile, esterase activity and viable counts of PBSA-degrading fungi were found to be stable in soils without PBSA film. These results suggest that the higher the distribution ratio of native PBSA-degrading fungi in the soil, the faster the film degradation is. This could be due to the rapid accumulation of secreted esterases in these soils.

  2. The effect of EDTA and metal cations on the 5-bromoindoxyl acetate esterase activity in the thyroid of the guinea pig

    DEFF Research Database (Denmark)

    Kirkeby, S

    1976-01-01

    Miscellaneous metal cations and EDTA have been used as activators and inhibitors of esterase activity in the thyroid of the guinea-pig. The results indicate that the 5-bromoiondoxyl acetate esterase in the epithelial cells probably consists of two different A-esterase isoenzymes, one present...

  3. Insights into the effect of N-acetyl-L-cysteine-capped CdTe quantum dots on the structure and activity of human serum albumin by spectroscopic techniques

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Haoyu; Yang, Xudan; Li, Meng; Han, Songlin; Liu, Yingxue [School of Environmental Science and Engineering, Shandong University, China-America CRC for Environment & Health, Shandong Province, 27# Shanda South Road, Jinan 250100 (China); Tan, Xuejie [School of Chemistry and Pharmaceutical Engineering, Qilu University of Technology, Jinan, Shandong Province 250353 (China); Liu, Chunguang, E-mail: chunguangliu2013@sdu.edu.cn [School of Environmental Science and Engineering, Shandong University, China-America CRC for Environment & Health, Shandong Province, 27# Shanda South Road, Jinan 250100 (China); Liu, Rutao [School of Environmental Science and Engineering, Shandong University, China-America CRC for Environment & Health, Shandong Province, 27# Shanda South Road, Jinan 250100 (China)

    2015-11-15

    Quantum dots (QDs) are a kind of nanostructured semiconductor crystals with the size range of 1–10 nm. Their unique photophysical properties and potential toxicity to human health have aroused wide concern of scientists and general public. However, the interaction mechanism of QDs on human serum albumin (HSA, the vital protein in human blood) from both structural and functional perspectives is rarely reported. In the present work, effects of N-acetyl-L-cysteine-capped CdTe quantum dots with fluorescence emission peak at 612 nm (QDs-612) on the conformation and function of HSA were investigated by spectroscopic methods, molecular docking study and esterase activity assay. The hydrophobic interaction between HSA and QDs-612 was spontaneous with the binding constants calculated to be 6.85×10{sup 5} L mol{sup −1} (298 K) and 8.89×10{sup 5} L mol{sup −1} (308 K). The binding of QDs-612 to HSA induced the static quenching of fluorescence and the changes of secondary structure and microenvironment of Tyr-411 residue, which resulted in serious decrease on the hydrolysis of substrate p-nitrophenylacetate in esterase activity assay of HSA. This work confirms the possibility on direct interaction of QDs-612 with HSA and obtains a possible mechanism of relationship between conformation and function of HSA. - Highlights: • The interaction between CdTe QDs (QDs-612) and HSA is spontaneous. • The predominant force of the binding is hydrophobic interaction. • The interaction changes the secondary structure of HSA. • Tyr-411 residue of HSA expose to a hydrophilic environment. • The esterase activity of HSA decreases by adding QDs-612.

  4. Zymography Detection of a Bacterial Extracellular Thermoalkaline Esterase/Lipase Activity.

    Science.gov (United States)

    Tapizquent, María; Fernández, Marleny; Barreto, Georgina; Hernández, Zully; Contreras, Lellys M; Kurz, Liliana; Wilkesman, Jeff

    2017-01-01

    Lipases are esterases that occur widely in nature, yet those with commercial relevance are exclusively from microbial origin. Glycerol and long-chain fatty acids are the products after hydrolysis of esters bonds in saponifiable lipids catalyzed by lipases. In this work, we describe lipase/esterase activity contained in cell-free fractions from thermophilic bacteria, cultured in medium containing olive oil. Analysis of the cell-free fractions by electrotransference zymography, using tributyrin as substrate, revealed bands corresponding to lipase activity. The method is simple, fast, and inexpensive.

  5. Differential patterns of histone acetylation in inflammatory bowel diseases

    Directory of Open Access Journals (Sweden)

    Adcock Ian M

    2011-01-01

    Full Text Available Abstract Post-translational modifications of histones, particularly acetylation, are associated with the regulation of inflammatory gene expression. We used two animal models of inflammation of the bowel and biopsy samples from patients with Crohn's disease (CD to study the expression of acetylated histones (H 3 and 4 in inflamed mucosa. Acetylation of histone H4 was significantly elevated in the inflamed mucosa in the trinitrobenzene sulfonic acid model of colitis particularly on lysine residues (K 8 and 12 in contrast to non-inflamed tissue. In addition, acetylated H4 was localised to inflamed tissue and to Peyer's patches (PP in dextran sulfate sodium (DSS-treated rat models. Within the PP, H3 acetylation was detected in the mantle zone whereas H4 acetylation was seen in both the periphery and the germinal centre. Finally, acetylation of H4 was significantly upregulated in inflamed biopsies and PP from patients with CD. Enhanced acetylation of H4K5 and K16 was seen in the PP. These results demonstrate that histone acetylation is associated with inflammation and may provide a novel therapeutic target for mucosal inflammation.

  6. Structure, morphology and functionality of acetylated and oxidised barley starches.

    Science.gov (United States)

    El Halal, Shanise Lisie Mello; Colussi, Rosana; Pinto, Vânia Zanella; Bartz, Josiane; Radunz, Marjana; Carreño, Neftali Lenin Villarreal; Dias, Alvaro Renato Guerra; Zavareze, Elessandra da Rosa

    2015-02-01

    Acetylation and oxidation are chemical modifications which alter the properties of starch. The degree of modification of acetylated and oxidized starches is dependent on the catalyst and active chlorine concentrations, respectively. The objective of this study was to evaluate the effect of acetylation and oxidation on the structural, morphological, physical-chemical, thermal and pasting properties of barley starch. Barley starches were acetylated at different catalyst levels (11%, 17%, and 23% of NaOH solution) and oxidized at different sodium hypochlorite concentrations (1.0%, 1.5%, and 2.0% of active chlorine). Fourier-transformed infrared spectroscopy (FTIR), X-ray diffractograms, thermal, morphological, and pasting properties, swelling power and solubility of starches were evaluated. The degree of substitution (DS) of the acetylated starches increased with the rise in catalyst concentration. The percentage of carbonyl (CO) and carboxyl (COOH) groups in oxidized starches also increased with the rise of active chlorine level. The presence of hydrophobic acetyl groups, carbonyl and carboxyl groups caused a partial disorganization and depolymerization of starch granules. The structural, morphological and functional changes in acetylated and oxidized starches varied according to reaction conditions. Acetylation makes barley starch more hydrophobic by the insertion of acetyl groups. Also the oxidation promotes low retrogradation and viscosity. All these characteristics are important for biodegradable film production. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Comparison of esterase gene amplification, gene expression and esterase activity in insecticide susceptible and resistant strains of the brown planthopper, Nilaparvata lugens (Stål).

    Science.gov (United States)

    Vontas, J G; Small, G J; Hemingway, J

    2000-12-01

    Organophosphorus and carbamate insecticide resistance in Nilaparvata lugens is based on amplification of a carboxylesterase gene, Nl-EST1. An identical gene occurs in susceptible insects. Quantitative real-time PCR was used to demonstrate that Nl-EST1 is amplified 3-7-fold in the genome of resistant compared to susceptible planthoppers. Expression levels were similar to amplification levels, with 1-15-fold more Nl-EST1 mRNA in individual insects and 5-11-fold more Nl-EST1 mRNA in mass whole body homogenates of resistant females compared to susceptibles. These values corresponded to an 8-10-fold increase in esterase activity in the head and thorax of individual resistant insects. Although amplification, expression and activity levels of Nl-EST1 in resistant N. lugens were similar, the correlation between esterase activity and Nl-EST1 mRNA levels in resistant individuals was not linear.

  8. Differential bacterial capture and transport preferences facilitate co-growth on dietary xylan in the human gut

    DEFF Research Database (Denmark)

    Leth, Maria Louise; Ejby, Morten; Workman, Christopher

    2018-01-01

    Metabolism of dietary glycans is pivotal in shaping the human gut microbiota. However, the mechanisms that promote competition for glycans among gut commensals remain unclear. Roseburia intestinalis, an abundant butyrate-producing Firmicute, is a key degrader of the major dietary fibre xylan...... of capture and transport preferences as a possible strategy to facilitate co-growth on abundant dietary fibres and may offer a unique route to manipulate the microbiota based on glycan transport preferences in therapeutic interventions to boost distinct taxa....

  9. Thermophilic acetylxylan esterase genes and enzymes from alicyclobacillus acidocaldarius and related organisms and methods

    Science.gov (United States)

    Thompson, Vicki S.; Thompson, David N.; Reed, David W.; Lacey, Jeffrey A.; Apel, William A.

    2018-01-09

    A genetically modified organism comprising at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharide, lignocellulose, hemicellulose, lignin, chitin, heteroxylan, and/or xylan-decorating group; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  10. Extração e caracterização de xilanas de sabugos de milho Extraction and characterization of xylans from corncobs

    Directory of Open Access Journals (Sweden)

    Simone S. Silva

    1998-06-01

    Full Text Available Neste trabalho, duas frações de xilana, denominadas xilana A e xilana B, foram isoladas a partir de sabugos de milho através de três processos diferentes, combinando métodos de extração aquosa, remoção de lipídeos, deslignificação e extração alcalina. Os produtos obtidos durante os processos foram analisados por termogravimetria. A etapa de deslignificação foi responsável por uma acentuada degradação dos polímeros, evidenciada por queda de rendimento e resistência térmica. Os espectros obtidos no infravermelho evidenciaram a ausência de ácidos urônicos na cadeia polimérica. As viscosidades intrínsecas obtidas para a xilana A (56 mL/g e xilana B (75 mL/g associadas aos resultados do infravermelho sugerem um número maior de grupos substituintes, constituídos basicamente por resíduos de L-arabinose, para a xilana B.In this work, two fractions of xylan, named xylan A and xylan B, were isolated from corncobs through three different processes using aqueous extraction, lipid removal, delignification, and alkaline extraction. The products obtained during the processes were analysed by thermogravimetry. The delignification step was responsible for the occurrence of an accentuated polymer degradation, evidenced by yield and thermal resistance decrease. Infrared spectra indicated the absence of uronic acids in the polymeric chains. Intrinsic viscosities obtained for xylan A (56 mL/g and xylan B (75 mL/g, associated to the results from i.r. analysis, suggested a higher number of substituents, basically constituted of L-arabinose residues, in the case of xylan B.

  11. Chaperone-like activities of α-synuclein: α-Synuclein assists enzyme activities of esterases

    International Nuclear Information System (INIS)

    Ahn, Misun; Kim, SeungBum; Kang, Mira; Ryu, Yeonwoo; Doohun Kim, T.

    2006-01-01

    α-Synuclein, a major constituent of Lewy bodies (LBs), has been implicated to play a critical role in the pathogenesis of Parkinson's disease (PD), although the physiological function of α-synuclein has not yet been known. Here we have shown that α-synuclein, which has no well-defined secondary or tertiary structure, can protect the enzyme activity of microbial esterases against stress conditions such as heat, pH, and organic solvents. In particular, the flexibility of α-synuclein and its C-terminal region seems to be important for complex formation, but the structural integrity of the C-terminal region may not be required for stabilization of enzyme activity. In addition, atomic force microscopy (AFM) and in vivo enzyme assays showed highly specific interactions of esterases with α-synuclein. Our results indicate that α-synuclein not only protects the enzyme activity of microbial esterases in vitro, but also can stabilize the active conformation of microbial esterases in vivo

  12. Esterase and protease activities of Bacillus spp. from afitin, iru and ...

    African Journals Online (AJOL)

    The electrophoretic profiles of fermented African locust bean protein (ALBP), using strains presenting the highest protease activities in casein agar, were analyzed by SDS-PAGE to select strains with good ability to be used as starter cultures. All the Bacillus spp. tested showed esterase activity against tributyrin with high ...

  13. Enrichment of maize and triticale bran with recombinant Aspergillus tubingensis ferulic acid esterase

    CSIR Research Space (South Africa)

    Zwane, EN

    2017-03-01

    Full Text Available acid, in particular for the enrichment of food substrates. A recombinant Aspergillus tubingensis ferulic acid esterase Type A (FAEA) was expressed in Aspergillus niger D15#26 and purified with anion-exchange chromatography (3487 U/mg, Km = 0.43 mM, Kcat...

  14. A novel esterase from Saccharomyces carlsbergensis, a possible function for the yeast TIP1 gene

    DEFF Research Database (Denmark)

    Horsted, M W; Dey, E S; Holmberg, S

    1998-01-01

    An extracellular esterase was isolated from the brewer's yeast, Saccharomyces carlsbergensis. Inhibition by diisopropyl fluorophosphate shows that the enzyme has a serine active site. By mass spectrometry, the molecular weight of the enzyme was 16.9 kDa. The optimal pH for activity was in the range...

  15. Lipase and esterase: to what extent can this classification be applied accurately?

    Directory of Open Access Journals (Sweden)

    Danielle Branta Lopes

    2011-09-01

    Full Text Available Enzyme technology is an ever-growing field of knowledge and, in recent years, this technology has raised renewed interest, due to the search for new paradigms in several productive processes. Lipases, esterases and cutinases are enzymes used in a wide range of processes involving synthesis and hydrolysis reactions. The objective of this work was to investigate and compare the specific lipase and esterase activities of five enzymes - four already classified as lipases and one classified as cutinase - in the presence of natural and synthetic substrates. All tested enzymes presented both esterase and lipase specific activities. The highest specific esterase activity was observed for Aspergillus 1068 lipase in natural substrate and for F. oxysporum cutinase in synthetic substrate, while the highest specific lipase activity was observed for Geotrichum sp. lipase in natural substrate and for F. oxysporum cutinase in synthetic substrate. These results display some interface-independent lipolytic activity for all lipases tested. This is in accordance with the rationale that a new and broader definition of lipases may be necessary.

  16. Esterase isozymes patterns of grape vine (Vitis vinifera L. are altered in response to fungicide exposure

    Directory of Open Access Journals (Sweden)

    Gleice Ribeiro Orasmo

    2015-10-01

    Full Text Available Current analysis characterizes the effect of different fungicides often applied for pest control on a-and b-esterase patterns of four economically important table-wine grape cultivars (Italia, Rubi, Benitaka and Brasil of Vitis vinifera. The a- and b-esterase patterns in bud leaves of the cultivars were assessed by native PAGE analysis. Cabrio Top® compound inhibited Est-2, Est-5, Est-6, Est-7, Est-8, Est-9 and Est-10 carboxylesterases, whereas Est-4, Est-11, Est-12, Est-13, Est-14 acetylesterases and Est-16 carboxylesterase were detected as weakly stained bands. Carboxylesterases and acetylesterases were also detected as weakly stained bands when exposed to fungicides Orthocide 500®, Positron Duo® and Folicur PM®. No changes in a- and b-esterase patterns were reported when the vines were exposed to the fungicides Rovral SC®, Kumulus DF®, Curzate M®, Score® or Cuprogarb 500®. The evidence of functional changes in carboxylesterase and acetylesterase levels in current study is a warning to grape producers on the dangers inherent in the indiscriminate use of potent and modern fungicides extensively used in agriculture. The inhibition effect of fungicides on esterase isozyme molecules seems to be independent of the fungicide chemical.

  17. Fungal glucuronoyl esterases : Genome mining based enzyme discovery and biochemical characterization

    NARCIS (Netherlands)

    Dilokpimol, Adiphol; Mäkelä, Miia R; Cerullo, Gabriella; Zhou, Miaomiao; Varriale, Simona; Gidijala, Loknath; Brás, Joana L A; Jütten, Peter; Piechot, Alexander; Verhaert, Raymond; Faraco, Vincenza; Hilden, Kristiina S.; de Vries, Ronald P

    2018-01-01

    4-O-Methyl-d-glucuronic acid (MeGlcA) is a side-residue of glucuronoarabinoxylan and can form ester linkages to lignin, contributing significantly to the strength and rigidity of the plant cell wall. Glucuronoyl esterases (4-O-methyl-glucuronoyl methylesterases, GEs) can cleave this ester bond, and

  18. Cholesterol esterase inhibitory activity of bioactives from leaves of Mangifera indica L

    Science.gov (United States)

    Gururaja, G. M.; Mundkinajeddu, Deepak; Dethe, Shekhar M.; Sangli, Gopala K.; Abhilash, K.; Agarwal, Amit

    2015-01-01

    Background: In the earlier studies, methanolic extract of Mangifera indica L leaf was exhibited hypocholesterol activity. However, the bioactive compounds responsible for the same are not reported so far. Objective: To isolate the bioactive compounds with hypocholesterol activity from the leaf extract using cholesterol esterase inhibition assay which can be used for the standardization of extract. Materials and Methods: The leaf methanolic extract of M. indica (Sindoora variety) was partitioned with ethyl acetate and chromatographed on silica gel to yield twelve fractions and the activity was monitored by using cholesterol esterase inhibition assay. Active fractions were re-chromatographed to yield individual compounds. Results and Discussion: A major compound mangiferin present in the extract was screened along with other varieties of mango leaves for cholesterol esterase inhibition assay. However, the result indicates that compounds other than mangiferin may be active in the extract. Invitro pancreatic cholesterol esterase inhibition assay was used for bioactivity guided fractionation (BAGF) to yield bioactive compound for standardization of extract. Bioactivity guided fractionation afford the active fraction containing 3b-taraxerol with an IC50 value of 0.86μg/ml. Conclusion: This study demonstrates that M. indica methanol extract of leaf have significant hypocholesterol activity which is standardized with 3b-taraxerol, a standardized extract for hypocholesterol activity resulted in development of dietary supplement from leaves of Mangifera indica. PMID:26692750

  19. Dampak Hipoksia Sistemik terhadap Malondialdehida, Glial Fibrillary Acidic Protein dan Aktivitas Asetilkolin Esterase Otak Tikus

    OpenAIRE

    Andriani Andriani; Ani Retno Prijanti; Ninik Mudjihartini; Sri Widia A. Jusman

    2016-01-01

    Hipoksia sistemik menyebabkan berkurangnya oksigen dan energi di otak sehingga memicupenglepasan neurotransmiter asetilkolin, meningkatkan radikal bebas dan glial fibrillary acidic protein (GFAP)yang berfungsi menjaga kekuatan membran. Tujuan penelitian untuk melihat gambaran adaptasi otak padahipoksia sistemik terhadap fungsi asetilkolin esterase, kerusakan membran sel neuron dan astrosit. Penelitiandilakukan di Laboratorium Biokimia & Biologi Molekuler FK Universitas Indonesia, pada ta...

  20. Production and partial characterisation of feruloyl esterase by Sporotrichum thermophile in solid-state fermentation

    DEFF Research Database (Denmark)

    Topakas, E.; Kalogeris, E.; Kekos, D.

    2003-01-01

    A number of factors affecting production of feruloyl esterase an enzyme that hydrolyse ester linkages of ferulic acid (FA) in plant cell walls, by the thermophylic fungus Sporotrichum thermophile under solid state fermentation (SSF) were investigated. Initial moisture content and type of carbon...

  1. Usefulness of Leukocyte Esterase Test Versus Rapid Strep Test for Diagnosis of Acute Strep Pharyngitis

    Directory of Open Access Journals (Sweden)

    Kumara V. Nibhanipudi MD

    2015-08-01

    Full Text Available Objective: A study to compare the usage of throat swab testing for leukocyte esterase on a test strip(urine dip stick-multi stick to rapid strep test for rapid diagnosis of Group A Beta hemolytic streptococci in cases of acute pharyngitis in children. Hypothesis: The testing of throat swab for leukocyte esterase on test strip currently used for urine testing may be used to detect throat infection and might be as useful as rapid strep. Methods: All patients who come with a complaint of sore throat and fever were examined clinically for erythema of pharynx, tonsils and also for any exudates. Informed consent was obtained from the parents and assent from the subjects. 3 swabs were taken from pharyngo-tonsillar region, testing for culture, rapid strep & Leukocyte Esterase. Results: Total number is 100. Cultures 9(+; for rapid strep== 84(- and16 (+; For LE== 80(- and 20(+ Statistics: From data configuration Rapid Strep versus LE test don’t seem to be a random (independent assignment but extremely aligned. The Statistical results show rapid and LE show very agreeable results. Calculated Value of Chi Squared Exceeds Tabulated under 1 Degree Of Freedom (P<.0.0001 reject Null Hypothesis and Conclude Alternative Conclusions: Leukocyte esterase on throat swab is as useful as rapid strep test for rapid diagnosis of strep pharyngitis on test strip currently used for urine dip stick causing acute pharyngitis in children.

  2. Crystallization and preliminary X-ray diffraction studies of the pneumococcal teichoic acid phosphorylcholine esterase Pce

    Energy Technology Data Exchange (ETDEWEB)

    Lagartera, Laura; González, Ana; Stelter, Meike; García, Pedro; Kahn, Richard; Menéndez, Margarita; Hermoso, Juan A., E-mail: xjuan@iqfr.csic.es

    2005-02-01

    The modular choline-binding protein Pce, the phosphorylcholine esterase from S. pneumoniae, has been crystallized by the hanging-drop vapour-diffusion method. A SAD data set from a derivative with a gadolinium complex has been collected to 2.7 Å resolution.

  3. Cloning, Characterization, Controlled Overexpression, and Inactivation of the Major Tributyrin Esterase Gene of Lactococcus lactis

    NARCIS (Netherlands)

    Fernández, Leonides; Beerthuyzen, Marke M.; Brown, Julie; Siezen, Roland J.; Coolbear, Tim; Holland, Ross; Kuipers, Oscar P.

    2000-01-01

    The gene encoding the major intracellular tributyrin esterase of Lactococcus lactis was cloned using degenerate DNA probes based on 19 known N-terminal amino acid residues of the purified enzyme. The gene, named estA, was sequenced and found to encode a protein of 258 amino acid residues. The

  4. C1-esterase inhibitor blocks T lymphocyte proliferation and cytotoxic T lymphocyte generation in vitro

    DEFF Research Database (Denmark)

    Nissen, Mogens Holst; Bregenholt, S; Nording, J A

    1998-01-01

    We have previously shown that activated C1s complement and activated T cells cleave beta2-microglobulin (beta2m) in vitro leading to the formation of desLys58 beta2m. This process can specifically be inhibited by C1-esterase inhibitor (C1-inh). Furthermore we showed that exogenously added desLys58...

  5. Preliminary X-ray Study of Naproxen Esterase from Bacillus subtilis

    NARCIS (Netherlands)

    van der Laan, Jan; Teplyakov, A.V.; Lammers, A.A.; Dijkstra, B.W.

    1993-01-01

    Single crystals of naproxen esterase from Bacillus subtilis have been obtained from PEG6000 solutions at pH 8.0 by liquid-liquid diffusion while applying a temperature gradient from 4°C to room temperature over a period of four weeks. The crystals belong to the trigonal space group P3121 or P3221

  6. A New Functional Classification of Glucuronoyl Esterases by Peptide Pattern Recognition

    DEFF Research Database (Denmark)

    Wittrup Agger, Jane; Busk, Peter Kamp; Pilgaard, Bo

    2017-01-01

    of characterized enzymes exist and the exact activity is still uncertain. Here peptide pattern recognition is used as a bioinformatic tool to identify and group new CE15 proteins that are likely to have glucuronoyl esterase activity. 1024 CE15-like sequences were drawn from GenBank and grouped into 24 groups...

  7. Molecular population genetics of the β-esterase gene cluster of ...

    Indian Academy of Sciences (India)

    We suggest that the demographic history (bottleneck and admixture of genetically differentiated populations) is the major factor shaping the pattern of nucleotide polymorphism in the -esterase gene cluster. However there are some 'footprints' of directional and balancing selection shaping specific distribution of nucleotide ...

  8. Bacteroides intestinalis DSM 17393, a member of the human colonic microbiome, upregulates multiple endoxylanases during growth on xylan.

    Science.gov (United States)

    Wang, Kui; Pereira, Gabriel V; Cavalcante, Janaina J V; Zhang, Meiling; Mackie, Roderick; Cann, Isaac

    2016-09-29

    Many human diets contain arabinoxylan, and the ease of genome sequencing coupled with reduced cost have led to unraveling the arsenal of genes utilized by the colonic Bacteroidetes to depolymerize this polysaccharide. The colonic Bacteroidetes with potential to ferment arabinoxylans include Bacteroides intestinalis. In this study, we analyzed the hydrolytic activities of members of a xylan degradation cluster encoded on the genome of Bacteroides intestinalis DSM 17393. Here, it is demonstrated that a cocktail of the xylanolytic enzymes completely hydrolyze arabinoxylans found in human diets. We show that this bacterium and relatives have evolved and secrete a unique bifunctional endoxylanase/arabinofuranosidase in the same polypeptide. The bifunctional enzyme and other secreted enzymes attack the polysaccharides extracellularly to remove the side-chains, exposing the xylan backbone for cleavage to xylo-oligosaccharides and xylose. These end products are transported into the cell where a β-xylosidase cleaves the oligosaccharides to fermentable sugars. While our experiments focused on B. intestinalis, it is likely that the extracellular enzymes also release nutrients to members of the colonic microbial community that practice cross-feeding. The presence of the genes characterized in this study in other colonic Bacteroidetes suggests a conserved strategy for energy acquisition from arabinoxylan, a component of human diets.

  9. Carbon Nanotubes Reinforced Maleic Anhydride-Modified Xylan-g-Poly(N-isopropylacrylamide) Hydrogel with Multifunctional Properties

    Science.gov (United States)

    Liu, Xinxin; Song, Tao; Chang, Minmin; Meng, Ling; Wang, Xiaohui; Sun, Runcang; Ren, Junli

    2018-01-01

    Introducing multifunctional groups and inorganic material imparts xylan-based hydrogels with excellent properties, such as responsiveness to pH, temperature, light, and external magnetic field. In this work, a composite hydrogel was synthesized by introducing acid treated carbon nanotubes (AT-CNTs) into the maleic anhydride modified xylan grafted with poly(N-isopropylacrylamide) (MAX-g-PNIPAM) hydrogels network. It was found that the addition of AT-CNTs affected the MAX-g-PNIPAM hydrogel structure, the swelling ratio and mechanical properties, and imparted the hydrogel with new properties of electrical conductivity and near infrared region (NIR) photothermal conversion. AT-CNTs could reinforce the mechanical properties of MAX-g-PNIPAM hydrogels, being up to 83 kPa for the compressive strength when the amount was 11 wt %, which was eight times than that of PNIPAM hydrogel and four times than that of MAX-g-PNIPAM hydrogel. The electroconductibility was enhanced by the increase of AT-CNTs amounts. Meanwhile, the composite hydrogel also exhibited multiple shape memory and NIR photothermal conversion properties, and water temperature was increased from 26 °C to 56 °C within 8 min under the NIR irradiation. Thus, the AT-CNTs reinforced MAX-g-PNIPAM hydrogel possessed promising multifunctional properties, which offered many potential applications in the fields of biosensors, thermal-arrest technology, and drug-controlled release. PMID:29495611

  10. Discovery and characterization of Ku acetylation in Mycobacterium smegmatis.

    Science.gov (United States)

    Zhou, Ying; Chen, Tao; Zhou, Lin; Fleming, Joy; Deng, Jiaoyu; Wang, Xude; Wang, Liwei; Wang, Yingying; Zhang, Xiaoli; Wei, Wenjing; Bi, Lijun

    2015-03-01

    Lysine acetylation is an important post-translational modification and is known to regulate many eukaryotic cellular processes. Little, however, is known about acetylated proteins in prokaryotes. Here, using immunoblotting, mass spectrometry and mutagenesis studies, we investigate the acetylation dynamics of the DNA repair protein Ku and its relationship with the deacetylase protein Sir2 and the non-homologous end joining (NHEJ) pathway in Mycobacterium smegmatis. We report that acetylation of Ku increases with growth, while NHEJ activity decreases, providing support for the hypothesis that acetylation of Ku may be involved in the DNA damage response in bacteria. Ku has multiple lysine sites. Our results indicate that K29 is an important acetylation site and that deficiency of Sir2 or mutation of K29 affects the quantity of Ku and its acetylation dynamics. Our findings expand knowledge of acetylation targets in prokaryotes and indicate a new direction for further research on bacterial DNA repair mechanisms. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Influence of acetylation on the physicochemical properties of ...

    African Journals Online (AJOL)

    The study investigates the effect of acetylation on the physicochemical properties of composited starches from sweet potato and water yam. Starch was respectively isolated from both sources, dried and subjected to acetylation at different combination. The result shows that the modified starches were of low percentage of ...

  12. The kinetics of the acetylation of gelatinised potato starch

    NARCIS (Netherlands)

    de Graaf, R.A.; Broekroelofs, G.A.; Janssen, L.P.B.M.; Beenackers, A.A C M

    1995-01-01

    The reaction rates, in the base-catalysed acetylation of gelatinised aqueous starch (4 wt%), by vinylacetate (ViAc), were investigated in a semibatch reactor at temperatures ranging from 20 to 50 degrees C. The desired starch acetylation reaction is accompanied by an undesired parallel

  13. Trichostatin A induced histone acetylation causes decondensation of interphase chromatin.

    NARCIS (Netherlands)

    T.A. Knoch (Tobias); M. Wachsmuth (Malte); M. Frank-Stöhr (Monika); M. Stöhr (Michael); C.P. Bacher (Christian); K. Rippe (Karsten)

    2004-01-01

    textabstractThe effect of trichostatin A (TSA)-induced histone acetylation on the interphase chromatin structure was visualized in vivo with a HeLa cell line stably expressing histone H2A, which was fused to enhanced yellow fluorescent protein. The globally increased histone acetylation caused a

  14. 21 CFR 172.372 - N-Acetyl-L-methionine.

    Science.gov (United States)

    2010-04-01

    ... and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN CONSUMPTION Special Dietary and Nutritional Additives § 172.372 N-Acetyl-L-methionine. The food additive N-acetyl-L...

  15. Acetylated starch of Ofada rice as a sustained polymer in ...

    African Journals Online (AJOL)

    Objectives: To formulate and evaluate repaglinide microspheres using acetylated starch of the indigenous rice species Oryza glaberrima Steud (Ofada) as polymer. Materials and Methods: Ofada rice starch was acetylated with acetic anhydride in pyridine (DS 2.68) and characterized for morphology (Scanning electron ...

  16. Regulation of autophagy by cytosolic acetyl-coenzyme A

    DEFF Research Database (Denmark)

    Mariño, Guillermo; Pietrocola, Federico; Eisenberg, Tobias

    2014-01-01

    Acetyl-coenzyme A (AcCoA) is a major integrator of the nutritional status at the crossroads of fat, sugar, and protein catabolism. Here we show that nutrient starvation causes rapid depletion of AcCoA. AcCoA depletion entailed the commensurate reduction in the overall acetylation of cytoplasmic p...

  17. Efficient acetylation of primary amines and amino acids in ...

    Indian Academy of Sciences (India)

    This effort represents the first efficient use of this most reactive but cheap acetylating agent to acetylate amines in excellent yields in aqueous medium. This is a potentially useful green chemical transformation where reaction takes place in environment-friendly brine solution leading to easy work-up and isolation of the ...

  18. Effects of Model Salivary Esterases and MMP Inhibition on the Restoration's Marginal Integrity and Potential Degradative Contribution of Cariogenic Bacteria

    Science.gov (United States)

    Huang, Bo

    Enzyme-catalyzed degradation of the restoration-tooth interface compromises interfacial integrity, thereby contributing to secondary caries, which is a major cause of resin-based restoration failure. It is hypothesized that in addition to salivary esterases, the cariogenic bacterium Streptococcus mutans has specific esterases that degrade the resin-dentin interface, releasing biodegradation by- products (BBPs) such as bis-hydroxy-propoxy-phenyl-propane (BisHPPP). In turn, BisHPPP affects S. mutans by stimulating the expression of esterases. Another hypothesis is that the biostability of the resin-dentin interface is affected by simulated salivary esterases, dentinal matrix metalloproteinase (MMP) inhibition, and restorative materials. To test the first hypothesis, putative esterase genes in S. mutans UA159 were identified, purified, and characterized. SMU_118c was identified as the dominant esterase in S. mutans UA159 and showed a similar hydrolytic activity profile to salivary esterases. BisHPPP upregulated expression of the SMU_118c gene and related protein in a concentration-dependent manner. This positive feedback process could accelerate the degradation of the restoration-tooth interface and lead to premature restoration failure. To test the second hypothesis, an in vitro model was established to evaluate the effects of salivary esterases, MMP inhibition and restorative materials on interfacial integrity. It was confirmed that interfacial integrity was compromised with time and was further deteriorated by simulated salivary esterases, as indicated by the greater depth of bacterial ingress and more bacterial biomass of biofilm along the interface. However, this process could be modulated by using different restorative materials and MMPs inhibition. This project elucidated the mechanistic interaction between oral bacteria and restorative materials and established a new, in vitro, and physiologically relevant model to assess the effect of material chemistry

  19. The biology of lysine acetylation integrates transcriptional programming and metabolism

    Directory of Open Access Journals (Sweden)

    Mujtaba Shiraz

    2011-03-01

    Full Text Available Abstract The biochemical landscape of lysine acetylation has expanded from a small number of proteins in the nucleus to a multitude of proteins in the cytoplasm. Since the first report confirming acetylation of the tumor suppressor protein p53 by a lysine acetyltransferase (KAT, there has been a surge in the identification of new, non-histone targets of KATs. Added to the known substrates of KATs are metabolic enzymes, cytoskeletal proteins, molecular chaperones, ribosomal proteins and nuclear import factors. Emerging studies demonstrate that no fewer than 2000 proteins in any particular cell type may undergo lysine acetylation. As described in this review, our analyses of cellular acetylated proteins using DAVID 6.7 bioinformatics resources have facilitated organization of acetylated proteins into functional clusters integral to cell signaling, the stress response, proteolysis, apoptosis, metabolism, and neuronal development. In addition, these clusters also depict association of acetylated proteins with human diseases. These findings not only support lysine acetylation as a widespread cellular phenomenon, but also impel questions to clarify the underlying molecular and cellular mechanisms governing target selectivity by KATs. Present challenges are to understand the molecular basis for the overlapping roles of KAT-containing co-activators, to differentiate between global versus dynamic acetylation marks, and to elucidate the physiological roles of acetylated proteins in biochemical pathways. In addition to discussing the cellular 'acetylome', a focus of this work is to present the widespread and dynamic nature of lysine acetylation and highlight the nexus that exists between epigenetic-directed transcriptional regulation and metabolism.

  20. Supplementation with xylanase and β-xylosidase to reduce xylo-oligomer and xylan inhibition of enzymatic hydrolysis of cellulose and pretreated corn stover

    Science.gov (United States)

    2011-01-01

    Background Hemicellulose is often credited with being one of the important physical barriers to enzymatic hydrolysis of cellulose, and acts by blocking enzyme access to the cellulose surface. In addition, our recent research has suggested that hemicelluloses, particularly in the form of xylan and its oligomers, can more strongly inhibit cellulase activity than do glucose and cellobiose. Removal of hemicelluloses or elimination of their negative effects can therefore become especially pivotal to achieving higher cellulose conversion with lower enzyme doses. Results In this study, cellulase was supplemented with xylanase and β-xylosidase to boost conversion of both cellulose and hemicellulose in pretreated biomass through conversion of xylan and xylo-oligomers to the less inhibitory xylose. Although addition of xylanase and β-xylosidase did not necessarily enhance Avicel hydrolysis, glucan conversions increased by 27% and 8% for corn stover pretreated with ammonia fiber expansion (AFEX) and dilute acid, respectively. In addition, adding hemicellulase several hours before adding cellulase was more beneficial than later addition, possibly as a result of a higher adsorption affinity of cellulase and xylanase to xylan than glucan. Conclusions This key finding elucidates a possible mechanism for cellulase inhibition by xylan and xylo-oligomers and emphasizes the need to optimize the enzyme formulation for each pretreated substrate. More research is needed to identify advanced enzyme systems designed to hydrolyze different substrates with maximum overall enzyme efficacy. PMID:21702938

  1. Comparative Analysis of End Point Enzymatic Digests of Arabino-Xylan Isolated from Switchgrass (Panicum virgatum L of Varying Maturities using LC-MSn

    Directory of Open Access Journals (Sweden)

    Michael J. Bowman

    2012-11-01

    Full Text Available Switchgrass (Panicum virgatum L., SG is a perennial grass presently used for forage and being developed as a bioenergy crop for conversion of cell wall carbohydrates to biofuels. Up to 50% of the cell wall associated carbohydrates are xylan. SG was analyzed for xylan structural features at variable harvest maturities. Xylan from each of three maturities was isolated using classical alkaline extraction to yield fractions (Xyl A and B with varying compositional ratios. The Xyl B fraction was observed to decrease with plant age. Xylan samples were subsequently prepared for structure analysis by digesting with pure endo-xylanase, which preserved side-groups, or a commercial carbohydrase preparation favored for biomass conversion work. Enzymatic digestion products were successfully permethylated and analyzed by reverse-phase liquid chromatography with mass spectrometric detection (RP-HPLC-MSn. This method is advantageous compared to prior work on plant biomass because it avoids isolation of individual arabinoxylan oligomers. The use of RP-HPLC- MSn differentiated 14 structural oligosaccharides (d.p. 3–9 from the monocomponent enzyme digestion and nine oligosaccharide structures (d.p. 3–9 from hydrolysis with a cellulase enzyme cocktail. The distribution of arabinoxylan oligomers varied depending upon the enzyme(s applied but did not vary with harvest maturity.

  2. Supplementation with xylanase and β-xylosidase to reduce xylo-oligomer and xylan inhibition of enzymatic hydrolysis of cellulose and pretreated corn stover

    Directory of Open Access Journals (Sweden)

    Qing Qing

    2011-06-01

    Full Text Available Abstract Background Hemicellulose is often credited with being one of the important physical barriers to enzymatic hydrolysis of cellulose, and acts by blocking enzyme access to the cellulose surface. In addition, our recent research has suggested that hemicelluloses, particularly in the form of xylan and its oligomers, can more strongly inhibit cellulase activity than do glucose and cellobiose. Removal of hemicelluloses or elimination of their negative effects can therefore become especially pivotal to achieving higher cellulose conversion with lower enzyme doses. Results In this study, cellulase was supplemented with xylanase and β-xylosidase to boost conversion of both cellulose and hemicellulose in pretreated biomass through conversion of xylan and xylo-oligomers to the less inhibitory xylose. Although addition of xylanase and β-xylosidase did not necessarily enhance Avicel hydrolysis, glucan conversions increased by 27% and 8% for corn stover pretreated with ammonia fiber expansion (AFEX and dilute acid, respectively. In addition, adding hemicellulase several hours before adding cellulase was more beneficial than later addition, possibly as a result of a higher adsorption affinity of cellulase and xylanase to xylan than glucan. Conclusions This key finding elucidates a possible mechanism for cellulase inhibition by xylan and xylo-oligomers and emphasizes the need to optimize the enzyme formulation for each pretreated substrate. More research is needed to identify advanced enzyme systems designed to hydrolyze different substrates with maximum overall enzyme efficacy.

  3. Insight into Aluminum Sulfate-Catalyzed Xylan Conversion into Furfural in a γ-Valerolactone/Water Biphasic Solvent under Microwave Conditions.

    Science.gov (United States)

    Yang, Tao; Zhou, Yi-Han; Zhu, Sheng-Zhen; Pan, Hui; Huang, Yao-Bing

    2017-10-23

    A simple and efficient biphasic system with an earth-abundant metal salt catalyst was used to produce furfural from xylan with a high yield of up to 87.8 % under microwave conditions. Strikingly, the metal salt Al 2 (SO 4 ) 3 exhibited excellent catalytic activity for xylan conversion, owing to a combination of Lewis and Brønsted acidity and its ability to promote good phase separation. The critical role of the SO 4 2- anion was first analyzed, which resulted in the aforementioned characteristics when combined with the Al 3+ cation. The mixed solvent system with γ-valerolactone (GVL) as the organic phase provided the highest furfural yield, resulting from its good dielectric properties and dissolving capacity, which facilitated the absorption of microwave energy and promoted mass transfer. Mechanistic studies suggested that the xylan-to-furfural conversion proceeded mainly through a hydrolysis-isomerization-dehydration pathway and the hexa-coordinated Lewis acidic [Al(OH) 2 (aq)] + species were the active sites for xylose-xylulose isomerization. Detailed kinetic studies of the subreaction for the xylan conversion revealed that GVL regulates the reaction rates and pathways by promoting the rates of the key steps involved for furfural production and suppressing the side reactions for humin production. Finally, the Al 2 (SO 4 ) 3 catalyst was used for the production of furfural from several lignocellulosic feedstocks, revealing its great potential for other biomass conversions. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Direct Kinetic Evidence for the Formation of an Acylpyridinium Intermediate in Synthetic p-Nitrophenyl Esterase-Catalyzed Hydrolysis Reactions

    National Research Council Canada - National Science Library

    Wang, Guang-Jia

    1996-01-01

    .... The deacylation rate was also found to exhibit a maximum for the same substrate 2 (n=6). These results are similar to those previously reported with cholesterol esterase as catalyst for the same hydrolysis reaction...

  5. Dampak Hipoksia Sistemik terhadap Malondialdehida, Glial Fibrillary Acidic Protein dan Aktivitas Asetilkolin Esterase Otak Tikus

    Directory of Open Access Journals (Sweden)

    Andriani Andriani

    2016-09-01

    Full Text Available Hipoksia sistemik menyebabkan berkurangnya oksigen dan energi di otak sehingga memicupenglepasan neurotransmiter asetilkolin, meningkatkan radikal bebas dan glial fibrillary acidic protein (GFAPyang berfungsi menjaga kekuatan membran. Tujuan penelitian untuk melihat gambaran adaptasi otak padahipoksia sistemik terhadap fungsi asetilkolin esterase, kerusakan membran sel neuron dan astrosit. Penelitiandilakukan di Laboratorium Biokimia & Biologi Molekuler FK Universitas Indonesia, pada tahun 2013.Penelitian ekperimental ini menggunakan hewan coba tikus spraque dawley yang diinduksi hipoksia sistemikyang diambil jaringan otak bagian korteks dan plasma tikus. Kelompok tikus terdiri atas kelompok kontrol,kelompok perlakuan induksi hipoksia hari ke-1, 3 hari, 5 hari dan hari ke-7. Parameter yang diukur adalahkadar malondialdehida (MDA otak dan plasma, aktivitas spesifik enzim AChE jaringan otak serta kadar GFAPjaringan otak. Hasil menunjukkan bahwa hipoksia sistemik tidak meningkatkankadar MDA otak dan plasma.Induksi hipoksia sistemik meningkatkan aktivitas spesifik enzim AChE dan kadar GFAP jaringan otak secarabermakna. Pada plasma tidak terjadi peningkatan kadar GFAP. Hipoksia sistemik selama hari ke-7 belummenyebabkan kerusakan oksidatif, namun memperlihatkan peningkatan aktivitas AChe dan adaptasi astrositmelalui peningkatan GFAP. Kata kunci: hipoksia, astrosit, glial fibrillary acidic protein, malondialdehida, asetilkolin esterase   Systemic Hypoxia Effect on Rat Brain Malondialdehyde, Glial FibrillaryAcidic Protein, and Acetylcholine Esterase Activity Abstract Sistemic hypoxia causes lack of oxygen and energy in brain that trigger the release of acetylcholine,free radical and Glial fibrillary acidic protein (GFAP, a specific protein in astrocyte cells that act to strenghtenastrocite membrane. The aim of the research was to evaluate the damages of brain in systemic hypoxiathrough activity of acetylcholine esterase, neuron and astrocyte membran

  6. Histone acetylation regulates the time of replication origin firing.

    Science.gov (United States)

    Vogelauer, Maria; Rubbi, Liudmilla; Lucas, Isabelle; Brewer, Bonita J; Grunstein, Michael

    2002-11-01

    The temporal firing of replication origins throughout S phase in yeast depends on unknown determinants within the adjacent chromosomal environment. We demonstrate here that the state of histone acetylation of surrounding chromatin is an important regulator of temporal firing. Deletion of RPD3 histone deacetylase causes earlier origin firing and concurrent binding of the replication factor Cdc45p to origins. In addition, increased acetylation of histones in the vicinity of the late origin ARS1412 by recruitment of the histone acetyltransferase Gcn5p causes ARS1412 alone to fire earlier. These data indicate that histone acetylation is a direct determinant of the timing of origin firing.

  7. Phosphorylation and Acetylation of Acyl-CoA Synthetase- I

    DEFF Research Database (Denmark)

    Frahm, Jennifer L; Li, Lei O; Grevengoed, Trisha J

    2011-01-01

    Long chain acyl-CoA synthetase 1 (ACSL1) contributes 50 to 90% of total ACSL activity in liver, adipose tissue, and heart and appears to direct the use of long chain fatty acids for energy. Although the functional importance of ACSL1 is becoming clear, little is understood about its post...... and acetylated amino acids by mass spectrometry. We then compared these results to the post-translational modifications observed in vivo in liver and brown adipose tissue after mice were fasted or exposed to a cold environment. We identified universal N-terminal acetylation, 15 acetylated lysines, and 25...

  8. Synthetic biology for engineering acetyl coenzyme a metabolism in yeast

    DEFF Research Database (Denmark)

    Nielsen, Jens

    2014-01-01

    The yeast Saccharomyces cerevisiae is a widely used cell factory for the production of fuels, chemicals, and pharmaceuticals. The use of this cell factory for cost-efficient production of novel fuels and chemicals requires high yields and low by-product production. Many industrially interesting...... chemicals are biosynthesized from acetyl coenzyme A (acetyl-CoA), which serves as a central precursor metabolite in yeast. To ensure high yields in production of these chemicals, it is necessary to engineer the central carbon metabolism so that ethanol production is minimized (or eliminated) and acetyl...

  9. Using a simple HPLC approach to identify the enzymatic products of UTL-5g, a small molecule TNF-α inhibitor, from porcine esterase and from rabbit esterase.

    Science.gov (United States)

    Swartz, Kenneth; Zhang, Yiguan; Valeriote, Frederick; Chen, Ben; Shaw, Jiajiu

    2013-12-01

    UTL-5g is a novel small-molecule chemoprotector that lowers hepatotoxicity, nephrotoxicity, and myelotoxicity induced by cisplatin through TNF-α inhibition among other factors. As a prelude to investigating the metabolites of UTL-5g, we set out to identify the enzymatic products of UTL-5g under the treatment of both porcine liver esterase (PLE) and rabbit liver esterase (RLE). First, a number of mixtures made by UTL-5g and PLE were incubated at 25°C. At predetermined time points, individual samples were quenched by acetonitrile, vortexed, and centrifuged. The supernatants were then analyzed by reversed-phase HPLC (using a C18 column). The retention times and UV/vis spectra of individual peaks were compared to those of UTL-5g and its two postulated enzymatic products; thus the enzymatic products of UTL-5g were tentatively identified. Secondly, a different HPLC method (providing different retentions times) was used to cross-check and to confirm the identities of the two enzymatic products. Based on the observations, it was concluded that under the treatment of PLE, the major enzymatic products of UTL-5g were 5-methyliosxazole-3-carboxylic acid (ISOX) and 2,4-dichloroaniline (DCA). Treatment of UTL-5g by RLE also provided the same enzymatic products of UTL-5g from esterase. These results indicate that the peptide bond in UTL-5g was cleaved by PLE/RLE. Michaelis-Menten kinetics showed that the Km values of UTL-5g were 2.07mM with PLE and 0.37mM with RLE indicating that UTL-5g had a higher affinity with RLE. In summary, by a simple HPLC approach, we have concluded that the peptide bond in UTL-5g was cleaved by esterase from either porcine liver or rabbit liver in vitro and afforded DCA (at a mole ratio of 1:1) and ISOX. However, further studies are needed in order to determine whether UTL-5g is metabolized by microsomal enzymes to produce ISOX and DCA. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Esterase activities of intracellular extracts of wild strains of lactic acid bacteria isolated from Serra da Estrela cheese

    OpenAIRE

    Macedo, Angela C.; Tavares, Tânia G.; Malcata, F. Xavier

    2003-01-01

    Lactococcus lactis subsp. lactis strain ESB110019 and Lactobacillus plantarum strain ESB5004, novel strains that were previously isolated from the wild adventitious microflora of certified Serra da Estrela cheeses, were assayed for esterase activity using, as substrates, ortho- and para-nitrophenyl derivatives of fatty acids. Both strains preferentially hydrolyzed short-chain fatty acids; L. lactis ESB110019 exhibited a stronger esterase activity than Lb. plantarum ESB5004 and cleaved the p-n...

  11. Acetylation of Mammalian ADA3 Is Required for Its Functional Roles in Histone Acetylation and Cell Proliferation.

    Science.gov (United States)

    Mohibi, Shakur; Srivastava, Shashank; Bele, Aditya; Mirza, Sameer; Band, Hamid; Band, Vimla

    2016-10-01

    Alteration/deficiency in activation 3 (ADA3) is an essential component of specific histone acetyltransferase (HAT) complexes. We have previously shown that ADA3 is required for establishing global histone acetylation patterns and for normal cell cycle progression (S. Mohibi et al., J Biol Chem 287:29442-29456, 2012, http://dx.doi.org/10.1074/jbc.M112.378901). Here, we report that these functional roles of ADA3 require its acetylation. We show that ADA3 acetylation, which is dynamically regulated in a cell cycle-dependent manner, reflects a balance of coordinated actions of its associated HATs, GCN5, PCAF, and p300, and a new partner that we define, the deacetylase SIRT1. We use mass spectrometry and site-directed mutagenesis to identify major sites of ADA3 acetylated by GCN5 and p300. Acetylation-defective mutants are capable of interacting with HATs and other components of HAT complexes but are deficient in their ability to restore ADA3-dependent global or locus-specific histone acetylation marks and cell proliferation in Ada3-deleted murine embryonic fibroblasts (MEFs). Given the key importance of ADA3-containing HAT complexes in the regulation of various biological processes, including the cell cycle, our study presents a novel mechanism to regulate the function of these complexes through dynamic ADA3 acetylation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  12. A Transcriptomic Analysis of Xylan Mutants Does Not Support the Existence of a Secondary Cell Wall Integrity System in Arabidopsis.

    Science.gov (United States)

    Faria-Blanc, Nuno; Mortimer, Jenny C; Dupree, Paul

    2018-01-01

    Yeast have long been known to possess a cell wall integrity (CWI) system, and recently an analogous system has been described for the primary walls of plants (PCWI) that leads to changes in plant growth and cell wall composition. A similar system has been proposed to exist for secondary cell walls (SCWI). However, there is little data to support this. Here, we analyzed the stem transcriptome of a set of cell wall biosynthetic mutants in order to investigate whether cell wall damage, in this case caused by aberrant xylan synthesis, activates a signaling cascade or changes in cell wall synthesis gene expression. Our data revealed remarkably few changes to the transcriptome. We hypothesize that this is because cells undergoing secondary cell wall thickening have entered a committed programme leading to cell death, and therefore a SCWI system would have limited impact. The absence of transcriptomic responses to secondary cell wall alterations may facilitate engineering of the secondary cell wall of plants.

  13. Properties of Two Novel Esterases Identified from Culture Supernatant of Penicillium purpurogenum Grown on Sugar Beet Pulp.

    Science.gov (United States)

    Oleas, Gabriela; Callegari, Eduardo; Sepulveda, Romina; Eyzaguirre, Jaime

    2016-01-01

    The filamentous fungus Penicillium purpurogenum grows on a variety of natural carbon sources, such as sugar beet pulp, and secretes to the medium a large number of enzymes that degrade the carbohydrate components of lignocellulose. Sugar beet pulp is rich in pectin, and the purpose of this work is to identify novel esterases produced by the fungus, which may participate in pectin degradation. Partially purified culture supernatants of the fungus grown on sugar beet pulp were subjected to mass spectrometry analysis. Peptides thus identified, which may be part of potential esterases were probed against the proteins deduced from the fungal genome sequence. The cDNAs of two putative esterases identified were expressed in Pichia pastoris and their properties studied. One of these enzymes, named FAET, is a feruloyl esterase, while the other, PE, is classified as a pectin methyl esterase. These findings add to our knowledge of the enzymology of pectin degradation by Penicillium purpurogenum, and define properties of two novel esterases acting on de-esterification of pectin. Their availability may be useful as tools for the study of pectin structure and degradation.

  14. Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes.

    Science.gov (United States)

    Zhang, Meiling; Chekan, Jonathan R; Dodd, Dylan; Hong, Pei-Ying; Radlinski, Lauren; Revindran, Vanessa; Nair, Satish K; Mackie, Roderick I; Cann, Isaac

    2014-09-02

    Enzymes that degrade dietary and host-derived glycans represent the most abundant functional activities encoded by genes unique to the human gut microbiome. However, the biochemical activities of a vast majority of the glycan-degrading enzymes are poorly understood. Here, we use transcriptome sequencing to understand the diversity of genes expressed by the human gut bacteria Bacteroides intestinalis and Bacteroides ovatus grown in monoculture with the abundant dietary polysaccharide xylan. The most highly induced carbohydrate active genes encode a unique glycoside hydrolase (GH) family 10 endoxylanase (BiXyn10A or BACINT_04215 and BACOVA_04390) that is highly conserved in the Bacteroidetes xylan utilization system. The BiXyn10A modular architecture consists of a GH10 catalytic module disrupted by a 250 amino acid sequence of unknown function. Biochemical analysis of BiXyn10A demonstrated that such insertion sequences encode a new family of carbohydrate-binding modules (CBMs) that binds to xylose-configured oligosaccharide/polysaccharide ligands, the substrate of the BiXyn10A enzymatic activity. The crystal structures of CBM1 from BiXyn10A (1.8 Å), a cocomplex of BiXyn10A CBM1 with xylohexaose (1.14 Å), and the CBM from its homolog in the Prevotella bryantii B14 Xyn10C (1.68 Å) reveal an unanticipated mode for ligand binding. A minimal enzyme mix, composed of the gene products of four of the most highly up-regulated genes during growth on wheat arabinoxylan, depolymerizes the polysaccharide into its component sugars. The combined biochemical and biophysical studies presented here provide a framework for understanding fiber metabolism by an important group within the commensal bacterial population known to influence human health.

  15. Xylan utilization in human gut commensal bacteria is orchestrated by unique modular organization of polysaccharide-degrading enzymes

    KAUST Repository

    Zhang, Meiling

    2014-08-18

    Enzymes that degrade dietary and host-derived glycans represent the most abundant functional activities encoded by genes unique to the human gut microbiome. However, the biochemical activities of a vast majority of the glycan-degrading enzymes are poorly understood. Here, we use transcriptome sequencing to understand the diversity of genes expressed by the human gut bacteria Bacteroides intestinalis and Bacteroides ovatus grown in monoculture with the abundant dietary polysaccharide xylan. The most highly induced carbohydrate active genes encode a unique glycoside hydrolase (GH) family 10 endoxylanase (BiXyn10A or BACINT-04215 and BACOVA-04390) that is highly conserved in the Bacteroidetes xylan utilization system. The BiXyn10A modular architecture consists of a GH10 catalytic module disrupted by a 250 amino acid sequence of unknown function. Biochemical analysis of BiXyn10A demonstrated that such insertion sequences encode a new family of carbohydrate-binding modules (CBMs) that binds to xy-lose- configured oligosaccharide/polysaccharide ligands, the substrate of the BiXyn10A enzymatic activity. The crystal structures of CBM1 from BiXyn10A (1.8 Å), a cocomplex of BiXyn10A CBM1 with xylohexaose (1.14 Å), and the CBM fromits homolog in the Prevotella bryantii B 14 Xyn10C (1.68 Å) reveal an unanticipated mode for ligand binding. Aminimal enzyme mix, composed of the gene products of four of the most highly up-regulated genes during growth on wheat arabinoxylan, depolymerizes the polysaccharide into its component sugars. The combined biochemical and biophysical studies presented here provide a framework for understanding fiber metabolism by an important group within the commensal bacterial population known to influence human health.

  16. Prospect for Developing a Consolidated Bioprocessing (CBP) Strain Using Xylan as the Substrate: the Case Study of Yarrowia lipolytica

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Wei; Wei, Hui; Alahuhta, Markus; Zhang, Min; Himmel, Michael E.

    2016-07-08

    To achieve the goal of developing a direct microbial sugar conversion platform for the production of lipids and drop-in fuels from cellulosic biomass substrate, Yarrowia lipolytica was used to investigate its potential for being developed as CBP strain by expressing cellulase and xylanase enzymes. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing glucose and xylose to produce lipids; however, due to the lack of the biomass degrading enzymes, it cannot directly utilize lignocellulosic substrates as carbon sources. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. To the best of our knowledge, this is the first study introducing heterologous hemicellulose genes into the genome of Y. lipolytica. SDS-PAGE and western blotting analysis showed that the endo-xylanase gene XynII and exo-xylosidase gene XlnD were successfully expressed and secreted, and the expressed xylanases were likely either not or sparsely glycosylated, which is advantageous for expression of heterologous proteins from any species. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action on converting xylan to xylose was observed when XlnD worked in concert with XynII. XlnD was able to work on the xylo-oligomers generated by XynII, enhancing the xylan conversion to monomeric xylose. The successful expression of these xylanases in Yarrowia further advances us towards our goal to develop a direct microbial conversion process using this organism. and xylose to produce lipids; however, due to the lack of the biomass degrading enzymes, it cannot directly utilize lignocellulosic substrates as carbon sources. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of

  17. Chromatographic study of highly methoxylated lime pectins deesterified by different pectin methyl-esterases.

    Science.gov (United States)

    Ralet, M C; Bonnin, E; Thibault, J F

    2001-03-25

    The inter-molecular distribution of free carboxyl groups of two highly methoxylated pectins enzymatically deesterified by plant and fungus pectin methyl-esterases were investigated by size-exclusion (SEC) and ion-exchange chromatography (IEC). "Homogeneous" populations with respect to molar mass or charge density were thereby obtained and their chemical composition and physico-chemical properties (transport parameter for monovalent cations and calcium, calcium activity coefficient) were studied. Chemical analysis showed that the composition varies from one SEC fraction to another, the highest molar mass fraction being richer in rhamnose and galactose and exhibiting a slightly higher degree of methylation. Separation of pectins by IEC revealed a quite homogeneous charge density distribution for F58 contrary to P60 which exhibited a large distribution of methoxyl groups. The free carboxyl groups distributions and calcium binding behaviours of SEC and IEC fractions were shown to differ widely for highly methoxylated pectins deesterified by plant and fungus pectin methyl-esterases.

  18. Production and partial characterization of alkaline feruloyl esterases by Fusarium oxysporum during submerged batch cultivation

    DEFF Research Database (Denmark)

    Topakas, E.; Christakopoulos, Paul

    2004-01-01

    Production of feruloyl esterases (FAEs) by Fusarium oxysporum was enhanced by optimization of initial pH of the culture medium, the type and concentration of nitrogen and carbon source. Submerged batch cultivation in a laboratory bioreactor (17 1) produced activity at 82 nkat g(-1) dry substrate....... Production of FAE does not therefore, require FA, however, production is diminished by the removal of esterified FA from the growth substrate. Optimal FAE activity was observed at pH 7 and 50 degreesC with 68 and 55% activity at pH 8 and pH 9, respectively. The esterase was fully stable at pH 5-8 and up...

  19. Efficient acetylation of primary amines and amino acids in ...

    Indian Academy of Sciences (India)

    bDepartment of Clinical and Experimental Pharmacology, School of Tropical Medicine ... As a result ... methods of acetylation of amines are known using ace- ... vents we report here, environmentally benign, eco- ... It was filtered under suction,.

  20. Characterization of a cold-adapted esterase and mutants from a psychotolerant Pseudomonas sp. strain.

    Science.gov (United States)

    Dong, Juan; Gasmalla, Mohammed A A; Zhao, Wei; Sun, Jingtao; Liu, Wenyu; Wang, Mingming; Han, Liang; Yang, Ruijin

    2017-09-01

    A cold-adapted esterase-producing strain named T1-39 was isolated from Glacier No. 1, Tianshan, People's Republic of China and identified as Pseudomonas sp. from 16S rRNA sequence analysis. The esterase (EstT1-39) secreted by this strain preferentially hydrolyzed esters of glycerol with short- and medium-chain fatty acids. Mutants of T1-39 were generated by the atmospheric and room temperature plasma method and screened for enhanced esterase activity. Among all the mutants, strain TB11 had 4.45-fold higher esterase productivity than T1-39, with high genetic stability over 10 generations of continuous cultivation. Maximum activity of EstT1-39 and EstTB11 was observed at 30 ℃, pH 9.0 and 25 ℃, pH 8.5, respectively. EstTB11 was thermally more stable (50 ℃ for 1 H) and active over a broader pH range than EstT1-39. EstTB11 also retained 38% of its maximal activity at 0 ℃ and was found to be able to hydrolyze milk fats into short- and medium-chain fatty acids at 4 ℃. The characteristics of EstT1-39 made it a cold-adapted enzyme and the EstTB11 from the mutant, with its higher activity at lower temperatures, may be suitable for the production of aromas and flavors in the dairy industry. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  1. Biochemical Characterization of a Family 15 Carbohydrate Esterase from a Bacterial Marine Arctic Metagenome.

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    Concetta De Santi

    Full Text Available The glucuronoyl esterase enzymes of wood-degrading fungi (Carbohydrate Esterase family 15; CE15 form part of the hemicellulolytic and cellulolytic enzyme systems that break down plant biomass, and have possible applications in biotechnology. Homologous enzymes are predicted in the genomes of several bacteria, however these have been much less studied than their fungal counterparts. Here we describe the recombinant production and biochemical characterization of a bacterial CE15 enzyme denoted MZ0003, which was identified by in silico screening of a prokaryotic metagenome library derived from marine Arctic sediment. MZ0003 has high similarity to several uncharacterized gene products of polysaccharide-degrading bacterial species, and phylogenetic analysis indicates a deep evolutionary split between these CE15s and fungal homologs.MZ0003 appears to differ from previously-studied CE15s in some aspects. Some glucuronoyl esterase activity could be measured by qualitative thin-layer chromatography which confirms its assignment as a CE15, however MZ0003 can also hydrolyze a range of other esters, including p-nitrophenyl acetate, which is not acted upon by some fungal homologs. The structure of MZ0003 also appears to differ as it is predicted to have several large loop regions that are absent in previously studied CE15s, and a combination of homology-based modelling and site-directed mutagenesis indicate its catalytic residues deviate from the conserved Ser-His-Glu triad of many fungal CE15s. Taken together, these results indicate that potentially unexplored diversity exists among bacterial CE15s, and this may be accessed by investigation of the microbial metagenome. The combination of low activity on typical glucuronoyl esterase substrates, and the lack of glucuronic acid esters in the marine environment suggest that the physiological substrate of MZ0003 and its homologs is likely to be different from that of related fungal enzymes.

  2. A Novel Halotolerant Thermoalkaliphilic Esterase from Marine Bacterium Erythrobacter seohaensis SW-135

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    Ying-Yi Huo

    2017-11-01

    Full Text Available A novel esterase gene, e69, was cloned from Erythrobacter seohaensis SW-135, which was isolated from a tidal flat sediment of the Yellow Sea in Korea. This gene is 825 bp in length and codes for a 29.54 kDa protein containing 274 amino acids. Phylogenetic analysis showed that E69 is a new member of the bacterial lipolytic enzyme family IV. This enzyme exhibited the highest level of activity toward p-nitrophenyl (NP butyrate but little or no activity toward the other p-NP esters tested. The optimum temperature and pH of the catalytic activity of E69 were 60°C and pH 10.5, respectively. The enzyme exhibited stable activity over a wide range of alkaline pH values (7.5–9.5. In addition, E69 was found to be a halotolerant esterase as it exhibited the highest hydrolytic activity in the presence of 0.5 M NaCl and was still active in the presence of 3 M NaCl. Moreover, it possessed some degree of tolerance to Triton X-100 and several organic solvents. Through homology modeling and comparison with other esterases, it was suggested that the absence of the cap domain and its narrow substrate-binding pocket might be responsible for its narrow substrate specificity. Sequence and structural analysis results suggested that its high ratio of negatively to positively charged residues, large hydrophobic surface area, and negative electrostatic potential on the surface may be responsible for its alkaline adaptation. The results of this study provide insight into marine alkaliphilic esterases, and the unique properties of E69 make it a promising candidate as a biocatalyst for industrial applications.

  3. Environmental Factors Modulating the Stability and Enzymatic Activity of the Petrotoga mobilis Esterase (PmEst.

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    Jose L S Lopes

    Full Text Available Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/β protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required.

  4. EVALUATION OF ESTERASE POLYMORPHISMS IN MATURE SEEDS OF RADISH (RAPHANUS SATIVUS L. ACCESSIONS OF VIR COLLECTION

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    A. S. Rudakova

    2017-01-01

    Full Text Available A biochemical evaluation of 25 radish accessions (Raphanus sativus L. on esterase isozymes of mature seeds has been carried out. The results of the experiments showed a wide range of diversity among the genotypes based on electrophoretic zones of esterase isoenzymes. The revealed isoenzyme complex of esterases was represented by eight isoforms with molecular weights from 37.7 kD to 57.6 kD. All accessions were divided into 13 electrophoretic zymotypes, differing from each other by the presence or absence of definite zones. The most often observed electrophoretic zymo-type is Gr. 1, which includes 24% of the total number of accessions evaluated. There are 8 zymotypes (Gr. 6 Gr. 13 with a frequency of occurrence 4%. Three groups (Gr. 2 – Gr. 4 had the same frequency of occurrence – 12%. Zimotype of Gr. 5 containes the maximum number of zones – 8. 2 zimotypes – Gr. 3 and Gr. 12 had the smallest number of 4 zones. Two zones of esterases – zones 7 and 8 (Мr 39.7кD and Мr 37.7 kD, respectively were monomorphic. The remaining six zones were polymorphic, i.e. could be absent in some zimotypes. The frequency of occurrence of each zone in different zymotypes has varied from 6.58% to 17.11%. As results of this research the accessions that were selected can become the most promising parent forms for future genetic and selection studies of this culture.

  5. Common and Distant Structural Characteristics of Feruloyl Esterase Families from Aspergillus oryzae

    OpenAIRE

    Udatha, D. B. R. K. Gupta; Mapelli, Valeria; Panagiotou, Gianni; Olsson, Lisbeth

    2012-01-01

    Background: Feruloyl esterases (FAEs) are important biomass degrading accessory enzymes due to their capability of cleaving the ester links between hemicellulose and pectin to aromatic compounds of lignin, thus enhancing the accessibility of plant tissues to cellulolytic and hemicellulolytic enzymes. FAEs have gained increased attention in the area of biocatalytic transformations for the synthesis of value added compounds with medicinal and nutritional applications. Following the increasing a...

  6. Interactions between resin monomers and commercial composite resins with human saliva derived esterases.

    Science.gov (United States)

    Jaffer, F; Finer, Y; Santerre, J P

    2002-04-01

    Cholesterol esterase (CE) and pseudocholinesterase (PCE) have been reported to degrade commercial and model composite resins containing bisphenylglycidyl dimethacrylate (BisGMA), triethylene glycol dimethacrylate (TEGDMA) or the latter in combination with urethane modified BisGMA monomer systems. In addition, human saliva has been shown to contain esterase like activities similar to CE and PCE. Hence, it was the aim of the current study to determine to what extent human saliva could degrade two common commercial composite resins (Z250 from 3M Inc. and Spectrum TPH from L.D. Caulk) which contain the above monomer systems. Saliva samples from different volunteers were collected, processed, pooled, and freeze-dried. TEGDMA and BisGMA monomers were incubated with human saliva derived esterase activity (HSDEA) and their respective hydrolysis was monitored using high performance liquid chromatography (HPLC). Both monomers were completely hydrolyzed within 25 h by HSDEA. Photopolymerized composites were incubated with buffer or human saliva (pH 7.0 and 37 C) for 2, 8 and 16 days. The incubation solutions were analyzed using HPLC and mass spectrometry. Surface morphology characterization was carried out using scanning electron microscopy. Upon biodegradation, the Z250 composite yielded higher amounts of BisGMA and TEGDMA related products relative to the TPH composite. However, there were higher amounts of ethoxylated bis-phenol A released from the TPH material. In terms of total mass of products released, human saliva demonstrated a greater ability to degrade Z250. In summary, HSDEA has been shown to contain esterase activities that can readily catalyze the biodegradation of current commercial composite resins.

  7. A New Esterase from Thermobifida halotolerans Hydrolyses Polyethylene Terephthalate (PET and Polylactic Acid (PLA

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    Georg Steinkellner

    2012-02-01

    Full Text Available A new esterase from Thermobifida halotolerans (Thh_Est was cloned and expressed in E. coli and investigated for surface hydrolysis of polylactic acid (PLA and polyethylene terephthalate (PET. Thh_Est is a member of the serine hydrolases superfamily containing the -GxSxG- motif with 85–87% homology to an esterase from T. alba, to an acetylxylan esterase from T. fusca and to various Thermobifida cutinases. Thh_Est hydrolyzed the PET model substrate bis(benzoyloxyethylterephthalate and PET releasing terephthalic acid and mono-(2-hydroxyethyl terephthalate in comparable amounts (19.8 and 21.5 mmol/mol of enzyme while no higher oligomers like bis-(2-hydroxyethyl terephthalate were detected. Similarly, PLA was hydrolyzed as indicated by the release of lactic acid. Enzymatic surface hydrolysis of PET and PLA led to a strong hydrophilicity increase, as quantified with a WCA decrease from 90.8° and 75.5° to 50.4° and to a complete spread of the water drop on the surface, respectively.

  8. Novel Cold-Adapted Esterase MHlip from an Antarctic Soil Metagenome

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    Moreno Galleni

    2013-01-01

    Full Text Available An Antarctic soil metagenomic library was screened for lipolytic enzymes and allowed for the isolation of a new cytosolic esterase from the a/b hydrolase family 6, named MHlip. This enzyme is related to hypothetical genes coding esterases, aryl-esterases and peroxydases, among others. MHlip was produced, purified and its activity was determined. The substrate profile of MHlip reveals a high specificity for short p-nitrophenyl-esters. The apparent optimal activity of MHlip was measured for p-nitrophenyl-acetate, at 33 °C, in the pH range of 6–9. The MHlip thermal unfolding was investigated by spectrophotometric methods, highlighting a transition (Tm at 50 °C. The biochemical characterization of this enzyme showed its adaptation to cold temperatures, even when it did not present evident signatures associated with cold-adapted proteins. Thus, MHlip adaptation to cold probably results from many discrete structural modifications, allowing the protein to remain active at low temperatures. Functional metagenomics is a powerful approach to isolate new enzymes with tailored biophysical properties (e.g., cold adaptation. In addition, beside the ever growing amount of sequenced DNA, the functional characterization of new catalysts derived from environment is still required, especially for poorly characterized protein families like α/b hydrolases.

  9. Purification and biochemical characterization of feruloyl esterases from Aspergillus terreus MTCC 11096.

    Science.gov (United States)

    Kumar, C Ganesh; Kamle, Avijeet; Kamal, Ahmed

    2013-01-01

    Aspergillus terreus MTCC 11096 isolated from the soils of agricultural fields cultivating sweet sorghum was previously identified to produce feruloyl esterases (FAEs). The enzymes responsible for feruloyl esterase activity were purified to homogeneity and named as AtFAE-1, AtFAE-2, and AtFAE-3. The enzymes were monomeric having molecular masses of 74, 23 and 36 kDa, respectively. Active protein bands were identified by a developed pH-dependent zymogram on native PAGE. The three enzymes exhibited variation in pH tolerance ranging between pH 5-8 and thermostability of up to 55°C. Inhibition studies revealed that the serine residue was essential for feruloyl esterase activity; moreover aspartyl and glutamyl residues are not totally involved at the active site. Metal ions such as Ca(2+), K(+), and Mg(2+) stabilized the enzyme activity for all three FAEs. Kinetic data indicated that all three enzymes showed catalytic efficiencies (k(cat) /K(m)) against different synthesized alkyl and aryl esters indicating their broad substrate specificity. The peptide mass fingerprinting by MALDI/TOF-MS analysis and enzyme affinity toward methoxy and hydroxy substituents on the benzene ring revealed that the AtFAE-1 belonged to type A while AtFAE-2 and AtFAE-3 were type C FAE. The FAEs could release 65 to 90% of ferulic acid from agrowaste substrates in the presence of xylanase. © 2013 American Institute of Chemical Engineers.

  10. Mechanism-Guided Discovery of an Esterase Scaffold with Promiscuous Amidase Activity

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    Charlotte Kürten

    2016-06-01

    Full Text Available The discovery and generation of biocatalysts with extended catalytic versatilities are of immense relevance in both chemistry and biotechnology. An enhanced atomistic understanding of enzyme promiscuity, a mechanism through which living systems acquire novel catalytic functions and specificities by evolution, would thus be of central interest. Using esterase-catalyzed amide bond hydrolysis as a model system, we pursued a simplistic in silico discovery program aiming for the identification of enzymes with an internal backbone hydrogen bond acceptor that could act as a reaction specificity shifter in hydrolytic enzymes. Focusing on stabilization of the rate limiting transition state of nitrogen inversion, our mechanism-guided approach predicted that the acyl hydrolase patatin of the α/β phospholipase fold would display reaction promiscuity. Experimental analysis confirmed previously unknown high amidase over esterase activity displayed by the first described esterase machinery with a protein backbone hydrogen bond acceptor to the reacting NH-group of amides. The present work highlights the importance of a fundamental understanding of enzymatic reactions and its potential for predicting enzyme scaffolds displaying alternative chemistries amenable to further evolution by enzyme engineering.

  11. Discovery of potential cholesterol esterase inhibitors using in silico docking studies

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    Thirumalaisamy Sivashanmugam

    2013-08-01

    Full Text Available New drug discovery is considered broadly in terms of two kinds of investiga-tional activities such as exploration and exploitation. This study deals with the evaluation of the cholesterol esterase inhibitory activity of flavonoids apigenin, biochanin, curcumin, diosmetin, epipervilline, glycitein, okanin, rhamnazin and tangeritin using in silico docking studies. In silico docking studies were carried out using AutoDock 4.2, based on the Lamarckian genetic algorithm principle. The results showed that all the selected flavonoids showed binding energy ranging between -7.08 kcal/mol to -5.64 kcal/mol when compared with that of the standard compound gallic acid (-4.11 kcal/mol. Intermolecular energy (-9.13 kcal/mol to -7.09 kcal/mol and inhibition constant (6.48 µM to 73.18 µM of the ligands also coincide with the binding energy. All the selected flavonoids contributed cholesterol esterase inhibitory activity, these molecular docking analyses could lead to the further develop-ment of potent cholesterol esterase inhibitors for the treatment of obesity.

  12. Cloning, expression and characterization of a novel esterase from a South China Sea sediment metagenome

    Science.gov (United States)

    Zhang, Hao; Li, Fuchao; Chen, Huaxin; Zhao, Jin; Yan, Jinfei; Jiang, Peng; Li, Ronggui; Zhu, Baoli

    2015-07-01

    Lipolytic enzymes, including esterases and lipases, represent a group of hydrolases that catalyze the cleavage and formation of ester bonds. A novel esterase gene, scsEst01, was cloned from a South China Sea sediment metagenome. The scsEst01 gene consisted of 921 bp encoding 307 amino acid residues. The predicted amino acid sequence shared less than 90% identity with other lipolytic enzymes in the NCBI nonredundant protein database. ScsEst01 was successfully co-expressed in Escherichia coli BL21 (DE3) with chaperones (dnaK-dnaJ-grpE) to prevent the formation of inclusion bodies. The recombinant protein was purified on an immobilized metal ion affinity column containing chelating Sepharose charged with Ni2+. The enzyme was characterized using p -nitrophenol butyrate as a substrate. ScsEst01 had the highest lipolytic activity at 35°C and pH 8.0, indicative of a meso-thermophilic alkaline esterase. ScsEst01 was thermostable at 20°C. The lipolytic activity of scsEst01 was strongly increased by Fe2+, Mn2+ and 1% Tween 80 or Tween 20.

  13. Eco-friendly surface modification on polyester fabrics by esterase treatment

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Jindan; Cai, Guoqiang; Liu, Jinqiang; Ge, Huayun; Wang, Jiping, E-mail: jipingwanghz@gmail.com

    2014-03-01

    Graphical abstract: - Highlights: • We used a simple and easy way to measure the enzyme activity. • We studied the mechanism by characterizing the chemical changes in the surface of fabric. • We studied the advantages in surface wettability, fiber integrity and mechanical performance of cutinase treated fabrics. • Cutinase pretreated fibers exhibited much improved fabric wicking and better fiber integrity comparing to alkali treated ones. • Cutinase pretreatment technology promotes energy conservation and emission reduction. - Abstract: Currently, traditional alkali deweighting technology is widely used to improve the hydrophilicity of polyester fabrics. However, the wastewater and heavy chemicals in the effluent cause enormous damage to the environment. Esterase treatment, which is feasible in mild conditions with high selectivity, can provide a clean and efficient way for polyester modification. Under the optimum conditions, the polyester fabric hydrolysis process of esterase had a linear kinetics. X-ray photoelectron spectrometry (XPS) results showed that hydroxyl and carboxyl groups were produced only on the surface of modified fiber without changing the chemical composition of the bulk. These fibers exhibited much improved fabric wicking, as well as greatly improved oily stain removal performance. Compared to the harsh alkali hydrolysis, the enzyme treatment led to smaller weight loss and better fiber integrity. The esterase treatment technology is promising to produce higher-quality polyester textiles with an environmental friendly approach.

  14. Purification and properties of an O-acetyl-transferase from Escherichia coli that can O-acetylate polysialic acid sequences

    International Nuclear Information System (INIS)

    Higa, H.; Varki, A.

    1986-01-01

    Certain strains of bacteria synthesize an outer polysialic acid (K1) capsule. Some strains of K1 + E.coli are also capable of adding O-acetyl-esters to the exocyclic hydroxyl groups of the sialic acid residues. Both the capsule and the O-acetyl modification have been correlated with differences in antigenicity and pathogenicity. The authors have developed an assay for an O-acetyl-transferase in E.coli that transfers O-[ 3 H]acetyl groups from [ 3 H]acetyl-Coenzyme A to colominic acid (fragments of the polysialic acid capsule). Using this assay, the enzyme was solubilized, and purified ∼ 600-fold using a single affinity chromatography step with Procion Red-A Agarose. The enzyme also binds to Coenzyme A Sepharose, and can be eluted with high salt or Coenzyme A. The partially purified enzyme has a pH optimum of 7.0 - 7.5, is unaffected by divalent cations, is inhibited by high salt concentrations, is inhibited by Coenzyme A (50% inhibition at 100 μM), and shows an apparent Km for colominic acid of 3.7 mM (sialic acid concentration). This enzyme could be involved in the O-acetyl +/- form variation seen in some strains of K1 + E.coli

  15. Furfural Production from d-Xylose and Xylan by Using Stable Nafion NR50 and NaCl in a Microwave-Assisted Biphasic Reaction

    Directory of Open Access Journals (Sweden)

    Sarah Le Guenic

    2016-08-01

    Full Text Available Pentose dehydration and direct transformation of xylan into furfural were performed in a water-cyclopentyl methyl ether (CPME biphasic system under microwave irradiation. Heated up between 170 and 190 °C in the presence of Nafion NR50 and NaCl, d-xylose, l-arabinose and xylan gave furfural with maximum yields of 80%, 42% and 55%, respectively. The influence of temperature and reaction time on the reaction kinetics was discussed. This study was also completed by the survey of different reactant ratios, such as organic layer-water or catalyst-inorganic salt ratios. The exchange between proton and cation induced by an excess of NaCl was monitored, and a synergetic effect between the remaining protons and the released HCl was also discovered.

  16. Furfural Production from d-Xylose and Xylan by Using Stable Nafion NR50 and NaCl in a Microwave-Assisted Biphasic Reaction.

    Science.gov (United States)

    Le Guenic, Sarah; Gergela, David; Ceballos, Claire; Delbecq, Frederic; Len, Christophe

    2016-08-22

    Pentose dehydration and direct transformation of xylan into furfural were performed in a water-cyclopentyl methyl ether (CPME) biphasic system under microwave irradiation. Heated up between 170 and 190 °C in the presence of Nafion NR50 and NaCl, d-xylose, l-arabinose and xylan gave furfural with maximum yields of 80%, 42% and 55%, respectively. The influence of temperature and reaction time on the reaction kinetics was discussed. This study was also completed by the survey of different reactant ratios, such as organic layer-water or catalyst-inorganic salt ratios. The exchange between proton and cation induced by an excess of NaCl was monitored, and a synergetic effect between the remaining protons and the released HCl was also discovered.

  17. Asparagus IRX9, IRX10, and IRX14A Are Components of an Active Xylan Backbone Synthase Complex that Forms in the Golgi Apparatus1[OPEN

    Science.gov (United States)

    Zeng, Wei; Picard, Kelsey L.; Song, Lili; Wu, Ai-Min; Farion, Isabela M.; Zhao, Jia; Ford, Kris; Bacic, Antony

    2016-01-01

    Heteroxylans are abundant components of plant cell walls and provide important raw materials for the food, pharmaceutical, and biofuel industries. A number of studies in Arabidopsis (Arabidopsis thaliana) have suggested that the IRREGULAR XYLEM9 (IRX9), IRX10, and IRX14 proteins, as well as their homologs, are involved in xylan synthesis via a Golgi-localized complex termed the xylan synthase complex (XSC). However, both the biochemical and cell biological research lags the genetic and molecular evidence. In this study, we characterized garden asparagus (Asparagus officinalis) stem xylan biosynthesis genes (AoIRX9, AoIRX9L, AoIRX10, AoIRX14A, and AoIRX14B) by heterologous expression in Nicotiana benthamiana. We reconstituted and partially purified an active XSC and showed that three proteins, AoIRX9, AoIRX10, and AoIRX14A, are necessary for xylan xylosyltranferase activity in planta. To better understand the XSC structure and its composition, we carried out coimmunoprecipitation and bimolecular fluorescence complementation analysis to show the molecular interactions between these three IRX proteins. Using a site-directed mutagenesis approach, we showed that the DxD motifs of AoIRX10 and AoIRX14A are crucial for the catalytic activity. These data provide, to our knowledge, the first lines of biochemical and cell biological evidence that AoIRX9, AoIRX10, and AoIRX14A are core components of a Golgi-localized XSC, each with distinct roles for effective heteroxylan biosynthesis. PMID:26951434

  18. Asparagus IRX9, IRX10, and IRX14A Are Components of an Active Xylan Backbone Synthase Complex that Forms in the Golgi Apparatus.

    Science.gov (United States)

    Zeng, Wei; Lampugnani, Edwin R; Picard, Kelsey L; Song, Lili; Wu, Ai-Min; Farion, Isabela M; Zhao, Jia; Ford, Kris; Doblin, Monika S; Bacic, Antony

    2016-05-01

    Heteroxylans are abundant components of plant cell walls and provide important raw materials for the food, pharmaceutical, and biofuel industries. A number of studies in Arabidopsis (Arabidopsis thaliana) have suggested that the IRREGULAR XYLEM9 (IRX9), IRX10, and IRX14 proteins, as well as their homologs, are involved in xylan synthesis via a Golgi-localized complex termed the xylan synthase complex (XSC). However, both the biochemical and cell biological research lags the genetic and molecular evidence. In this study, we characterized garden asparagus (Asparagus officinalis) stem xylan biosynthesis genes (AoIRX9, AoIRX9L, AoIRX10, AoIRX14A, and AoIRX14B) by heterologous expression in Nicotiana benthamiana We reconstituted and partially purified an active XSC and showed that three proteins, AoIRX9, AoIRX10, and AoIRX14A, are necessary for xylan xylosyltranferase activity in planta. To better understand the XSC structure and its composition, we carried out coimmunoprecipitation and bimolecular fluorescence complementation analysis to show the molecular interactions between these three IRX proteins. Using a site-directed mutagenesis approach, we showed that the DxD motifs of AoIRX10 and AoIRX14A are crucial for the catalytic activity. These data provide, to our knowledge, the first lines of biochemical and cell biological evidence that AoIRX9, AoIRX10, and AoIRX14A are core components of a Golgi-localized XSC, each with distinct roles for effective heteroxylan biosynthesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

  19. Determination of the barrier height for acetyl radical dissociation from acetyl chloride photodissociation at 235 nm using velocity map imaging.

    Science.gov (United States)

    Tang, Xiaonan; Ratliff, Britni J; FitzPatrick, Benjamin L; Butler, Laurie J

    2008-12-18

    This work uses velocity map imaging to determine the barrier height for acetyl radical, CH3CO, dissociation to CH3 + CO. Photodissociation of acetyl chloride at 235 nm generates acetyl radicals with an internal energy distribution spanning this barrier. We determine the velocity and internal energy distribution of all nascent acetyl radicals, stable and unstable, by measuring the velocities of the Cl(2P3/2) and Cl(2P1/2) cofragments. These Cl cofragments are detected with 2 + 1 resonance-enhanced multiphoton ionization (REMPI) in a spin-orbit branching ratio Cl(2P3/2):Cl(2P1/2) of 3.3 +/- 0.2. Using 157 nm photoionization, we then detect the recoil velocities of the energetically stable acetyl radicals. The radicals and momentum matched Cl atoms evidence parallel angular distributions. Comparison of the total recoil translational energy distribution P(E(T)) for all radicals to that obtained from the detection of stable radicals yields an onset for dissociation at a translational energy of 25.0 +/- 0.4 kcal/mol. From this onset we can calculate the barrier height for CH3CO --> CH3 + CO, but this relies on prior determinations of the C-Cl bond energy of acetyl chloride. Using an experimental bond dissociation energy of 83.4 +/- 0.2 kcal/mol yields a dissociation barrier of 14.2 +/- 0.5 kcal/mol. Our data evidence that a portion of the acetyl radicals formed with total internal energy above the barrier are stable due to the partitioning of energy into rotation during the C-Cl bond fission of the precursor. Thus, the internal energy onset for dissociation is not as sharp as was assumed in prior determinations of the barrier. The experimentally determined onset is compared with that predicted from electronic structure calculations at the G3//B3LYP and CCSD(T) levels of theory.

  20. Histones of Chlamydomonas reinhardtii. Synthesis, acetylation, and methylation

    International Nuclear Information System (INIS)

    Waterborg, J.H.; Robertson, A.J.; Tatar, D.L.; Borza, C.M.; Davie, J.R.

    1995-01-01

    Histones of the green alga Chlamydomonas reinhardtii were prepared by a new method and fractionated by reversed-phase high-performance liquid chromatography. Acid-urea-Triton gel analysis and tritiated acetate labeling demonstrated high levels of steady-state acetylation for the single histone H3 protein, in contrast to low levels on histones H4 and H2B. Twenty percent of histone H3 is subject to dynamic acetylation with, on average, three acetylated lysine residues per protein molecule. Histone synthesis in light-dark-synchronized cultures was biphasic with pattern differences between two histone H1 variants, between two H2A variants, and between H2B and ubiquitinated H2B. Automated protein sequence analysis of histone H3 demonstrated a site-specific pattern of steady-state acetylation between 7 and 17% at five of the six amino-terminal lysines and of monomethylation between 5 and 81% at five of the eight amino-terminal lysines in a pattern that may limit dynamic acetylation. An algal histone H3 sequence was confirmed by protein sequencing with a since threonine as residue 28 instead of the serine(28)-alanine(29) sequence, present in all other known plant and animal H3 histones

  1. Genome-wide analysis of esterase-like genes in the striped rice stem borer, Chilo suppressalis.

    Science.gov (United States)

    Wang, Baoju; Wang, Ying; Zhang, Yang; Han, Ping; Li, Fei; Han, Zhaojun

    2015-06-01

    The striped rice stem borer, Chilo suppressalis, a destructive pest of rice, has developed high levels of resistance to certain insecticides. Esterases are reported to be involved in insecticide resistance in several insects. Therefore, this study systematically analyzed esterase-like genes in C. suppressalis. Fifty-one esterase-like genes were identified in the draft genomic sequences of the species, and 20 cDNA sequences were derived which encoded full- or nearly full-length proteins. The putative esterase proteins derived from these full-length genes are overall highly diversified. However, key residues that are functionally important including the serine residue in the active site are conserved in 18 out of the 20 proteins. Phylogenetic analysis revealed that most of these genes have homologues in other lepidoptera insects. Genes CsuEst6, CsuEst10, CsuEst11, and CsuEst51 were induced by the insecticide triazophos, and genes CsuEst9, CsuEst11, CsuEst14, and CsuEst51 were induced by the insecticide chlorantraniliprole. Our results provide a foundation for future studies of insecticide resistance in C. suppressalis and for comparative research with esterase genes from other insect species.

  2. The effect of C1-esterase inhibitor in definite and suspected streptococcal toxic shock syndrome. Report of seven patients.

    Science.gov (United States)

    Fronhoffs, S; Luyken, J; Steuer, K; Hansis, M; Vetter, H; Walger, P

    2000-10-01

    To evaluate the effect of adjunctive C1-esterase inhibitor substitution therapy on clinical characteristics and outcome of patients with streptococcal toxic shock syndrome (TSS). Observational. Medizinische Poliklinik, University of Bonn, Germany. Seven patients with direct or indirect evidence of streptococcal TSS. In addition to conventional and supportive therapy, all patients received 2-3 single doses of C1-esterase inhibitor totaling 6,000-10,000 U within the first 24 h after admission. All patients developed fulminant septic shock, multiorgan failure and/or capillary leak syndrome and necrotizing fasciitis within 10-72 h following the onset of first symptoms. Between 1 and 4 days following administration of C1-esterase inhibitor, a marked shift of fluid from extravascular to intravascular compartments took place in all but one patient, accompanied by a transient intra-alveolar lung edema and rapidly decreasing need for adrenergic agents. Six of seven patients survived. These clinical observations in a small series of patients and the favorable outcome point towards a positive effect of early and high-dose administration of C1-esterase inhibitor as adjunctive therapy in streptococcal TSS. The possible mechanism involved may be the attenuation of capillary leak syndrome (CLS) via early inactivation of complement and contact systems. Controlled studies are needed to establish an improvement of the survival rates of patients with streptococcal TSS following administration of C1-esterase inhibitor.

  3. Synthesis of Acylated Xylan-Based Magnetic Fe3O4 Hydrogels and Their Application for H2O2 Detection

    Directory of Open Access Journals (Sweden)

    Qing-Qing Dai

    2016-08-01

    Full Text Available Acylated xylan-based magnetic Fe3O4 nanocomposite hydrogels (ACX-MNP-gels were prepared by fabricating Fe3O4 nanoctahedra in situ within a hydrogel matrix which was synthesized by the copolymerization of acylated xylan (ACX with acrylamide and N-isopropylacrylamide under ultraviolet irradiation. The size of the Fe3O4 fabricated within the hydrogel matrix could be adjusted through controlling the crosslinking concentrations (C. The magnetic hydrogels showed desirable magnetic and mechanical properties, which were confirmed by XRD, Raman spectroscopy, physical property measurement system, SEM, TGA, and compression test. Moreover, the catalytic performance of the magnetic hydrogels was explored. The magnetic hydrogels (C = 7.5 wt % presented excellent catalytic activity and provided a sensitive response to H2O2 detection even at a concentration level of 5 × 10−6 mol·L−1. This approach to preparing magnetic hydrogels loaded with Fe3O4 nanoparticles endows xylan-based hydrogels with new promising applications in biotechnology and environmental chemistry.

  4. Mechanism of action of clostridial glycine reductase: Isolation and characterization of a covalent acetyl enzyme intermediate

    International Nuclear Information System (INIS)

    Arkowitz, R.A.; Abeles, R.H.

    1991-01-01

    Clostridial glycine reductase consists of proteins A, B, and C and catalyzes the reaction glycine + P i + 2e - → acetyl phosphate + NH 4 + . Evidence was previously obtained that is consistent with the involvement of an acyl enzyme intermediate in this reaction. The authors now demonstrate that protein C catalyzes exchange of [ 32 P]P i into acetyl phosphate, providing additional support for an acetyl enzyme intermediate on protein C. Furthermore, they have isolated acetyl protein C and shown that it is qualitatively, catalytically competent. Acetyl protein C can be obtained through the forward reaction from protein C and Se-(carboxymethyl)selenocysteine-protein A, which is generated by the reaction of glycine with proteins A and B. Acetyl protein C can also be generated through the reverse reaction by the addition of acetyl phosphate to protein C. Both procedures lead to the same acetyl enzyme. The acetyl enzyme reacts with P i to give acetyl phosphate. When [ 14 C]acetyl protein C is denaturated with TCA and redissolved with urea, radioactivity remained associated with the protein. Treatment with KBH 4 removes all the radioactivity associated with protein C, resulting in the formation of [ 14 C]ethanol. They conclude that a thiol group on protein C is acetylated. Proteins A and C together catalyze the exchange of tritium atoms from [ 3 H]H 2 O into acetyl phosphate. This exchange reaction supports the proposal that an enol of the acetyl enzyme is an intermediate in the reaction sequence

  5. Molecular cloning and characterization of a novel pyrethroid-hydrolyzing esterase originating from the Metagenome

    Directory of Open Access Journals (Sweden)

    Liu Yu

    2008-12-01

    Full Text Available Abstract Background Pyrethroids and pyrethrins are widely used insecticides. Extensive applications not only result in pest resistance to these insecticides, but also may lead to environmental issues and human exposure. Numerous studies have shown that very high exposure to pyrethroids might cause potential problems to man and aquatic organisms. Therefore, it is important to develop a rapid and efficient disposal process to eliminate or minimize contamination of surface water, groundwater and agricultural products by pyrethroid insecticides. Bioremediation is considered to be a reliable and cost-effective technique for pesticides abatement and a major factor determining the fate of pyrethroid pesticides in the environment, and suitable esterase is expected to be useful for potential application for detoxification of pyrethroid residues. Soil is a complex environment considered as one of the main reservoirs of microbial diversity on the planet. However, most of the microorganisms in nature are inaccessible as they are uncultivable in the laboratory. Metagenomic approaches provide a powerful tool for accessing novel valuable genetic resources (novel enzymes and developing various biotechnological applications. Results The pyrethroid pesticides residues on foods and the environmental contamination are a public safety concern. Pretreatment with pyrethroid-hydrolyzing esterase has the potential to alleviate the conditions. To this end, a pyrethroid-hydrolyzing esterase gene was successfully cloned using metagenomic DNA combined with activity-based functional screening from soil, sequence analysis of the DNA responsible for the pye3 gene revealed an open reading frame of 819 bp encoding for a protein of 272 amino acid residues. Extensive multiple sequence alignments of the deduced amino acid of Pye3 with the most homologous carboxylesterases revealed moderate identity (45–49%. The recombinant Pye3 was heterologously expressed in E. coli BL21(DE3

  6. Small molecule inhibitors of bromodomain-acetyl-lysine interactions.

    Science.gov (United States)

    Brand, Michael; Measures, Angelina R; Measures, Angelina M; Wilson, Brian G; Cortopassi, Wilian A; Alexander, Rikki; Höss, Matthias; Hewings, David S; Rooney, Timothy P C; Paton, Robert S; Conway, Stuart J

    2015-01-16

    Bromodomains are protein modules that bind to acetylated lysine residues. Their interaction with histone proteins suggests that they function as "readers" of histone lysine acetylation, a component of the proposed "histone code". Bromodomain-containing proteins are often found as components of larger protein complexes with roles in fundamental cellular process including transcription. The publication of two potent ligands for the BET bromodomains in 2010 demonstrated that small molecules can inhibit the bromodomain-acetyl-lysine protein-protein interaction. These molecules display strong phenotypic effects in a number of cell lines and affect a range of cancers in vivo. This work stimulated intense interest in developing further ligands for the BET bromodomains and the design of ligands for non-BET bromodomains. Here we review the recent progress in the field with particular attention paid to ligand design, the assays employed in early ligand discovery, and the use of computational approaches to inform ligand design.

  7. Synthesis of polyrotaxanes from acetyl-β-cyclodextrin

    Science.gov (United States)

    Ristić, I. S.; Nikolić, L.; Nikolić, V.; Ilić, D.; Budinski-Simendić, J.

    2011-12-01

    Polyrotaxanes are intermediary products in the synthesis of topological gels. They are created by inclusion complex formation of hydrophobic linear macromolecules with cyclodextrins or their derivatives. Then, pairs of cyclodextrin molecules with covalently linkage were practically forming the nodes of the semi-flexible polymer network. Such gels are called topological gels and they can absorb huge quantities of water due to the net flexibility allowing the poly(ethylene oxide) chains to slide through the cyclodextrin cavities, without being pulled out altogether. For polyrotaxane formation poly(ethylene oxide) was used like linear macromolecules. There are hydroxyl groups at poly(ethylene oxide) chains, whereby the linking of the voluminous molecules should be made. To avoid the reaction of cyclodextrin OH groups with stoppers, they should be protected by, e.g., acetylation. In this work, the acetylation of the OH groups of β-cyclodextrin was performed by acetic acid anhydride with iodine as the catalyst. The acetylation reaction was assessed by the FTIR and HPLC method. By the HPLC analysis was found that the acetylation was completed in 20 minutes. Inserting of poly(ethylene oxide) with 4000 g/mol molecule mass into acetyl-β-cyclodextrin with 2:1 poly(ethylene oxide) monomer unit to acetyl-β-cyclodextrin ratio was also monitored by FTIR, and it was found that the process was completed in 12 h at the temperature of 10°C. If the process is performed at temperatures above 10°C, or for periods longer than 12 hours, the process of uncontrolled hydrolysis of acetate groups was initiated.

  8. A novel feruloyl esterase from rumen microbial metagenome: Gene cloning and enzyme characterization in the release of mono- and diferulic acids

    Science.gov (United States)

    A feruloyl esterase (FAE) gene was isolated from a rumen microbial metagenome, cloned into E. coli, and expressed in active form. The enzyme (RuFae4) was classified as a Type D feruloyl esterase based on its action on synthetic substrates and ability to release diferulates. The RuFae4 alone releas...

  9. Histochemical studies on genetical control of hormonal enzyme inducibility in the mouse. IV: Cellular localization of androgen sensitive nonspecific esterase in the epididymis

    DEFF Research Database (Denmark)

    Kirkeby, S; Blecher, S R

    1981-01-01

    Nonspecific esterase of mouse epididymis has previously been studied histochemically, using alpha naphthyl-acetate and 5-bromoindoxyl acetate techniques, as well as certain inhibitors. Epithelial cell types of the epididymis have been characterized, and certain esterase isozymes in a particular...

  10. Crystallization and preliminary X-ray diffraction analysis of the glucuronoyl esterase catalytic domain from Hypocrea jecorina

    International Nuclear Information System (INIS)

    Wood, S. J.; Li, X.-L.; Cotta, M. A.; Biely, P.; Duke, N. E. C.; Schiffer, M.; Pokkuluri, P. R.

    2008-01-01

    The catalytic domain of the glucuronoyl esterase from H. jecorina was overexpresssed, purified and crystallized in space group P2 1 2 1 2 1 . X-ray diffraction data were collected to 1.9 Å resolution. The catalytic domain of the glucuronoyl esterase from Hypocrea jecorina (anamorph Trichoderma reesei) was overexpresssed, purified and crystallized by the sitting-drop vapor-diffusion method using 1.4 M sodium/potassium phosphate pH 6.9. The crystals belonged to space group P2 1 2 1 2 1 and X-ray diffraction data were collected to 1.9 Å resolution. This is the first enzyme with glucoronoyl esterase activity to be crystallized; its structure will be valuable in lignocellulose-degradation research

  11. Crystallization and preliminary X-ray diffraction analysis of the glucuronoyl esterase catalytic domain from Hypocrea jecorina

    Energy Technology Data Exchange (ETDEWEB)

    Wood, S. J. [Biosciences Division, Argonne National Laboratory, Argonne, IL 60439 (United States); Li, X.-L.; Cotta, M. A. [Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, USDA-ARS, Peoria, Illinois 61604 (United States); Biely, P. [Institute of Chemistry, Slovak Academy of Sciences, 845 38 Bratislava (Slovakia); Duke, N. E. C.; Schiffer, M.; Pokkuluri, P. R., E-mail: rajp@anl.gov [Biosciences Division, Argonne National Laboratory, Argonne, IL 60439 (United States)

    2008-04-01

    The catalytic domain of the glucuronoyl esterase from H. jecorina was overexpresssed, purified and crystallized in space group P2{sub 1}2{sub 1}2{sub 1}. X-ray diffraction data were collected to 1.9 Å resolution. The catalytic domain of the glucuronoyl esterase from Hypocrea jecorina (anamorph Trichoderma reesei) was overexpresssed, purified and crystallized by the sitting-drop vapor-diffusion method using 1.4 M sodium/potassium phosphate pH 6.9. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1} and X-ray diffraction data were collected to 1.9 Å resolution. This is the first enzyme with glucoronoyl esterase activity to be crystallized; its structure will be valuable in lignocellulose-degradation research.

  12. Molecular characterization of a novel family VIII esterase from burkholderia multivorans UWC10

    CSIR Research Space (South Africa)

    Rashamuse, KJ

    2007-02-01

    Full Text Available et al., 1992). Here we report the cloning, purification, and 3D model of a novel family VIII esterase from Burkholderia multivorans UWC10. To our knowledge no report of esterolytic activity from B. multivorans is currently available. METHODOLOGY... stream_source_info Rashamuse1_2007_d.pdf.txt stream_content_type text/plain stream_size 9884 Content-Encoding UTF-8 stream_name Rashamuse1_2007_d.pdf.txt Content-Type text/plain; charset=UTF-8 Molecular Characterization...

  13. Esterase activity able to hydrolyze dietary antioxidant hydroxycinnamates is distributed along the intestine of mammals

    DEFF Research Database (Denmark)

    Andreasen, Mette Findal; Kroon, P A; Williamson, G

    2001-01-01

    and may contribute to the beneficial effects derived from consumption of cereal bran. However, these compounds are ester linked to the main polymers in the plant cell wall and cannot be absorbed in this complex form. The present work shows that esterases with activity toward esters of the major dietary......Hydroxycinnamic acids are effective antioxidants and are abundant components of plant cell walls, especially in cereal bran. For example, wheat and rye brans are rich sources of the hydroxycinnamates ferulic acid, sinapic acid, and p-coumaric acid. These phenolics are part of human and animal diets...

  14. Fibulin-1C, C1 esterase inhibitor and glucose regulated protein 75 interact with the CREC proteins, calumenin and reticulocalbin

    DEFF Research Database (Denmark)

    Hansen, Gry Aune Westergaard; Ludvigsen, Maja; Jacobsen, Christian

    2015-01-01

    Affinity purification, immunoprecipitation, gel electrophoresis and mass spectrometry were used to identify fibulin-1C, C1 esterase inhibitor and glucose regulated protein 75, grp75, as binding partners of the CREC proteins, calumenin and reticulocalbin. Surface plasmon resonance was used to verify...... the interaction of all three proteins with each of the CREC proteins. Fibulin-1C interacts with calumenin and reticulocalbin with an estimated dissociation constant around 50-60 nM. The interaction, at least for reticulocalbin, was not dependent upon the presence of Ca2+. C1 esterase inhibitor interacted...

  15. Arginine-esterase activity of kallikrein in the sera of whole-body irradiated rats and guinea-pigs

    Energy Technology Data Exchange (ETDEWEB)

    Pouckova, P; Pospisil, J; Dienstbier, Z [Karlova Univ., Prague (Czechoslovakia). Biofyzikalni Ustav

    1977-09-01

    In whole-body irradiated rats (800 R=LDsub(50/30)) and guinea pigs (300 R=LDsub(50/30)) changes were investigated in the arginine esterase activity of kallikrein in native serum as well as in serum exposed to contact with a clay suspension. From the values obtained the activity of prekallikrein was calculated. While in the rat serum significant changes in the arginine esterase activity of kallikrein were found, in the guinea pig serum the kallikrein activity did not change markedly. The activity of prekallikrein immediately after irradiation assumes a similar course in both types of laboratory animals while during later intervals a reverse pattern was observed.

  16. Characterisation of esterases as potential biomarkers of pesticide exposure in the lugworm Arenicola marina (Annelida: Polychaeta)

    International Nuclear Information System (INIS)

    Hannam, Marie L.; Hagger, Josephine A.; Jones, Malcolm B.; Galloway, Tamara S.

    2008-01-01

    Here, we identify and characterise cholinesterase (ChE) and carboxylesterase (CbE) activities in the body tissues of the sediment dwelling worm Arenicola marina. Exposure to the organophosphorus pesticide azamethiphos yielded an in vitro IC 50 of 5 μg l -1 for propionylcholinesterase (PChE). PChE was significantly inhibited in vivo after a 10 day exposure to 100 μg l -1 azamethiphos, equivalent to the recommended aquatic application rate (ANOVA; F = 2.75, P = 0.033). To determine sensitivity to environmental conditions, A. marina were exposed for 10 days to field collected sediments. PChE activity was significantly lower in worms exposed to sediments from an estuary classified to be at high risk from point source pollution by the UK Environment Agency (ANOVA; F = 15.33, P < 0.001). Whilst causality cannot be directly attributed from these latter exposures, they provide an important illustration of the potential utility of esterase activity as a biomarker of environmental quality in this ecologically relevant sentinel species. - This paper provides a preliminary characterisation of esterase enzyme activities in the tissues and body fluids of the sediment dwelling worm Arenicola marina and explores their potential use as biomarkers of organophosphorus pesticide exposure in the marine environment

  17. Characterisation of esterases as potential biomarkers of pesticide exposure in the lugworm Arenicola marina (Annelida: Polychaeta)

    Energy Technology Data Exchange (ETDEWEB)

    Hannam, Marie L. [Ecotoxicology and Stress Biology Research Centre, School of Biological Sciences, University of Plymouth, Drake Circus, Plymouth, Devon, PL4 8AA (United Kingdom)], E-mail: marie.hannam@plymouth.ac.uk; Hagger, Josephine A.; Jones, Malcolm B.; Galloway, Tamara S. [Ecotoxicology and Stress Biology Research Centre, School of Biological Sciences, University of Plymouth, Drake Circus, Plymouth, Devon, PL4 8AA (United Kingdom)

    2008-03-15

    Here, we identify and characterise cholinesterase (ChE) and carboxylesterase (CbE) activities in the body tissues of the sediment dwelling worm Arenicola marina. Exposure to the organophosphorus pesticide azamethiphos yielded an in vitro IC{sub 50} of 5 {mu}g l{sup -1} for propionylcholinesterase (PChE). PChE was significantly inhibited in vivo after a 10 day exposure to 100 {mu}g l{sup -1} azamethiphos, equivalent to the recommended aquatic application rate (ANOVA; F = 2.75, P = 0.033). To determine sensitivity to environmental conditions, A. marina were exposed for 10 days to field collected sediments. PChE activity was significantly lower in worms exposed to sediments from an estuary classified to be at high risk from point source pollution by the UK Environment Agency (ANOVA; F = 15.33, P < 0.001). Whilst causality cannot be directly attributed from these latter exposures, they provide an important illustration of the potential utility of esterase activity as a biomarker of environmental quality in this ecologically relevant sentinel species. - This paper provides a preliminary characterisation of esterase enzyme activities in the tissues and body fluids of the sediment dwelling worm Arenicola marina and explores their potential use as biomarkers of organophosphorus pesticide exposure in the marine environment.

  18. Crystallization and preliminary crystallographic studies of the metalloglycoprotein esterase A4 using a baculovirus expression system

    Energy Technology Data Exchange (ETDEWEB)

    Hiraki, Toshiki [Protein Design Laboratory, Yokohama City University, 1-7-29 Suehiro, Tsurumi, Yokohama 230-0045 (Japan); Shibayama, Naoya [Department of Physiology, Division of Biophysics, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498 (Japan); Yoon, Young-Ho [Protein Design Laboratory, Yokohama City University, 1-7-29 Suehiro, Tsurumi, Yokohama 230-0045 (Japan); Yun, Kyung-Mook [Department of Physiology, Division of Biophysics, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498 (Japan); Hamamoto, Toshiro [Department of Biochemistry, Jichi Medical University, 3311-1 Yakushiji, Shimotsuke, Tochigi 329-0498 (Japan); Tame, Jeremy R. H.; Park, Sam-Yong, E-mail: park@tsurumi.yokohama-cu.ac.jp [Protein Design Laboratory, Yokohama City University, 1-7-29 Suehiro, Tsurumi, Yokohama 230-0045 (Japan)

    2007-09-01

    Esterase A4 (EA4) is a timer protein found in diapause eggs of the silkworm Bombyx mori. The gene for this metalloglycoprotein was cloned from B. mori eggs and expressed using a baculovirus expression system in silkworm pupae. Crystals of the purified protein have been grown that diffract to beyond 2.1 Å resolution at 100 K using synchrotron radiation. Esterase A4 (EA4) is a timer protein found in diapause eggs of the silkworm Bombyx mori. The gene for this metalloglycoprotein was cloned from B. mori eggs and expressed using a baculovirus expression system in silkworm pupae. Crystals of the purified protein have been grown that diffract to beyond 2.1 Å resolution at 100 K using synchrotron radiation. The protein crystals belong to space group P2{sub 1}, with unit-cell parameters a = 47.1, b = 73.9, c = 47.4 Å, β = 104.1°. With one dimer per asymmetric unit, the crystal volume per unit protein weight (V{sub M}) is 2.3 Å{sup 3} Da{sup −1} and the solvent content is 47%.

  19. Analysis of esterase isozyme and SSR for mutagenic progenies induced by space mutation in mustard

    International Nuclear Information System (INIS)

    Shen Jinjuan; Liu Yihua; Zhang Zhaorong; Ran Guangkui; Zhao Shouzhong; Xiao Li

    2012-01-01

    Seeds of five mustard (Brassica juncea Coss) varieties were carried into outer space by 'Shijian No.8' satellite. After five years' consecutive planting and selection, ten relatively stable mutant lines were obtained, which had significant variation in agronomic and economic characters. The mutant lines and their original varieties without space mutation treatment as control were studied by esterase isozyme and SSR analyses. Electrophoresis analysis of esterase isozymes indicated that there were differences between mutant lines and their controls in enzyme types and enzyme activity. Different mustard varieties had different enzymographs, and so did the mutants induced by space mutation, which shows different sensitivity among different mustard varieties. The SSR analysis showed that large differences were found in the SSR loci between mutant lines and their original variety, the variation frequency was between 9.52% and 57.14% with an average frequency of 26.19% for all the mutant lines. Among the mutant SSR loci, about 56.36% showed changes in band number and 43.64% in molecular weight. These results indicated that the ten mutant lines had large genetic difference in phenotype, genomic sequence and gene expression, and the outer space mutation would be an effective method to develop new mustard germplasm and variety. (authors)

  20. Predicting post-translational lysine acetylation using support vector machines

    DEFF Research Database (Denmark)

    Gnad, Florian; Ren, Shubin; Choudhary, Chunaram

    2010-01-01

    spectrometry to identify 3600 lysine acetylation sites on 1750 human proteins covering most of the previously annotated sites and providing the most comprehensive acetylome so far. This dataset should provide an excellent source to train support vector machines (SVMs) allowing the high accuracy in silico...

  1. High regioselective acetylation of vitamin A precursors using lipase ...

    African Journals Online (AJOL)

    Administrator

    2011-09-26

    Sep 26, 2011 ... High regioselective acetylation of vitamin A precursors using lipase B from Candida antarctica in organic media. Jingpeng Sun, Keju Jing* and Yinghua Lu. Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen. University, Xiamen 361005, P. R. ...

  2. Surface effects in the acetylation of granular potato starch

    NARCIS (Netherlands)

    Steeneken, P.A.M.; Woortman, A.J.J.

    2008-01-01

    The occurrence of surface effects in the acetylation of granular potato starch with acetic anhydride to degrees of substitution 0.04-0.2 was studied by two different approaches. The first approach involved the fractionation of granular starch acetates into five different size classes and analysis of

  3. Effect Of Nicotine And Tobacco Consumption On Brain Acetyl ...

    African Journals Online (AJOL)

    The effect of nicotine and tobacco consumption on brain acetyl cholinesterase and serum alkaline phosphatase in rats was studied. Rats were divided into three groups and the first group was fed rat chow and water ad libitum and an oral administration of 2ml of 0.1%(v/v) nicotine per 100g body weight of rats per day.

  4. Genetic control of differential acetylation in diabetic rats.

    Directory of Open Access Journals (Sweden)

    Pamela J Kaisaki

    Full Text Available Post-translational protein modifications such as acetylation have significant regulatory roles in metabolic processes, but their relationship to both variation in gene expression and DNA sequence is unclear. We address this question in the Goto-Kakizaki (GK rat inbred strain, a model of polygenic type 2 diabetes. Expression of the NAD-dependent deacetylase Sirtuin-3 is down-regulated in GK rats compared to normoglycemic Brown Norway (BN rats. We show first that a promoter SNP causes down-regulation of Sirtuin-3 expression in GK rats. We then use mass-spectrometry to identify proteome-wide differential lysine acetylation of putative Sirtuin-3 protein targets in livers of GK and BN rats. These include many proteins in pathways connected to diabetes and metabolic syndrome. We finally sequence GK and BN liver transcriptomes and find that mRNA expression of these targets does not differ significantly between GK and BN rats, in contrast to other components of the same pathways. We conclude that physiological differences between GK and BN rats are mediated by a combination of differential protein acetylation and gene transcription and that genetic variation can modulate acetylation independently of expression.

  5. Effect of acetyl groups of wood on furfural preparation

    Energy Technology Data Exchange (ETDEWEB)

    Skvortsov, S.V.; Kolchina, N.P.; Miroshnichenko, V.G.

    1981-01-01

    The deacetylation (4% Na/sub 2/CO/sub 3/, 60 degrees) of birch sawdust prior to hydrolysis decreased the yield of furfural, presumably due to thermal degradation of pentosans and buildup of HCOOH in the wood stock. Thus, while untreated birch sawdust gave 6.6% furfural, the acetylated sawdust gave only 4.5% furfural.

  6. Effect of acetylation and varietal differences on the pasting ...

    African Journals Online (AJOL)

    The pasting properties of starch from eight varieties of corn; Okomasa, Obatanpa, Dodzi, Mamaba, Dadaba, Dorke, Golden crystal, and CIDA-ba were studied to establish the effects of acetylation and varietal differences on the pasting properties. Native starches extracted from the corn varieties were modified with 10% v/v ...

  7. Acetylation of wood components and fourier transform infra-red ...

    African Journals Online (AJOL)

    In this study, the reactivity of wood components with acetic anhydride or vinyl acetate was studied. It was found that the reactivity of wood components was virgin wood flour > holocellulose >> a-cellulose. Acetylation of Turkish pine or cedar wood flour with acetic anhydride was significantly improved in the presence of ...

  8. DNA Damage-Induced Acetylation of Lysine 3016 of ATM Activates ATM Kinase Activity▿ †

    OpenAIRE

    Sun, Yingli; Xu, Ye; Roy, Kanaklata; Price, Brendan D.

    2007-01-01

    The ATM protein kinase is essential for cells to repair and survive genotoxic events. The activation of ATM's kinase activity involves acetylation of ATM by the Tip60 histone acetyltransferase. In this study, systematic mutagenesis of lysine residues was used to identify regulatory ATM acetylation sites. The results identify a single acetylation site at lysine 3016, which is located in the highly conserved C-terminal FATC domain adjacent to the kinase domain. Antibodies specific for acetyl-ly...

  9. The Acetyl Group Buffering Action of Carnitine Acetyltransferase Offsets Macronutrient-Induced Lysine Acetylation of Mitochondrial Proteins

    Directory of Open Access Journals (Sweden)

    Michael N. Davies

    2016-01-01

    Full Text Available Lysine acetylation (AcK, a posttranslational modification wherein a two-carbon acetyl group binds covalently to a lysine residue, occurs prominently on mitochondrial proteins and has been linked to metabolic dysfunction. An emergent theory suggests mitochondrial AcK occurs via mass action rather than targeted catalysis. To test this hypothesis, we performed mass spectrometry-based acetylproteomic analyses of quadriceps muscles from mice with skeletal muscle-specific deficiency of carnitine acetyltransferase (CrAT, an enzyme that buffers the mitochondrial acetyl-CoA pool by converting short-chain acyl-CoAs to their membrane permeant acylcarnitine counterparts. CrAT deficiency increased tissue acetyl-CoA levels and susceptibility to diet-induced AcK of broad-ranging mitochondrial proteins, coincident with diminished whole body glucose control. Sub-compartment acetylproteome analyses of muscles from obese mice and humans showed remarkable overrepresentation of mitochondrial matrix proteins. These findings reveal roles for CrAT and L-carnitine in modulating the muscle acetylproteome and provide strong experimental evidence favoring the nonenzymatic carbon pressure model of mitochondrial AcK.

  10. Comprehensive Proteomic Analysis of Lysine Acetylation in the Foodborne Pathogen Trichinella spiralis

    Directory of Open Access Journals (Sweden)

    Yong Yang

    2018-01-01

    Full Text Available Lysine acetylation is a dynamic and highly conserved post-translational modification that plays a critical role in regulating diverse cellular processes. Trichinella spiralis is a foodborne parasite with a considerable socio-economic impact. However, to date, little is known regarding the role of lysine acetylation in this parasitic nematode. In this study, we utilized a proteomic approach involving anti-acetyl lysine-based enrichment and highly sensitive mass spectrometry to identify the global acetylated proteome and investigate lysine acetylation in T. spiralis. In total, 3872 lysine modification sites were identified in 1592 proteins that are involved in a wide variety of biological processes. Consistent with the results of previous studies, a large number of the acetylated proteins appear to be involved in metabolic and biosynthetic processes. Interestingly, according to the functional enrichment analysis, 29 acetylated proteins were associated with phagocytosis, suggesting an important role of lysine acetylation in this process. Among the identified proteins, 15 putative acetylation motifs were detected. The presence of serine downstream of the lysine acetylation site was commonly observed in the regions surrounding the sites. Moreover, protein interaction network analysis revealed that various interactions are regulated by protein acetylation. These data represent the first report of the acetylome of T. spiralis and provide an important resource for further explorations of the role of lysine acetylation in this foodborne pathogen.

  11. In-gel detection of esterase-like albumin activity: Characterization of esterase-free sera albumin and its putative role as non-invasive biomarker of hepatic fibrosis

    Directory of Open Access Journals (Sweden)

    Areeba Ahmad

    2017-07-01

    Full Text Available Albumin is a globular and un-glycosylated multifunctional plasma protein and thus correlated with several human diseases. Owing to esterase contamination, albumin levels are usually misleading. In this study, we propose methodical accuracy for albumin estimation taking healthy and fibrotic rats. Liver fibrosis in rats was generated by N′-Nitrosodimethylamine (NDMA (10 mg/kg body weight within three weeks followed by its confirmation through H&E and immunohistochemical staining for α-SMA expression. Animal sera were screened by native polyacrylamide gel electrophoresis (native-PAGE (7.5%. In-gel esterase-like albumin activity was detected using α- and β-naphthyl acetate (5.58 × 10−3 mM; pH 7.5 as substrate. Sera albumin was purified from unstained PA gel-slices through electroelution. Subsequent to conformation of albumin purity by its molecular weight determination using SDS–PAGE (10% and peptide mass fingerprinting by MALDI-TOF-MS, samples were treated with different concentrations of urea. Urea-treated albumins were screened for esterase activity, conformational change and, albumin levels by immunoblotting. Our results demonstrate that esterase-like albumin activity in rat sera albumin is located in domain-III. The esterase-like activity remains detectable up to 4 M urea, which diminishes with increasing urea concentrations. Further, immunoblotting of urea-treated albumin samples displays a significant decline in purified protein bands, indicating hypoalbuminemia during hepatic fibrosis in rats. In conclusion, the present approach of albumin separation and estimation is of potential interest and may be recommended for diagnostic purposes.

  12. Sequential Dy(OTf)3 -Catalyzed Solvent-Free Per-O-Acetylation and Regioselective Anomeric De-O-Acetylation of Carbohydrates.

    Science.gov (United States)

    Yan, Yi-Ling; Guo, Jiun-Rung; Liang, Chien-Fu

    2017-09-19

    Dysprosium(III) trifluoromethanesulfonate-catalyzed per-O-acetylation and regioselective anomeric de-O-acetylation of carbohydrates can be tuned by adjusting the reaction medium. In this study, the per-O-acetylation of unprotected sugars by using a near-stoichiometric amount of acetic anhydride under solvent-free conditions resulted in the exclusive formation of acetylated saccharides as anomeric mixtures, whereas anomeric de-O-acetylation in methanol resulted in a moderate-to-excellent yield. Reactions with various unprotected monosaccharides or disaccharides followed by a semi-one-pot sequential conversion into the corresponding acetylated glycosyl hemiacetal also resulted in high yields. Furthermore, the obtained hemiacetals could be successfully transformed into trichloroimidates after Dy(OTf) 3 -catalyzed glycosylation. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Studies investigating the excretion of acetyl urea in the milk of dairy cows receiving oral doses of 14C acetyl urea

    International Nuclear Information System (INIS)

    Bergner, H.; Kijora, C.; Goersch, R.

    1976-01-01

    2 experimental cows were fed acetyl urea several weeks before the trial was started. The first cow received a daily amount of 200 g and the second cow 855 g. On the first day of experiment both cows were given 5 mCi of 14 C acetyl urea intraruminally. Up to 6 hrs after the beginning of the experiment acetyl urea in blood plasma was shown to contain a higher proportion of 14 C activity than urea. 0.21 g urea and 0.18 g acetyl urea were contained in 1 kg of milk from cow No 1 while 1 kg of milk from cow No 2 contained 0.18 g urea and 0.12 g acetyl urea. The feeding of acetyl urea to dairy cows is not recommended on the basis of the fact that any further contamination of human nutrition with foreign substances should be possibly avoided. (author)

  14. Studies investigating the excretion of acetyl urea in the milk of dairy cows receiving oral doses of /sup 14/C acetyl urea

    Energy Technology Data Exchange (ETDEWEB)

    Bergner, H; Kijora, C; Goersch, R [Humboldt-Universitaet, Berlin (German Democratic Republic). Sektion Tierproduktion und Veterinaermedizin

    1976-01-01

    2 experimental cows were fed acetyl urea several weeks before the trial was started. The first cow received a daily amount of 200 g and the second cow 855 g. On the first day of experiment both cows were given 5 mCi of /sup 14/C acetyl urea intraruminally. Up to 6 hrs after the beginning of the experiment acetyl urea in blood plasma was shown to contain a higher proportion of /sup 14/C activity than urea. 0.21 g urea and 0.18 g acetyl urea were contained in 1 kg of milk from cow No 1 while 1 kg of milk from cow No 2 contained 0.18 g urea and 0.12 g acetyl urea. The feeding of acetyl urea to dairy cows is not recommended on the basis of the fact that any further contamination of human nutrition with foreign substances should be possibly avoided.

  15. ISOLATION OF JUVENILE HORMONES ESTERASE AND ITS PARTIAL CDNA CLONE FROM THE BEETLE, TENEBRIO MOLITOR. (R825433)

    Science.gov (United States)

    Juvenile hormone esterase (JHE) plays an essential role in insect development. It is partially responsible for the clearance of juvenile hormone (JH) which regulates various aspects of insect development and reproduction. Because of its role in regulating JH titer, this enzyme...

  16. Effect of Temperature and High Pressure on the Activity and Mode of Action of Fungal Pectin Methyl Esterase

    NARCIS (Netherlands)

    Duvetter, T.; Fraeye, I.; Sila, D.N.; Verlent, I.; Smout, C.; Clynen, E.; Schoofs, L.; Schols, H.A.; Hendrickx, M.; Loey, van A.

    2006-01-01

    Pectin was de-esterified with purified recombinant Aspergillus aculeatus pectin methyl esterase (PME) during isothermal-isobaric treatments. By measuring the release of methanol as a function of treatment time, the rate of enzymatic pectin conversion was determined. Elevated temperature and pressure

  17. Generation of transgenic wheat (Triticum aestivum L.) accumulating heterologous endo-xylanase or ferulic acid esterase in the endosperm.

    Science.gov (United States)

    Harholt, Jesper; Bach, Inga C; Lind-Bouquin, Solveig; Nunan, Kylie J; Madrid, Susan M; Brinch-Pedersen, Henrik; Holm, Preben B; Scheller, Henrik V

    2010-04-01

    Endo-xylanase (from Bacillus subtilis) or ferulic acid esterase (from Aspergillus niger) were expressed in wheat under the control of the endosperm-specific 1DX5 glutenin promoter. Constructs both with and without the endoplasmic reticulum retention signal (Lys-Asp-Glu-Leu) KDEL were used. Transgenic plants were recovered in all four cases but no qualitative differences could be observed whether KDEL was added or not. Endo-xylanase activity in transgenic grains was increased between two and threefold relative to wild type. The grains were shrivelled and had a 25%-33% decrease in mass. Extensive analysis of the cell walls showed a 10%-15% increase in arabinose to xylose ratio, a 50% increase in the proportion of water-extractable arabinoxylan, and a shift in the MW of the water-extractable arabinoxylan from being mainly larger than 85 kD to being between 2 and 85 kD. Ferulic acid esterase-expressing grains were also shrivelled, and the seed weight was decreased by 20%-50%. No ferulic acid esterase activity could be detected in wild-type grains whereas ferulic acid esterase activity was detected in transgenic lines. The grain cell walls had 15%-40% increase in water-unextractable arabinoxylan and a decrease in monomeric ferulic acid between 13% and 34%. In all the plants, the observed changes are consistent with a plant response that serves to minimize the effect of the heterologously expressed enzymes by increasing arabinoxylan biosynthesis and cross-linking.

  18. Feruloyl esterases as a tool for the release of phenolic compounds from agro-industrial by-products

    NARCIS (Netherlands)

    Benoit, Isabelle; Navarro, David; Marnet, Nathalie; Rakotomanomana, Nnjara; Lesage-Meessen, Laurence; Sigoillot, Jean-Claude; Asther, Marcel; Asther, Michèle

    2006-01-01

    Agro-industrial by-products are a potential source of added-value phenolic acids with promising applications in the food and pharmaceutical industries. Here two purified feruloyl esterases from Aspergillus niger, FAEA and FAEB were tested for their ability to release phenolic acids such as caffeic

  19. Spatial distribution and esterase activity in populations of Aedes (Stegomyia aegypti (Linnaeus (Diptera: Culicidae resistant to temephos

    Directory of Open Access Journals (Sweden)

    Wanessa Porto Tito Gambarra

    2013-04-01

    Full Text Available INTRODUCTION: The need for studies that describe the resistance patterns in populations of Aedes aegypti (Linnaeus in function of their region of origin justified this research, which aimed to characterize the resistance to temephos and to obtain information on esterase activity in populations of Aedes aegypti collected in municipalities of the State of Paraíba. METHODS: Resistance to temephos was evaluated and characterized from the diagnostic dose of 0.352mg i.a./L and multiple concentrations that caused mortalities between 5% and 99%. Electrophoresis of isoenzymes was used to verify the patterns of esterase activity among populations of the vector. RESULTS: All populations of Aedes aegypti were resistant to temephos, presenting a resistance rate (RR greater than 20. The greatest lethal dose 50% of the sample (CL50 was found for the municipality of Lagoa Seca, approximately forty-one times the value of CL50 for the Rockefeller population. The populations characterized as resistant showed two to six regions of α and β-esterase, called EST-1 to EST-6, while the susceptible population was only seen in one region of activity. CONCLUSIONS: Aedes aegypti is widely distributed and shows a high degree of resistance to temephos in all municipalities studied. In all cases, esterases are involved in the metabolism and, consequently, in the resistance to temephos.

  20. Pig Liver Esterase (PLE) as Biocatalyst in Organic Synthesis: From Nature to Cloning and to Practical Applications

    NARCIS (Netherlands)

    Dominguez de Maria, Pablo; Garcia-Burgos, Carlos A.; Bargeman, Gerrald; van Gemert, Robert W.

    2007-01-01

    Pig liver esterase (PLE, EC 3.1.1.1) has been employed extensively for research purposes during the last three decades, especially in kinetic resolutions, in desymmetrizations of prochiral substrates, and in the synthesis of nucleosides. Its practical use, however, has been traditionally hampered

  1. Functional characterisation of a metagenome derived family VIII esterase with a deacetylation activity on ß-lactam antibiotics

    CSIR Research Space (South Africa)

    Mokoena, N

    2013-08-01

    Full Text Available characterisation of a novel esterase (Est22) derived from an acidic Leachate environment. The enzyme is 423 amino acids in length and contained 22aa signal peptide. Analysis of the Est22 primary structure revealed the presence of N-terminus S-x-x-K sequence, which...

  2. Crystallization and preliminary crystallographic analysis of an esterase with a novel domain from the hyperthermophile Thermotoga maritima

    NARCIS (Netherlands)

    Sun, Lei; Levisson, Mark; Hendriks, Sjon; Akveld, Twan; Kengen, Serve W. M.; Dijkstra, Bauke W.; van der Oost, John

    A predicted esterase ( EstA) with an unusual new domain from the hyperthermophilic bacterium Thermotoga maritima has been cloned and overexpressed in Escherichia coli. The purified protein was crystallized by the hanging-drop vapour-diffusion technique in the presence of lithium sulfate and

  3. Paralogous gene analysis reveals a highly enantioselective 1,2-O-isopropylideneglycerol caprylate esterase of Bacillus subtilis

    NARCIS (Netherlands)

    Droge, MJ; Bos, R; Quax, WJ

    Carboxylesterase NP of Bacillus subtilis Thai 1-8, characterized in 1992 as a very enantioselective (S)-naproxen esterase, was found to show no enantiopreference towards (S)-1,2-O-isopropylideneglycerol (IPG) esters. The ybfK gene was identified by the B. subtilis genome project as an unknown gene

  4. Generation of transgenic wheat (Triticum aestivum L.) accumulating heterologous endo-xylanase or ferulic acid esterase in the endosperm

    DEFF Research Database (Denmark)

    Harholt, Jesper; Bach, Inga Christensen; Lind Bouquin, Solveig

    2010-01-01

    Endo-xylanase (from Bacillus subtilis) or ferulic acid esterase (from Aspergillus niger) were expressed in wheat under the control of the endosperm-specific 1DX5 glutenin promoter. Constructs both with and without the endoplasmic reticulum retention signal (Lys-Asp-Glu-Leu) KDEL were used....... Extensive analysis of the cell walls showed a 10%-15% increase in arabinose to xylose ratio, a 50% increase in the proportion of water-extractable arabinoxylan, and a shift in the MW of the water-extractable arabinoxylan from being mainly larger than 85 kD to being between 2 and 85 kD. Ferulic acid esterase......-expressing grains were also shrivelled, and the seed weight was decreased by 20%-50%. No ferulic acid esterase activity could be detected in wild-type grains whereas ferulic acid esterase activity was detected in transgenic lines. The grain cell walls had 15%-40% increase in water-unextractable arabinoxylan...

  5. Crystallization and preliminary X-ray diffraction analysis of ybfF, a new esterase from Escherichia coli K12

    Energy Technology Data Exchange (ETDEWEB)

    Park, Suk-Youl; Lee, Sang-Hak; Lee, Jieun; Jung, Che-Hun; Kim, Jeong-Sun, E-mail: jsunkim@chonnam.ac.kr [Department of Chemistry and Institute of Basic Sciences, Chonnam National University, Gwangju 500-757 (Korea, Republic of)

    2007-12-01

    The crystallization of ybfF, a new esterase from E. coli, and the collection of diffraction data to 1.1 Å resolution are reported. The product of the recently discovered ybfF gene, which belongs to the esterase family, does not show high sequence similarity to other esterases. To provide the molecular background to the enzymatic mechanism of the ybfF esterase, the ybfF protein from Escherichia coli K12 (Ec-ybfF) was cloned, expressed and purified. The Ec-ybfF protein was crystallized from 60% Tacsimate and 0.1 M bis-Tris propane buffer pH 7.0. Diffraction data were collected to 1.10 Å resolution using synchrotron radiation. The crystal belongs to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 66.09, b = 90.71, c = 92.88 Å. With two Ec-ybfF molecules in the asymmetric unit, the crystal volume per unit protein weight is 2.17 Å{sup 3} Da{sup −1}, corresponding to a solvent content of 42%.

  6. Combining substrate specificity analysis with support vector classifiers reveals feruloyl esterase as a phylogenetically informative protein group

    DEFF Research Database (Denmark)

    Olivares Hernandez, Roberto; Sunner, Hampus; Frisvad, Jens Christian

    2010-01-01

    Background Our understanding of how fungi evolved to develop a variety of ecological niches, is limited but of fundamental biological importance. Specifically, the evolution of enzymes affects how well species can adapt to new environmental conditions. Feruloyl esterases (FAEs) are enzymes able...

  7. Evaluation of the nitrite and leukocyte esterase activity tests for the diagnosis of acute symptomatic urinary tract infection in men.

    NARCIS (Netherlands)

    Koeijers, J.J.; Kessels, A.G.H.; Nys, S.; Bartelds, A.; Donker, G.; Stobberingh, E.; Verbon, A.

    2007-01-01

    For 422 male patients with symptoms indicative of a urinary tract infection, nitrite and leukocyte esterase activity dipstick test results were compared with results of culture of urine samples. The positive predictive value of a positive nitrite test result was 96%. Addition of results of the

  8. Generation of transgenic wheat (Triticum aestivum L.) accumulating heterologous endo-xylanase or ferulic acid esterase in the endosperm

    Energy Technology Data Exchange (ETDEWEB)

    Harholt, Jesper; Bach, Inga C; Lind-Bouquin, Solveig; Nunan, Kylie J.; Madrid, Susan M.; Brinch-Pedersen, Henrik; Holm, Preben B.; Scheller, Henrik V.

    2009-12-08

    Endo-xylanase (from Bacillus subtilis) or ferulic acid esterase (from Aspergillus niger) were expressed in wheat under the control of the endosperm specific 1DX5 glutenin promoter. Constructs both with and without the endoplasmic reticulum retention signal KDEL were used. Transgenic plants were recovered in all four cases but no qualitative differences could be observed whether KDEL was added or not. Endo-xylanase activity in transgenic grains was increased between two and three fold relative to wild type. The grains were shriveled and had a 25-33% decrease in mass. Extensive analysis of the cell walls showed a 10-15% increase in arabinose to xylose ratio, a 50% increase in the proportion of water extractable arabinoxylan, and a shift in the MW of the water extractable arabinoxylan from being mainly larger than 85 kD to being between 2 kD and 85 kD. Ferulic acid esterase expressing grains were also shriveled and the seed weight was decreased by 20-50%. No ferulic acid esterase activity could be detected in wild type grains whereas ferulic acid esterase activity was detected in transgenic lines. The grain cell walls had 15-40% increase in water unextractable arabinoxylan and a decrease in monomeric ferulic acid between 13 and 34%. In all the plants the observed changes are consistent with a plant response that serves to minimize the effect of the heterologously expressed enzymes by increasing arabinoxylan biosynthesis and cross-linking.

  9. Evaluating the efficacy of subcutaneous C1-esterase inhibitor administration for use in rat models of inflammatory diseases

    NARCIS (Netherlands)

    Emmens, Reindert W.; Naaijkens, Benno A.; Roem, Dorina; Kramer, Klaas; Wouters, Diana; Zeerleder, Sacha; van Ham, Marieke S.; Niessen, Hans W.; Krijnen, Paul A.

    2014-01-01

    Context: C1-esterase inhibitor (C1-inh) therapy is currently administered to patients with C1-inh deficiency through intravenous injections. The possibility of subcutaneous administration is currently being explored since this would alleviate need for hospitalization and increase mobility and

  10. 2-Acetylthiamin pyrophosphate (acetyl-TPP) pH-rate profile for hydrolysis of acetyl-TPP and isolation of acetyl-TPP as a transient species in pyruvate dehydrogenase catalyzed reactions

    International Nuclear Information System (INIS)

    Gruys, K.J.; Datta, A.; Frey, P.A.

    1989-01-01

    Rate constants for the hydrolysis of acetyl-TPP were measured pH values of 2.5 and 7.5 and plotted as log k obs versus pH. The pH-rate profile defined two legs, each with a slope of +1 but separated by a region of decreased slope between pH 4 and pH 6. The rates were insensitive to buffer concentrations. Each leg of the profile reflected specific-base-catalyzed hydrolysis of acetyl-TPP, analogous to the hydrolysis of 2-acetyl-3,4-dimethylthiazolium ion. The separation of the two legs of this profile has been shown to be caused by the ionization of a group exhibiting a pK a of 4.73 within acetyl-TPP that is remote from the acetyl group, the aminopyrimidine ring, which is promoted below pH 4.73. The protonation level of this ring has been shown to control the equilibrium partitioning of acetyl-TPP among its carbinolamine, keto, and hydrate forms. The differential partitioning of these species is a major factor causing the separation between the two legs of the pH-rate profile. The characteristic pH-rate profile and the availability of synthetic acetyl-TPP have facilitated the isolation and identification of [1- 14 C]acetyl-TPP from acid-quenched enymatic reaction mixtures at steady states. [1- 14 C]Acetyl-TPP was identified as a transient species in reactions catalyzed by the PDH complex or the pyruvate dehydrogenase component of the complex (E 1 ). The pH-rate profile for hydrolysis of [1- 14 C]-acetyl-TPP, isolated from enzymatic reactions was found to be indistinguishable from that for authentic acetyl-TPP, which constituted positive identification of the 14 C-labeled enzymic species

  11. Functional characterization of salt-tolerant microbial esterase WDEst17 and its use in the generation of optically pure ethyl (R)-3-hydroxybutyrate.

    Science.gov (United States)

    Wang, Yilong; Xu, Yongkai; Zhang, Yun; Sun, Aijun; Hu, Yunfeng

    2018-06-01

    The two enantiomers of ethyl 3-hydroxybutyrate are important intermediates for the synthesis of a great variety of valuable chiral drugs. The preparation of chiral drug intermediates through kinetic resolution reactions catalyzed by esterases/lipases has been demonstrated to be an efficient and environmentally friendly method. We previously functionally characterized microbial esterase PHE21 and used PHE21 as a biocatalyst to generate optically pure ethyl (S)-3-hydroxybutyrate. Herein, we also functionally characterized one novel salt-tolerant microbial esterase WDEst17 from the genome of Dactylosporangium aurantiacum subsp. Hamdenensis NRRL 18085. Esterase WDEst17 was further developed as an efficient biocatalyst to generate (R)-3-hydroxybutyrate, an important chiral drug intermediate, with the enantiomeric excess being 99% and the conversion rate being 65.05%, respectively, after process optimization. Notably, the enantio-selectivity of esterase WDEst17 was opposite than that of esterase PHE21. The identification of esterases WDEst17 and PHE21 through genome mining of microorganisms provides useful biocatalysts for the preparation of valuable chiral drug intermediates. © 2018 Wiley Periodicals, Inc.

  12. Acetyl coenzyme A synthetase is acetylated on multiple lysine residues by a protein acetyltransferase with a single Gcn5-type N-acetyltransferase (GNAT) domain in Saccharopolyspora erythraea.

    Science.gov (United States)

    You, Di; Yao, Li-Li; Huang, Dan; Escalante-Semerena, Jorge C; Ye, Bang-Ce

    2014-09-01

    Reversible lysine acetylation (RLA) is used by cells of all domains of life to modulate protein function. To date, bacterial acetylation/deacetylation systems have been studied in a few bacteria (e.g., Salmonella enterica, Bacillus subtilis, Escherichia coli, Erwinia amylovora, Mycobacterium tuberculosis, and Geobacillus kaustophilus), but little is known about RLA in antibiotic-producing actinomycetes. Here, we identify the Gcn5-like protein acetyltransferase AcuA of Saccharopolyspora erythraea (SacAcuA, SACE_5148) as the enzyme responsible for the acetylation of the AMP-forming acetyl coenzyme A synthetase (SacAcsA, SACE_2375). Acetylated SacAcsA was deacetylated by a sirtuin-type NAD(+)-dependent consuming deacetylase (SacSrtN, SACE_3798). In vitro acetylation/deacetylation of SacAcsA enzyme was studied by Western blotting, and acetylation of lysine residues Lys(237), Lys(380), Lys(611), and Lys(628) was confirmed by mass spectrometry. In a strain devoid of SacAcuA, none of the above-mentioned Lys residues of SacAcsA was acetylated. To our knowledge, the ability of SacAcuA to acetylate multiple Lys residues is unique among AcuA-type acetyltransferases. Results from site-specific mutagenesis experiments showed that the activity of SacAcsA was controlled by lysine acetylation. Lastly, immunoprecipitation data showed that in vivo acetylation of SacAcsA was influenced by glucose and acetate availability. These results suggested that reversible acetylation may also be a conserved regulatory posttranslational modification strategy in antibiotic-producing actinomycetes. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  13. Esterases activity in the axolotl Ambystoma mexicanum exposed to chlorpyrifos and its implication to motor activity.

    Science.gov (United States)

    Robles-Mendoza, Cecilia; Zúñiga-Lagunes, Sebastian R; Ponce de León-Hill, Claudia A; Hernández-Soto, Jesús; Vanegas-Pérez, Cecilia

    2011-10-01

    The axolotl Ambystoma mexicanum is a neotenic salamander considered a good biological model due to its ability to regenerate limbs, tail, brain and heart cells. Nevertheless, severe reduction of A. mexicanum wild populations in the lacustrine area of Xochimilco, the natural habitat of the axolotl, could be related to several environmental pressures as the presence of organophosphate pesticides (OPPs), intensively applied in agricultural activities in Xochimilco. Thus the aim of this study was to evaluate the effect of environmentally realistic chlorpyrifos (CPF) concentrations, a OPP commonly used in this zone, on esterases activity (acetylcholinesterase and carboxylesterase) and bioconcentration of CPF and to relate them with the motor activity of A. mexicanum juveniles. Axolotls were exposed 48 h to 0.05 and 0.1mg CPF/L, and the responses were evaluated at the end of the CPF exposure. Results suggest that CPF is bioconcentrated into axolotls and that the CPF internal concentrations are related with the observed inhibition activity of AChE (>50%) and CbE (≈ 50%). CPF concentration responsible of the inhibition of the 50% of AChE activity (IC50) was estimated in 0.04 mg CPF/L; however IC50 for CbE activity was not possible to calculate since inhibition levels were lower than 50%, results that suggest a higher resistance of CbE enzymatic activity to CPF. However, motor activity was a more sensitive endpoint to CPF poisoning since time that axolotls spent active and walking, frequency and speed of swimming, frequency of prey attack were reduced >90% of control groups. The motor activity alterations in the axolotl could be related with the registered esterases inhibition. Thus important alterations on axolotls were identified even at short time and low concentrations of CPF exposure. Also, it was possible to link biochemical responses as esterases activity with higher levels of biological organization as behavior. This study provides tools for the regulation of the

  14. Analysing deltamethrin susceptibility and pyrethroid esterase activity variations in sylvatic and domestic Triatoma infestans at the embryonic stage

    Directory of Open Access Journals (Sweden)

    Pablo Luis Santo-Orihuela

    2013-12-01

    Full Text Available The aim of the present work was to study the deltamethrin susceptibility of eggs from Triatoma infestans populations and the contribution of pyrethroid esterases to deltamethrin degradation. Insects were collected from sylvatic areas, including Veinte de Octubre and Kirus-Mayu (Bolivia and from domiciliary areas, including El Palmar (Bolivia and La Pista (Argentina. Deltamethrin susceptibility was determined by dose-response bioassays. Serial dilutions of deltamethrin (0.0005-1 mg/mL were topically applied to 12-day-old eggs. Samples from El Palmar had the highest lethal dose ratio (LDR value (44.90 compared to the susceptible reference strain (NFS, whereas the Veinte de Octubre samples had the lowest value (0.50. Pyrethroid esterases were evaluated using 7-coumaryl permethrate (7-CP on individually homogenised eggs from each population and from NFS. The El Palmar and La Pista samples contained 40.11 and 36.64 pmol/min/mg protein, respectively, and these values were statistically similar to NFS (34.92 pmol/min/mg protein and different from Kirus-Mayu and Veinte de Octubre (27.49 and 22.69 pmol/min/mg protein, respectively. The toxicological data indicate that the domestic populations were resistant to deltamethrin, but no statistical contribution of 7-CP esterases was observed. The sylvatic populations had similar LDR values to NFS, but lower 7-CP esterase activities. Moreover, this is the first study of the pyrethroid esterases on T. infestans eggs employing a specific substrate (7-CP.

  15. Chemical Pretreatment-Independent Saccharifications of Xylan and Cellulose of Rice Straw by Bacterial Weak Lignin-Binding Xylanolytic and Cellulolytic Enzymes.

    Science.gov (United States)

    Teeravivattanakit, Thitiporn; Baramee, Sirilak; Phitsuwan, Paripok; Sornyotha, Somphit; Waeonukul, Rattiya; Pason, Patthra; Tachaapaikoon, Chakrit; Poomputsa, Kanokwan; Kosugi, Akihiko; Sakka, Kazuo; Ratanakhanokchai, Khanok

    2017-11-15

    Complete utilization of carbohydrate fractions is one of the prerequisites for obtaining economically favorable lignocellulosic biomass conversion. This study shows that xylan in untreated rice straw was saccharified to xylose in one step without chemical pretreatment, yielding 58.2% of the theoretically maximum value by Paenibacillus curdlanolyticus B-6 PcAxy43A, a weak lignin-binding trifunctional xylanolytic enzyme, endoxylanase/β-xylosidase/arabinoxylan arabinofuranohydrolase. Moreover, xylose yield from untreated rice straw was enhanced to 78.9% by adding endoxylanases PcXyn10C and PcXyn11A from the same bacterium, resulting in improvement of cellulose accessibility to cellulolytic enzyme. After autoclaving the xylanolytic enzyme-treated rice straw, it was subjected to subsequent saccharification by a combination of the Clostridium thermocellum endoglucanase CtCel9R and Thermoanaerobacter brockii β-glucosidase TbCglT, yielding 88.5% of the maximum glucose yield, which was higher than the glucose yield obtained from ammonia-treated rice straw saccharification (59.6%). Moreover, this work presents a new environment-friendly xylanolytic enzyme pretreatment for beneficial hydrolysis of xylan in various agricultural residues, such as rice straw and corn hull. It not only could improve cellulose saccharification but also produced xylose, leading to an improvement of the overall fermentable sugar yields without chemical pretreatment. IMPORTANCE Ongoing research is focused on improving "green" pretreatment technologies in order to reduce energy demands and environmental impact and to develop an economically feasible biorefinery. The present study showed that PcAxy43A, a weak lignin-binding trifunctional xylanolytic enzyme, endoxylanase/β-xylosidase/arabinoxylan arabinofuranohydrolase from P. curdlanolyticus B-6, was capable of conversion of xylan in lignocellulosic biomass such as untreated rice straw to xylose in one step without chemical pretreatment. It

  16. Two-dimensional NMR evidence for cleavage of lignin and xylan substituents in wheat straw through hydrothermal pretreatment and enzymatic hydrolysis

    DEFF Research Database (Denmark)

    Yelle, Daniel J.; Kaparaju, Laxmi-Narasimha Prasad; Hunt, Christopher G.

    2013-01-01

    correlation spectroscopy, via an heteronuclear single quantum coherence experiment, revealed substantial lignin β-aryl ether cleavage, deacetylation via cleavage of the natural acetates at the 2-O- and 3-O-positions of xylan, and uronic acid depletion via cleavage of the (1 → 2)-linked 4-O....... g., further deacylation revealed by the depletion in ferulate and p-coumarate structures). Supplementary chemical analyses showed that the hydrothermal pretreatment increased the cellulose and lignin concentration with partial removal of extractives and hemicelluloses. The subsequent enzymatic...

  17. Lipids Reprogram Metabolism to Become a Major Carbon Source for Histone Acetylation

    DEFF Research Database (Denmark)

    McDonnell, Eoin; Crown, Scott B; Fox, Douglas B

    2016-01-01

    Cells integrate nutrient sensing and metabolism to coordinate proper cellular responses to a particular nutrient source. For example, glucose drives a gene expression program characterized by activating genes involved in its metabolism, in part by increasing glucose-derived histone acetylation....... Here, we find that lipid-derived acetyl-CoA is a major source of carbon for histone acetylation. Using (13)C-carbon tracing combined with acetyl-proteomics, we show that up to 90% of acetylation on certain histone lysines can be derived from fatty acid carbon, even in the presence of excess glucose...

  18. Acetylation Increases EWS-FLI1 DNA Binding and Transcriptional Activity

    International Nuclear Information System (INIS)

    Schlottmann, Silke; Erkizan, Hayriye V.; Barber-Rotenberg, Julie S.; Knights, Chad; Cheema, Amrita; Üren, Aykut; Avantaggiati, Maria L.; Toretsky, Jeffrey A.

    2012-01-01

    Ewing Sarcoma (ES) is associated with a balanced chromosomal translocation that in most cases leads to the expression of the oncogenic fusion protein and transcription factor EWS-FLI1. EWS-FLI1 has been shown to be crucial for ES cell survival and tumor growth. However, its regulation is still enigmatic. To date, no functionally significant post-translational modifications of EWS-FLI1 have been shown. Since ES are sensitive to histone deacetylase inhibitors (HDI), and these inhibitors are advancing in clinical trials, we sought to identify if EWS-FLI1 is directly acetylated. We convincingly show acetylation of the C-terminal FLI1 (FLI1-CTD) domain, which is the DNA binding domain of EWS-FLI1. In vitro acetylation studies showed that acetylated FLI1-CTD has higher DNA binding activity than the non-acetylated protein. Over-expression of PCAF or treatment with HDI increased the transcriptional activity of EWS-FLI1, when co-expressed in Cos7 cells. However, our data that evaluates the acetylation of full-length EWS-FLI1 in ES cells remains unclear, despite creating acetylation specific antibodies to four potential acetylation sites. We conclude that EWS-FLI1 may either gain access to chromatin as a result of histone acetylation or undergo regulation by direct acetylation. These data should be considered when patients are treated with HDAC inhibitors. Further investigation of this phenomenon will reveal if this potential acetylation has an impact on tumor response.

  19. Acetylated tubulin is essential for touch sensation in mice.

    Science.gov (United States)

    Morley, Shane J; Qi, Yanmei; Iovino, Loredana; Andolfi, Laura; Guo, Da; Kalebic, Nereo; Castaldi, Laura; Tischer, Christian; Portulano, Carla; Bolasco, Giulia; Shirlekar, Kalyanee; Fusco, Claudia M; Asaro, Antonino; Fermani, Federica; Sundukova, Mayya; Matti, Ulf; Reymond, Luc; De Ninno, Adele; Businaro, Luca; Johnsson, Kai; Lazzarino, Marco; Ries, Jonas; Schwab, Yannick; Hu, Jing; Heppenstall, Paul A

    2016-12-13

    At its most fundamental level, touch sensation requires the translation of mechanical energy into mechanosensitive ion channel opening, thereby generating electro-chemical signals. Our understanding of this process, especially how the cytoskeleton influences it, remains unknown. Here we demonstrate that mice lacking the α-tubulin acetyltransferase Atat1 in sensory neurons display profound deficits in their ability to detect mechanical stimuli. We show that all cutaneous afferent subtypes, including nociceptors have strongly reduced mechanosensitivity upon Atat1 deletion, and that consequently, mice are largely insensitive to mechanical touch and pain. We establish that this broad loss of mechanosensitivity is dependent upon the acetyltransferase activity of Atat1, which when absent leads to a decrease in cellular elasticity. By mimicking α-tubulin acetylation genetically, we show both cellular rigidity and mechanosensitivity can be restored in Atat1 deficient sensory neurons. Hence, our results indicate that by influencing cellular stiffness, α-tubulin acetylation sets the force required for touch.

  20. Acetyl-L-carnitine improves aged brain function.

    Science.gov (United States)

    Kobayashi, Satoru; Iwamoto, Machiko; Kon, Kazuo; Waki, Hatsue; Ando, Susumu; Tanaka, Yasukazu

    2010-07-01

    The effects of acetyl-L-carnitine (ALCAR), an acetyl derivative of L-carnitine, on memory and learning capacity and on brain synaptic functions of aged rats were examined. Male Fischer 344 rats were given ALCAR (100 mg/kg bodyweight) per os for 3 months and were subjected to the Hebb-Williams tasks and AKON-1 task to assess their learning capacity. Cholinergic activities were determined with synaptosomes isolated from brain cortices of the rats. Choline parameters, the high-affinity choline uptake, acetylcholine (ACh) synthesis and depolarization-evoked ACh release were all enhanced in the ALCAR group. An increment of depolarization-induced calcium ion influx into synaptosomes was also evident in rats given ALCAR. Electrophysiological studies using hippocampus slices indicated that the excitatory postsynaptic potential slope and population spike size were both increased in ALCAR-treated rats. These results indicate that ALCAR increases synaptic neurotransmission in the brain and consequently improves learning capacity in aging rats.

  1. Effects of temperature, ultraviolet radiation and pectin methyl esterase on aerobic methane release from plant material.

    Science.gov (United States)

    Bruhn, D; Mikkelsen, T N; Obro, J; Willats, W G T; Ambus, P

    2009-11-01

    This study examines the effects of different irradiance types on aerobic methane (CH(4)) efflux rates from terrestrial plant material. Furthermore, the role of the enzyme pectin methyl esterase (PME) on CH(4) efflux potential was also examined. Different types of plant tissue and purified pectin were incubated in glass vials with different combinations of irradiation and/or temperature. Purified dry pectin was incubated in solution, and with or without PME. Before and after incubation, the concentration of CH(4) was measured with a gas chromatograph. Rates of CH(4) emission were found to depend exponentially on temperature and linearly on UV-B irradiance. UV-B had a greater stimulating effect than UV-A, while visible light had no effect on emission rates. PME was found to substantially reduce the potential for aerobic CH(4) emissions upon demethylation of pectin.

  2. Synthesis activity-based zymography for detection of lipases and esterases.

    Science.gov (United States)

    Kwon, Min-A; Kim, Hyun Suk; Hahm, Dae-Hyun; Song, Jae Kwang

    2011-04-01

    A new zymography method for lipases and esterases was developed on the basis of the esterification reaction between fatty acids and alcohols. The enzymes were separated by SDS-PAGE and native PAGE. The gel was washed and then incubated in an aqueous solution containing fatty acids (oleic acid 18:1 or caprylic acid 8:0) and dodecanol. Synthesis was visualized by in situ precipitation of water-insoluble and non-diffusible fatty acid esters, such as dodecyl oleate and dodecyl octanoate. The synthesis activity-based zymography was confirmed with different enzyme samples, including commercial lipase preparations, purified recombinant lipase and cutinase, and crude culture supernatants of lipolytic enzyme-producing soil bacteria.

  3. Reactivity of Acetylcholine Esterase in inner Ear Maculae of Fish after Development at Hypergravity

    Science.gov (United States)

    Feucht, I.; Hilbig, R.; Anken, R.

    It has been shown earlier that the growth of inner ear otoliths of larval fish is (among other environmental factors) guided by the gravity vector. This guidance most probably is effected by the efferent vestibular system in the brainstem, because a transection of the nervus vestibularis has been shown to effect a cessation of the supply of calcium to the otoliths. The efferent innervation of fish inner ear maculae uses the synaptic transmitter acetylcholine (ACh). Therefore, we were - in order to further assess the role of the efferent system for otolith growth - prompted to determine ACh esterase-reactivity in the sensory epithelium of the utricle and the saccule (as well as in a non-gravity relevant brain region for control) in larval cichlid fish (Oreochromis mossambicus), which had been maintained at hypergravity during their development. The respective data will be communicated at the meeting. Acknowledgement: This work was financially supported by the German Aerospace Center (DLR) (FKZ: 50 WB 9997).

  4. Exceptional thermal stability and organic solvent tolerance of an esterase expressed from a thermophilic host

    DEFF Research Database (Denmark)

    Mei, Yuxia; Peng, Nan; Zhao, Shumiao

    2012-01-01

    , giving SisEstA. Upon Escherichia coli expression, only the thioredoxin-tagged EstA recombinant protein was soluble. The fusion protein was then purified, and removing the protein tag yielded EcSisEstA. Both forms of the thermophilic EstA enzyme were characterized. We found that SisEstA formed dimer...... that of EcSisEstA at 90°C. This indicated that thermophilic enzymes yielded from homologous expression should be better biocatalysts than those obtained from mesophilic expression.......A protein expression system recently developed for the thermophilic crenarchaeon Sulfolobus islandicus was employed to produce recombinant protein for EstA, a thermophilic esterase encoded in the same organism. Large amounts of protein were readily obtained by an affinity protein purification...

  5. Cytological localization of adenosine kinase, nucleoside phosphorylase-1, and esterase-10 genes on mouse chromosome 14

    International Nuclear Information System (INIS)

    Samuelson, L.C.; Farber, R.A.

    1985-01-01

    The authors have determined the regional locations on mouse chromosome 14 of the genes for mouse adenosine kinase (ADK), nucleoside phosphorylase- 1 (NP-1), and esterase-10 (ES-10) by analysis of rearranged mouse chromosomes in gamma-irradiated Chinese hamster X mouse hybrid cell lines. Irradiated clones were screened for expression of the murine forms of these enzymes; segregant clones that expressed only one or two of the three markers were karyotyped. The patterns of enzyme expression in these segregants were correlated with the presence of rearranged chromosomes. The Adk gene was localized to bands A2 to B, Np-1 to bands B to C1, and Es-10 to bands D2 to E2

  6. From Classical to High Throughput Screening Methods for Feruloyl Esterases: A Review.

    Science.gov (United States)

    Ramírez-Velasco, Lorena; Armendáriz-Ruiz, Mariana; Rodríguez-González, Jorge Alberto; Müller-Santos, Marcelo; Asaff-Torres, Ali; Mateos-Díaz, Juan Carlos

    2016-01-01

    Feruloyl esterases (FAEs) are a diverse group of hydrolases widely distributed in plants and microorganisms which catalyzes the cleavage and formation of ester bonds between plant cell wall polysaccharides and phenolic acids. FAEs have gained importance in biofuel, medicine and food industries due to their capability of acting on a large range of substrates for cleaving ester bonds and synthesizing highadded value molecules through esterification and transesterification reactions. During the past two decades extensive studies have been carried out on the production, characterization and classification of FAEs, however only a few reports of suitable High Throughput Screening assays for this kind of enzymes have been reported. This review is focused on a concise but complete revision of classical to High Throughput Screening methods for FAEs, highlighting its advantages and disadvantages, and finally suggesting future perspectives for this important research field.

  7. Acetylation site specificities of lysine deacetylase inhibitors in human cells

    DEFF Research Database (Denmark)

    Schölz, Christian; Weinert, Brian Tate; Wagner, Sebastian A

    2015-01-01

    Lysine deacetylases inhibitors (KDACIs) are used in basic research, and many are being investigated in clinical trials for treatment of cancer and other diseases. However, their specificities in cells are incompletely characterized. Here we used quantitative mass spectrometry (MS) to obtain acety......1-α, providing a possible mechanistic explanation of its adverse, pro-inflammatory effects. Our results offer a systems view of KDACI specificities, providing a framework for studying function of acetylation and deacetylases....

  8. Effect of phenobarbital on inducing insecticide tolerance and esterase changes in Aedes aegypti (Diptera: Culicidae

    Directory of Open Access Journals (Sweden)

    Rita de Cássia Sousa-Polezzi

    2004-01-01

    Full Text Available The effect of phenobarbital (PB on the induction of tolerance to the organophosphorous insecticide temephos (TE was investigated in Aedes aegypti L4 larvae submitted to two different PB-treatments:(1 continuous treatment from the egg to the larval L4 stage and (2 discontinuous treatment in which L4 larvae were exposed for 30 h. Mosquitoes from two Brazilian cities were studied: São José do Rio Preto (SJ in São Paulo State and Goiânia (GO in Goiás State. According to criterions established by World Health Organization (WHO mosquitoes from SJ are organophosphate-susceptible while mosquitoes from GO are organophosphate-resistant. For both SJ and GO larvae the two different PB-treatments resulted in significantly increased tolerance (measured by reduced mortality to 0.01mg/L TE while for larvae exposed to 0.02 mg/L TE only continuous PB-treatment resulted in significantly increased TE-tolerance. The reduction of mortality rate was greater in SJ larvae than in GO larvae, confirming data from other organisms indicating that the effect of PB is more pronounced in susceptible strains. To test if oxidase enzymes were involved in PB-induced tolerance we treated PB-pretreated SJ and GO larvae with the oxidase inhibitor piperonyl butoxide (PBO before exposure to TE and observed increased (rather than decreased tolerance, suggesting that oxidases are not involved in the tolerance process and that PB and PBO can act in concert or synergistically. Esterase patterns of PB-pretreated larvae indicated that the cholinesterases EST-13 and EST-14 are involved in the PB-induced TE- tolerance, reinforcing a previous study carried out in our laboratory which suggested that increased esterase synthesis is the mechanism responsible for the development of insecticide resistance in Aedes aegypti.

  9. Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

    Science.gov (United States)

    Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

    2013-01-01

    Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0. PMID:23685703

  10. Direct imaging of ER calcium with targeted-esterase induced dye loading (TED).

    Science.gov (United States)

    Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

    2013-05-07

    Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca(2+) indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca(2+) indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca(2+) indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca(2+) indicator and a hydrophilic fluorescent dye/Ca(2+) complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0.

  11. EWSR1 regulates mitosis by dynamically influencing microtubule acetylation.

    Science.gov (United States)

    Wang, Yi-Long; Chen, Hui; Zhan, Yi-Qun; Yin, Rong-Hua; Li, Chang-Yan; Ge, Chang-Hui; Yu, Miao; Yang, Xiao-Ming

    2016-08-17

    EWSR1, participating in transcription and splicing, has been identified as a translocation partner for various transcription factors, resulting in translocation, which in turn plays crucial roles in tumorigenesis. Recent studies have investigated the role of EWSR1 in mitosis. However, the effect of EWSR1 on mitosis is poorly understood. Here, we observed that depletion of EWSR1 resulted in cell cycle arrest in the mitotic phase, mainly due to an increase in the time from nuclear envelope breakdown to metaphase, resulting in a high percentage of unaligned chromosomes and multipolar spindles. We also demonstrated that EWSR1 is a spindle-associated protein that interacts with α-tubulin during mitosis. EWSR1 depletion increased the cold-sensitivity of spindle microtubules, and decreased the rate of spindle assembly. EWSR1 regulated the level of microtubule acetylation in the mitotic spindle; microtubule acetylation was rescued in EWSR1-depleted mitotic cells following suppression of HDAC6 activity by its specific inhibitor or siRNA treatment. In summary, these results suggest that EWSR1 regulates the acetylation of microtubules in a cell cycle-dependent manner through its dynamic location on spindle MTs, and may be a novel regulator for mitosis progress independent of its translocation.

  12. Chromatin decondensed by acetylation shows an elevated radiation response

    International Nuclear Information System (INIS)

    Nackerdien, Z.; Michie, J.; Boehm, L.

    1989-01-01

    V-79 Chinese hamster lung fibroblasts exposed to 5 mM n-sodium butyrate were irradiated with 60Co gamma rays and cell survival was determined by the cell colony assay. In a separate set of experiments the acetylated chromatin obtained from these cells was irradiated and the change of molecular weight of the DNA was evaluated by alkaline sucrose density centrifugation. At a survival level of 10(-2) to 10(-4) cells exposed to butyrate were found to be 1.3-1.4 times more radiosensitive than control cells. Exposure of isolated chromatin to 100 Gy of 60Co gamma irradiation generated 0.9 +/- 0.03 single-strand breaks (ssb) per 10 Gy per 10(8) Da and 2.0 +/- 0.3 ssb/10 Gy/10(8) Da for control and acetylated chromatin, respectively. The elevated radiation sensitivity of chromatin relaxed by acetylation is in good agreement with previous results on chromatin expanded by histone H1 depletion. Packing and accessibility of DNA in chromatin appear to be major factors which influence the radiation sensitivity. The intrinsic radiation sensitivity of chromatin in various packing states is discussed in light of the variation of radiation sensitivity of whole cells in the cell cycle which incorporates repair

  13. Autoradiographic study of nuclear protein acetylation during Locust spermiogenesis

    International Nuclear Information System (INIS)

    Bouvier, D.; Chevaillier, P.

    1975-01-01

    Autoradiographic studies, at the light and electron microscope level, demonstrate that spermatid nuclei of the Locust Locusta migratoria incorporate 3 H-acetate, especially during the first stages of spermiogenesis. The highest level of acetate incorporation is observed during stage II of spermiogenesis. During this stage and the following, the spermatid nucleus undergoes a number of structural and chemical modifications: chromatin decondenses and somatic histones are progressively replaced by newly synthesized arginine-rich proteins. Therefore, the higher degree of acetylation of nuclear components coincides with chromatin decondensation and precedes the protein transition occurring in later stages. Cytochemical and autoradiographic tests have been realized so as to localize 3 H-acetate in the nuclear components. Trichloracetic acid was used at various concentrations: the action of hydrochloric acid, pronase and DNase was also tested. The results support the idea that proteins, and among them histones, are the only nuclear components to be acetylated during spermiogenesis. Thus, histone acetylation seems to play an important role in modulating histone-DNA interactions and allowing histone replacement [fr

  14. Substituent-specific antibody against glucuronoxylan reveals close association of glucuronic acid and acetyl substituents and distinct labeling patterns in tree species

    DEFF Research Database (Denmark)

    Koutaniemi, Sanna; Guillon, Fabienne; Tranquet, Olivier

    2012-01-01

    Immunolabeling can be used to locate plant cell wall carbohydrates or other components to specific cell types or to specific regions of the wall. Some antibodies against xylans exist; however, many partly react with the xylan backbone and thus provide limited information on the type of substituen...

  15. The effect of rabbit age on in vitro caecal fermentation of starch, pectin, xylan, cellulose, compound feed and its fibre.

    Science.gov (United States)

    Lavrenčič, A

    2007-03-01

    In vitro gas production kinetics of six different substrates, pectin (PEC), xylan (XYL), starch (STA), cellulose (CEL), commercial compound feed (FEED; 201 g crude protein per kg, 155 g crude fibre per kg, 334 g neutral-detergent fibre (NDF) per kg and 190 g acid-detergent fibre (ADF) per kg) and an NDF prepared from commercial compound feed (NDFFEED) were determined using the caecum contents of weaned rabbits (36 days of age) and of rabbits at slaughter age (78 days of age) as inoculums. The cumulated gas production over 96 h of incubation was modelled with Gompertz model, and the kinetic parameters compared. The total potential gas production (parameter 'B' of the Gompertz model) was not affected (P>0.05) by the inoculum source, except with STA, where rabbits at slaughter weight had significantly higher total potential fermentability (314 ml/g dry matter (DM)) than those at weaning age (189 ml/g DM). Intensities of fermentation (maximum fermentation rate; MFR) of PEC (32.2 ml/h) and XYL (24.4 ml/h) were significantly greater in rabbits at weaning, while that of STA (45 ml/h) was significantly lower than at slaughter age (23.0, 14.3 and 14.0 ml/h for PEC, XYL and STA, respectively). The MFRs of CEL and NDFFEED were very similar between inoculum sources. In the first 10 h of fermentation which correspond to the normal retention time of the substrates in the caecum, the highest amount of gas was produced from PEC, followed by FEED and XYL. These substrates had a time of maximum fermentation rate (TMFR) at both rabbit ages short enough (8.0 and 9.5 h for PEC, 9.5 and 6.6 h for FEED, 13.7 and 14.2 h for XYL at weaning and at slaughter age, respectively) to be almost completely fermented in vivo.

  16. Crystal structure of tabtoxin resistance protein complexed with acetyl coenzyme A reveals the mechanism for beta-lactam acetylation.

    Science.gov (United States)

    He, Hongzhen; Ding, Yi; Bartlam, Mark; Sun, Fei; Le, Yi; Qin, Xincheng; Tang, Hong; Zhang, Rongguang; Joachimiak, Andrzej; Liu, Jinyuan; Zhao, Nanming; Rao, Zihe

    2003-01-31

    Tabtoxin resistance protein (TTR) is an enzyme that renders tabtoxin-producing pathogens, such as Pseudomonas syringae, tolerant to their own phytotoxins. Here, we report the crystal structure of TTR complexed with its natural cofactor, acetyl coenzyme A (AcCoA), to 1.55A resolution. The binary complex forms a characteristic "V" shape for substrate binding and contains the four motifs conserved in the GCN5-related N-acetyltransferase (GNAT) superfamily, which also includes the histone acetyltransferases (HATs). A single-step mechanism is proposed to explain the function of three conserved residues, Glu92, Asp130 and Tyr141, in catalyzing the acetyl group transfer to its substrate. We also report that TTR possesses HAT activity and suggest an evolutionary relationship between TTR and other GNAT members.

  17. Crystal structure of tabtoxin resistance protein complexed with acetyl coenzyme A reveals the mechanism for {beta}-lactam acetylation.

    Energy Technology Data Exchange (ETDEWEB)

    He, H.; Ding, Y.; Bartlam, M.; Sun, F.; Le, Y.; Qin, X.; Tang, H.; Zhang, R.; Joachimiak, A.; Liu, J.; Zhao, N.; Rao, Z.; Biosciences Division; Tsinghua Univ.; Chinese Academy of Science

    2003-01-31

    Tabtoxin resistance protein (TTR) is an enzyme that renders tabtoxin-producing pathogens, such as Pseudomonas syringae, tolerant to their own phytotoxins. Here, we report the crystal structure of TTR complexed with its natural cofactor, acetyl coenzyme A (AcCoA), to 1.55 {angstrom} resolution. The binary complex forms a characteristic 'V' shape for substrate binding and contains the four motifs conserved in the GCN5-related N-acetyltransferase (GNAT) superfamily, which also includes the histone acetyltransferases (HATs). A single-step mechanism is proposed to explain the function of three conserved residues, Glu92, Asp130 and Tyr141, in catalyzing the acetyl group transfer to its substrate. We also report that TTR possesses HAT activity and suggest an evolutionary relationship between TTR and other GNAT members.

  18. Density functional and ab initio study of the tautomeric forms of 3-acetyl tetronic and 3-acetyl tetramic acids

    International Nuclear Information System (INIS)

    Skylaris, Chris-Kriton; Igglessi-Markopoulou, Olga; Detsi, Anastasia; Markopoulos, John

    2003-01-01

    We propose all the accessible paths of interconversion between the tautomers of 3-acetyl tetronic and 3-acetyl tetramic acids by performing calculations with the density functional B3LYP method and the ab initio MP2 method. Our findings clarify at the atomic level the mechanisms of the equilibria between these tautomers, a topic so far only partially understood on the basis of studies by nuclear magnetic resonance (NMR) spectroscopy. We show that thermal effects via relative Gibbs free energies ΔG must be taken into account in order to reach good quantitative agreement with the available experimental information on the ratios of the most stable tautomers. The calculated 1 H and 13 C chemical shifts are in agreement with the experimental values from NMR spectroscopy

  19. Enhanced Agarose and Xylan Degradation for Production of Polyhydroxyalkanoates by Co-Culture of Marine Bacterium, Saccharophagus degradans and Its Contaminant, Bacillus cereus

    Directory of Open Access Journals (Sweden)

    Shailesh S. Sawant

    2017-02-01

    Full Text Available Over reliance on energy or petroleum products has raised concerns both in regards to the depletion of their associated natural resources as well as their increasing costs. Bioplastics derived from microbes are emerging as promising alternatives to fossil fuel derived petroleum plastics. The development of a simple and eco-friendly strategy for bioplastic production with high productivity and yield, which is produced in a cost effective manner utilising abundantly available renewable carbon sources, would have the potential to result in an inexhaustible global energy source. Here we report the biosynthesis of bioplastic polyhydroxyalkanoates (PHAs in pure cultures of marine bacterium, Saccharophagus degradans 2-40 (Sde 2-40, its contaminant, Bacillus cereus, and a co-culture of these bacteria (Sde 2-40 and B. cereus degrading plant and algae derived complex polysaccharides. Sde 2-40 degraded the complex polysaccharides agarose and xylan as sole carbon sources for biosynthesis of PHAs. The ability of Sde 2-40 to degrade agarose increased after co-culturing with B. cereus. The association of Sde 2-40 with B. cereus resulted in increased cell growth and higher PHA production (34.5% of dry cell weight from xylan as a carbon source in comparison to Sde 2-40 alone (22.7% of dry cell weight. The present study offers an innovative prototype for production of PHA through consolidated bioprocessing of complex carbon sources by pure and co-culture of microorganisms.

  20. Effect of degradation of xylan constituent in Mitsumata (Edgeworthia papyrifera Sieb. et Zucc. ) bast on its pulping by pectinolytic enzymes form Erwinia carotovora

    Energy Technology Data Exchange (ETDEWEB)

    Tanabe, Hiroyuki; Matsuo, Ryukichi; Kobayashi, Yoshinari

    1988-01-01

    Pulping of mitsumata (Edgeworthia papyrifera Sieb. et Zucc.) bast by the crude enzyme from a bacterium Erwinia carotovora FERM P-7576, was more effective by a stepwise treatment at pH 6.5 and subsequently at pH 9.5 and eluted greater amount of xylose constituent than a constant pH treatment at pH 9.5 where only the maceration enzymes, endo-pectate lyase and endo-pectin lyase, among the crude enzyme are operative. The crude enzymes obtained from the cultivation of this bacterial strain on mitsumata bast fibers were more effective for the stepwise pH pulping method than those from the cultivation on soluble pectin. Xylanase activity in the mitsumata bast-induced enzyme at pH 6.5 was twice as high as that in the soluble pectin-induced one. The activities of other hemicellulases and cellulase were, high as that in the soluble pectin-induced one. The activities of other hemicellulases and cellulase were, however, independent on the inducing materials. Purified exo-type xylanase prepared from the crude enzyme acted comparably to the entire crude enzyme in the first step of the combination pulping, but the xylanase per se showed no maceration activity. These results suggests that the degradation of xylan constituent within the bast fibers effects the acceleration of the subsequent enzymatic pulping by the pectinolytic maceration enzymes. The maceration mechanism involving xylan degradation was also discussed.

  1. Production of furfural from xylose, xylan and corncob in gamma-valerolactone using FeCl3·6H2O as catalyst.

    Science.gov (United States)

    Zhang, Luxin; Yu, Hongbing; Wang, Pan; Li, Yong

    2014-01-01

    An efficient and simple one-pot monophasic reaction system with small carbon footprint for converting xylose, xylan and corncob into furfural was developed in gamma-valerolactone (GVL, an ideal sustainable solvent derived from lignocelluloses) by using FeCl3·6H2O as catalyst. Good yields of furfural from xylose were obtained, and the system was shown to work for xylan and corncob as well. A surprisingly high furfural yield of 79.6% from untreated corncob was achieved at 458 K for 100 min. Contrary to what was generally believed, the addition of water, reduced the rate of the reactions, but showed positive effect on preventing the furfural from degradation in GVL. Besides, the C6 sugars (glucose and cellulose) afforded 11.4-24.5% furfural yields when employing this catalytic approach. The reaction system proposed in this manuscript showed great potential for optimizing the catalytic process in furfural production. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Effects of the cellulose, xylan and lignin constituents on biomass pyrolysis characteristics and bio-oil composition using the Simplex Lattice Mixture Design method

    International Nuclear Information System (INIS)

    Fan, Yongsheng; Cai, Yixi; Li, Xiaohua; Jiao, Lihua; Xia, Jisheng; Deng, Xiuli

    2017-01-01

    Highlights: • Simplex Lattice Mixture Design was firstly applied to study biomass pyrolysis process. • Interactions between the constituents had effects on the biomass pyrolysis behavior. • Biomass pyrolysis behavior can be predicted based on the ratios of three constituents. • Bio-oil composition was affected by the constituents and their pyrolysis products. - Abstract: In order to clarify the relationships between biomass pyrolysis mechanism and its main constituents. The effects of main constituents on biomass pyrolysis characteristics were firstly determined by thermo-gravimetric analysis based on the Simplex Lattice Mixture Design to investigate that whether the prediction of the pyrolysis behavior of a certain lignocellulosic biomass is possible when its main constituent contents are known. The results showed that there are constituent interactions in the pyrolysis process, which can be intuitively reflected through the change laws of kinetics parameters. The mathematical models for calculating kinetics values were established, and the models were proved to be valid for predicting lignocellulosic biomass pyrolysis behavior. In addition, the effects of biomass constituents on bio-oil compositions were explored by subsequent vacuum pyrolysis experiments. The xylan pyrolysis had a certain inhibitory effect on the pyrolysis of cellulose, and the pyrolysis products of lignin might promote the further decomposition of sugars from cellulose pyrolysis, while the interaction between xylan and lignin had a little effect on the bio-oil composition.

  3. A new method for the quantification of monosaccharides, uronic acids and oligosaccharides in partially hydrolyzed xylans by HPAEC-UV/VIS.

    Science.gov (United States)

    Lorenz, Dominic; Erasmy, Nicole; Akil, Youssef; Saake, Bodo

    2016-04-20

    A new method for the chemical characterization of xylans is presented, to overcome the difficulties in quantification of 4-O-methyl-α-D-glucuronic acid (meGlcA). In this regard, the hydrolysis behavior of xylans from beech and birch wood was investigated to obtain the optimum conditions for hydrolysis, using sulfuric acid. Due to varying linkage strengths and degradation, no general method for complete hydrolysis can be designed. Therefore, partial hydrolysis was applied, yielding monosaccharides and small meGlcA containing oligosaccharides. For a new method by HPAEC-UV/VIS, these samples were reductively aminated by 2-aminobenzoic acid. By quantification of monosaccharides and oligosaccharides, as well as comparison with borate-HPAEC and (13)C NMR-spectroscopy, we revealed that the concentrations meGlcA are significantly underestimated compared to conventional methods. The detected concentrations are 85.4% (beech) and 76.3% (birch) higher with the new procedure. Furthermore, the quantified concentrations of xylose were 9.3% (beech) and 6.5% (birch) higher by considering the unhydrolyzed oligosaccharides as well. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. The activity of non-specific esterase in the thyroid epithelial cells of the guinea pig as influenced by various inhibitors and activators. A histochemical study

    DEFF Research Database (Denmark)

    Kirkeby, S

    1976-01-01

    The action of various inhibitors and activators upon esterase activity in the thyroid epithelial cells is demonstrated. The agents used were triorthocresylphosphate (TOCP), parachloromercuribenzoate (PCMB), Arsanillic acid, p-nitrophenyl dimethyl carbamate and bis p-nitrophenyl phosphate. TOCP wa...

  5. Enzymatic Xylose Release from Pretreated Corn Bran Arabinoxylan: Differential Effects of Deacetylation and Deferuloylation on Insoluble and Soluble Substrate Fractions

    DEFF Research Database (Denmark)

    Agger, Jane; Viksø-Nielsen, Ander; Meyer, Anne S.

    2010-01-01

    In the present work enzymatic hydrolysis of arabinoxylan from pretreated corn bran (190 °C, 10 min) was evaluated by measuring the release of xylose and arabinose after treatment with a designed minimal mixture of monocomponent enzymes consisting of α-l-arabinofuranosidases, an endoxylanase......, and a β-xylosidase. The pretreatment divided the corn bran material 50:50 into soluble and insoluble fractions having A:X ratios of 0.66 and 0.40, respectively. Addition of acetyl xylan esterase to the monocomponent enzyme mixture almost doubled the xylose release from the insoluble substrate fraction...

  6. Isolation and characterization of a heavy metal-resistant, thermophilic esterase from a Red Sea Brine Pool

    KAUST Repository

    Mohamed, Yasmine M.; Ghazy, Mohamed A.; Sayed, Ahmed; Ouf, Amged; El-Dorry, Hamza; Siam, Rania

    2013-01-01

    The Red Sea Atlantis II brine pool is an extreme environment that displays multiple harsh conditions such as high temperature, high salinity and high concentrations of multiple, toxic heavy metals. The survival of microbes in such an environment by utilizing resistant enzymes makes them an excellent source of extremophilic enzymes. We constructed a fosmid metagenomic library using DNA isolated from the deepest and most secluded layer of this pool. We report the isolation and biochemical characterization of an unusual esterase: EstATII. EstATII is thermophilic (optimum temperature, 65 C), halotolerant (maintains its activity in up to 4.5â€...M NaCl) and maintains at least 60% of its activity in the presence of a wide spectrum of heavy metals. The combination of biochemical characteristics of the Red Sea Atlantis II brine pool esterase, i.e., halotolerance, thermophilicity and resistance to heavy metals, makes it a potentially useful biocatalyst.

  7. Isolation and characterization of a heavy metal-resistant, thermophilic esterase from a Red Sea Brine Pool

    KAUST Repository

    Mohamed, Yasmine M.

    2013-11-28

    The Red Sea Atlantis II brine pool is an extreme environment that displays multiple harsh conditions such as high temperature, high salinity and high concentrations of multiple, toxic heavy metals. The survival of microbes in such an environment by utilizing resistant enzymes makes them an excellent source of extremophilic enzymes. We constructed a fosmid metagenomic library using DNA isolated from the deepest and most secluded layer of this pool. We report the isolation and biochemical characterization of an unusual esterase: EstATII. EstATII is thermophilic (optimum temperature, 65 C), halotolerant (maintains its activity in up to 4.5â€...M NaCl) and maintains at least 60% of its activity in the presence of a wide spectrum of heavy metals. The combination of biochemical characteristics of the Red Sea Atlantis II brine pool esterase, i.e., halotolerance, thermophilicity and resistance to heavy metals, makes it a potentially useful biocatalyst.

  8. Whole-Cell Biocatalytic Synthesis of Cinnamyl Acetate with a Novel Esterase from the DNA Library of Acinetobacter hemolyticus.

    Science.gov (United States)

    Dong, Hao; Secundo, Francesco; Xue, Changhu; Mao, Xiangzhao

    2017-03-15

    Cinnamyl acetate has a wide application in the flavor and fragrance industry because of its sweet, balsamic, and floral odor. Up to now, lipases have been mainly used in enzyme-mediated synthesis of cinnamyl acetate, whereas esterases are used in only a few cases. Moreover, the use of purified enzymes is often a disadvantage, which leads to increases of the production costs. In this paper, a genomic DNA library of Acinetobacter hemolyticus was constructed, and a novel esterase (EstK1) was identified. After expression in Escherichia coli, the whole-cell catalyst of EstK1 displayed high transesterification activity to produce cinnamyl acetate in nonaqueous systems. Furthermore, under optimal conditions (vinyl acetate as acyl donor, isooctane as solvent, molar ratio 1:4, temperature 40 °C), the conversion ratio of cinnamyl alcohol could be up to 94.1% at 1 h, and it reached an even higher level (97.1%) at 2 h.

  9. Isolated angioedema of the bowel due to C1 esterase inhibitor deficiency: a case report and review of literature

    Directory of Open Access Journals (Sweden)

    Kothari Shivangi T

    2011-02-01

    Full Text Available Abstract Introduction We report a rare, classic case of isolated angioedema of the bowel due to C1-esterase inhibitor deficiency. It is a rare presentation and very few cases have been reported worldwide. Angioedema has been classified into three categories. Case presentation A 66-year-old Caucasian man presented with a ten-month history of episodic severe cramping abdominal pain, associated with loose stools. A colonoscopy performed during an acute attack revealed nonspecific colitis. Computed tomography of the abdomen performed at the same time showed a thickened small bowel and ascending colon with a moderate amount of free fluid in the abdomen. Levels of C4 ( Conclusion In addition to a detailed comprehensive medical history, laboratory data and imaging studies are required to confirm a diagnosis of angioedema due to C1 esterase inhibitor deficiency.

  10. Identification of a Marine Bacillus Strain C5 and Parathion-Methyl Degradation Characteristics of the Extracellular Esterase B1

    Directory of Open Access Journals (Sweden)

    Jianhua Hao

    2014-01-01

    Full Text Available A bacterial strain C5 that can produce new type of marine esterase was isolated and screened from marine sludge. According to 16S rRNA sequence analysis and physiological and biochemical experiments, the strain was identified as Bacillus subtilis. A single isozyme with a molecular weight of 86 kDa was observed by SDS-PAGE and native-PAGE. On this basis, the mechanism of esterase B1 secreted by strain C5 degrading parathion-methyl was explored, and the effects of temperature and pH on the degradation rate were investigated. From the results, p-nitrophenol was one of the degradation products of B1 degrading parathion-methyl, and the best degradation effect could be achieved at the temperature of 40°C and the neutral pH value.

  11. Potato tuber pectin structure is influenced by pectin methyl esterase activity and impacts on cooked potato texture

    OpenAIRE

    Ross, Heather A.; Wright, Kathryn M.; McDougall, Gordon J.; Roberts, Alison G.; Chapman, Sean N.; Morris, Wayne L.; Hancock, Robert D.; Stewart, Derek; Tucker, Gregory A.; James, Euan K.; Taylor, Mark A.

    2010-01-01

    Although cooked potato tuber texture is an important trait that influences consumer preference, a detailed understanding of tuber textural properties at the molecular level is lacking. Previous work has identified tuber pectin methyl esterase activity (PME) as a potential factor impacting on textural properties. In this study, tuber PME isoform and gene expression profiles have been determined in potato germplasm with differing textural properties as assessed using an amended wedge fracture m...

  12. Multiple resistance to pirimiphos-methyl and bifenthrin in Tribolium castaneum involves the activity of lipases, esterases, and laccase2.

    Science.gov (United States)

    Julio, Alison Henrique Ferreira; Gigliolli, Adriana Aparecida Sinópolis; Cardoso, Kátia Aparecida Kern; Drosdoski, Sandro Daniel; Kulza, Rodrigo Amaral; Seixas, Flávio Augusto Vicente; Ruvolo-Takasusuki, Maria Claudia Colla; de Souza, Cristina Giatti Marques; Lapenta, Ana Silvia

    2017-05-01

    Several recent studies have elucidated the molecular mechanisms that confer insecticide resistance on insect pests. However, little is known about multiple resistance in red flour beetle (Tribolium castaneum) at molecular level. The multiple resistance is characterized as resistance to different classes of insecticides that have different target sites, and is mediated by several enzymatic systems. In this study, we investigated the biochemical and molecular mechanisms involved in multiple resistance of T. castaneum to bifenthrin (pyrethroid [Pyr]) and pirimiphos-methyl (organophosphate [Org]). We used artificial selection, biochemical and in silico approaches including structural computational biology. After five generations of artificial selection in the presence of bifenthrin (F5Pyr) or pirimiphos-methyl (F5Org), we found high levels of multiple resistance. The hierarchical enzymatic cluster revealed a pool of esterases (E), lipases (LIPs) and laccase2 (LAC2) potentially contributing to the resistance in different ways throughout development, after one or more generations in the presence of insecticides. The enzyme-insecticide interaction network indicated that E2, E3, LIP3, and LAC2 are enzymes potentially required for multiple resistance phenotype. Kinetic analysis of esterases from F5Pyr and F5Org showed that pirimiphos-methyl and specially bifenthrin promote enzyme inhibition, indicating that esterases mediate resistance by sequestering bifenthrin and pirimiphos-methyl. Our computational data were in accordance with kinetic results, indicating that bifenthrin has higher affinity at the active site of esterase than pirimiphos-methyl. We also report the capability of these insecticides to modify the development in T. castaneum. Our study provide insights into the biochemical mechanisms employed by T. castaneum to acquire multiple resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Identification of novel esterase-active enzymes from hot environments by use of the host bacterium Thermus thermophilus

    Directory of Open Access Journals (Sweden)

    Benedikt eLeis

    2015-04-01

    Full Text Available Functional metagenomic screening strategies, which are independent of known sequence information, can lead to the identification of truly novel genes and enzymes. Since E. coli has been used exhaustively for this purpose as a host, it is important to establish alternative expression hosts and to use them for functional metagenomic screening for new enzymes. In this study we show that Thermus thermophilus HB27 is an excellent screening host and can be used as an alternative provider of truly novel biocatalysts. In a previous study we constructed the mutant strain BL03 that was no longer able to grow on defined minimal medium supplemented with tributyrin as the sole carbon source and could be used as a host to screen for metagenomic DNA fragments that could complement growth on tributyrin. Several thousand single fosmid clones from thermophilic metagenomic libraries from heated compost and hot spring water samples were subjected to a comparative screening for esterase activity in both T. thermophilus strain BL03 and E. coli EPI300. We scored a greater number of active clones in the thermophilic bacterium than in the mesophilic E. coli. From all clones functionally screened in E. coli, only two thermostable α/β-fold hydrolase enzymes with high amino acid sequence similarity to already characterized enzymes were identifiable. In contrast, five further fosmids were found that conferred lipolytic activities in T. thermophilus. Four open reading frames (ORFs were found which did not share significant similarity to known esterase enzymes. Two of the genes were expressed in both hosts and the novel thermophilic esterases, which based on their primary structures could not be assigned to known esterase or lipase families, were purified and preliminarily characterized. Our work underscores the benefit of using additional screening hosts other than E. coli for the identification of novel biocatalysts with industrial relevance.

  14. Acetyl Groups in Typha capensis: Fate of Acetates during Organosolv and Ionosolv Pulping

    Directory of Open Access Journals (Sweden)

    Idi Guga Audu

    2018-06-01

    Full Text Available During biomass fractionation, any native acetylation of lignin and heteropolysaccharide may affect the process and the resulting lignin structure. In this study, Typha capensis (TC and its lignin isolated by milling (MWL, ionosolv (ILL and organosolv (EOL methods were investigated for acetyl group content using FT-Raman, 1H NMR, 2D-NMR, back-titration, and Zemplén transesterification analytical methods. The study revealed that TC is a highly acetylated grass; extractive free TC (TCextr and TC MWL exhibited similar values of acetyl content: 6 wt % and 8 wt % by Zemplén transesterification, respectively, and 11 wt % by back-titration. In contrast, lignin extracted from organosolv and [EMIm][OAc] pulping lost 80% of the original acetyl groups. With a high acetyl content in the natural state, TC could be an interesting raw material in biorefinery in which acetic acid could become an important by-product.

  15. Novel myelin penta- and hexa-acetyl-galactosyl-ceramides: structural characterization and immunoreactivity in cerebrospinal fluid

    DEFF Research Database (Denmark)

    Podbielska, Maria; Dasgupta, Somsankar; Levery, Steven B

    2010-01-01

    Fast migrating cerebrosides (FMC) are derivatives of galactosylceramide (GalCer). The structures of the most hydrophobic FMC-5, FMC-6, and FMC-7 were determined by electrospray ionization linear ion-trap mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy complementing previous...... NMR spectroscopy and gas chromatography-mass spectrometry to be 3-O-acetyl-sphingosine-GalCer derivatives with galactose O-acetyl modifications. FMC-5 and FMC-6 are 3-O-acetyl-sphingosine-2,3,4,6-tetra-O-acetyl-GalCer with nonhydroxy and hydroxy-N-fatty-acids, while FMC-7 has an additional O...... Mycoplasma fermentans. The cross-reactivity of highly acetylated GalCer with microbial acyl-glycolipid raises the possibility that myelin-O-acetyl-cerebrosides, bacterial infection, and neurological disease are linked....

  16. Evaluation of gels obtained from acetylation of chitosan in heterogeneous medium

    International Nuclear Information System (INIS)

    Garcia, Rosangela Balaban; Silva, Dayse Luzia Pinheiro da; Costa, Marta

    2008-01-01

    Chitosan was acetylated during 2, 5 and 10 h and physical gels were obtained at different polymer concentrations in N,N-dimethylacetamide containing 5% of LiCl. Acetylation was confirmed by infrared spectroscopy and 13 C NMR, and degrees of acetylation in the range of 0.82-0.91 were determined by NMR. The O-acetylation degree (0.12-0.15) was exclusively determined by a volumetric method. Rheological studies showed that the storage modulus values were smaller for the more acetylated samples and increased with the temperature and the polymer concentration. All the gels presented storage modulus superior to loss modulus, evidencing more elastic than viscous characteristics. The results obtained in this work suggest a gelation process based on a balance between O and N-acetylation and intermolecular bonds. (author)

  17. Est16, a New Esterase Isolated from a Metagenomic Library of a Microbial Consortium Specializing in Diesel Oil Degradation.

    Science.gov (United States)

    Pereira, Mariana Rangel; Mercaldi, Gustavo Fernando; Maester, Thaís Carvalho; Balan, Andrea; Lemos, Eliana Gertrudes de Macedo

    2015-01-01

    Lipolytic enzymes have attracted attention from a global market because they show enormous biotechnological potential for applications such as detergent production, leather processing, cosmetics production, and use in perfumes and biodiesel. Due to the intense demand for biocatalysts, a metagenomic approach provides methods of identifying new enzymes. In this study, an esterase designated as Est16 was selected from 4224 clones of a fosmid metagenomic library, revealing an 87% amino acid identity with an esterase/lipase (accession number ADM63076.1) from an uncultured bacterium. Phylogenetic studies showed that the enzyme belongs to family V of bacterial lipolytic enzymes and has sequence and structural similarities with an aryl-esterase from Pseudomonas fluorescens and a patented Anti-Kazlauskas lipase (patent number US20050153404). The protein was expressed and purified as a highly soluble, thermally stable enzyme that showed a preference for basic pH. Est16 exhibited activity toward a wide range of substrates and the highest catalytic efficiency against p-nitrophenyl butyrate and p-nitrophenyl valerate. Est16 also showed tolerance to the presence of organic solvents, detergents and metals. Based on molecular modeling, we showed that the large alpha-beta domain is conserved in the patented enzymes but not the substrate pocket. Here, it was demonstrated that a metagenomic approach is suitable for discovering the lipolytic enzyme diversity and that Est16 has the biotechnological potential for use in industrial processes.

  18. Effects of high hydrostatic pressure and temperature increase on Escherichia coli spp. and pectin methyl esterase inactivation in orange juice.

    Science.gov (United States)

    Torres, E F; González-M, G; Klotz, B; Rodrigo, D

    2016-03-01

    The aim of this study was to evaluate the effect of high hydrostatic pressure treatment combined with moderate processing temperatures (25 ℃-50 ℃) on the inactivation of Escherichia coli O157: H7 (ATCC 700728), E. coli K12 (ATCC 23716), and pectin methyl esterase in orange juice, using pressures of 250 to 500 MPa with times ranging between 1 and 30 min. Loss of viability of E. coli O157:H7 increased significantly as pressure and treatment time increased, achieving a 6.5 log cycle reduction at 400 MPa for 3 min at 25 ℃ of treatment. With regard to the inactivation of pectin methyl esterase, the greatest reduction obtained was 90.05 ± 0.01% at 50 ℃ and 500 MPa of pressure for 15 min; therefore, the pectin methyl esterase enzyme was highly resistant to the treatments by high hydrostatic pressure. The results obtained in this study showed a synergistic effect between the high pressure and moderate temperatures in inactivating E. coli cells. © The Author(s) 2016.

  19. A cold active (2R,3R)-(-)-di-O-benzoyl-tartrate hydrolyzing esterase from Rhodotorula mucilaginosa.

    Science.gov (United States)

    Zimmer, Christian; Platz, Tanja; Cadez, Neza; Giffhorn, Friedrich; Kohring, Gert-Wieland

    2006-11-01

    In a screening procedure a pink-colored yeast was isolated from enrichment cultures with (2R,3R)-(-)-di-O-benzoyl-tartrate (benzoyl-tartrate) as the sole carbon source. The organism saar1 was identified by morphological, physiological, and 18S ribosomal DNA/internal transcribed spacer analysis as Rhodotorula mucilaginosa, a basidiomycetous yeast. During growth the yeast hydrolyzed the dibenzoyl ester stoichiometrically to the monoester using the separated benzoate as the growth substrate, before the monoester was further cleaved into benzoate and tartrate, which were both metabolized. The corresponding benzoyl esterase was purified from the culture supernatant and characterized as a monomeric glycosylated 86-kDa protein with an optimum pH of 7.5 and an optimum temperature of 45 degrees C. At 0 degrees C the esterase still exhibited 20% of the corresponding activity at 30 degrees C, which correlates it to psychrophilic enzymes. The esterase could hydrolyze short chain p-nitrophenyl-alkyl esters and several benzoyl esters like benzoyl-methyl ester, ethylene-glycol-dibenzoyl ester, phenyl-benzoyl ester, cocaine, and 1,5-anhydro-D: -fructose-tribenzoyl ester. However feruloyl-ethyl ester was not hydrolyzed. The activity characteristics let the enzyme appear as a promising tool for synthesis of benzoylated compounds for pharmaceutical, cosmetic, or fine chemical applications, even at low temperatures.

  20. Cloning and characterization of a pyrethroid pesticide decomposing esterase gene, Est3385, from Rhodopseudomonas palustris PSB-S.

    Science.gov (United States)

    Luo, Xiangwen; Zhang, Deyong; Zhou, Xuguo; Du, Jiao; Zhang, Songbai; Liu, Yong

    2018-05-09

    Full length open reading frame of pyrethroid detoxification gene, Est3385, contains 963 nucleotides. This gene was identified and cloned based on the genome sequence of Rhodopseudomonas palustris PSB-S available at the GneBank. The predicted amino acid sequence of Est3385 shared moderate identities (30-46%) with the known homologous esterases. Phylogenetic analysis revealed that Est3385 was a member in the esterase family I. Recombinant Est3385 was heterologous expressed in E. coli, purified and characterized for its substrate specificity, kinetics and stability under various conditions. The optimal temperature and pH for Est3385 were 35 °C and 6.0, respectively. This enzyme could detoxify various pyrethroid pesticides and degrade the optimal substrate fenpropathrin with a Km and Vmax value of 0.734 ± 0.013 mmol·l -1 and 0.918 ± 0.025 U·µg -1 , respectively. No cofactor was found to affect Est3385 activity but substantial reduction of enzymatic activity was observed when metal ions were applied. Taken together, a new pyrethroid degradation esterase was identified and characterized. Modification of Est3385 with protein engineering toolsets should enhance its potential for field application to reduce the pesticide residue from agroecosystems.

  1. Est16, a New Esterase Isolated from a Metagenomic Library of a Microbial Consortium Specializing in Diesel Oil Degradation.

    Directory of Open Access Journals (Sweden)

    Mariana Rangel Pereira

    Full Text Available Lipolytic enzymes have attracted attention from a global market because they show enormous biotechnological potential for applications such as detergent production, leather processing, cosmetics production, and use in perfumes and biodiesel. Due to the intense demand for biocatalysts, a metagenomic approach provides methods of identifying new enzymes. In this study, an esterase designated as Est16 was selected from 4224 clones of a fosmid metagenomic library, revealing an 87% amino acid identity with an esterase/lipase (accession number ADM63076.1 from an uncultured bacterium. Phylogenetic studies showed that the enzyme belongs to family V of bacterial lipolytic enzymes and has sequence and structural similarities with an aryl-esterase from Pseudomonas fluorescens and a patented Anti-Kazlauskas lipase (patent number US20050153404. The protein was expressed and purified as a highly soluble, thermally stable enzyme that showed a preference for basic pH. Est16 exhibited activity toward a wide range of substrates and the highest catalytic efficiency against p-nitrophenyl butyrate and p-nitrophenyl valerate. Est16 also showed tolerance to the presence of organic solvents, detergents and metals. Based on molecular modeling, we showed that the large alpha-beta domain is conserved in the patented enzymes but not the substrate pocket. Here, it was demonstrated that a metagenomic approach is suitable for discovering the lipolytic enzyme diversity and that Est16 has the biotechnological potential for use in industrial processes.

  2. Solution Behavior and Activity of a Halophilic Esterase under High Salt Concentration

    Science.gov (United States)

    Rao, Lang; Zhao, Xiubo; Pan, Fang; Li, Yin; Xue, Yanfen; Ma, Yanhe; Lu, Jian R.

    2009-01-01

    Background Halophiles are extremophiles that thrive in environments with very high concentrations of salt. Although the salt reliance and physiology of these extremophiles have been widely investigated, the molecular working mechanisms of their enzymes under salty conditions have been little explored. Methodology/Principal Findings A halophilic esterolytic enzyme LipC derived from archeaon Haloarcula marismortui was overexpressed from Escherichia coli BL21. The purified enzyme showed a range of hydrolytic activity towards the substrates of p-nitrophenyl esters with different alkyl chains (n = 2−16), with the highest activity being observed for p-nitrophenyl acetate, consistent with the basic character of an esterase. The optimal esterase activities were found to be at pH 9.5 and [NaCl] = 3.4 M or [KCl] = 3.0 M and at around 45°C. Interestingly, the hydrolysis activity showed a clear reversibility against changes in salt concentration. At the ambient temperature of 22°C, enzyme systems working under the optimal salt concentrations were very stable against time. Increase in temperature increased the activity but reduced its stability. Circular dichroism (CD), dynamic light scattering (DLS) and small angle neutron scattering (SANS) were deployed to determine the physical states of LipC in solution. As the salt concentration increased, DLS revealed substantial increase in aggregate sizes, but CD measurements revealed the maximal retention of the α-helical structure at the salt concentration matching the optimal activity. These observations were supported by SANS analysis that revealed the highest proportion of unimers and dimers around the optimal salt concentration, although the coexistent larger aggregates showed a trend of increasing size with salt concentration, consistent with the DLS data. Conclusions/Significance The solution α-helical structure and activity relation also matched the highest proportion of enzyme unimers and dimers. Given that

  3. NEW EMBO MEMBER'S REVIEW: Acetylation: a regulatory modification to rival phosphorylation?

    OpenAIRE

    Kouzarides, Tony

    2000-01-01

    The fact that histones are modified by acetylation has been known for almost 30 years. The recent identification of enzymes that regulate histone acetylation has revealed a broader use of this modification than was suspected previously. Acetylases are now known to modify a variety of proteins, including transcription factors, nuclear import factors and α–tubulin. Acetylation regulates many diverse functions, including DNA recognition, protein–protein interaction and protein stability. There i...

  4. Autoimmune regulator is acetylated by transcription coactivator CBP/p300

    Energy Technology Data Exchange (ETDEWEB)

    Saare, Mario, E-mail: mario.saare@ut.ee [Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, 19th Ravila Str, Tartu (Estonia); Rebane, Ana [Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, 19th Ravila Str, Tartu (Estonia); SIAF, Swiss Institute of Allergy and Asthma Research, University of Zuerich, Davos (Switzerland); Rajashekar, Balaji; Vilo, Jaak [BIIT, Bioinformatics, Algorithmics and Data Mining group, Institute of Computer Science, University of Tartu, Tartu (Estonia); Peterson, Paert [Molecular Pathology, Institute of General and Molecular Pathology, University of Tartu, 19th Ravila Str, Tartu (Estonia)

    2012-08-15

    The Autoimmune Regulator (AIRE) is a regulator of transcription in the thymic medulla, where it controls the expression of a large set of peripheral-tissue specific genes. AIRE interacts with the transcriptional coactivator and acetyltransferase CBP and synergistically cooperates with it in transcriptional activation. Here, we aimed to study a possible role of AIRE acetylation in the modulation of its activity. We found that AIRE is acetylated in tissue culture cells and this acetylation is enhanced by overexpression of CBP and the CBP paralog p300. The acetylated lysines were located within nuclear localization signal and SAND domain. AIRE with mutations that mimicked acetylated K243 and K253 in the SAND domain had reduced transactivation activity and accumulated into fewer and larger nuclear bodies, whereas mutations that mimicked the unacetylated lysines were functionally similar to wild-type AIRE. Analogously to CBP, p300 localized to AIRE-containing nuclear bodies, however, the overexpression of p300 did not enhance the transcriptional activation of AIRE-regulated genes. Further studies showed that overexpression of p300 stabilized the AIRE protein. Interestingly, gene expression profiling revealed that AIRE, with mutations mimicking K243/K253 acetylation in SAND, was able to activate gene expression, although the affected genes were different and the activation level was lower from those regulated by wild-type AIRE. Our results suggest that the AIRE acetylation can influence the selection of AIRE activated genes. -- Highlights: Black-Right-Pointing-Pointer AIRE is acetylated by the acetyltransferases p300 and CBP. Black-Right-Pointing-Pointer Acetylation occurs between CARD and SAND domains and within the SAND domain. Black-Right-Pointing-Pointer Acetylation increases the size of AIRE nuclear dots. Black-Right-Pointing-Pointer Acetylation increases AIRE protein stability. Black-Right-Pointing-Pointer AIRE acetylation mimic regulates a different set of AIRE

  5. Analysis of functional xylanases in xylan degradation by Aspergillus niger E-1 and characterization of the GH family 10 xylanase XynVII.

    Science.gov (United States)

    Takahashi, Yui; Kawabata, Hiroaki; Murakami, Shuichiro

    2013-01-01

    Xylanases produced by Aspergillus niger are industrially important and many types of xylanases have been reported. Individual xylanases have been well studied for their enzymatic properties, gene cloning, and heterologous expression. However, less attention has been paid to the relationship between xylanase genes carried on the A. niger genome and xylanases produced by A. niger strains. Therefore, we examined xylanase genes encoded on the genome of A. niger E-1 and xylanases produced in culture. Seven putative xylanase genes, xynI-VII (named in ascending order of the molecular masses of the deduced amino acid sequences), were amplified from the strain E-1 genome using primers designed from the genome sequence of A. niger CBS 513.88 by PCR and phylogenetically classified into three clusters. Additionally, culture supernatant analysis by DE52 anion-exchange column chromatography revealed that this strain produced three xylanases, XynII, XynIII, and XynVII, which were identified by N-terminal amino acid sequencing and MALDI-TOF-MS analyses, in culture when gown in 0.5% xylan medium supplemented with 50 mM succinate. Furthermore, XynVII, the only GH family 10 xylanase in A. niger E-1, was purified and characterized. The purified enzyme showed a single band with a molecular mass of 35 kDa by SDS-PAGE. The highest activity of purified XynVII was observed at 55°C and pH 5.5. The enzyme was stable in the broad pH range of 3-10 and up to 60°C and was resistant to most metal ions and modifying regents. XynVII showed high specificity against beechwood xylan with K m and V max values of 2.8 mg mL(-1) and 127 μmol min(-1)mg(-1), respectively. TLC and MALDI-TOF-MS analyses showed that the final hydrolyzed products of the enzyme from beechwood xylan were xylose, xylobiose, and xylotriose substituted with a 4-o-metylglucuronic acid residue.

  6. Kinetic Modelling and Experimental Studies for the Effects of Fe2+ Ions on Xylan Hydrolysis with Dilute-Acid Pretreatment and Subsequent Enzymatic Hydrolysis

    Directory of Open Access Journals (Sweden)

    Hui Wei

    2018-01-01

    Full Text Available High-temperature (150–170 °C pretreatment of lignocellulosic biomass with mineral acids is well established for xylan breakdown. Fe2+ is known to be a cocatalyst of this process although kinetics of its action remains unknown. The present work addresses the effect of ferrous ion concentration on sugar yield and degradation product formation from corn stover for the entire two-step treatment, including the subsequent enzymatic cellulose hydrolysis. The feedstock was impregnated with 0.5% acid and 0.75 mM iron cocatalyst, which was found to be optimal in preliminary experiments. The detailed kinetic data of acid pretreatment, with and without iron, was satisfactorily modelled with a four-step linear sequence of first-order irreversible reactions accounting for the formation of xylooligomers, xylose and furfural as intermediates to provide the values of Arrhenius activation energy. Based on this kinetic modelling, Fe2+ turned out to accelerate all four reactions, with a significant alteration of the last two steps, that is, xylose degradation. Consistent with this model, the greatest xylan conversion occurred at the highest severity tested under 170 °C/30 min with 0.75 mM Fe2+, with a total of 8% xylan remaining in the pretreated solids, whereas the operational conditions leading to the highest xylose monomer yield, 63%, were milder, 150 °C with 0.75 mM Fe2+ for 20 min. Furthermore, the subsequent enzymatic hydrolysis with the prior addition of 0.75 mM of iron(II increased the glucose production to 56.3% from 46.3% in the control (iron-free acid. The detailed analysis indicated that conducting the process at lower temperatures yet long residence times benefits the yield of sugars. The above kinetic modelling results of Fe2+ accelerating all four reactions are in line with our previous mechanistic research showing that the pretreatment likely targets multiple chemistries in plant cell wall polymer networks, including those represented by the C

  7. Lysine acetylation targets protein complexes and co-regulates major cellular functions

    DEFF Research Database (Denmark)

    Choudhary, Chuna Ram; Kumar, Chanchal; Gnad, Florian

    2009-01-01

    Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation's cellular roles. We used high-resolution mass spectrometry to identify 3600......, cell cycle, splicing, nuclear transport, and actin nucleation. Acetylation impaired phosphorylation-dependent interactions of 14-3-3 and regulated the yeast cyclin-dependent kinase Cdc28. Our data demonstrate that the regulatory scope of lysine acetylation is broad and comparable with that of other...

  8. Metabolism of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in the human neutrophil

    International Nuclear Information System (INIS)

    Triggiani, M.; D'Souza, D.M.; Chilton, F.H.

    1991-01-01

    The biosynthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl-GPC) together with that of 1-alkyl-2-acetyl-GPC (platelet-activating factor) has been demonstrated in a variety of inflammatory cells and tissues. It has been hypothesized that the relative proportion of these phospholipids produced upon cell activation may be influenced by their rates of catabolism. We studied the catabolism of 1-acyl-2-acetyl-GPC in resting and activated human neutrophils and compared it to that of 1-alkyl-2-acetyl-GPC. Neutrophils rapidly catabolize both 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC; however, the rate of catabolism of 1-acyl-2-acetyl-GPC is approximately 2-fold higher than that of 1-alkyl-2-acetyl-GPC. In addition, most of 1-acyl-2-acetyl-GPC is catabolized through a pathway different from that of 1-alkyl-2-acetyl-GPC. The main step in the catabolism of 1-acyl-2-acetyl-GPC is the removal of the long chain at the sn-1 position; the long chain residue is subsequently incorporated either into triglycerides or into phosphatidylcholine. The 1-lyso-2-acetyl-GPC formed in this reaction is then further degraded to glycerophosphocholine, choline, or phosphocholine. 1-Acyl-2-acetyl-GPC is also catabolized, to a lesser extent, through deacetylation at the sn-2 position and reacylation with a long chain fatty acid. Stimulation of neutrophils by A23187 results in a higher rate of catabolism of 1-acyl-2-acetyl-GPC by increasing both the removal of the long chain at the sn-1 position and the deacetylation-reacylation at the sn-2 position. In a broken cell preparation, the cytosolic fraction of the neutrophil was shown to contain an enzyme activity which cleaved the sn-1 position of 1-acyl-2-acetyl-GPC and 1-acyl-2-lyso-GPC but not of 1,2-diacyl-GPC

  9. Preparation of radioactive acetyl-l-carnitine by an enzymatic exchange reaction

    International Nuclear Information System (INIS)

    Emaus, R.; Bieber, L.L.

    1982-01-01

    A rapid method for the preparation of [1- 14 C]acetyl-L-carnitine is described. The method involves exchange of [1- 14 C]acetic acid into a pool of unlabeled acetyl-L-carnitine using the enzymes acetyl-CoA synthetase and carnitine acetyltransferase. After isotopic equilibrium is attained, radioactive acetylcarnitine is separated from the other reaction components by chromatography on Dowex 1 (C1 - ) anion exchange resin. One of the procedures used to verify the product [1- 14 C]acetyl-L-carnitine can be used to synthesize (3S)-[5- 14 C]citric acid

  10. Lysine Acetylation of CREBH Regulates Fasting-Induced Hepatic Lipid Metabolism

    Science.gov (United States)

    Kim, Hyunbae; Mendez, Roberto; Chen, Xuequn; Fang, Deyu

    2015-01-01

    Cyclic AMP-responsive element-binding protein 3-like 3, hepatocyte specific (CREBH), is a hepatic transcription factor that functions as a key regulator of energy homeostasis. Here, we defined a regulatory CREBH posttranslational modification process, namely, lysine-specific acetylation, and its functional involvement in fasting-induced hepatic lipid metabolism. Fasting induces CREBH acetylation in mouse livers in a time-dependent manner, and this event is critical for CREBH transcriptional activity in regulating hepatic lipid homeostasis. The histone acetyltransferase PCAF-mediated acetylation and the deacetylase sirtuin-1-mediated deacetylation coexist to maintain CREBH acetylation states under fasting conditions. Site-directed mutagenesis and functional analyses revealed that the lysine (K) residue at position 294 (K294) within the bZIP domain of the CREBH protein is the site where fasting-induced acetylation/deacetylation occurs. Introduction of the acetylation-deficient (K294R) or acetylation-mimicking (K294Q) mutation inhibited or enhanced CREBH transcriptional activity, respectively. Importantly, CREBH acetylation at lysine 294 was required for the interaction and synergy between CREBH and peroxisome proliferator-activated receptor α (PPARα) in activating their target genes upon fasting or glucagon stimulation. Introduction of the CREBH lysine 294 mutation in the liver leads to hepatic steatosis and hyperlipidemia in animals under prolonged fasting. In summary, our study reveals a molecular mechanism by which fasting or glucagon stimulation modulates lipid homeostasis through acetylation of CREBH. PMID:26438600

  11. Comparative analysis of pharmacological treatments with N-acetyl-DL-leucine (Tanganil) and its two isomers (N-acetyl-L-leucine and N-acetyl-D-leucine) on vestibular compensation: Behavioral investigation in the cat.

    Science.gov (United States)

    Tighilet, Brahim; Leonard, Jacques; Bernard-Demanze, Laurence; Lacour, Michel

    2015-12-15

    Head roll tilt, postural imbalance and spontaneous nystagmus are the main static vestibular deficits observed after an acute unilateral vestibular loss (UVL). In the UVL cat model, these deficits are fully compensated over 6 weeks as the result of central vestibular compensation. N-Acetyl-dl-leucine is a drug prescribed in clinical practice for the symptomatic treatment of acute UVL patients. The present study investigated the effects of N-acetyl-dl-leucine on the behavioral recovery after unilateral vestibular neurectomy (UVN) in the cat, and compared the effects of each of its two isomers N-acetyl-L-leucine and N-acetyl-D-leucine. Efficacy of these three drug treatments has been evaluated with respect to a placebo group (UVN+saline water) on the global sensorimotor activity (observation grids), the posture control (support surface measurement), the locomotor balance (maximum performance at the rotating beam test), and the spontaneous vestibular nystagmus (recorded in the light). Whatever the parameters tested, the behavioral recovery was strongly and significantly accelerated under pharmacological treatments with N-acetyl-dl-leucine and N-acetyl-L-leucine. In contrast, the N-acetyl-D-leucine isomer had no effect at all on the behavioral recovery, and animals of this group showed the same recovery profile as those receiving a placebo. It is concluded that the N-acetyl-L-leucine isomer is the active part of the racemate component since it induces a significant acceleration of the vestibular compensation process similar (and even better) to that observed under treatment with the racemate component only. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. The Metabolic Fate of Deoxynivalenol and Its Acetylated Derivatives in a Wheat Suspension Culture: Identification and Detection of DON-15-O-Glucoside, 15-Acetyl-DON-3-O-Glucoside and 15-Acetyl-DON-3-Sulfate

    Directory of Open Access Journals (Sweden)

    Clemens Schmeitzl

    2015-08-01

    Full Text Available Deoxynivalenol (DON is a protein synthesis inhibitor produced by the Fusarium species, which frequently contaminates grains used for human or animal consumption. We treated a wheat suspension culture with DON or one of its acetylated derivatives, 3-acetyl-DON (3-ADON, 15-acetyl-DON (15-ADON and 3,15-diacetyl-DON (3,15-diADON, and monitored the metabolization over a course of 96 h. Supernatant and cell extract samples were analyzed using a tailored LC-MS/MS method for the quantification of DON metabolites. We report the formation of tentatively identified DON-15-O-β-D-glucoside (D15G and of 15-acetyl-DON-3-sulfate (15-ADON3S as novel deoxynivalenol metabolites in wheat. Furthermore, we found that the recently identified 15-acetyl-DON-3-O-β-D-glucoside (15-ADON3G is the major metabolite produced after 15-ADON challenge. 3-ADON treatment led to a higher intracellular content of toxic metabolites after six hours compared to all other treatments. 3-ADON was exclusively metabolized into DON before phase II reactions occurred. In contrast, we found that 15-ADON was directly converted into 15-ADON3G and 15-ADON3S in addition to metabolization into deoxynivalenol-3-O-β-D-glucoside (D3G. This study highlights significant differences in the metabolization of DON and its acetylated derivatives.

  13. The dynamic organization of fungal acetyl-CoA carboxylase

    Science.gov (United States)

    Hunkeler, Moritz; Stuttfeld, Edward; Hagmann, Anna; Imseng, Stefan; Maier, Timm

    2016-04-01

    Acetyl-CoA carboxylases (ACCs) catalyse the committed step in fatty-acid biosynthesis: the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA. They are important regulatory hubs for metabolic control and relevant drug targets for the treatment of the metabolic syndrome and cancer. Eukaryotic ACCs are single-chain multienzymes characterized by a large, non-catalytic central domain (CD), whose role in ACC regulation remains poorly characterized. Here we report the crystal structure of the yeast ACC CD, revealing a unique four-domain organization. A regulatory loop, which is phosphorylated at the key functional phosphorylation site of fungal ACC, wedges into a crevice between two domains of CD. Combining the yeast CD structure with intermediate and low-resolution data of larger fragments up to intact ACCs provides a comprehensive characterization of the dynamic fungal ACC architecture. In contrast to related carboxylases, large-scale conformational changes are required for substrate turnover, and are mediated by the CD under phosphorylation control.

  14. Autotrophic acetyl coenzyme A biosynthesis in Methanococcus maripaludis

    International Nuclear Information System (INIS)

    Shieh, J.; Whitman, W.B.

    1988-01-01

    To detect autotrophic CO 2 assimilation in cell extracts of Methanococcus maripaludis, lactate dehydrogenase and NADH were added to convert pyruvate formed from autotropically synthesized acetyl coenzyme A to lactate. The lactate produced was determined spectrophotometrically. When CO 2 fixation was pulled in the direction of lactate synthesis, CO 2 reduction to methane was inhibited. Bromoethanesulfonate (BES), a potent inhibitor of methanogenesis, enhanced lactate synthesis, and methyl coenzyme M inhibited it in the absence of BES. Lactate synthesis was dependent on CO 2 and H 2 , but H 2 + CO 2 -independent synthesis was also observed. In cell extracts, the rate of lactate synthesis was about 1.2 nmol min -1 mg of protein -1 . When BES was added, the rate of lactate synthesis increased to 2.1 nmol min -1 mg of protein -1 . Because acetyl coenzyme A did not stimulate lactate synthesis, pyruvate synthase may have been the limiting activity in these assays. Radiolabel from 14 CO 2 was incorporated into lactate. The percentages of radiolabel in the C-1, C-2, and C-3 positions of lactate were 73, 33, and 11%, respectively. Both carbon monoxide and formaldehyde stimulated lactate synthesis. 14 CH 2 O was specifically incorporated into the C-3 of lactate, and 14 CO was incorporated into the C-1 and C-2 positions. Low concentrations of cyanide also inhibited autotrophic growth, CO dehydrogenase activity, and autotrophic lactate synthesis. These observations are in agreement with the acetogenic pathway of autotrophic CO 2 assimilation

  15. Protein acetylation sites mediated by Schistosoma mansoni GCN5

    International Nuclear Information System (INIS)

    Moraes Maciel, Renata de; Furtado Madeiro da Costa, Rodrigo; Meirelles Bastosde Oliveira, Francisco; Rumjanek, Franklin David; Fantappie, Marcelo Rosado

    2008-01-01

    The transcriptional co-activator GCN5, a histone acetyltransferase (HAT), is part of large multimeric complexes that are required for chromatin remodeling and transcription activation. As in other eukaryotes, the DNA from the parasite Schistosome mansoni is organized into nucleosomes and the genome encodes components of chromatin-remodeling complexes. Using a series of synthetic peptides we determined that Lys-14 of histone H3 was acetylated by the recombinant SmGCN5-HAT domain. SmGCN5 was also able to acetylate schistosome non-histone proteins, such as the nuclear receptors SmRXR1 and SmNR1, and the co-activator SmNCoA-62. Electron microscopy revealed the presence of SmGCN5 protein in the nuclei of vitelline cells. Within the nucleus, SmGCN5 was found to be located in interchromatin granule clusters (IGCs), which are transcriptionally active structures. The data suggest that SmGCN5 is involved in transcription activation

  16. The role of low levels of juvenile hormone Esterase in the metamorphosis of Manduca sexta

    Directory of Open Access Journals (Sweden)

    M.H. Browder

    2001-10-01

    Full Text Available The activity of juvenile hormone esterase (JHE in feeding fifth instar larvae of Manduca sexta increases gradually with larval weight and rises to a peak after larvae pass the critical weight when juvenile hormone secretion ceases. Starvation of larvae of Manduca sexta (L. that had exceeded the critical weight inhibited peak levels of JHE, but did not delay entry into the wandering stage when larvae leave the plant in search of a pupation site. This suggests that peak levels of JHE may not be essential for the normal timing of metamorphosis. Starved larvae pupated normally, indicating the peak of JHE was not necessary for a morphologically normal pupation. Treatments of larvae with the selective JHE inhibitor O-ethyl-S-phenyl phosphoramidothiolate (EPPAT that began immediately after larvae achieved the critical weight (6.0 to 6.5 grams for our strain of Manduca delayed entry into the wandering stage. By contrast, EPPAT treatment of larvae at weights above 8.0g had no effect on the subsequent timing of the onset of wandering. Therefore, although the normal timing of the onset of wandering does not require peak levels of JHE, it requires low to moderate levels of JHE to be present until larvae reach a weight of about 8.0g.

  17. Subunit Stabilization and Polyethylene Glycolation of Cocaine Esterase Improves In Vivo Residence Time

    Energy Technology Data Exchange (ETDEWEB)

    Narasimhan, Diwahar; Collins, Gregory T.; Nance, Mark R.; Nichols, Joseph; Edwald, Elin; Chan, Jimmy; Ko, Mei-Chuan; Woods, James H.; Tesmer, John J.G.; Sunahara, Roger K. (Michigan)

    2012-03-15

    No small-molecule therapeutic is available to treat cocaine addiction, but enzyme-based therapy to accelerate cocaine hydrolysis in serum has gained momentum. Bacterial cocaine esterase (CocE) is the fastest known native enzyme that hydrolyzes cocaine. However, its lability at 37 C has limited its therapeutic potential. Cross-linking subunits through disulfide bridging is commonly used to stabilize multimeric enzymes. Herein we use structural methods to guide the introduction of two cysteine residues within dimer interface of CocE to facilitate intermolecular disulfide bond formation. The disulfide-crosslinked enzyme displays improved thermostability, particularly when combined with previously described mutations that enhance stability (T172R-G173Q). The newly modified enzyme yielded an extremely stable form of CocE (CCRQ-CocE) that retained greater than 90% of its activity after 41 days at 37 C, representing an improvement of more than 4700-fold over the wild-type enzyme. CCRQ-CocE could also be modified by polyethylene glycol (PEG) polymers, which improved its in vivo residence time from 24 to 72 h, as measured by a cocaine lethality assay, by self-administration in rodents, and by measurement of inhibition of cocaine-induced cardiovascular effects in rhesus monkeys. PEG-CCRQ elicited negligible immune response in rodents. Subunit stabilization and PEGylation has thus produced a potential protein therapeutic with markedly higher stability both in vitro and in vivo.

  18. Expression and characterization of a new esterase with GCSAG motif from a permafrost metagenomic library.

    Science.gov (United States)

    Petrovskaya, Lada E; Novototskaya-Vlasova, Ksenia A; Spirina, Elena V; Durdenko, Ekaterina V; Lomakina, Galina Yu; Zavialova, Maria G; Nikolaev, Evgeny N; Rivkina, Elizaveta M

    2016-05-01

    As a result of construction and screening of a metagenomic library prepared from a permafrost-derived microcosm, we have isolated a novel gene coding for a putative lipolytic enzyme that belongs to the hormone-sensitive lipase family. It encodes a polypeptide of 343 amino acid residues whose amino acid sequence displays maximum likelihood with uncharacterized proteins from Sphingomonas species. A putative catalytic serine residue of PMGL2 resides in a new variant of a recently discovered GTSAG sequence in which a Thr residue is replaced by a Cys residue (GCSAG). The recombinant PMGL2 was produced in Escherichia coli cells and purified by Ni-affinity chromatography. The resulting protein preferably utilizes short-chain p-nitrophenyl esters (C4 and C8) and therefore is an esterase. It possesses maximum activity at 45°C in slightly alkaline conditions and has limited thermostability at higher temperatures. Activity of PMGL2 is stimulated in the presence of 0.25-1.5 M NaCl indicating the good salt tolerance of the new enzyme. Mass spectrometric analysis demonstrated that N-terminal methionine in PMGL2 is processed and cysteine residues do not form a disulfide bond. The results of the study demonstrate the significance of the permafrost environment as a unique genetic reservoir and its potential for metagenomic exploration. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Pectin methyl esterase inhibits intrusive and symplastic cell growth in developing wood cells of Populus.

    Science.gov (United States)

    Siedlecka, Anna; Wiklund, Susanne; Péronne, Marie-Amélie; Micheli, Fabienne; Lesniewska, Joanna; Sethson, Ingmar; Edlund, Ulf; Richard, Luc; Sundberg, Björn; Mellerowicz, Ewa J

    2008-02-01

    Wood cells, unlike most other cells in plants, grow by a unique combination of intrusive and symplastic growth. Fibers grow in diameter by diffuse symplastic growth, but they elongate solely by intrusive apical growth penetrating the pectin-rich middle lamella that cements neighboring cells together. In contrast, vessel elements grow in diameter by a combination of intrusive and symplastic growth. We demonstrate that an abundant pectin methyl esterase (PME; EC 3.1.1.11) from wood-forming tissues of hybrid aspen (Populus tremula x tremuloides) acts as a negative regulator of both symplastic and intrusive growth of developing wood cells. When PttPME1 expression was up- and down-regulated in transgenic aspen trees, the PME activity in wood-forming tissues was correspondingly altered. PME removes methyl ester groups from homogalacturonan (HG) and transgenic trees had modified HG methylesterification patterns, as demonstrated by two-dimensional nuclear magnetic resonance and immunostaining using PAM1 and LM7 antibodies. In situ distributions of PAM1 and LM7 epitopes revealed changes in pectin methylesterification in transgenic trees that were specifically localized in expanding wood cells. The results show that en block deesterification of HG by PttPME1 inhibits both symplastic growth and intrusive growth. PttPME1 is therefore involved in mechanisms determining fiber width and length in the wood of aspen trees.

  20. HIGHLY METHYL ESTERIFIED SEEDS is a pectin methyl esterase involved in embryo development.

    Science.gov (United States)

    Levesque-Tremblay, Gabriel; Müller, Kerstin; Mansfield, Shawn D; Haughn, George W

    2015-03-01

    Homogalacturonan pectin domains are synthesized in a highly methyl-esterified form that later can be differentially demethyl esterified by pectin methyl esterase (PME) to strengthen or loosen plant cell walls that contain pectin, including seed coat mucilage, a specialized secondary cell wall of seed coat epidermal cells. As a means to identify the active PMEs in seed coat mucilage, we identified seven PMEs expressed during seed coat development. One of these, HIGHLY METHYL ESTERIFIED SEEDS (HMS), is abundant during mucilage secretion, peaking at 7 d postanthesis in both the seed coat and the embryo. We have determined that this gene is required for normal levels of PME activity and homogalacturonan methyl esterification in the seed. The hms-1 mutant displays altered embryo morphology and mucilage extrusion, both of which are a consequence of defects in embryo development. A significant decrease in the size of cells in the embryo suggests that the changes in embryo morphology are a consequence of lack of cell expansion. Progeny from a cross between hms-1 and the previously characterized PME inhibitor5 overexpression line suggest that HMS acts independently from other cell wall-modifying enzymes in the embryo. We propose that HMS is required for cell wall loosening in the embryo to facilitate cell expansion during the accumulation of storage reserves and that its role in the seed coat is masked by redundancy. © 2015 American Society of Plant Biologists. All Rights Reserved.

  1. Pectin Methyl Esterase Activity Change in Intermediate Moisture Sun-Dried Figs after Storage

    Directory of Open Access Journals (Sweden)

    Dilek Demirbüker Kavak

    2015-12-01

    Full Text Available Intermediate moisture fruits can be obtained by rehydrating dried fruits. Intermediate moisture fruits are suitable for direct consumption compared to dry fruits and can be directly used in the production of various products such as bakery products, dairy products and candies. Aim of this study is to compare the pectin methyl esterase (PME activity of intermediate moisture figs which causes softening of the texture and to compare their microbial stability after 3 months storage period. For this purpose, dried figs were rehydrated in 30 and 80° C water until they reach 30% moisture content. Rehydrated samples were stored for 3 months at +4°C. Results showed that there was no statistically significant difference between the control samples and the samples rehydrated at 80°C according to the total viable counts. At the end of the storage period, results of residual PME activity in control samples was 24.1 μmol COOH min-1g-1, while it was found 17.4 μmol COOH min-1g-1 in samples rehydrated at 80°C. As a result rehydration conducted at 80°C provided 28% reduction in PME activity compared to the control samples rehydrated at 30°C, although it did not affect the microbial load significantly after storage.

  2. An increase in pectin methyl esterase activity accompanies dormancy breakage and germination of yellow cedar seeds.

    Science.gov (United States)

    Ren, C; Kermode, A R

    2000-09-01

    Pectin methyl esterase (PME) (EC 3.1.1.11) catalyzes the hydrolysis of methylester groups of cell wall pectins. We investigated the role of this enzyme in dormancy termination and germination of yellow cedar (Chamaecyparis nootkatensis [D. Don] Spach) seeds. PME activity was not detected in dormant seeds of yellow cedar but was induced and gradually increased during moist chilling; high activity coincided with dormancy breakage and germination. PME activity was positively correlated to the degree of dormancy breakage of yellow cedar seeds. The enzyme produced in different seed parts and in seeds at different times during moist chilling, germination, and early post-germinative growth consisted of two isoforms, both basic with isoelectric points of 8.7 and 8.9 and the same molecular mass of 62 kD. The pH optimum for the enzyme was between 7.4 and 8.4. In intact yellow cedar seeds, activities of the two basic isoforms of PME that were induced in embryos and in megagametophytes following dormancy breakage were significantly suppressed by abscisic acid. Gibberellic acid had a stimulatory effect on the activities of these isoforms in embryos and megagametophytes of intact seeds at the germinative stage. We hypothesize that PME plays a role in weakening of the megagametophyte, allowing radicle emergence and the completion of germination.

  3. High-pressure processing of apple juice: kinetics of pectin methyl esterase inactivation.

    Science.gov (United States)

    Riahi, Esmaeil; Ramaswamy, Hosahalli S

    2003-01-01

    High-pressure (HP) inactivation kinetics of pectin methyl esterase (PME) in apple juice were evaluated. Commercial PME was dispensed in clarified apple juice, sealed in dual peel sterilizable plastic bags, and subjected to different high-pressure processing conditions (200-400 MPa, 0-180 min). Residual enzyme activity was determined by a titration method estimating the rate of free carboxyl group released by the enzyme acting on pectin substrate at pH 7.5 (30 degrees C). The effects of pressure level and pressure holding time on enzyme inactivation were significant (p < 0.05). PME from the microbial source was found to be more resistant (p < 0.05) to pressure inactivation than PME from the orange peel. Almost a full decimal reduction in the activity of commercial PME was achieved by HP treatment at 400 MPa for 25 min. Inactivation kinetics were evaluated on the basis of a dual effect model involving a pressure pulse effect and a first-order rate model, and the pressure sensitivity of rate constants was modeled by using the z-value concept.

  4. Separating esterase targets of organophosphorus compounds in the brain by preparative chromatography.

    Science.gov (United States)

    Mangas, I; Vilanova, E; Benabent, M; Estévez, J

    2014-02-10

    Low level exposure to organophosphorus esters (OPs) may cause long-term neurological effects and affect specific cognition domains in experimental animals and humans. Action on known targets cannot explain most of these effects by. Soluble carboxylesterases (EC 3.1.1.1) of chicken brain have been kinetically discriminated using paraoxon, mipafox and phenylmethyl sulfonylfluoride as inhibitors and phenyl valerate as a substrate. Three different enzymatic components were discriminated and called Eα, Eβ and Eγ. In this work, a fractionation procedure with various steps was developed using protein native separation methods by preparative HPLC. Gel permeation chromatography followed by ion exchange chromatography allowed enriched fractions with different kinetic behaviors. The soluble chicken brain fraction was fractionated, while total esterase activity, proteins and enzymatic components Eα, Eβ and Eγ were monitored in each subfraction. After the analysis, 13 fractions were pooled and conserved. Preincubation of the soluble chicken brain fraction of with the organophosphorus mipafox gave rise to a major change in the ion exchange chromatography profile, but not in the molecular exchanged chromatography profile, which suggest that mipafox permanently modifies the ionic properties of numerous proteins. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  5. Glucuronoyl Esterase Screening and Characterization Assays Utilizing Commercially Available Benzyl Glucuronic Acid Ester

    Directory of Open Access Journals (Sweden)

    Hampus Sunner

    2015-09-01

    Full Text Available Research on glucuronoyl esterases (GEs has been hampered by the lack of enzyme assays based on easily obtainable substrates. While benzyl d-glucuronic acid ester (BnGlcA is a commercially available substrate that can be used for GE assays, several considerations regarding substrate instability, limited solubility and low apparent affinities should be made. In this work we discuss the factors that are important when using BnGlcA for assaying GE activity and show how these can be applied when designing BnGlcA-based GE assays for different applications: a thin-layer chromatography assay for qualitative activity detection, a coupled-enzyme spectrophotometric assay that can be used for high-throughput screening or general activity determinations and a HPLC-based detection method allowing kinetic determinations. The three-level experimental procedure not merely facilitates routine, fast and simple biochemical characterizations but it can also give rise to the discovery of different GEs through an extensive screening of heterologous Genomic and Metagenomic expression libraries.

  6. A Lactobacillus plantarum Esterase Active on a Broad Range of Phenolic Esters

    Science.gov (United States)

    Esteban-Torres, María; Landete, José María; Reverón, Inés; Santamaría, Laura; de las Rivas, Blanca

    2015-01-01

    Lactobacillus plantarum is the lactic acid bacterial species most frequently found in the fermentation of food products of plant origin on which phenolic compounds are abundant. L. plantarum strains showed great flexibility in their ability to adapt to different environments and growth substrates. Of 28 L. plantarum strains analyzed, only cultures from 7 strains were able to hydrolyze hydroxycinnamic esters, such as methyl ferulate or methyl caffeate. As revealed by PCR, only these seven strains possessed the est_1092 gene. When the est_1092 gene was introduced into L. plantarum WCFS1 or L. lactis MG1363, their cultures acquired the ability to degrade hydroxycinnamic esters. These results support the suggestion that Est_1092 is the enzyme responsible for the degradation of hydroxycinnamic esters on the L. plantarum strains analyzed. The Est_1092 protein was recombinantly produced and biochemically characterized. Surprisingly, Est_1092 was able to hydrolyze not only hydroxycinnamic esters, since all the phenolic esters assayed were hydrolyzed. Quantitative PCR experiments revealed that the expression of est_1092 was induced in the presence of methyl ferulate, an hydroxycinnamic ester, but was inhibited on methyl gallate, an hydroxybenzoic ester. As Est_1092 is an enzyme active on a broad range of phenolic esters, simultaneously possessing feruloyl esterase and tannase activities, its presence on some L. plantarum strains provides them with additional advantages to survive and grow on plant environments. PMID:25746986

  7. Construction of an Immobilized Thermophilic Esterase on Epoxy Support for Poly(ε-caprolactone Synthesis

    Directory of Open Access Journals (Sweden)

    Hui Ren

    2016-06-01

    Full Text Available Developing an efficient immobilized enzyme is of great significance for improving the operational stability of enzymes in poly(ε-caprolactone synthesis. In this paper, a thermophilic esterase AFEST from the archaeon Archaeoglobus fulgidus was successfully immobilized on the epoxy support Sepabeads EC-EP via covalent attachment, and the immobilized enzyme was then employed as a biocatalyst for poly(ε-caprolactone synthesis. The enzyme loading and recovered activity of immobilized enzyme was measured to be 72 mg/g and 10.4 U/mg using p-nitrophenyl caprylate as the substrate at 80 °C, respectively. Through the optimization of reaction conditions (enzyme concentration, temperature, reaction time and medium, poly(ε-caprolactone was obtained with 100% monomer conversion and low number-average molecular weight (Mn < 1300 g/mol. Further, the immobilized enzyme exhibited excellent reusability, with monomer conversion values exceeding 75% during 15 batch reactions. Finally, poly(ε-caprolactone was enzymatically synthesized with an isolated yield of 75% and Mn value of 3005 g/mol in a gram-scale reaction.

  8. Construction of an Immobilized Thermophilic Esterase on Epoxy Support for Poly(ε-caprolactone) Synthesis.

    Science.gov (United States)

    Ren, Hui; Xing, Zhen; Yang, Jiebing; Jiang, Wei; Zhang, Gang; Tang, Jun; Li, Quanshun

    2016-06-18

    Developing an efficient immobilized enzyme is of great significance for improving the operational stability of enzymes in poly(ε-caprolactone) synthesis. In this paper, a thermophilic esterase AFEST from the archaeon Archaeoglobus fulgidus was successfully immobilized on the epoxy support Sepabeads EC-EP via covalent attachment, and the immobilized enzyme was then employed as a biocatalyst for poly(ε-caprolactone) synthesis. The enzyme loading and recovered activity of immobilized enzyme was measured to be 72 mg/g and 10.4 U/mg using p-nitrophenyl caprylate as the substrate at 80 °C, respectively. Through the optimization of reaction conditions (enzyme concentration, temperature, reaction time and medium), poly(ε-caprolactone) was obtained with 100% monomer conversion and low number-average molecular weight (Mn enzyme exhibited excellent reusability, with monomer conversion values exceeding 75% during 15 batch reactions. Finally, poly(ε-caprolactone) was enzymatically synthesized with an isolated yield of 75% and Mn value of 3005 g/mol in a gram-scale reaction.

  9. Design, characterisation and application of alginate-based encapsulated pig liver esterase.

    Science.gov (United States)

    Pauly, Jan; Gröger, Harald; Patel, Anant V

    2018-06-05

    Encapsulation of hydrolases in biopolymer-based hydrogels often suffers from low activities and encapsulation efficiencies along with high leaching and unsatisfactory recycling properties. Exemplified for the encapsulation of pig liver esterase the coating of alginate and chitosan beads have been studied by creating various biopolymer hydrogel beads. Enzyme activity and encapsulation efficiency were notably enhanced by chitosan coating of alginate beads while leaching remained nearly unchanged. This was caused by the enzymatic reaction acidifying the matrix, which increased enzyme retention through enhanced electrostatic enzyme-alginate interaction but decreased activity through enzyme deactivation. A practical and ready-to-use method for visualising pH in beads during reaction by co-encapsulation of a conventional pH indicator was also found. Our method proves that pH control inside the beads can only be realised by buffering. The resulting beads provided a specific activity of 0.267 μmol ∙ min -1 ∙ mg -1 , effectiveness factor 0.88, encapsulation efficiency of 88%, 5% leaching and good recycling properties. This work will contribute towards better understanding and application of encapsulated hydrolases for enzymatic syntheses. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Acetylcholine esterase activity in mild cognitive impairment and Alzheimer's disease

    Energy Technology Data Exchange (ETDEWEB)

    Herholz, Karl [University of Manchester, Wolfson Molecular Imaging Centre, Clinical Neuroscience, Manchester (United Kingdom); University of Cologne, Cologne (Germany)

    2008-03-15

    Impairment of cholinergic neurotransmission is a well-established fact in Alzheimer's disease (AD), but there is controversy about its relevance at the early stages of the disease and in mild cognitive impairment (MCI). In vivo positron emission tomography imaging of cortical acetylcholine esterase (AChE) activity as a marker of cholinergic innervation that is expressed by cholinergic axons and cholinoceptive neurons has demonstrated a reduction of this enzyme activity in manifest AD. The technique is also useful to measure the inhibition of cerebral AChE induced by cholinesterase inhibitors for treatment of dementia symptoms. A reduction of cortical AchE activity was found consistently in all studies of AD and in few cases of MCI who later concerted to AD. The in vivo findings in MCI and very mild AD are still preliminary, and studies seem to suggest that cholinergic innervation and AChE as the main degrading enzyme are both reduced, which might result in partial compensation of their effect. (orig.)

  11. Covalent immobilization of cholesterol esterase and cholesterol oxidase on polyaniline films for application to cholesterol biosensor

    Energy Technology Data Exchange (ETDEWEB)

    Singh, Suman [Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi-110012 (India); Solanki, Pratima R. [Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi-110012 (India); Pandey, M.K. [Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi-110012 (India); Malhotra, B.D. [Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi-110012 (India)]. E-mail: bansi@mail.nplindia.ernet.in

    2006-05-24

    Cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) have been covalently immobilized on electrochemically prepared polyaniline (PANI) films. These PANI/ChEt/ChOx enzyme films have been characterized using UV-visible, Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). Electrochemical behavior of these films has been studied using cyclic voltammetry (CV) and amperometric techniques, respectively. The PANI/ChEt/ChOx enzyme films show broad oxidation peak from 0.2 to 0.5 V. These PANI/ChEt/ChOx biosensing electrodes have a response time of about 40 s, linearity from 50 to 500 mg/dl of cholesterol oleate concentration. These PANI/ChEt/ChOx films are thermally stable up to 46 deg. C. This polyaniline based cholesterol biosensor has optimum pH in the range of 6.5-7.5, sensitivity as 7.5 x 10{sup -4} nA/mg dl and a lifetime of about 6 weeks.

  12. Pectin methyl esterases and pectins in normal and hyperhydric shoots of carnation cultured in vitro.

    Science.gov (United States)

    Saher, Shady; Piqueras, Abel; Hellin, Eladio; Olmos, Enrique

    2005-02-01

    Control and hyperhydric micropropagated plantlets from three carnation cultivars have been used to study their pectin composition and the activity of pectin methyl esterases (PMEs; EC 3.1.1.11). Pectins are a highly heterogeneous group of polymers that contribute to cell adhesion, cell wall architecture, and cell wall mechanical strength. Pectins control cell wall porosity and cell wall ionic status and are implicated in intercellular space development. The degree of esterification of pectins is controlled by the activity of cell wall PMEs; their different actions can affect the properties of the cell wall, which have been considered important with respect to controlling the development of hyperhydricity. The total pectins of hyperhydric leaves of the three varieties were significantly reduced in comparison with controls. The pectate fraction was significantly increased in hyperhydric leaves of all varieties while soluble pectins and protopectins were significantly lower. The PME activity of hyperhydric leaves was higher (4-10 times) compared to controls of the three varieties. Isoelectric focusing of PME isozymes revealed the presence of three isoforms; neutral PME activity was the major isozyme in control and hyperhydric leaves of the three varieties, whilst a decrease in the activity of the acidic isoforms was observed in hyperhydric leaves. The different PME activities could regulate some of the structural changes related to hyperhydricity in micropropagated carnation plants.

  13. A Lactobacillus plantarum esterase active on a broad range of phenolic esters.

    Science.gov (United States)

    Esteban-Torres, María; Landete, José María; Reverón, Inés; Santamaría, Laura; de las Rivas, Blanca; Muñoz, Rosario

    2015-05-01

    Lactobacillus plantarum is the lactic acid bacterial species most frequently found in the fermentation of food products of plant origin on which phenolic compounds are abundant. L. plantarum strains showed great flexibility in their ability to adapt to different environments and growth substrates. Of 28 L. plantarum strains analyzed, only cultures from 7 strains were able to hydrolyze hydroxycinnamic esters, such as methyl ferulate or methyl caffeate. As revealed by PCR, only these seven strains possessed the est_1092 gene. When the est_1092 gene was introduced into L. plantarum WCFS1 or L. lactis MG1363, their cultures acquired the ability to degrade hydroxycinnamic esters. These results support the suggestion that Est_1092 is the enzyme responsible for the degradation of hydroxycinnamic esters on the L. plantarum strains analyzed. The Est_1092 protein was recombinantly produced and biochemically characterized. Surprisingly, Est_1092 was able to hydrolyze not only hydroxycinnamic esters, since all the phenolic esters assayed were hydrolyzed. Quantitative PCR experiments revealed that the expression of est_1092 was induced in the presence of methyl ferulate, an hydroxycinnamic ester, but was inhibited on methyl gallate, an hydroxybenzoic ester. As Est_1092 is an enzyme active on a broad range of phenolic esters, simultaneously possessing feruloyl esterase and tannase activities, its presence on some L. plantarum strains provides them with additional advantages to survive and grow on plant environments. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  14. [Anaesthesic management of vaginal delivery in a parturient with C1 esterase deficiency].

    Science.gov (United States)

    Libert, N; Schérier, S; Dubost, C; Franck, L; Rouquette, I; Tortosa, J-C; Rousseau, J-M

    2009-04-01

    Hereditary and acquired angioedema (HAE/AAE) are the clinical translation of a qualitative or a quantitative deficit of C1 esterase inhibitor (C1 INH). The frequency and severity of clinical manifestations vary greatly, ranging from a moderate swelling of the extremities to obstruction of upper airway. Anaesthesiologists and intensivists must be prepared to manage acute manifestations of this disease in case of life-threatening laryngeal edema. Surgery, physical trauma and labour are classical triggers of the disease. The anaesthesiologists should be aware of the drugs used as prophylaxis and treatment of acute attacks when considering labour and caesarean section. Androgens are contraindicated during pregnancy. If prophylaxis is required, tranexamic acid may be used with caution. The safest obstetric approach appears to be to administer a predelivery infusion of C1 INH concentrate. It is important to avoid manipulation of the airway as much as possible by relying on regional techniques. We report the case of a patient suffering from an HAE discovered during pregnancy. The management included administration of C1 INH during labor and early epidural analgesia for pain relief. A short review of the pathophysiology and therapeutic options follows.

  15. β-D-(1→4), β-D-(1→3) 'mixed linkage' xylans from red seaweeds of the order Nemaliales and Palmariales.

    Science.gov (United States)

    Viana, Adriano G; Noseda, Miguel D; Gonçalves, Alan G; Duarte, Maria Eugênia R; Yokoya, Nair; Matulewicz, Maria C; Cerezo, Alberto S

    2011-06-01

    Xylans from five seaweeds belonging to the order Nemaliales (Galaxaura marginata, Galaxaura obtusata, Tricleocarpacylindrica, Tricleocarpa fragilis, and Scinaia halliae) and one of the order Palmariales (Palmaria palmata) collected on the Brazilian coasts were extracted with hot water and purified from acid xylomannans and/or xylogalactans through Cetavlon precipitation of the acid polysaccharides. The β-D-(1→4), β-D-(1→3) 'mixed linkage' structures were determined using methylation analysis and 1D and 2D NMR spectroscopy. The presence of large sequences of β-(1→4)-linked units suggests transient aggregates of ribbon- or helical-ordered structures that would explain the low optical rotations. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. Characterization and purification of a bacterial chlorogenic acid esterase detected during the extraction of chlorogenic acid from arbuscular mycorrhizal tomato roots.

    Science.gov (United States)

    Negrel, Jonathan; Javelle, Francine; Morandi, Dominique; Lucchi, Géraldine

    2016-12-01

    A Gram-negative bacterium able to grow using chlorogenic acid (5-caffeoylquinic acid) as sole carbon source has been isolated from the roots of tomato plants inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis. An intracellular esterase exhibiting very high affinity (K m  = 2 μM) for chlorogenic acid has been extracted and purified by FPLC from the chlorogenate-grown cultures of this bacterium. The molecular mass of the purified esterase determined by SDS-PAGE was 61 kDa and its isoelectric point determined by chromatofocusing was 7.75. The esterase hydrolysed chlorogenic acid analogues (caffeoylshikimate, and the 4- and 3-caffeoylquinic acid isomers), feruloyl esterases substrates (methyl caffeate and methyl ferulate), and even caffeoyl-CoA in vitro but all of them were less active than chlorogenic acid, demonstrating that the esterase is a genuine chlorogenic acid esterase. It was also induced when the bacterial strain was cultured in the presence of hydroxycinnamic acids (caffeic, p-coumaric or ferulic acid) as sole carbon source, but not in the presence of simple phenolics such as catechol or protocatechuic acid, nor in the presence of organic acids such as succinic or quinic acids. The purified esterase was remarkably stable in the presence of methanol, rapid formation of methyl caffeate occurring when its activity was measured in aqueous solutions containing 10-60% methanol. Our results therefore show that this bacterial chlorogenase can catalyse the transesterification reaction previously detected during the methanolic extraction of chlorogenic acid from arbuscular mycorrhizal tomato roots. Data are presented suggesting that colonisation by Rhizophagus irregularis could increase chlorogenic acid exudation from tomato roots, especially in nutrient-deprived plants, and thus favour the growth of chlorogenate-metabolizing bacteria on the root surface or in the mycorhizosphere. Copyright © 2016 Elsevier Masson SAS. All rights

  17. PRODUCTION AND CHARACTERIZATION OF AN ALKALOTHERMOSTABLE, ORGANIC SOLVENT TOLERANT AND SURFACTANT TOLERANT ESTERASE PRODUCED BY A THERMOPHILIC BACTERIUM GEOBACILLUS SP. AGP-04, ISOLATED FROM BAKRESHWAR HOT SPRING, INDIA

    Directory of Open Access Journals (Sweden)

    Amit Ghati

    2013-10-01

    Full Text Available A thermophilic bacteria, Geobacillus sp. AGP-04, isolated from Surya Kund hot spring, Bakreshwar, West Bengal, India was studied in terms of capability of tributyrin hydrolysis and characterization of its thermostable esterase activity using p-nitrophenyl butyrate (PNPB as substrate. The extracellular crude preparation was characterized in terms of pH and temperature optima and stability, organic solvent tolerance capacity and stability, substrate specificity, surfactant tolerance capacity, kinetic parameters and activation/inhibition behavior towards some metal ions and chemicals. Tributyrin agar assay exhibited that Geobacillus sp. AGP-04 secretes an extracellular esterase. The Vmax and Km values of the esterase were found to be 5099 U/Land 103.5µM, respectively in the presence of PNPB as substrate. The optimum temperature and pH, for Geobacillus sp. AGP-04 esterase was 60oC and 8.0, respectively. Although the enzyme activity was not significantly altered by incubating crude extract solution at 20-70oC for 1 hour, the enzyme activity was fully lost at 90oC for same incubation period. The pH stability profile showed that original crude esterase activity is stable at a broad range (pH 5.0-10.0. Moreover, the enzyme was highly organic solvent and surfactant tolerant. The effect of some chemical on crude esterase activity indicated that Geobacillus sp. AGP-04 produce an esterase which contains a serine residue in active site and for its activity -SH groups are essential. Besides, enzyme production was highly induced if fermentation medium contain polysaccharides and oil as carbon source.

  18. Comparative Analysis of Proteome-Wide Lysine Acetylation in Juvenile and Adult Schistosoma japonicum

    Directory of Open Access Journals (Sweden)

    Qing Li

    2017-11-01

    Full Text Available Schistosomiasis is a devastating parasitic disease caused by tremotodes of the genus Schistosoma. Eggs produced by sexually mature schistosomes are the causative agents of for pathogenesis and transmission. Elucidating the molecular mechanism of schistosome development and sexual maturation would facilitate the prevention and control of schistosomiasis. Acetylation of lysine is a dynamic and reversible post-translational modification playing keys role in many biological processes including development in both eukaryotes and prokaryotes. To investigate the impacts of lysine acetylation on Schistosoma japonicum (S. japonicum development and sexual maturation, we used immunoaffinity-based acetyllysine peptide enrichment combined with mass spectrometry (MS, to perform the first comparative analysis of proteome-wide lysine acetylation in both female and male, juvenile (18 days post infection, 18 dpi and adult (28 dpi schistosome samples. In total, we identified 874 unique acetylated sites in 494 acetylated proteins. The four samples shared 47 acetylated sites and 46 proteins. More acetylated sites and proteins shared by both females and males were identified in 28 dpi adults (189 and 143, respectively than in 18 dpi schistosomula (76 and 59, respectively. More stage-unique acetylated sites and proteins were also identified in 28 dpi adults (494 and 210, respectively than in 18 dpi schistosomula (73 and 44, respectively. Functional annotation showed that in different developmental stages and genders, a number of proteins involving in muscle movement, glycometabolism, lipid metabolism, energy metabolism, environmental stress resistance, antioxidation, etc., displayed distinct acetylation profiles, which was in accordance with the changes of their biological functions during schistosome development, suggesting that lysine acetylation modification exerted important regulatory roles in schistosome development. Taken together, our data provided the first

  19. N-Acetyl-4-aminophenol (paracetamol), N-acetyl-2-aminophenol and acetanilide in urine samples from the general population, individuals exposed to aniline and paracetamol users.

    Science.gov (United States)

    Dierkes, Georg; Weiss, Tobias; Modick, Hendrik; Käfferlein, Heiko Udo; Brüning, Thomas; Koch, Holger M

    2014-01-01

    Epidemiological studies suggest associations between the use of N-acetyl-4-aminophenol (paracetamol) during pregnancy and increased risks of reproductive disorders in the male offspring. Previously we have reported a ubiquitous urinary excretion of N-acetyl-4-aminophenol in the general population. Possible sources are (1) direct intake of paracetamol through medication, (2) paracetamol residues in the food chain and (3) environmental exposure to aniline or related substances that are metabolized into N-acetyl-4-aminophenol. In order to elucidate the origins of the excretion of N-acetyl-4-aminophenol in urine and to contribute to the understanding of paracetamol and aniline metabolism in humans we developed a rapid, turbulent-flow HPLC-MS/MS method with isotope dilution for the simultaneous quantification of N-acetyl-4-aminophenol and two other aniline related metabolites, N-acetyl-2-aminophenol and acetanilide. We applied this method to three sets of urine samples: (1) individuals with no known exposure to aniline and also no recent paracetamol medication; (2) individuals after occupational exposure to aniline but no paracetamol medication and (3) paracetamol users. We confirmed the omnipresent excretion of N-acetyl-4-aminophenol. Additionally we revealed an omnipresent excretion of N-acetyl-2-aminophenol. In contrast, acetanilide was only found after occupational exposure to aniline, not in the general population or after paracetamol use. The results lead to four preliminary conclusions: (1) other sources than aniline seem to be responsible for the major part of urinary N-acetyl-4-aminophenol in the general population; (2) acetanilide is a metabolite of aniline in man and a valuable biomarker for aniline in occupational settings; (3) aniline baseline levels in the general population measured after chemical hydrolysis do not seem to originate from acetanilide and hence not from a direct exposure to aniline itself and (4) N-acetyl-2-aminophenol does not seem to be

  20. The Effect of Hypochlorite Oxidation and Acetylation on Some of the ...

    African Journals Online (AJOL)

    The study evaluated the effect of hypochlorite oxidation and acetylation on some physicochemical properties of Icacina trichantha starch. The native and modified (oxidized and acetylated) starches were studied with respect to Infrared spectroscopy(IR), microscopy, gelatinization, swelling power, solubility index, amylose ...

  1. Kinetic and Thermodynamic Analysis of Acetyl-CoA Activation of Staphylococcus aureus Pyruvate Carboxylase.

    Science.gov (United States)

    Westerhold, Lauren E; Bridges, Lance C; Shaikh, Saame Raza; Zeczycki, Tonya N

    2017-07-11

    Allosteric regulation of pyruvate carboxylase (PC) activity is pivotal to maintaining metabolic homeostasis. In contrast, dysregulated PC activity contributes to the pathogenesis of numerous diseases, rendering PC a possible target for allosteric therapeutic development. Recent research efforts have focused on demarcating the role of acetyl-CoA, one of the most potent activators of PC, in coordinating catalytic events within the multifunctional enzyme. Herein, we report a kinetic and thermodynamic analysis of acetyl-CoA activation of the Staphylococcus aureus PC (SaPC)-catalyzed carboxylation of pyruvate to identify novel means by which acetyl-CoA synchronizes catalytic events within the PC tetramer. Kinetic and linked-function analysis, or thermodynamic linkage analysis, indicates that the substrates of the biotin carboxylase and carboxyl transferase domain are energetically coupled in the presence of acetyl-CoA. In contrast, both kinetic and energetic coupling between the two domains is lost in the absence of acetyl-CoA, suggesting a functional role for acetyl-CoA in facilitating the long-range transmission of substrate-induced conformational changes within the PC tetramer. Interestingly, thermodynamic activation parameters for the SaPC-catalyzed carboxylation of pyruvate are largely independent of acetyl-CoA. Our results also reveal the possibility that global conformational changes give rise to observed species-specific thermodynamic activation parameters. Taken together, our kinetic and thermodynamic results provide a possible allosteric mechanism by which acetyl-CoA coordinates catalysis within the PC tetramer.

  2. Transport and metabolism of indole-3-acetyl-myo-inositol-galactoside in seedlings of Zea mays

    International Nuclear Information System (INIS)

    Komoszynski, M.; Bandurski, R.S.

    1986-01-01

    Indole-3-acetyl-myo-inositol galactoside labeled with 3 H in the indole and 14 C in the galactose moieties was applied to kernels of 5 day old germinating seedlings of Zea mays. Indole-3-acetyl-myo-inositol galactoside was not transported into either the shoot or root tissue as the intact molecule but was instead hydrolyzed to yield [ 3 H]indole-3-acetyl-myo-inositol and [ 3 H]indole-3-acetic acid which were then transported to the shoot with little radioactivity going to the root. With certain assumptions concerning the equilibration of applied [ 3 H]indole-3-acetyl-myo-inositol-[U- 14 C]galactose with the endogenous pool, it may be concluded that indole-3-acetyl-myo-inositol galactoside in the endosperm supplies about 2 picomoles per plant per hour of indole-3-acetic acid to the shoot and thus is comparable to indole-3-acetyl-myo-inositol as a source of indoleacetic acid for the shoot. Quantitative estimates of the amount of galactose in the kernels suggest that [ 3 H]indole-3-acetyl-myo-inositol-[ 14 C] galactose is hydrolyzed after the compound leaves the endosperm but before it reaches the shoot. In addition, [ 3 H]indole-3-acetyl-myo-inositol-[ 14 C]galactose supplies appreciable amounts of 14 C to the shoot and both 14 C and 3 H to an uncharacterized insoluble fraction of the endosperm

  3. CPLA 1.0: an integrated database of protein lysine acetylation.

    Science.gov (United States)

    Liu, Zexian; Cao, Jun; Gao, Xinjiao; Zhou, Yanhong; Wen, Longping; Yang, Xiangjiao; Yao, Xuebiao; Ren, Jian; Xue, Yu

    2011-01-01

    As a reversible post-translational modification (PTM) discovered decades ago, protein lysine acetylation was known for its regulation of transcription through the modification of histones. Recent studies discovered that lysine acetylation targets broad substrates and especially plays an essential role in cellular metabolic regulation. Although acetylation is comparable with other major PTMs such as phosphorylation, an integrated resource still remains to be developed. In this work, we presented the compendium of protein lysine acetylation (CPLA) database for lysine acetylated substrates with their sites. From the scientific literature, we manually collected 7151 experimentally identified acetylation sites in 3311 targets. We statistically studied the regulatory roles of lysine acetylation by analyzing the Gene Ontology (GO) and InterPro annotations. Combined with protein-protein interaction information, we systematically discovered a potential human lysine acetylation network (HLAN) among histone acetyltransferases (HATs), substrates and histone deacetylases (HDACs). In particular, there are 1862 triplet relationships of HAT-substrate-HDAC retrieved from the HLAN, at least 13 of which were previously experimentally verified. The online services of CPLA database was implemented in PHP + MySQL + JavaScript, while the local packages were developed in JAVA 1.5 (J2SE 5.0). The CPLA database is freely available for all users at: http://cpla.biocuckoo.org.

  4. A bioinformatics-based overview of protein Lys-Ne-acetylation

    Science.gov (United States)

    Among posttranslational modifications, there are some conceptual similarities between Lys-N'-acetylation and Ser/Thr/Tyr O-phosphorylation. Herein we present a bioinformatics-based overview of reversible protein Lys-acetylation, including some comparisons with reversible protein phosphorylation. T...

  5. A proteome-scale study on in vivo protein Nα-acetylation using an optimized method

    DEFF Research Database (Denmark)

    Zhang, Xumin; Ye, Juanying; Engholm-Keller, Kasper

    2011-01-01

    Protein N-terminal acetylation (N(α) -acetylation) is among the most common modifications in eukaryotes. We previously described a simple method to enrich N(α) -modified peptides using CNBr-activated Sepharose resin. A limitation of this method is that an optimal ratio of sample to resin had to b...

  6. The Effect of Acetyl-L-Carnitine Administration on Persons with Down Syndrome

    Science.gov (United States)

    Pueschel, Siegfried M.

    2006-01-01

    Since previous investigations reported improvements in cognition of patients with dementia after acetyl-L-carnitine therapy and since there is an increased risk for persons with Down syndrome to develop Alzheimer disease, this study was designed to investigate the effect of acetyl-L-carnitine administration on neurological, intellectual, and…

  7. Protective Effects of Acetylation on the Pathological Reactions of the Lens Crystallins with Homocysteine Thiolactone.

    Directory of Open Access Journals (Sweden)

    Zeinab Moafian

    Full Text Available Various post-translational lens crystallins modifications result in structural and functional insults, contributing to the development of lens opacity and cataract disorders. Lens crystallins are potential targets of homocysteinylation, particularly under hyperhomocysteinemia which has been indicated in various eye diseases. Since both homocysteinylation and acetylation primarily occur on protein free amino groups, we applied different spectroscopic methods and gel mobility shift analysis to examine the possible preventive role of acetylation against homocysteinylation. Lens crystallins were extensively acetylated in the presence of acetic anhydride and then subjected to homocysteinylation in the presence of homocysteine thiolactone (HCTL. Extensive acetylation of the lens crystallins results in partial structural alteration and enhancement of their stability, as well as improvement of α-crystallin chaperone-like activity. In addition, acetylation partially prevents HCTL-induced structural alteration and aggregation of lens crystallins. Also, acetylation protects against HCTL-induced loss of α-crystallin chaperone activity. Additionally, subsequent acetylation and homocysteinylation cause significant proteolytic degradation of crystallins. Therefore, further experimentation is required in order to judge effectively the preventative role of acetylation on the structural and functional insults induced by homocysteinylation of lens crystallins.

  8. Prediction of Nepsilon-acetylation on internal lysines implemented in Bayesian Discriminant Method.

    Science.gov (United States)

    Li, Ao; Xue, Yu; Jin, Changjiang; Wang, Minghui; Yao, Xuebiao

    2006-12-01

    Protein acetylation is an important and reversible post-translational modification (PTM), and it governs a variety of cellular dynamics and plasticity. Experimental identification of acetylation sites is labor-intensive and often limited by the availability of reagents such as acetyl-specific antibodies and optimization of enzymatic reactions. Computational analyses may facilitate the identification of potential acetylation sites and provide insights into further experimentation. In this manuscript, we present a novel protein acetylation prediction program named PAIL, prediction of acetylation on internal lysines, implemented in a BDM (Bayesian Discriminant Method) algorithm. The accuracies of PAIL are 85.13%, 87.97%, and 89.21% at low, medium, and high thresholds, respectively. Both Jack-Knife validation and n-fold cross-validation have been performed to show that PAIL is accurate and robust. Taken together, we propose that PAIL is a novel predictor for identification of protein acetylation sites and may serve as an important tool to study the function of protein acetylation. PAIL has been implemented in PHP and is freely available on a web server at: http://bioinformatics.lcd-ustc.org/pail.

  9. Prediction of Nε-acetylation on internal lysines implemented in Bayesian Discriminant Method

    Science.gov (United States)

    Li, Ao; Xue, Yu; Jin, Changjiang; Wang, Minghui; Yao, Xuebiao

    2007-01-01

    Protein acetylation is an important and reversible post-translational modification (PTM), and it governs a variety of cellular dynamics and plasticity. Experimental identification of acetylation sites is labor-intensive and often limited by the availability reagents such as acetyl-specific antibodies and optimization of enzymatic reactions. Computational analyses may facilitate the identification of potential acetylation sites and provide insights into further experimentation. In this manuscript, we present a novel protein acetylation prediction program named PAIL, prediction of acetylation on internal lysines, implemented in a BDM (Bayesian Discriminant Method) algorithm. The accuracies of PAIL are 85.13%, 87.97% and 89.21% at low, medium and high thresholds, respectively. Both Jack-Knife validation and n-fold cross validation have been performed to show that PAIL is accurate and robust. Taken together, we propose that PAIL is a novel predictor for identification of protein acetylation sites and may serve as an important tool to study the function of protein acetylation. PAIL has been implemented in PHP and is freely available on a web server at: http://bioinformatics.lcd-ustc.org/pail. PMID:17045240

  10. Total levels of hippocampal histone acetylation predict normal variability in mouse behavior.

    Directory of Open Access Journals (Sweden)

    Addie May I Nesbitt

    Full Text Available Genetic, pharmacological, and environmental interventions that alter total levels of histone acetylation in specific brain regions can modulate behaviors and treatment responses. Efforts have been made to identify specific genes that are affected by alterations in total histone acetylation and to propose that such gene specific modulation could explain the effects of total histone acetylation levels on behavior - the implication being that under naturalistic conditions variability in histone acetylation occurs primarily around the promoters of specific genes.Here we challenge this hypothesis by demonstrating with a novel flow cytometry based technique that normal variability in open field exploration, a hippocampus-related behavior, was associated with total levels of histone acetylation in the hippocampus but not in other brain regions.Results suggest that modulation of total levels of histone acetylation may play a role in regulating biological processes. We speculate in the discussion that endogenous regulation of total levels of histone acetylation may be a mechanism through which organisms regulate cellular plasticity. Flow cytometry provides a useful approach to measure total levels of histone acetylation at the single cell level. Relating such information to behavioral measures and treatment responses could inform drug delivery strategies to target histone deacetylase inhibitors and other chromatin modulators to places where they may be of benefit while avoiding areas where correction is not needed and could be harmful.

  11. Histone Acetylation is Recruited in Consolidation as a Molecular Feature of Stronger Memories

    Science.gov (United States)

    Federman, Noel; Fustinana, Maria Sol; Romano, Arturo

    2009-01-01

    Gene expression is a key process for memory consolidation. Recently, the participation of epigenetic mechanisms like histone acetylation was evidenced in long-term memories. However, until now the training strength required and the persistence of the chromatin acetylation recruited are not well characterized. Here we studied whether histone…

  12. Multi-step rearrangement mechanism for acetyl cedrene to the hydrocarbon follower

    DEFF Research Database (Denmark)

    Paknikar, Shashikumar Keshav; Kamounah, Fadhil S.; Hansen, Poul Erik

    2017-01-01

    Conversion of acetyl cedrene (2) to its follower (3) using acetic anhydride and polyphosphoric acid involves a multi-step cationic molecular rearrangement, which is consistent with deuteriation and 1-13C labeling studies of acetyl cedrene. The key step involves cyclopropylcarbinyl cation-cyclopro...

  13. Cyclic AMP Inhibits the Activity and Promotes the Acetylation of Acetyl-CoA Synthetase through Competitive Binding to the ATP/AMP Pocket.

    Science.gov (United States)

    Han, Xiaobiao; Shen, Liqiang; Wang, Qijun; Cen, Xufeng; Wang, Jin; Wu, Meng; Li, Peng; Zhao, Wei; Zhang, Yu; Zhao, Guoping

    2017-01-27

    The high-affinity biosynthetic pathway for converting acetate to acetyl-coenzyme A (acetyl-CoA) is catalyzed by the central metabolic enzyme acetyl-coenzyme A synthetase (Acs), which is finely regulated both at the transcriptional level via cyclic AMP (cAMP)-driven trans-activation and at the post-translational level via acetylation inhibition. In this study, we discovered that cAMP directly binds to Salmonella enterica Acs (SeAcs) and inhibits its activity in a substrate-competitive manner. In addition, cAMP binding increases SeAcs acetylation by simultaneously promoting Pat-dependent acetylation and inhibiting CobB-dependent deacetylation, resulting in enhanced SeAcs inhibition. A crystal structure study and site-directed mutagenesis analyses confirmed that cAMP binds to the ATP/AMP pocket of SeAcs, and restrains SeAcs in an open conformation. The cAMP contact residues are well conserved from prokaryotes to eukaryotes, suggesting a general regulatory mechanism of cAMP on Acs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Histone Deacetylase 1 Plays an Acetylation-Independent Role in Influenza A Virus Replication

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    Lin Chen

    2017-12-01

    Full Text Available Influenza A viruses (IAVs take advantage of the host acetylation system for their own benefit. Whether the nucleoprotein (NP of IAVs undergoes acetylation and the interaction between the NP and the class I histone deacetylases (HDACs were largely unknown. Here, we showed that the NP protein of IAV interacted with HDAC1, which downregulated the acetylation level of NP. Using mass spectrometry, we identified lysine 103 as an acetylation site of the NP. Compared with wild-type protein, two K103 NP mutants, K103A and K103R, enhanced replication efficiency of the recombinant viruses in vitro. We further demonstrated that HDAC1 facilitated viral replication via two paths: promoting the nuclear retention of NP and inhibiting TBK1-IRF3 pathway. Our results lead to a new mechanism for regulating NP acetylation, indicating that HDAC1 may be a possible target for antiviral drugs.

  15. Effect of Acetyl Group on Mechanical Properties of Chitin/Chitosan Nanocrystal: A Molecular Dynamics Study

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    Junhe Cui

    2016-01-01

    Full Text Available Chitin fiber is the load-bearing component in natural chitin-based materials. In these materials, chitin is always partially deacetylated to different levels, leading to diverse material properties. In order to understand how the acetyl group enhances the fracture resistance capability of chitin fiber, we constructed atomistic models of chitin with varied acetylation degree and analyzed the hydrogen bonding pattern, fracture, and stress-strain behavior of these models. We notice that the acetyl group can contribute to the formation of hydrogen bonds that can stabilize the crystalline structure. In addition, it is found that the specimen with a higher acetylation degree presents a greater resistance against fracture. This study describes the role of the functional group, acetyl groups, in crystalline chitin. Such information could provide preliminary understanding of nanomaterials when similar functional groups are encountered.

  16. Isolation and characterization of a thermolysin peptide containing acetyllysine from enzymatically acetylated f2al histone

    International Nuclear Information System (INIS)

    Horiuchi, Kentaro; Fujimoto, Daisaburo

    1973-01-01

    Previous studies (vol. 72, 433, '72) in this laboratory showed that histone acetylase in the cytosol of calf thymus introduced acetyl groups primarily into the epsilon-amino groups of lysine residues in a histone fraction, f2al. In an attempt to examine the site of acetylation in f2al by the enzyme, 14 C-acetylated f2al was isolated and digested by thermolysin. A radioactive peptide, which accounted for 50 - 60% of the total radioactivity, was obtained from the thermolysin digest and identified as the fragment containing amino acid residues 10-21. It appears, therefore, that the major sites of acetylation by the enzyme are the lysine 12 or 16 or both, which are known to be acetylated in vivo. It was also shown that the peptide was not deacetylated by histone deacetylase, in contrast with the whole f2al molecule. (author)

  17. Partially acetylated sugarcane bagasse for wicking oil from contaminated wetlands

    Energy Technology Data Exchange (ETDEWEB)

    Chung, S. [Samsung Engineering Co. Ltd., R and D Center, Suwon, Gyeonggi (Korea, Republic of); Suidan, M.T. [University of Cincinnati, School of Energy, Environmental, Biological and Medical Engineering, Cincinnati, OH (United States); Venosa, A.D. [NRMRL, U.S. EPA, Cincinnati, OH (United States)

    2011-12-15

    Sugarcane bagasse was partially acetylated to enhance its oil-wicking ability in saturated environments while holding moisture for hydrocarbon biodegradation. The water sorption capacity of raw bagasse was reduced fourfold after treatment, which indicated considerably increased hydrophobicity but not a limited capability to hold moisture for hydrocarbon biodegradation. Characterization results by Fourier transform infrared (FT-IR), scanning electron microscopy (SEM), X-ray diffraction (XRD), and surface area analyzer suggested that treated bagasse exhibited enhanced hydrophobicity and surface area. Oil wicking test results indicate that treated bagasse is more effective in wicking oil from highly saturated environments than raw bagasse and suggest that application of this material in remediation of oil spills in highly saturated wetlands is promising. (Copyright copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  18. Acetylated Hyaluronic Acid: Enhanced Bioavailability and Biological Studies

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    Carmela Saturnino

    2014-01-01

    Full Text Available Hyaluronic acid (HA, a macropolysaccharidic component of the extracellular matrix, is common to most species and it is found in many sites of the human body, including skin and soft tissue. Not only does HA play a variety of roles in physiologic and in pathologic events, but it also has been extensively employed in cosmetic and skin-care products as drug delivery agent or for several biomedical applications. The most important limitations of HA are due to its short half-life and quick degradation in vivo and its consequently poor bioavailability. In the aim to overcome these difficulties, HA is generally subjected to several chemical changes. In this paper we obtained an acetylated form of HA with increased bioavailability with respect to the HA free form. Furthermore, an improved radical scavenging and anti-inflammatory activity has been evidenced, respectively, on ABTS radical cation and murine monocyte/macrophage cell lines (J774.A1.

  19. Acetylated flavonoid glycosides potentiating NGF action from Scoparia dulcis.

    Science.gov (United States)

    Li, Yushan; Chen, Xigui; Satake, Masayuki; Oshima, Yasukatsu; Ohizumi, Yasushi

    2004-04-01

    Three new acetylated flavonoid glycosides, 5,6,4'-trihydroxyflavone 7-O-alpha-L-2,3-di-O-acetylrhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (1), apigenin 7-O-alpha-L-3-O-acetylrhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (2), and apigenin 7-O-alpha-L-2,3-di-O-acetylrhamnopyranosyl-(1-->6)-beta-D-glucopyranoside (3), were isolated from Scoparia dulcis together with the known compound eugenyl beta-D-glucopyranoside (4). Their structures were elucidated by spectroscopic analyses. Compounds 2 and 3 showed an enhancing activity of nerve growth factor-mediated neurite outgrowth in PC12D cells.

  20. Mechanism of the lysosomal membrane enzyme acetyl coenzyme A: alpha-glucosaminide N-acetyltransferase

    International Nuclear Information System (INIS)

    Bame, K.J.

    1986-01-01

    Acetyl-CoA:α-glucosaminide N-acetyltransferase is a lysosomal membrane enzyme, deficient in the genetic disease Sanfilippo C syndrome. The enzyme catalyzes the transfer of an acetyl group from cytoplasmic acetyl-CoA to terminal α-glucosamine residues of heparan sulfate within the organelle. The reaction mechanism was examined using high purified lysosomal membranes from rat liver and human fibroblasts. The N-acetyltransferase reaction is optimal above pH 5.5 and a 2-3 fold stimulation of activity is observed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicate that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. The binding of acetyl-CoA to the enzyme is measured by exchange label from [ 3 H]CoA to acetyl-CoA, and is optimal at pH's above 7.0. The acetyl-enzyme intermediate is formed by incubating membranes with [ 3 H]acetyl-CoA. The acetyl group can be transferred to glucosamine, forming [ 3 H]N-acetylglucosamine; the transfer is optimal between pH 4 and 5. Lysosomal membranes from Sanfilippo C fibroblasts confirm that these half reactions carried out by the N-acetyltransferase. The enzyme is inactivated by N-bromosuccinimide and diethylpyrocarbonate, indicating that a histidine is involved in the reaction. These results suggest that the histidine residue is at the active site of the enzyme. The properties of the N-acetyltransferase in the membrane, the characterization of the enzyme kinetics, the chemistry of a histidine mediated acetylation and the pH difference across the lysosomal membrane all support a transmembrane acetylation mechanism

  1. Avoidance behaviour response and esterase inhibition in the earthworm, Lumbricus terrestris, after exposure to chlorpyrifos.

    Science.gov (United States)

    Martínez Morcillo, S; Yela, J L; Capowiez, Y; Mazzia, C; Rault, M; Sanchez-Hernandez, Juan C

    2013-05-01

    The avoidance response of earthworms to polluted soils has been standardised using a simple and low-cost test, which facilitates soil toxicity screening. In this study, the avoidance response of Lumbricus terrestris was quantified in chlorpyrifos-spiked soils, depending on the pesticide concentration and exposure duration. The inhibition of acetylcholinesterase (AChE) and carboxylesterase (CbE) activities was also determined as indirect measures of pesticide bioavailability. The effects of different chlorpyrifos concentrations were examined in a standardised test (two-chamber system) with 0.6, 3 and 15 mg/kg chlorpyrifos. A modification of the test involved a pre-exposure step (24, 48 or 72 h) in soils spiked with 15 mg/kg. In both protocols, earthworms were unable to avoid the contaminated soils. However, the esterase activities showed that all earthworms were exposed to chlorpyrifos. Acetylcholinesterase activity did not change in earthworms in the standardised behavioural test (0.58 ± 0.20 U/mg protein, mean ± SD; n = 72), whereas the CbE activity was significantly inhibited (62-87 % inhibition) in earthworms exposed to 3 and 15 mg/kg. In the modified test, earthworms had greatly inhibited AChE activity (0.088 ± 0.034 U/mg protein, n = 72), which was supported by reactivation of the inhibited enzyme activity in the presence of pralidoxime (2-PAM). Similarly, the CbE activity was significantly inhibited in earthworms with all treatments. This study suggests that the avoidance behaviour test for organophosphorus-contaminated soils could be supported by specific biomarkers to facilitate a better understanding of pesticide exposure and toxicity during this test.

  2. Functional tuning of the catalytic residue pKa in a de novo designed esterase.

    Science.gov (United States)

    Hiebler, Katharina; Lengyel, Zsófia; Castañeda, Carlos A; Makhlynets, Olga V

    2017-09-01

    AlleyCatE is a de novo designed esterase that can be allosterically regulated by calcium ions. This artificial enzyme has been shown to hydrolyze p-nitrophenyl acetate (pNPA) and 4-nitrophenyl-(2-phenyl)-propanoate (pNPP) with high catalytic efficiency. AlleyCatE was created by introducing a single-histidine residue (His 144 ) into a hydrophobic pocket of calmodulin. In this work, we explore the determinants of catalytic properties of AlleyCatE. We obtained the pK a value of the catalytic histidine using experimental measurements by NMR and pH rate profile and compared these values to those predicted from electrostatics pK a calculations (from both empirical and continuum electrostatics calculations). Surprisingly, the pK a value of the catalytic histidine inside the hydrophobic pocket of calmodulin is elevated as compared to the model compound pK a value of this residue in water. We determined that a short-range favorable interaction with Glu 127 contributes to the elevated pK a of His 144 . We have rationally modulated local electrostatic potential in AlleyCatE to decrease the pK a of its active nucleophile, His 144 , by 0.7 units. As a direct result of the decrease in the His 144 pK a value, catalytic efficiency of the enzyme increased by 45% at pH 6. This work shows that a series of simple NMR experiments that can be performed using low field spectrometers, combined with straightforward computational analysis, provide rapid and accurate guidance to rationally improve catalytic efficiency of histidine-promoted catalysis. Proteins 2017; 85:1656-1665. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  3. Depletion of juvenile hormone esterase extends larval growth in Bombyx mori.

    Science.gov (United States)

    Zhang, Zhongjie; Liu, Xiaojing; Shiotsuki, Takahiro; Wang, Zhisheng; Xu, Xia; Huang, Yongping; Li, Muwang; Li, Kai; Tan, Anjiang

    2017-02-01

    Two major hormones, juvenile hormone (JH) and 20-hydroxyecdysone (20E), regulate insect growth and development according to their precisely coordinated titres, which are controlled by both biosynthesis and degradation pathways. Juvenile hormone esterase (JHE) is the primary JH-specific degradation enzyme that plays a key role in regulating JH titers, along with JH epoxide hydrolase (JHEH) and JH diol kinase (JHDK). In the current study, a loss-of-function analysis of JHE in the silkworm, Bombyx mori, was performed by targeted gene disruption using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system. Depletion of B. mori JHE (BmJHE) resulted in the extension of larval stages, especially the penultimate and ultimate larval stages, without deleterious effects to silkworm physiology. The expression of JHEH and JHDK was upregulated in mutant animals, indicating the existence of complementary routes in the JH metabolism pathway in which inactivation of one enzyme will activate other enzymes. RNA-Seq analysis of mutant animals revealed that genes involved in protein processing in the endoplasmic reticulum and in amino acid metabolism were affected by BmJHE depletion. Depletion of JHE and subsequent delayed JH metabolism activated genes in the TOR pathway, which are ultimately responsible for extending larval growth. The transgenic Cas9 system used in the current study provides a promising approach for analysing the actions of JH, especially in nondrosophilid insects. Furthermore, prolonging larval stages produced larger larvae and cocoons, which is greatly beneficial to silk production. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. A stereospecific carboxyl esterase from Bacillus coagulans hosting nonlipase activity within a lipase-like fold.

    Science.gov (United States)

    De Vitis, Valerio; Nakhnoukh, Cristina; Pinto, Andrea; Contente, Martina L; Barbiroli, Alberto; Milani, Mario; Bolognesi, Martino; Molinari, Francesco; Gourlay, Louise J; Romano, Diego

    2018-03-01

    Microbial carboxylesterases are important biocatalysts that selectively hydrolyze an extensive range of esters. Here, we report the biochemical and structural characterization of an atypical carboxylesterase from Bacillus coagulans (BCE), endowed with high enantioselectivity toward different 1,2-O-isopropylideneglycerol (IPG or solketal) esters. BCE efficiently catalyzes the production of enantiopure (S)-IPG, a chiral building block for the synthesis of β-blockers, glycerophospholipids, and prostaglandins; efficient hydrolysis was observed up to 65 °C. To gain insight into the mechanistic bases of such enantioselectivity, we solved the crystal structures of BCE in apo- and glycerol-bound forms at resolutions of 1.9 and 1.8 Å, respectively. In silico docking studies on the BCE structure confirmed that IPG esters with small acyl chains (≤ C6) were easily accommodated in the active site pocket, indicating that small conformational changes are necessary to accept longer substrates. Furthermore, docking studies suggested that enantioselectivity may be due to an improved stabilization of the tetrahedral reaction intermediate for the S-enantiomer. Contrary to the above functional data implying nonlipolytic functions, BCE displays a lipase-like 3D structure that hosts a "lid" domain capping the main entrance to the active site. In lipases the lid mediates catalysis through interfacial activation, a process that we did not observe for BCE. Overall, we present the functional-structural properties of an atypical carboxyl esterase that has nonlipase-like functions, yet possesses a lipase-like 3D fold. Our data provide original enzymatic information in view of BCE applications as an inexpensive, efficient biocatalyst for the production of enantiopure (S)-IPG. Coordinates and structure factors have been deposited in the Protein Data Bank (www.rcsb.org) under accession numbers 5O7G (apo-BCE) and 5OLU (glycerol-bound BCE). © 2017 Federation of European Biochemical

  5. Vaginal Fornix Discharge Cellularity and Its Leukocyte Esterase Activity for Diagnosis of Endometritis in Dairy Cows

    Directory of Open Access Journals (Sweden)

    Abolfazl HAJIBEMANI

    2016-01-01

    Full Text Available The objective of the present study was to evaluate the application of some strip test markers (i.e., leukocyte esterase (LE activity, protein, nitrate and pH for diagnosis of endometritis in dairy cows using vaginal fornix discharge. Also, the total white blood cell count (t-WBC/l of this secretion and degenerative changes of neutrophils in cervical cytology were used as alternative methods to predict progression of the endometritis severity. Holstein cows (n=215 between 30-40 days in milk (DIM were included and examined. Giemsa-stained smear was prepared from cervical mucus. Cervical cytology test was considered as reference screening method for the detection of subclinical endometritis. The LE activity and t-WBC in the vaginal fornix discharge of subclinical endometritis cows were significantly higher than those from healthy cows. Sensitivity and specificity were 78% and 73% for LE10 activity (10 minutes after contacting with discharges and 60% and 69% for t-WBC (cut off point=210 cells/l for diagnosis of subclinical endometritis, respectively. There was a good agreement between LE10 activity, t-WBC and cervical cytology test with a Kappa coefficient of 0.4 and 0.42, respectively (P<0.0001. Total WBC count in discharge and degenerative neutrophils (DN percentages increase simultaneously with the degree and severity of endometritis. There was a highly significant (P<0.01 correlation between t-WBC and some reagent strip test markers (LE activity, protein and nitrate in clear discharge of studied cows. In conclusion, the present results suggest the LE activity and t-WBC in vaginal fornix discharge could be used as non-invasive reliable and valid methods for screening of subclinical endometritis in postpartum dairy herds.

  6. Immobilization of cholesterol esterase in mesoporous silica materials and its hydrolytic activity toward diethyl phthalate

    Energy Technology Data Exchange (ETDEWEB)

    Orita, Toru, E-mail: nqj45366@nifty.com [Division of Chemistry for Materials, Graduate School of Engineering, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie 514-8570 (Japan); Taiyo Kagaku Co. Ltd., 800 Yamada-cho, Yokkaichi, Mie 512-1111 (Japan); Tomita, Masahiro [Division of Chemistry for Materials, Graduate School of Engineering, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie 514-8570 (Japan); Saito, Takao; Nishida, Nasakazu; Kato, Katsuya [National Institute of Advanced Industrial Science and Technology, 2266-78 Anagahora, Moriyamaku, Nagoya, Aichi 463-8560 (Japan)

    2012-05-01

    Cholesterol esterase (CE, cholesteryl ester hydrolase, EC 3.1.1.13) from porcine pancreas (molecular weight 400-500 kDa) exhibits hydrolytic activity toward various toxic organic phthalate esters. CE was confined in the nanospace (diameter 3-30 nm) of five types of mesoporous silica (MPS) that differ in structural properties such as pore diameter, pore volume, and particle morphology. These structural properties were characterized by transmission electron microscopy, small-angle X-ray diffraction, N{sub 2} adsorption-desorption experiments, solid-state {sup 13}C nuclear magnetic resonance (NMR), and solid-state {sup 29}Si NMR. Catalytic activities of immobilized and free CE were evaluated by the hydrolysis of diethyl phthalate in phosphate buffer solutions containing an organic cosolvent. Optimal activity recovery was achieved when CE was immobilized in n-decane-functionalized MPS, which had a large pore size (22.5 nm). The immobilization also protected against effects of temperature within the range 30 Degree-Sign C-60 Degree-Sign C; CE immobilized in n-decyl-functionalized MPS exhibited better thermal stability than in non-functionalized MPS or free CE. Moreover, it retained approximately 60% of its catalytic activity even after six catalytic cycles. - Highlights: Black-Right-Pointing-Pointer The highest activity of immobilized CE was shown in MPS with a pore size of 22.5 nm. Black-Right-Pointing-Pointer Catalytic efficiency improved when MPS was functionalized by n-decyl substitution. Black-Right-Pointing-Pointer Immobilized CE exhibited good thermal stability and reusability. Black-Right-Pointing-Pointer Organic co-solvent and the substrate structures affected enzyme activities.

  7. C1-esterase inhibitor protects against early vein graft remodeling under arterial blood pressure.

    Science.gov (United States)

    Krijnen, Paul A J; Kupreishvili, Koba; de Vries, Margreet R; Schepers, Abbey; Stooker, Wim; Vonk, Alexander B A; Eijsman, Leon; Van Hinsbergh, Victor W M; Zeerleder, Sacha; Wouters, Diana; van Ham, Marieke; Quax, Paul H A; Niessen, Hans W M

    2012-01-01

    Arterial pressure induced vein graft injury can result in endothelial loss, accelerated atherosclerosis and vein graft failure. Inflammation, including complement activation, is assumed to play a pivotal role herein. Here, we analyzed the effects of C1-esterase inhibitor (C1inh) on early vein graft remodeling. Human saphenous vein graft segments (n=8) were perfused in vitro with autologous blood either supplemented or not with purified human C1inh at arterial pressure for 6h. The vein segments and perfusion blood were analyzed for cell damage and complement activation. In addition, the effect of purified C1inh on vein graft remodeling was analyzed in vivo in atherosclerotic C57Bl6/ApoE3 Leiden mice, wherein donor caval veins were interpositioned in the common carotid artery. Application of C1inh in the in vitro perfusion model resulted in significantly higher blood levels and significantly more depositions of C1inh in the vein wall. This coincided with a significant reduction in endothelial loss and deposition of C3d and C4d in the vein wall, especially in the circular layer, compared to vein segments perfused without supplemented C1inh. Administration of purified C1inh significantly inhibited vein graft intimal thickening in vivo in atherosclerotic C57Bl6/ApoE3 Leiden mice, wherein donor caval veins were interpositioned in the common carotid artery. C1inh significantly protects against early vein graft remodeling, including loss of endothelium and intimal thickening. These data suggest that it may be worth considering its use in patients undergoing coronary artery bypass grafting. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  8. Common and distant structural characteristics of feruloyl esterase families from Aspergillus oryzae.

    Directory of Open Access Journals (Sweden)

    D B R K Gupta Udatha

    Full Text Available BACKGROUND: Feruloyl esterases (FAEs are important biomass degrading accessory enzymes due to their capability of cleaving the ester links between hemicellulose and pectin to aromatic compounds of lignin, thus enhancing the accessibility of plant tissues to cellulolytic and hemicellulolytic enzymes. FAEs have gained increased attention in the area of biocatalytic transformations for the synthesis of value added compounds with medicinal and nutritional applications. Following the increasing attention on these enzymes, a novel descriptor based classification system has been proposed for FAEs resulting into 12 distinct families and pharmacophore models for three FAE sub-families have been developed. METHODOLOGY/PRINCIPAL FINDINGS: The feruloylome of Aspergillus oryzae contains 13 predicted FAEs belonging to six sub-families based on our recently developed descriptor-based classification system. The three-dimensional structures of the 13 FAEs were modeled for structural analysis of the feruloylome. The three genes coding for three enzymes, viz., A.O.2, A.O.8 and A.O.10 from the feruloylome of A. oryzae, representing sub-families with unknown functional features, were heterologously expressed in Pichia pastoris, characterized for substrate specificity and structural characterization through CD spectroscopy. Common feature-based pharamacophore models were developed according to substrate specificity characteristics of the three enzymes. The active site residues were identified for the three expressed FAEs by determining the titration curves of amino acid residues as a function of the pH by applying molecular simulations. CONCLUSIONS/SIGNIFICANCE: Our findings on the structure-function relationships and substrate specificity of the FAEs of A. oryzae will be instrumental for further understanding of the FAE families in the novel classification system. The developed pharmacophore models could be applied for virtual screening of compound databases for

  9. Common and distant structural characteristics of feruloyl esterase families from Aspergillus oryzae.

    Science.gov (United States)

    Udatha, D B R K Gupta; Mapelli, Valeria; Panagiotou, Gianni; Olsson, Lisbeth

    2012-01-01

    Feruloyl esterases (FAEs) are important biomass degrading accessory enzymes due to their capability of cleaving the ester links between hemicellulose and pectin to aromatic compounds of lignin, thus enhancing the accessibility of plant tissues to cellulolytic and hemicellulolytic enzymes. FAEs have gained increased attention in the area of biocatalytic transformations for the synthesis of value added compounds with medicinal and nutritional applications. Following the increasing attention on these enzymes, a novel descriptor based classification system has been proposed for FAEs resulting into 12 distinct families and pharmacophore models for three FAE sub-families have been developed. The feruloylome of Aspergillus oryzae contains 13 predicted FAEs belonging to six sub-families based on our recently developed descriptor-based classification system. The three-dimensional structures of the 13 FAEs were modeled for structural analysis of the feruloylome. The three genes coding for three enzymes, viz., A.O.2, A.O.8 and A.O.10 from the feruloylome of A. oryzae, representing sub-families with unknown functional features, were heterologously expressed in Pichia pastoris, characterized for substrate specificity and structural characterization through CD spectroscopy. Common feature-based pharamacophore models were developed according to substrate specificity characteristics of the three enzymes. The active site residues were identified for the three expressed FAEs by determining the titration curves of amino acid residues as a function of the pH by applying molecular simulations. Our findings on the structure-function relationships and substrate specificity of the FAEs of A. oryzae will be instrumental for further understanding of the FAE families in the novel classification system. The developed pharmacophore models could be applied for virtual screening of compound databases for short listing the putative substrates prior to docking studies or for post

  10. A novel esterase gene cloned from a metagenomic library from neritic sediments of the South China Sea

    Science.gov (United States)

    2011-01-01

    Background Marine microbes are a large and diverse group, which are exposed to a wide variety of pressure, temperature, salinity, nutrient availability and other environmental conditions. They provide a huge potential source of novel enzymes with unique properties that may be useful in industry and biotechnology. To explore the lipolytic genetic resources in the South China Sea, 23 sediment samples were collected in the depth South China Sea sediments assemblage in plasmid vector containing about 194 Mb of community DNA was prepared. Screening of a part of the unamplified library resulted in isolation of 15 unique lipolytic clones with the ability to hydrolyze tributyrin. A positive recombinant clone (pNLE1), containing a novel esterase (Est_p1), was successfully expressed in E. coli and purified. In a series of assays, Est_p1 displayed maximal activity at pH 8.57, 40°C, with ρ-Nitrophenyl butyrate (C4) as substrate. Compared to other metagenomic esterases, Est_p1 played a notable role in specificity for substrate C4 (kcat/Km value 11,500 S-1m M-1) and showed no inhibited by phenylmethylsulfonyl fluoride, suggested that the substrate binding pocket was suitable for substrate C4 and the serine active-site residue was buried at the bottom of substrate binding pocket which sheltered by a lid structure. Conclusions Esterase, which specificity towards short chain fatty acids, especially butanoic acid, is commercially available as potent flavoring tools. According the outstanding activity and specificity for substrate C4, Est_p1 has potential application in flavor industries requiring hydrolysis of short chain esters. PMID:22067554

  11. Crystal structure of hyperthermophilic esterase EstE1 and the relationship between its dimerization and thermostability properties

    Directory of Open Access Journals (Sweden)

    Koh Eunhee

    2007-07-01

    Full Text Available Abstract Background EstE1 is a hyperthermophilic esterase belonging to the hormone-sensitive lipase family and was originally isolated by functional screening of a metagenomic library constructed from a thermal environmental sample. Dimers and oligomers may have been evolutionally selected in thermophiles because intersubunit interactions can confer thermostability on the proteins. The molecular mechanisms of thermostabilization of this extremely thermostable esterase are not well understood due to the lack of structural information. Results Here we report for the first time the 2.1-Å resolution crystal structure of EstE1. The three-dimensional structure of EstE1 exhibits a classic α/β hydrolase fold with a central parallel-stranded beta sheet surrounded by alpha helices on both sides. The residues Ser154, Asp251, and His281 form the catalytic triad motif commonly found in other α/β hydrolases. EstE1 exists as a dimer that is formed by hydrophobic interactions and salt bridges. Circular dichroism spectroscopy and heat inactivation kinetic analysis of EstE1 mutants, which were generated by structure-based site-directed mutagenesis of amino acid residues participating in EstE1 dimerization, revealed that hydrophobic interactions through Val274 and Phe276 on the β8 strand of each monomer play a major role in the dimerization of EstE1. In contrast, the intermolecular salt bridges contribute less significantly to the dimerization and thermostability of EstE1. Conclusion Our results suggest that intermolecular hydrophobic interactions are essential for the hyperthermostability of EstE1. The molecular mechanism that allows EstE1 to endure high temperature will provide guideline for rational design of a thermostable esterase/lipase using the lipolytic enzymes showing structural similarity to EstE1.

  12. The Alpha-Defensin Immunoassay and Leukocyte Esterase Colorimetric Strip Test for the Diagnosis of Periprosthetic Infection

    Science.gov (United States)

    Wyatt, M.C.; Beswick, A.D.; Kunutsor, S.K.; Wilson, M.J.; Whitehouse, M.R.; Blom, A.W.

    2016-01-01

    Background: Synovial biomarkers have recently been adopted as diagnostic tools for periprosthetic joint infection (PJI), but their utility is uncertain. The purpose of this systematic review and meta-analysis was to synthesize the evidence on the accuracy of the alpha-defensin immunoassay and leukocyte esterase colorimetric strip test for the diagnosis of PJI compared with the Musculoskeletal Infection Society diagnostic criteria. Methods: We performed a systematic review to identify diagnostic technique studies evaluating the accuracy of alpha-defensin or leukocyte esterase in the diagnosis of PJI. MEDLINE and Embase on Ovid, ACM, ADS, arXiv, CERN DS (Conseil Européen pour la Recherche Nucléaire Document Server), CrossRef DOI (Digital Object Identifier), DBLP (Digital Bibliography & Library Project), Espacenet, Google Scholar, Gutenberg, HighWire, IEEE Xplore (Institute of Electrical and Electronics Engineers digital library), INSPIRE, JSTOR (Journal Storage), OAlster (Open Archives Initiative Protocol for Metadata Harvesting), Open Content, Pubget, PubMed, and Web of Science were searched for appropriate studies indexed from inception until May 30, 2015, along with unpublished or gray literature. The classification of studies and data extraction were performed independently by 2 reviewers. Data extraction permitted meta-analysis of sensitivity and specificity with construction of receiver operating characteristic curves for each test. Results: We included 11 eligible studies. The pooled diagnostic sensitivity and specificity of alpha-defensin (6 studies) for PJI were 1.00 (95% confidence interval [CI], 0.82 to 1.00) and 0.96 (95% CI, 0.89 to 0.99), respectively. The area under the curve (AUC) for alpha-defensin and PJI was 0.99 (95% CI, 0.98 to 1.00). The pooled diagnostic sensitivity and specificity of leukocyte esterase (5 studies) for PJI were 0.81 (95% CI, 0.49 to 0.95) and 0.97 (95% CI, 0.82 to 0.99), respectively. The AUC for leukocyte esterase and PJI

  13. Estrogenic and esterase-inhibiting potency in rainwater in relation to pesticide concentrations, sampling season and location

    International Nuclear Information System (INIS)

    Hamers, T.; Brink, P.J. van den; Mos, L.; Linden, S.C. van der; Legler, J.; Koeman, J.H.; Murk, A.J.

    2003-01-01

    Estrogenic potency of rainwater correlated well with organochlorine concentrations, but could not be attributed to specific pesticides. - In a year-round monitoring program (1998), pesticide composition and toxic potency of the mix of pollutants present in rainwater were measured. The goal of the study was to relate atmospheric deposition of toxic potency and pesticide composition to each other and to sampling period and local agricultural activity. Rainwater was collected in 26 consecutive periods of 14 days in a background location (BACK) and in two locations representative for different agricultural practices, i.e. intensive greenhouse horticulture (HORT) and flower bulb culture (BULB). Samples were chemically analyzed for carbamate (CARB), organophosphate (OP) and organochlorine (OC) pesticides and metabolites. Esterase inhibiting potency of rainwater extracts was measured in a specially developed bio-assay with honeybee esterases and was expressed as an equivalent concentration of the model inhibitor dichlorvos. Estrogenic potency of the extracts was measured in the ER-CALUX reporter gene assay and was expressed as an equivalent concentration of estradiol. Multivariate principal component analysis (PCA) techniques proved to be valuable tools to analyze the numerous pesticide concentrations in relation to toxic potency, sampling location, and sampling season. Pesticide composition in rainwater depended much more on sampling season than on sampling location, but differences between SPRING and SUMMER were mainly attributed to local differences in agricultural practice. On average, the esterase inhibiting potency exceeded the maximum permissible concentration set for dichlorvos in The Netherlands, and was significantly higher in HORT than in BACK and BULB. Esterase inhibition correlated significantly with OP and CARB concentrations, as expected given the working mechanism of these insecticides. The estrogenic potency incidentally exceeded NOEC levels reported for

  14. Estrogenic and esterase-inhibiting potency in rainwater in relation to pesticide concentrations, sampling season and location

    Energy Technology Data Exchange (ETDEWEB)

    Hamers, T.; Brink, P.J. van den; Mos, L.; Linden, S.C. van der; Legler, J.; Koeman, J.H.; Murk, A.J

    2003-05-01

    Estrogenic potency of rainwater correlated well with organochlorine concentrations, but could not be attributed to specific pesticides. - In a year-round monitoring program (1998), pesticide composition and toxic potency of the mix of pollutants present in rainwater were measured. The goal of the study was to relate atmospheric deposition of toxic potency and pesticide composition to each other and to sampling period and local agricultural activity. Rainwater was collected in 26 consecutive periods of 14 days in a background location (BACK) and in two locations representative for different agricultural practices, i.e. intensive greenhouse horticulture (HORT) and flower bulb culture (BULB). Samples were chemically analyzed for carbamate (CARB), organophosphate (OP) and organochlorine (OC) pesticides and metabolites. Esterase inhibiting potency of rainwater extracts was measured in a specially developed bio-assay with honeybee esterases and was expressed as an equivalent concentration of the model inhibitor dichlorvos. Estrogenic potency of the extracts was measured in the ER-CALUX reporter gene assay and was expressed as an equivalent concentration of estradiol. Multivariate principal component analysis (PCA) techniques proved to be valuable tools to analyze the numerous pesticide concentrations in relation to toxic potency, sampling location, and sampling season. Pesticide composition in rainwater depended much more on sampling season than on sampling location, but differences between SPRING and SUMMER were mainly attributed to local differences in agricultural practice. On average, the esterase inhibiting potency exceeded the maximum permissible concentration set for dichlorvos in The Netherlands, and was significantly higher in HORT than in BACK and BULB. Esterase inhibition correlated significantly with OP and CARB concentrations, as expected given the working mechanism of these insecticides. The estrogenic potency incidentally exceeded NOEC levels reported for

  15. Ach1 is involved in shuttling mitochondrial acetyl units for cytosolic C2 provision in Saccharomyces cerevisiae lacking pyruvate decarboxylase

    DEFF Research Database (Denmark)

    Chen, Yun; Zhang, Yiming; Siewers, Verena

    2015-01-01

    Saccharomyces cerevisiae, acetyl-CoA is compartmentalized in the cytosol, mitochondrion, peroxisome and nucleus, and cannot be directly transported between these compartments. With the acetyl-carnitine or glyoxylate shuttle, acetyl-CoA produced in peroxisomes or the cytoplasm can be transported...

  16. Comparative Study of Esterase and Hemolytic Activities in Clinically Important Candida Species, Isolated From Oral Cavity of Diabetic and Non-diabetic Individuals.

    Science.gov (United States)

    Fatahinia, Mahnaz; Poormohamadi, Farzad; Zarei Mahmoudabadi, Ali

    2015-03-01

    Diabetes mellitus as a chronic metabolic disease occurs in patients with partial or complete deficiency of insulin secretion or disorder in action of insulin on tissue. The disease is known to provide conditions for overgrowth of Candida species. Candida spp. cause candidiasis by many virulence factors such as esterase, hemolysin and phospholipase. This study aimed to compare esterase and hemolytic activity in various Candida species isolated from oral cavity of diabetic and non-diabetic individuals. Swab samples were taken from 95 patients with diabetes (35 men and 60 women) and 95 normal persons (42 men and 53 women) and cultured on Sabouraud dextrose agar. Identification of isolated yeasts was performed by germ tube test, morphology on CHROMagar Candida medium, corn meal agar and ability to grow at 45°C. Hemolysin activity was evaluated using blood plate assay and esterase activity was determined using the Tween 80 opacity test. Different Candida species were isolated from 57 (60%) diabetic and 24 (25%) non-diabetic individuals. Esterase activity was detected in all Candida isolates. Only 21.6% of C. albicans from patients with diabetes had esterase activity as + 3, while it ranged from + 1 to + 2 in others. Hemolytic activity was determined in C. albicans, C. dubliniensis, C. glabrata and C. krusei as 0.79, 0.58, 0.66 and 0.74, respectively. Hemolytic activity was significantly different in the two groups of diabetics and non-diabetics. Oral carriage of C. albicans in the diabetic group (n = 42; 66.7%) was significantly greater than the control group (n = 16; 57.1%). Esterase activity of C. albicans in diabetic group was higher than non-diabetic group. Although C. albicans remains the most frequently pathogenic yeast for human, but other species are increasing.

  17. Esterases no exame da estrutura populacional de Camu-camu (Myrciaria dubia (Kunth McVaugh-Myrtaceae Esterases for examining the population structure of Camu-camu (Myrciaria dubia (Kunth McVaugh-Myrtaceae

    Directory of Open Access Journals (Sweden)

    Aylton Saturnino Teixeira

    2004-01-01

    Full Text Available Dois sistemas enzimáticos (esterase e esterase-D, analisados pela técnica de eletroforese em gel de amido, em folhas jovens de plantas cultivadas em terra firme, de sementes provenientes de três amostras de populações naturais de camu-camu, Myrciaria dubia (Kunth McVaugh-Myrtaceae, procedentes de Iquitos, Boa Vista e Uatumã, revelaram a presença de 6 locos: Est-1, Est-2, Est-3, Est-4, Est-D1 e Est-D2. Dois dos seis locos gênicos examinados no presente estudo (Est-3 e Est-D2 mostraram-se polimórficos, sendo desse modo considerados valiosos no estudo de caracterização da estrutura populacional da espécie. Os padrões de polimorfismo revelados nos locos Est-3 e Est-D2 de camu-camu, são típicos de enzimas monoméricas e diméricas, respectivamente. O loco Est-3 apresentou um grande desbalanço genético dentro e entre as amostras populacionais examinadas, devido ao excessivo número observado de plantas heterozigóticas em relação ao número esperado. O loco Est-D2 apresentou um polimorfismo exclusivo para os alelos Est-D2¹,Est-D2² e Est-D2³, e um bom balanço genético na amostra populacional de Uatumã. Em função disso, dentre os demais locos gênicos aqui investigados, o loco Est-D2 parece ser o mais adequado para identificação e delimitação de prováveis estoques de camu-camu. Portanto, recomenda-se que esse loco esteja presente na lista dos marcadores isoenzimáticos a serem usados em futuras prospecções sobre genética populacional dessa espécie na região amazônica. Dados sobre a distribuição das freqüências alélicas, estimativas das distâncias genéticas, e estimativas de variação genética nos 6 locos de esterases examinados, foram eficazes na demonstração de diferenças genéticas entre as amostras populacionais examinadas da espécie. Os maiores valores de heterozigozidade média (0,1353; proporção de locos polimórficos (0,33 e número médio de alelos por loco (1,33 revelados na amostra

  18. Isolation and Expression analysis of OsPME1, encoding for a putative Pectin Methyl Esterase from Oryza sativa (subsp. indica)

    OpenAIRE

    Kanneganti, Vydehi; Gupta, Aditya Kumar

    2009-01-01

    Pectin Methyl Esterases (PMEs) play an essential role during plant development by affecting the mechanical properties of the plant cell walls. Recent studies indicated that PMEs play important role in pollen tube development. In this study, we isolated a 1.3 kb cDNA clone from rice panicle cDNA library. It contained a 1038 bp of open reading frame (ORF) encoding for a putative pectin methyl esterase of 345 aminoacids with a 20 aminoacid signal peptide and was hence designated as OsPME1 (Oryza...

  19. Acidic-alkaline ferulic acid esterase from Chaetomium thermophilum var. dissitum: Molecular cloning and characterization of recombinant enzyme expressed in Pichia pastoris

    DEFF Research Database (Denmark)

    Dotsenko, Gleb; Tong, Xiaoxue; Pilgaard, Bo

    2016-01-01

    A novel ferulic acid esterase encoding gene CtFae, was successfully cloned from a highly esterase active strain of the thermophile ascomycetous fungus Chaetomium thermophilum var. dissitum; the gene was heterologously expressed in Pichia pastoris KM71H. The recombinant enzyme (CtFae) was purified...... to homogeneity and subsequently characterized. CtFae was active towards synthetic esters of ferulic, p-coumaric, and caffeic acids, as well as towards wide range of p-nitrophenyl substrates. Its temperature and pH optima were 55 °C and pH 6.0, respectively. Enzyme rare features were broad pH optimum, high...

  20. The Use of Plasma-Derived Complement C1-Esterase Inhibitor Concentrate (Berinert®) in the Treatment of Angiotensin Converting Enzyme-Inhibitor Related Angioedema

    DEFF Research Database (Denmark)

    Hermanrud, Thorbjørn; Duus, Nicolaj; Bygum, Anette

    2016-01-01

    Angioedema of the upper airways is a severe and potentially life-threatening condition. The incidence has been increasing in the past two decades, primarily due to pharmaceuticals influencing the generation or degradation of the vasoactive molecule bradykinin. Plasma-derived C1-esterase inhibitor...... concentrate is a well-established treatment option of hereditary and acquired complement C1-esterase inhibitor deficiency, which are also mediated by an increased level of bradykinin resulting in recurrent angioedema. We here present a case of severe angiotensin converting enzyme-inhibitor related angioedema...

  1. Identification and characterization of a GDSL esterase gene located proximal to the swr quorum-sensing system of Serratia liquefaciens MG1

    DEFF Research Database (Denmark)

    Riedel, K.; Talker-Huiber, D.; Givskov, Michael Christian

    2003-01-01

    direction, designated estA, which encodes an esterase that belongs to family II of lipolytic enzymes. EstA was heterologously expressed in Escherichia coli, and the substrate specificity of the enzyme was determined in crude extracts. With the aid of zymograms visualizing EstA on polyacrylamide gels...... and by the analysis of a transcriptional fusion of the estA promoter to the promoterless lux4B genes, we showed that expression of the esterase is not regulated by the swr quorum-sensing system. An estA mutant was generated and was found to exhibit growth defects on minimal medium containing Tween 20 or Tween 80...

  2. Prevalence of positive urinary dipstick analysis (leucocyte esterase, nitrite, haemoglobin, or glucose) in a population of 3645 adult subjects--consequence for measurement of urinary albumin excretion rate

    DEFF Research Database (Denmark)

    Clausen, P; Jensen, J S; Borch-Johnsen, K

    1998-01-01

    OBJECTIVES: To assess prevalence of positive urinary dipstick analysis for leucocyte esterase, nitrite, haemoglobin, or glucose in the general population and measure the urinary albumin excretion rate (UAER) in subjects with or without a positive dipstick analysis. DESIGN: A cross-sectional study...... of 3645 subjects. SETTING: An unselected urban population study. MAIN OUTCOME MEASURES: Prevalence data of positive dipstick analyses and UAER values. RESULTS: Prevalence data of a positive dipstick analysis were 12%, 4%, 3% and 6%, respectively, for leucocyte esterase, nitrite, haemoglobin, and glucose...

  3. Morphological, mechanical, barrier and properties of films based on acetylated starch and cellulose from barley.

    Science.gov (United States)

    El Halal, Shanise Lisie Mello; Colussi, Rosana; Biduski, Bárbara; Evangelho, Jarine Amaral do; Bruni, Graziella Pinheiro; Antunes, Mariana Dias; Dias, Alvaro Renato Guerra; Zavareze, Elessandra da Rosa

    2017-01-01

    Biodegradable films of native or acetylated starches with different concentrations of cellulose fibers (0%, 10% and 20%) were prepared. The films were characterized by morphological, mechanical, barrier, and thermal properties. The tensile strength of the acetylated starch film was lower than those of the native starch film, without fibers. The addition of fibers increased the tensile strength and decreased the elongation and the moisture of native and acetylated starches films. The acetylated starch film showed higher water solubility when compared to native starch film. The addition of cellulose fibers reduced the water solubility of the acetylated starch film. The films reinforced with cellulose fiber exhibited a higher initial decomposition temperature and thermal stability. The mechanical, barrier, solubility, and thermal properties are factors which direct the type of the film application in packaging for food products. The films elaborated with acetylated starches of low degree of substitution were not effective in a reduction of the water vapor permeability. The addition of the cellulose fiber in acetylated and native starches films can contribute to the development of more resistant films to be applied in food systems that need to maintain their integrity. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  4. Proteomic Analysis of Lysine Acetylation Sites in Rat Tissues Reveals Organ Specificity and Subcellular Patterns

    Directory of Open Access Journals (Sweden)

    Alicia Lundby

    2012-08-01

    Full Text Available Lysine acetylation is a major posttranslational modification involved in a broad array of physiological functions. Here, we provide an organ-wide map of lysine acetylation sites from 16 rat tissues analyzed by high-resolution tandem mass spectrometry. We quantify 15,474 modification sites on 4,541 proteins and provide the data set as a web-based database. We demonstrate that lysine acetylation displays site-specific sequence motifs that diverge between cellular compartments, with a significant fraction of nuclear sites conforming to the consensus motifs G-AcK and AcK-P. Our data set reveals that the subcellular acetylation distribution is tissue-type dependent and that acetylation targets tissue-specific pathways involved in fundamental physiological processes. We compare lysine acetylation patterns for rat as well as human skeletal muscle biopsies and demonstrate its general involvement in muscle contraction. Furthermore, we illustrate that acetylation of fructose-bisphosphate aldolase and glycerol-3-phosphate dehydrogenase serves as a cellular mechanism to switch off enzymatic activity.

  5. Mitochondrial storage form of acetyl CoA carboxylase in fasted and alloxan diabetic rats

    International Nuclear Information System (INIS)

    Roman-Lopez, C.R.; Allred, J.B.

    1986-01-01

    Sodium dodecyl sulfate-denatured biotinyl proteins will bind [ 14 C]methyl avidin which remains bound through polyacrylamide gel electrophoresis. The method has been used to demonstrate the presence of two high molecular weight subunit forms of acetyl CoA carboxylase in rat liver cytoplasm, both of which are precipitated by antibody to purifed rat liver acetyl CoA carboxylase prepared from sheep serum. Rat liver mitochondria contained five distinct biotinyl protein subunits, the two largest of which have been identified as acetyl CoA carboxylase subunits on the basis of precipitation by anti-acetyl CoA carboxylase antibody. The small quantity of acetyl CoA carboxylase associated with rat liver microsomes could be attributed to cytoplasmic contamination. The binding of radioactive avidin is sufficiently tight to use as a measure of the quantity of acetyl CoA carboxylase. The quantity and activity of the cytoplasmic enzyme was reduced in fasted and in alloxan diabetic rats compared to that in fed controls but the quantity of the enzyme associated with isolated mitochondria was not reduced. The results indicate that there is a mitochondrial storage form of acetyl CoA carboxylase

  6. Changes in nuclear protein acetylation in u. v. -damaged human cells

    Energy Technology Data Exchange (ETDEWEB)

    Ramanathan, B.; Smerdon, M.J.

    1986-07-01

    We have investigated the levels of nuclear protein acetylation in u.v.-irradiated human fibroblasts. We measured the levels of acetylation in total acid-soluble nuclear proteins and observed two distinct differences between the irradiated and unirradiated (control) cells. Immediately after irradiation, there is a wave of protein hyperacetylation (i.e. a total acetylation level greater than that of unirradiated cells) that lasts for 2-6 h depending on the experimental conditions. This hyperacetylation phase is then followed by a hypoacetylation phase, lasting for many hours, and the total level of acetylation does not return to that of control cells until 24-72 h after u.v. damage. Both the magnitude and duration of each phase is dependent on the dose of u.v. light used. The wave of hyperacetylation is more pronounced at low u.v. doses (i.e. less than 5 J/m2), while the wave of hypoacetylation is more pronounced at higher u.v. doses (greater than or equal to 8 J/m2). Furthermore, the duration of each phase is prolonged when cells are exposed to 2 mM hydroxyurea. Examination of the acetylation levels of the individual nuclear proteins indicated that acetylation of the core histones follows the same pattern observed for the total acid-soluble protein fractions. Furthermore, these were the only major proteins in the total acid-soluble fraction observed to undergo the early, rapid hyperacetylation immediately following u.v. damage. Acetylation of histone H1 was negligible in both damaged and control cells, while three prominent non-histone proteins were acetylated only after long labeling times (greater than 4 h) in each case, gradually becoming hyperacetylated in the u.v.-damaged cells. These results raise the possibility that a causal relationship exists between nuclear protein acetylation and nucleotide excision repair of DNA in human cells.

  7. Transport and metabolism of indole-3-acetyl-myo-inositol-galactoside in seedlings of Zea mays

    Science.gov (United States)

    Komoszynski, M.; Bandurski, R. S.

    1986-01-01

    Indole-3-acetyl-myo-inositol galactoside labeled with 3H in the indole and 14C in the galactose moieties was applied to kernels of 5 day old germinating seedlings of Zea mays. Indole-3-acetyl-myo-inositol galactoside was not transported into either the shoot or root tissue as the intact molecule but was instead hydrolyzed to yield [3H]indole-3-acetyl-myo-inositol and [3H]indole-3-acetic acid which were then transported to the shoot with little radioactivity going to the root. With certain assumption concerning the equilibration of applied [3H]indole-3-acetyl-myo-inositol-[U-14C]galactose with the endogenous pool, it may be concluded that indole-3-acetyl-myo-inositol galactoside in the endosperm supplies about 2 picomoles per plant per hour of indole-3-acetyl-myo-inositol and 1 picomole per plant per hour of indole-3-acetic acid to the shoot and thus is comparable to indole-3-acetyl-myo-inositol as a source of indole-acetic acid for the shoot. Quantitative estimates of the amount of galactose in the kernels suggest that [3H]indole-3-acetyl-myo-inositol-[14C]galactose is hydrolyzed after the compound leaves the endosperm but before it reaches the shoot. In addition, [3H]indole-3-acetyl-myo-inositol-[14C]galactose supplies appreciable amounts of 14C to the shoot and both 14C and 3H to an uncharacterized insoluble fraction of the endosperm.

  8. Aspirin-Mediated Acetylation Protects Against Multiple Neurodegenerative Pathologies by Impeding Protein Aggregation.

    Science.gov (United States)

    Ayyadevara, Srinivas; Balasubramaniam, Meenakshisundaram; Kakraba, Samuel; Alla, Ramani; Mehta, Jawahar L; Shmookler Reis, Robert J

    2017-12-10

    Many progressive neurological disorders, including Alzheimer's disease (AD), Huntington's disease, and Parkinson's disease (PD), are characterized by accumulation of insoluble protein aggregates. In prospective trials, the cyclooxygenase inhibitor aspirin (acetylsalicylic acid) reduced the risk of AD and PD, as well as cardiovascular events and many late-onset cancers. Considering the role played by protein hyperphosphorylation in aggregation and neurodegenerative diseases, and aspirin's known ability to donate acetyl groups, we asked whether aspirin might reduce both phosphorylation and aggregation by acetylating protein targets. Aspirin was substantially more effective than salicylate in reducing or delaying aggregation in human neuroblastoma cells grown in vitro, and in Caenorhabditis elegans models of human neurodegenerative diseases in vivo. Aspirin acetylates many proteins, while reducing phosphorylation, suggesting that acetylation may oppose phosphorylation. Surprisingly, acetylated proteins were largely excluded from compact aggregates. Molecular-dynamic simulations indicate that acetylation of amyloid peptide energetically disfavors its association into dimers and octamers, and oligomers that do form are less compact and stable than those comprising unacetylated peptides. Hyperphosphorylation predisposes certain proteins to aggregate (e.g., tau, α-synuclein, and transactive response DNA-binding protein 43 [TDP-43]), and it is a critical pathogenic marker in both cardiovascular and neurodegenerative diseases. We present novel evidence that acetylated proteins are underrepresented in protein aggregates, and that aggregation varies inversely with acetylation propensity after diverse genetic and pharmacologic interventions. These results are consistent with the hypothesis that aspirin inhibits protein aggregation and the ensuing toxicity of aggregates through its acetyl-donating activity. This mechanism may contribute to the neuro-protective, cardio

  9. The small delta antigen of hepatitis delta virus is an acetylated protein and acetylation of lysine 72 may influence its cellular localization and viral RNA synthesis

    International Nuclear Information System (INIS)

    Mu, J.-J.; Tsay, Y.-G.; Juan, L.-J.; Fu, T.-F.; Huang, W.-H.; Chen, D.-S.; Chen, P.-J.

    2004-01-01

    Hepatitis delta virus (HDV) is a single-stranded RNA virus that encodes two viral nucleocapsid proteins named small and large form hepatitis delta antigen (S-HDAg and L-HDAg). The S-HDAg is essential for viral RNA replication while the L-HDAg is required for viral assembly. In this study, we demonstrated that HDAg are acetylated proteins. Metabolic labeling with [ 3 H]acetate revealed that both forms of HDAg could be acetylated in vivo. The histone acetyltransferase (HAT) domain of cellular acetyltransferase p300 could acetylate the full-length and the N-terminal 88 amino acids of S-HDAg in vitro. By mass spectrometric analysis of the modified protein, Lys-72 of S-HDAg was identified as one of the acetylation sites. Substitution of Lys-72 to Arg caused the mutant S-HDAg to redistribute from the nucleus to the cytoplasm. The mutant reduced viral RNA accumulation and resulted in the earlier appearance of L-HDAg. These results demonstrated that HDAg is an acetylated protein and mutation of HDAg at Lys-72 modulates HDAg subcellular localization and may participate in viral RNA nucleocytoplasmic shuttling and replication

  10. Proteomic investigations of lysine acetylation identify diverse substrates of mitochondrial deacetylase sirt3

    DEFF Research Database (Denmark)

    Sol, E-ri Maria; Wagner, Sebastian A; Weinert, Brian T

    2012-01-01

    Lysine acetylation is a posttranslational modification that is dynamically regulated by the activity of acetyltransferases and deacetylases. The human and mouse genomes encode 18 different lysine deacetylases (KDACs) which are key regulators of many cellular processes. Identifying substrates...... of KDACs and pinpointing the regulated acetylation sites on target proteins may provide important information about the molecular basis of their functions. Here we apply quantitative proteomics to identify endogenous substrates of the mitochondrial deacetylase Sirtuin 3 (Sirt3) by comparing site...... by modulating acetylation on diverse substrates. The experimental strategy described here is generic and can be applied to identify endogenous substrates of other lysine deacetylases....

  11. Identification and characterization of AckA-dependent protein acetylation in Neisseria gonorrhoeae.

    Directory of Open Access Journals (Sweden)

    Deborah M B Post

    Full Text Available Neisseria gonorrhoeae, the causative agent of gonorrhea, has a number of factors known to contribute to pathogenesis; however, a full understanding of these processes and their regulation has proven to be elusive. Post-translational modifications (PTMs of bacterial proteins are now recognized as one mechanism of protein regulation. In the present study, Western blot analyses, with an anti-acetyl-lysine antibody, indicated that a large number of gonococcal proteins are post-translationally modified. Previous work has shown that Nε-lysine acetylation can occur non-enzymatically with acetyl-phosphate (AcP as the acetyl donor. In the current study, an acetate kinase mutant (1291ackA, which accumulates AcP, was generated in N. gonorrhoeae. Broth cultures of N. gonorrhoeae 1291wt and 1291ackA were grown, proteins extracted and digested, and peptides containing acetylated-lysines (K-acetyl were affinity-enriched from both strains. Mass spectrometric analyses of these samples identified a total of 2686 unique acetylation sites. Label-free relative quantitation of the K-acetyl peptides derived from the ackA and wild-type (wt strains demonstrated that 109 acetylation sites had an ackA/wt ratio>2 and p-values <0.05 in at least 2/3 of the biological replicates and were designated as "AckA-dependent". Regulated K-acetyl sites were found in ribosomal proteins, central metabolism proteins, iron acquisition and regulation proteins, pilus assembly and regulation proteins, and a two-component response regulator. Since AckA is part of a metabolic pathway, comparative growth studies of the ackA mutant and wt strains were performed. The mutant showed a growth defect under aerobic conditions, an inability to grow anaerobically, and a defect in biofilm maturation. In conclusion, the current study identified AckA-dependent acetylation sites in N. gonorrhoeae and determined that these sites are found in a diverse group of proteins. This work lays the foundation for

  12. Crystallization and preliminary X-ray analysis of a novel esterase Rv0045c from Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Xu, Lipeng; Guo, Jiubiao; Zheng, Xiangdong; Wen, Tingyi; Sun, Fei; Liu, Siguo; Pang, Hai

    2010-01-01

    The novel esterase Rv0045c from M. tuberculosis was expressed and purified to homogeneity. The crystals of native and SeMet-labelled Rv0045c protein that were obtained diffracted to resolutions of 2.7 and 3.0 Å, respectively. The Rv0045c protein is predicted to be an esterase that is involved in lipid metabolism in Mycobacterium tuberculosis. The protein was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The Rv0045c protein crystals diffracted to a resolution of 2.7 Å using a synchrotron-radiation source and belonged to space group P3 1 or P3 2 , with unit-cell parameters a = b = 73.465, c = 48.064 Å, α = β = 90, γ = 120°. Purified SeMet-labelled Rv0045c protein was also crystallized and formed crystals that diffracted to a resolution of 3.0 Å using an in-house X-ray radiation source

  13. Inhibition, recovery and oxime-induced reactivation of muscle esterases following chlorpyrifos exposure in the earthworm Lumbricus terrestris

    International Nuclear Information System (INIS)

    Collange, B.; Wheelock, C.E.; Rault, M.; Mazzia, C.; Capowiez, Y.; Sanchez-Hernandez, J.C.

    2010-01-01

    Assessment of wildlife exposure to organophosphorus (OP) pesticides generally involves the measurement of cholinesterase (ChE) inhibition, and complementary biomarkers (or related endpoints) are rarely included. Herein, we investigated the time course inhibition and recovery of ChE and carboxylesterase (CE) activities in the earthworm Lumbricus terrestris exposed to chlorpyrifos, and the ability of oximes to reactivate the phosphorylated ChE activity. Results indicated that these esterase activities are a suitable multibiomarker scheme for monitoring OP exposure due to their high sensitivity to OP inhibition and slow recovery to full activity levels following pesticide exposure. Moreover, oximes reactivated the inhibited ChE activity of the earthworms exposed to 12 and 48 mg kg -1 chlorpyrifos during the first week following pesticide exposure. This methodology is useful for providing evidence for OP-mediated ChE inhibition in individuals with a short history of OP exposure (≤1 week); resulting a valuable approach for assessing multiple OP exposure episodes in the field. - Esterase inhibition combined with oxime reactivation methods is a suitable approach for monitoring organophosphate contamination

  14. Inhibition, recovery and oxime-induced reactivation of muscle esterases following chlorpyrifos exposure in the earthworm Lumbricus terrestris

    Energy Technology Data Exchange (ETDEWEB)

    Collange, B. [Universite d' Avignon et des Pays de Vaucluse, UMR 406 Abeilles et Environnement, Site AGROPARC, F-84914, Avignon Cede 09 (France); Wheelock, C.E. [Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE 171 77, Stockholm (Sweden); Rault, M.; Mazzia, C. [Universite d' Avignon et des Pays de Vaucluse, UMR 406 Abeilles et Environnement, Site AGROPARC, F-84914, Avignon Cede 09 (France); Capowiez, Y. [INRA, Unite PSH, Site AGROPARC, F-84914 Avignon Cedex 09 (France); Sanchez-Hernandez, J.C., E-mail: juancarlos.sanchez@uclm.e [Laboratory of Ecotoxicology, Faculty of Environmental Science, University of Castilla-La Mancha, Avda. Carlos III s/n, 45071, Toledo (Spain)

    2010-06-15

    Assessment of wildlife exposure to organophosphorus (OP) pesticides generally involves the measurement of cholinesterase (ChE) inhibition, and complementary biomarkers (or related endpoints) are rarely included. Herein, we investigated the time course inhibition and recovery of ChE and carboxylesterase (CE) activities in the earthworm Lumbricus terrestris exposed to chlorpyrifos, and the ability of oximes to reactivate the phosphorylated ChE activity. Results indicated that these esterase activities are a suitable multibiomarker scheme for monitoring OP exposure due to their high sensitivity to OP inhibition and slow recovery to full activity levels following pesticide exposure. Moreover, oximes reactivated the inhibited ChE activity of the earthworms exposed to 12 and 48 mg kg{sup -1} chlorpyrifos during the first week following pesticide exposure. This methodology is useful for providing evidence for OP-mediated ChE inhibition in individuals with a short history of OP exposure (<=1 week); resulting a valuable approach for assessing multiple OP exposure episodes in the field. - Esterase inhibition combined with oxime reactivation methods is a suitable approach for monitoring organophosphate contamination

  15. Evolution of the feruloyl esterase MtFae1a from Myceliophthora thermophila towards improved catalysts for antioxidants synthesis.

    Science.gov (United States)

    Varriale, Simona; Cerullo, Gabriella; Antonopoulou, Io; Christakopoulos, Paul; Rova, Ulrika; Tron, Thierry; Fauré, Régis; Jütten, Peter; Piechot, Alexander; Brás, Joana L A; Fontes, Carlos M G A; Faraco, Vincenza

    2018-04-23

    The chemical syntheses currently employed for industrial purposes, including in the manufacture of cosmetics, present limitations such as unwanted side reactions and the need for harsh chemical reaction conditions. In order to overcome these drawbacks, novel enzymes are developed to catalyze the targeted bioconversions. In the present study, a methodology for the construction and the automated screening of evolved variants library of a Type B feruloyl esterase from Myceliophthora thermophila (MtFae1a) was developed and applied to generation of 30,000 mutants and their screening for selecting the variants with higher activity than the wild-type enzyme. The library was generated by error-prone PCR of mtfae1a cDNA and expressed in Saccharomyces cerevisiae. Screening for extracellular enzymatic activity towards 4-nitrocatechol-1-yl ferulate, a new substrate developed ad hoc for high-throughput assays of feruloyl esterases, led to the selection of 30 improved enzyme variants. The best four variants and the wild-type MtFae1a were investigated in docking experiments with hydroxycinnamic acid esters using a model of 3D structure of MtFae1a. These variants were also used as biocatalysts in transesterification reactions leading to different target products in detergentless microemulsions and showed enhanced synthetic activities, although the screening strategy had been based on improved hydrolytic activity.

  16. Heterologous production and characterization of a chlorogenic acid esterase from Ustilago maydis with a potential use in baking.

    Science.gov (United States)

    Nieter, Annabel; Kelle, Sebastian; Takenberg, Meike; Linke, Diana; Bunzel, Mirko; Popper, Lutz; Berger, Ralf G

    2016-10-15

    Ustilago maydis, an edible mushroom growing on maize (Zea mays), is consumed as the food delicacy huitlacoche in Mexico. A chlorogenic acid esterase from this basidiomycete was expressed in good yields cultivating the heterologous host Pichia pastoris on the 5L bioreactor scale (reUmChlE; 45.9UL(-1)). In contrast to previously described chlorogenic acid esterases, the reUmChlE was also active towards feruloylated saccharides. The enzyme preferred substrates with the ferulic acid esterified to the O-5 position of arabinose residues, typical of graminaceous monocots, over the O-2 position of arabinose or the O-6 position of galactose residues. Determination of kcat/Km showed that the reUmChlE hydrolyzed chlorogenic acid 18-fold more efficiently than methyl ferulate, p-coumarate or caffeate. Phenolic acids were released by reUmChlE from natural substrates, such as destarched wheat bran, sugar beet pectin and coffee pulp. Treatment of wheat dough using reUmChlE resulted in a noticeable softening indicating a potential application of the enzyme in bakery and confectionery. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Identification of Morphological Character and Esterase Isozyme Pattern in Second-Generation Black Rice Plant Irradiated to Gamma Rays

    Science.gov (United States)

    Hartanti, R. S.; Putri, T. A. N.; Zulfa, F.; Sutarno; Suranto

    2017-04-01

    Black rice is one of the functional foods due to its high anthocyanin content. Black rice grain was irradiated by gamma rays with a dose of 200 Gy and 300 Gy. The main purpose of this irradiation is to induce mutation to the black rice plant in order to achieve the improved organism. This study was undertaken to elucidate the morphological character and esterase isozyme pattern of black rice plant after irradiated by gamma rays. There were morphological differences on leaves, stems and grains between irradiated and non irradiated black rice plant. Gamma radiation dose of 200 Gy showed the significant influence of the length of the stem, number of internodes, and length of leaves. The radiation dose of 300 Gy showed the significant influence of the decrease value of diameter of 3rd internodes, number of branches and width of leaves. Flowering time is getting faster as increasing radiation dose. At the age of 74 days after planting there are 9.15% plants of 200 Gy radiation dose that have flowered faster than normal plants. This value increased into 11.45% at the dose of radiation 300 Gy. There were differences in the esterase banding pattern between radiation dose of 200 Gy and 300 Gy than the control plants, indicated that randomly mutation has occurred.

  18. Esterase- and pH-responsive poly(β-amino ester)-capped mesoporous silica nanoparticles for drug delivery

    Science.gov (United States)

    Fernando, Isurika R.; Ferris, Daniel P.; Frasconi, Marco; Malin, Dmitry; Strekalova, Elena; Yilmaz, M. Deniz; Ambrogio, Michael W.; Algaradah, Mohammed M.; Hong, Michael P.; Chen, Xinqi; Nassar, Majed S.; Botros, Youssry Y.; Cryns, Vincent L.; Stoddart, J. Fraser

    2015-04-01

    Gating of mesoporous silica nanoparticles (MSNs) with the stimuli-responsive poly(β-amino ester) has been achieved. This hybrid nanocarrier releases doxorubicin (DOX) under acidic conditions or in the presence of porcine liver esterase. The DOX loaded poly(β-amino ester)-capped MSNs reduce cell viability when tested on MDA-MB-231 human breast cancer cells.Gating of mesoporous silica nanoparticles (MSNs) with the stimuli-responsive poly(β-amino ester) has been achieved. This hybrid nanocarrier releases doxorubicin (DOX) under acidic conditions or in the presence of porcine liver esterase. The DOX loaded poly(β-amino ester)-capped MSNs reduce cell viability when tested on MDA-MB-231 human breast cancer cells. Electronic supplementary information (ESI) available: Experimental details relating to (i) the synthesis and characterisation of the surface-functionalised MSN and POL (ii) cargo-loading and release studies in solution, (iii) cellular internalisation of nanomaterials, and (iv) cell viability tests. See DOI: 10.1039/c4nr07443b

  19. In-vivo measurements of regional acetylcholine esterase activity in degenerative dementia: comparison with blood flow and glucose metabolism.

    Science.gov (United States)

    Herholz, K; Bauer, B; Wienhard, K; Kracht, L; Mielke, R; Lenz, M O; Strotmann, T; Heiss, W D

    2000-01-01

    Memory and attention are cognitive functions that depend heavily on the cholinergic system. Local activity of acetylcholine esterase (AChE) is an indicator of its integrity. Using a recently developed tracer for positron emission tomography (PET), C-11-labeled N-methyl-4-piperidyl-acetate (C11-MP4A), we measured regional AChE activity in 4 non-demented subjects, 4 patients with dementia of Alzheimer type (DAT) and 1 patient with senile dementia of Lewy body type (SDLT), and compared the findings with measurements of blood flow (CBF) and glucose metabolism (CMRGlc). Initial tracer extraction was closely related to CBF. AChE activity was reduced significantly in all brain regions in demented subjects, whereas reduction of CMRGlc and CBF was more limited to temporo-parietal association areas. AChE activity in SDLT was in the lower range of values in DAT. Our results indicate that, compared to non-demented controls, there is a global reduction of cortical AChE activity in dementia. Dementia, cholinergic system, acetylcholine esterase, positron emission tomography, cerebral blood flow, cerebral glucose metabolism.

  20. Purification and characterization of a type B feruloyl esterase (StFAE-A) from the thermophilic fungus Sporotrichum thermophile

    DEFF Research Database (Denmark)

    Topakas, E.; Stamatis, H.; Biely, P.

    2004-01-01

    A feruloyl esterase (StFAE-A) produced by Sporotrichum thermophile was purified to homogeneity. The purified homogeneous preparation of native StFAE-A exhibited a molecular mass of 57.0+/-1.5 kDa, with a mass of 33+/-1 kDa on SDS-PAGE. The pI of the enzyme was estimated by cation......-exchange chromatofocusing to be at pH 3.1. The enzyme activity was optimal at pH 6.0 and 55-60 degreesC. The purified esterase was stable at the pH range 5.0-7.0. The enzyme retained 70% of activity after 7 h at 50 degreesC and lost 50% of its activity after 45 min at 55 degreesC and after 12 min at 60 degrees......C. Determination of k(cat)/K-m revealed that the enzyme hydrolyzed methyl p-coumarate 2.5- and 12-fold more efficiently than methyl caffeate and methyl ferulate, respectively. No activity on methyl sinapinate was detected. The enzyme was active on substrates containing ferulic acid ester linked to the C-5 and C-2...

  1. Fibulin-1C, C1 Esterase Inhibitor and Glucose Regulated Protein 75 Interact with the CREC Proteins, Calumenin and Reticulocalbin.

    Directory of Open Access Journals (Sweden)

    Gry Aune Westergaard Hansen

    Full Text Available Affinity purification, immunoprecipitation, gel electrophoresis and mass spectrometry were used to identify fibulin-1C, C1 esterase inhibitor and glucose regulated protein 75, grp75, as binding partners of the CREC proteins, calumenin and reticulocalbin. Surface plasmon resonance was used to verify the interaction of all three proteins with each of the CREC proteins. Fibulin-1C interacts with calumenin and reticulocalbin with an estimated dissociation constant around 50-60 nM. The interaction, at least for reticulocalbin, was not dependent upon the presence of Ca2+. C1 esterase inhibitor interacted with both proteins with an estimated dissociation constant at 1 μM for reticulocalbin and 150 nM for calumenin. The interaction, at least for calumenin, was dependent upon the presence of Ca2+ with strong interaction at 3.5 mM while no detectable interaction could be found at 0.1 mM. Grp75 binds with an affinity of approximately 3-7 nM with reticulocalbin as well as with calumenin. These interactions suggest functional participation of the CREC proteins in chaperone activity, cell proliferation and transformation, cellular aging, haemostasis and thrombosis as well as modulation of the complement system in fighting bacterial infection.

  2. Crystallization and preliminary crystallographic analysis of an esterase with a novel domain from the hyperthermophile Thermotoga maritima

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Lei [Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Dreijenplein 10, 6703 HB Wageningen (Netherlands); Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen (Netherlands); Levisson, Mark; Hendriks, Sjon; Akveld, Twan; Kengen, Servé W. M. [Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Dreijenplein 10, 6703 HB Wageningen (Netherlands); Dijkstra, Bauke W. [Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen (Netherlands); Oost, John van der, E-mail: john.vanderoost@wur.nl [Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Dreijenplein 10, 6703 HB Wageningen (Netherlands)

    2007-09-01

    A thermostable esterase (EstA) from Thermotoga maritima was cloned and purified. Crystals of EstA and its selenomethionine derivative were grown and diffract to beyond 2.6 Å resolution at 100 K using synchrotron radiation. A predicted esterase (EstA) with an unusual new domain from the hyperthermophilic bacterium Thermotoga maritima has been cloned and overexpressed in Escherichia coli. The purified protein was crystallized by the hanging-drop vapour-diffusion technique in the presence of lithium sulfate and polyethylene glycol 8000. Selenomethionine-substituted EstA crystals were obtained under the same conditions and three different-wavelength data sets were collected to 2.6 Å resolution. The crystal belongs to space group H32, with unit-cell parameters a = b = 130.2, c = 306.2 Å. There are two molecules in the asymmetric unit, with a V{sub M} of 2.9 Å{sup 3} Da{sup −1} and 58% solvent content.

  3. Crystallization and preliminary crystallographic studies of LipA, a secretory lipase/esterase from Xanthomonas oryzae pv. oryzae

    Energy Technology Data Exchange (ETDEWEB)

    Aparna, Gudlur; Chatterjee, Avradip; Jha, Gopaljee; Sonti, Ramesh V.; Sankaranarayanan, Rajan, E-mail: sankar@ccmb.res.in [Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007 (India)

    2007-08-01

    The crystallization and preliminary crystallographic studies of LipA, a lipase/esterase secreted by X. oryzae pv. oryzae during its infection of rice plants, are reported. Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight, a serious disease of rice. Several enzymes that are secreted through the type II secretion system of this bacterium play an important role in the plant–microbe interaction, being important for virulence and also being able to induce potent host defence responses. One of these enzymes is a secretory lipase/esterase, LipA, which shows a very weak homology to other bacterial lipases and gives a positive tributyrin plate assay. In this study, LipA was purified from the culture supernatant of an overexpressing clone of X. oryzae pv. oryzae and two types of crystals belonging to space group C2 but with two different unit-cell parameters were obtained using the hanging-drop vapour-diffusion method. Type I crystals diffract to a maximum resolution of 1.89 Å and have unit-cell parameters a = 93.1, b = 62.3, c = 66.1 Å, β = 90.8°. Type II crystals have unit-cell parameters a = 103.6, b = 54.6, c = 66.3 Å, β = 92.6° and diffract to 1.86 Å. Solvent-content analysis shows one monomer in the asymmetric unit in both the crystal forms.

  4. Endophytic fungi producing of esterases: evaluation in vitro of the enzymatic activity using pH indicator

    Directory of Open Access Journals (Sweden)

    Helen Cristina Fávero Lisboa

    2013-09-01

    Full Text Available A sensitive and efficient colorimetric method was optimized for detection of esterase enzymes produced by endophytic fungi for development of High-Throughput Screening (HTS. The fungi were isolated and obtained previously from plant species of Cerrado and Atlantic Forest located in areas of environmental preservation in the State of Sao Paulo / Brazil, as part of the project "Chemical and biological prospecting endophytic fungi associated to plant species of Cerrado and Atlantic Forest". The compounds ethyl butyrate, ethyl acetate and methyl propionate were used as standards esters which were hydrolyzed by extracellular enzyme from endophytic fungi (EC. 3.1.1.1 -carboxylesterases for production of carboxylic acids. Thus, the reduction of the pH increases the protonated indicator concentration (bromothymol blue, changing the color of the reaction medium (from blue to yellow, that can be observed and measured by spectrophotometry at 616 nm. The methodology with acid-base indicator was performed on 13 microorganisms, aiming Periconia atropurpurea asapotential source of esterase for biotransformation of short chain esters. The results also evidenced that this methodology showed to be efficient, fast, cheap, having low consumption of reagents and easy development, and can be applied to screen carboxylic-ester hydrolases in a large number of microorganisms.

  5. A carboxylesterase, Esterase-6, modulates sensory physiological and behavioral response dynamics to pheromone in Drosophila

    Directory of Open Access Journals (Sweden)

    Chertemps Thomas

    2012-06-01

    Full Text Available Abstract Background Insects respond to the spatial and temporal dynamics of a pheromone plume, which implies not only a strong response to 'odor on', but also to 'odor off'. This requires mechanisms geared toward a fast signal termination. Several mechanisms may contribute to signal termination, among which odorant-degrading enzymes. These enzymes putatively play a role in signal dynamics by a rapid inactivation of odorants in the vicinity of the sensory receptors, although direct in vivo experimental evidences are lacking. Here we verified the role of an extracellular carboxylesterase, esterase-6 (Est-6, in the sensory physiological and behavioral dynamics of Drosophila melanogaster response to its pheromone, cis-vaccenyl acetate (cVA. Est-6 was previously linked to post-mating effects in the reproductive system of females. As Est-6 is also known to hydrolyze cVA in vitro and is expressed in the main olfactory organ, the antenna, we tested here its role in olfaction as a putative odorant-degrading enzyme. Results We first confirm that Est-6 is highly expressed in olfactory sensilla, including cVA-sensitive sensilla, and we show that expression is likely associated with non-neuronal cells. Our electrophysiological approaches show that the dynamics of olfactory receptor neuron (ORN responses is strongly influenced by Est-6, as in Est-6° null mutants (lacking the Est-6 gene cVA-sensitive ORN showed increased firing rate and prolonged activity in response to cVA. Est-6° mutant males had a lower threshold of behavioral response to cVA, as revealed by the analysis of two cVA-induced behaviors. In particular, mutant males exhibited a strong decrease of male-male courtship, in association with a delay in courtship initiation. Conclusions Our study presents evidence that Est-6 plays a role in the physiological and behavioral dynamics of sex pheromone response in Drosophila males and supports a role of Est-6 as an odorant-degrading enzyme (ODE in male

  6. Medium Density Fibreboard Made of Acetylated Sludge from Paper Mill

    Directory of Open Access Journals (Sweden)

    Luthfi Hakim

    2013-03-01

    Full Text Available Research of using sludge as raw material for making medium density fibreboard (MDF was useful to create additional value of sludge. The objective of the research was to evaluate physical properties, mechanical properties, and durability of MDF from acetylated sludge in 4 levels of acetate anhydride (0%, 3%, 5%, and 7% with 3 replicates. The MDF was made using dry process. After materials were mixed with adhesives, they were pressed using hotpress under 170 oC temperature and 45 Pa pressure for 25 minutes. The size of the MDF sample was 25 cm x 20 cm x 1 cm with 0.8 g/cm3 density. The physical properties (density, moisture content, water absorption, thickness swelling and mechanical properties (modulus of elasticity, modulus of rupture, internal bond, screw holding power was tested based on JIS A 5905-2003 standard. The durability was evaluated using SNI 01-7207-2006. All physical properties of MDF fulfill JIS A 5905-2003. Acetate anhydride decreased the moisture content value of MDF. On the other hand, all mechanical properties did not fulfill the standard. That was caused by calcium carbonate in sludge that blocked the adhesion between sludge fibres. The durability of MDF tested here was classified Class I which is very resistant to termites.

  7. Experimental thermochemical study of 3-acetyl-2-methyl-5-phenylthiophene

    International Nuclear Information System (INIS)

    Ribeiro da Silva, Manuel A.V.; Santos, Ana Filipa L.O.M.

    2010-01-01

    The standard (p 0 =0.1MPa) massic energy of combustion, in oxygen, of the crystalline 3-acetyl-2-methyl-5-phenylthiophene was measured, at T = 298.15 K, by rotating-bomb combustion calorimetry, from which the standard molar enthalpy of formation, in the condensed phase, was calculated as Δ f H m 0 (cr)=-(104.3±3.1)kJ.mol -1 . The corresponding standard molar enthalpy of sublimation, at T = 298.15 K, Δ cr g H m 0 =(108.9±0.4)kJ.mol -1 , was derived by the Clausius-Clapeyron equation, from the temperature dependence of the vapour pressures of this compound, measured by the Knudsen effusion mass-loss technique. From the results presented above, the standard molar enthalpy of formation, in the gaseous phase, at T = 298.15 K, was derived, Δ f H m 0 (g)=(4.6±3.1)kJ.mol -1 . This value, in conjunction with the literature values of the experimental enthalpies of formation of thiophene, 2-methylthiophene, and 3-acetylthiophene, was used to predict the enthalpic increment due to the introduction of a phenyl group in the position 2- of the thiophene ring. The calculated increment was compared with the corresponding ones in benzene and pyridine derivatives.

  8. Acetyl analogs of combretastatin A-4: synthesis and biological studies.

    Science.gov (United States)

    Babu, Balaji; Lee, Megan; Lee, Lauren; Strobel, Raymond; Brockway, Olivia; Nickols, Alexis; Sjoholm, Robert; Tzou, Samuel; Chavda, Sameer; Desta, Dereje; Fraley, Gregory; Siegfried, Adam; Pennington, William; Hartley, Rachel M; Westbrook, Cara; Mooberry, Susan L; Kiakos, Konstantinos; Hartley, John A; Lee, Moses

    2011-04-01

    The combretastatins have received significant attention because of their simple chemical structures, excellent antitumor efficacy and novel antivascular mechanisms of action. Herein, we report the synthesis of 20 novel acetyl analogs of CA-4 (1), synthesized from 3,4,5-trimethoxyphenylacetone that comprises the A ring of CA-4 with different aromatic aldehydes as the B ring. Molecular modeling studies indicate that these new compounds possess a 'twisted' conformation similar to CA-4. The new analogs effectively inhibit the growth of human and murine cancer cells. The most potent compounds 6k, 6s and 6t, have IC(50) values in the sub-μM range. Analog 6t has an IC(50) of 182 nM in MDA-MB-435 cells and has advantages over earlier analogs due to its enhanced water solubility (456 μM). This compound initiates microtubule depolymerization with an EC(50) value of 1.8 μM in A-10 cells. In a murine L1210 syngeneic tumor model 6t had antitumor activity and no apparent toxicity. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. Isolation of Acetyl Aleuritolic Acid from Pimeleodendron Griffithianum (Euphorbiaceae)

    International Nuclear Information System (INIS)

    Rosmawati Abdul Aziz; Norizan Ahmat; Rosmawati Abdul Aziz; Norizan Ahmat

    2016-01-01

    Pimeleodendron griffithianum, locally known as Perah ikan is available in Thailand, Peninsular Malaysia and Sumatra. The plant can be found in wet forest, secondary forest, pole forest, semi-swamps, and along logging roads or on hill slopes. In Malaysia, the stem of P. griffithianum being used as commercial timber. So far, no chemical constituents have been reported from P. griffithianum. In this study, a phytochemical study was conducted on the stem bark of P. griffithianum. The stem bark was obtained from Sungai Siput, Perak. The cleaned, chopped and dried stem-bark was extracted using acetone for several times. The separation of the components was carried out using vacuum liquid chromatography (VLC) followed by recrystallization method. The structure of the pure compound was elucidated by spectroscopic methods including nuclear magnetic resonance (NMR), ultraviolate - visible (UV-Vis) and comparison with literature. One compound (1) was isolated and identified as acetyl aleuritolic acid. This compound was reported for the first time from this plant. (author)

  10. In vivo labelling of acetyl-aspartyl peptides in mouse brain from intracranially and intracranially and intraperitoneally administered acetyl-L-[U-14C]aspartate

    International Nuclear Information System (INIS)

    Sinichkin, A.; Sterri, S.; Edminson, P.D.; Reichelt, K.L.; Kvamme, E.

    1977-01-01

    Following intracranial and intraperitoneal injection of acetyl-L-[U- 14 C]aspartate into mice about 5% and 0.7% of the radioactivity, respectively, was recovered from the brain after 30 min. On chromatographic separation of the cationic and anionic compounds on a Dowex 50 column, the former fraction contained about 60% of the radioactivity, predominantly as labelled asparate and glutamate. The anionic compounds, containing 20% of the labelled compounds, were fractionated in several chromatographic systems and resolved into a great variety of labelled peptidic compounds of which five acetyl-[U 14 ]aspartyl peptides, containing two to four amino acids, were purified. One of these, acetyl-aspartyl glutamine, has not previously been found in brain. (author)

  11. Acetylated rice starches films with different levels of amylose: Mechanical, water vapor barrier, thermal, and biodegradability properties.

    Science.gov (United States)

    Colussi, Rosana; Pinto, Vânia Zanella; El Halal, Shanise Lisie Mello; Biduski, Bárbara; Prietto, Luciana; Castilhos, Danilo Dufech; Zavareze, Elessandra da Rosa; Dias, Alvaro Renato Guerra

    2017-04-15

    Biodegradable films from native or acetylated starches with different amylose levels were prepared. The films were characterized according to the mechanical, water vapor barrier, thermal, and biodegradability properties. The films from acetylated high amylose starches had higher moisture content and water solubility than the native high amylose starch film. However, the acetylation did not affect acid solubility of the films, regardless of the amylose content. Films made from high and medium amylose rice starches were obtained; however low amylose rice starches, whether native or acetylated, did not form films with desirable characteristics. The acetylation decreased the tensile strength and increased the elongation of the films. The acetylated starch-based films had a lower decomposition temperature and higher thermal stability than native starch films. Acetylated starches films exhibited more rapid degradation as compared with the native starches films. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Acetylation of spermidine and methylglyoxal bis(guanylhydrazone) in baby-hamster kidney cells (BHK-21/C13).

    Science.gov (United States)

    Wallace, H M; Nuttall, M E; Robinson, F C

    1988-01-01

    Treatment of BHK-21/C13 cells with methylglyoxal bis(guanylhydrazone) (MGBG) induced the cytosolic form of spermidine N1-acetyltransferase. It stabilized the enzyme against proteolytic degradation, but the drug did not affect the enzyme activity in vitro. MGBG was itself acetylated by BHK-21/C13 cells, but at only one-tenth the rate at which spermidine was acetylated. Acetylation occurred almost exclusively in the nuclear fraction. The product was identified as N-acetyl-MGBG by h.p.l.c., by using [3H]acetyl-CoA and [14C]MGBG as co-substrates. The results suggest that the acetylation of MGBG by BHK-21/C13 cells occurs by a different acetyltransferase enzyme from that which acetylates spermidine. PMID:3421945

  13. Proteome-wide analysis of lysine acetylation suggests its broad regulatory scope in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Henriksen, Peter; Wagner, Sebastian Alexander; Weinert, Brian Tate

    2012-01-01

    Post-translational modification of proteins by lysine acetylation plays important regulatory roles in living cells. The budding yeast Saccharomyces cerevisiae is a widely used unicellular eukaryotic model organism in biomedical research. S. cerevisiae contains several evolutionary conserved lysine...... acetyltransferases and deacetylases. However, only a few dozen acetylation sites in S. cerevisiae are known, presenting a major obstacle for further understanding the regulatory roles of acetylation in this organism. Here we use high resolution mass spectrometry to identify about 4000 lysine acetylation sites in S....... cerevisiae. Acetylated proteins are implicated in the regulation of diverse cytoplasmic and nuclear processes including chromatin organization, mitochondrial metabolism, and protein synthesis. Bioinformatic analysis of yeast acetylation sites shows that acetylated lysines are significantly more conserved...

  14. An MRM-based workflow for absolute quantitation of lysine-acetylated metabolic enzymes in mouse liver.

    Science.gov (United States)

    Xu, Leilei; Wang, Fang; Xu, Ying; Wang, Yi; Zhang, Cuiping; Qin, Xue; Yu, Hongxiu; Yang, Pengyuan

    2015-12-07

    As a key post-translational modification mechanism, protein acetylation plays critical roles in regulating and/or coordinating cell metabolism. Acetylation is a prevalent modification process in enzymes. Protein acetylation modification occurs in sub-stoichiometric amounts; therefore extracting biologically meaningful information from these acetylation sites requires an adaptable, sensitive, specific, and robust method for their quantification. In this work, we combine immunoassays and multiple reaction monitoring-mass spectrometry (MRM-MS) technology to develop an absolute quantification for acetylation modification. With this hybrid method, we quantified the acetylation level of metabolic enzymes, which could demonstrate the regulatory mechanisms of the studied enzymes. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of protein acetylation in physiology and pathophysiology.

  15. Identification and purification of O-acetyl-L-serine sulphhydrylase in Penicillium chrysogenum

    DEFF Research Database (Denmark)

    østergaard, Simon; Theilgaard, Hanne Birgitte; Nielsen, Jens Bredal

    1998-01-01

    We have demonstrated that Penicillium chrysogenum possesses the L-cysteine biosynthetic enzyme O-acetyI-L-serine sulphhydrylase (EC 4.2.99.8) of the direct sulphhydrylation pathway. The finding of this enzyme, and thus the presence of the direct sulphhydrylation pathway in P. chrysogenum, creates...... the potential for increasing the overall yield in penicillin production by enhancing the enzymatic activity of this microorganism. Only O-acetyl-L-serine sulphhydrylase and O-acetyl-L-homoserine sulphhydrylase (EC 4.2.99.10) have been demonstrated to use O-acetyl-L-serine as substrate for the formation of L-cysteine....... The purified enzyme did not catalyse the formation of L-homocysteine from O-acetyl-L-homoserine and sulphide, excluding the possibility that the purified enzyme was O-acetyI-L-homoserine sulphhydrylase with multiple substrate specificity. The purification enhanced the enzymatic specific activity 93-fold...

  16. Maintenance of Glucose Homeostasis through Acetylation of the Metabolic Transcriptional Coactivator PGC-1alpha

    National Research Council Canada - National Science Library

    Puigserver, Pere

    2008-01-01

    The main purpose of this proposal is to test the hypothesis that acetylation of PGC-1alpha by GCN5 and associated proteins, Pc3 and WDR18, is a key regulatory modification that controls hepatic glucose production...

  17. Maintenance of Glucose Homeostasis through Acetylation of the Metabolic Transcriptional Coactivator PGC-1alpha

    National Research Council Canada - National Science Library

    Puigserver, Pere

    2007-01-01

    ... hepatic glucose production. This investigation has a define scope to specifically test how these proteins control the acetylation status of PGC-1alpha and what is the functional effect in blood glucose levels...

  18. Interactions of acetylated histones with DNA as revealed by UV laser induced histone-DNA crosslinking

    International Nuclear Information System (INIS)

    Stefanovsky, V.Yu.; Dimitrov, S.I.; Angelov, D.; Pashev, I.G.

    1989-01-01

    The interaction of acetylated histones with DNA in chromatin has been studied by UV laser-induced crosslinking histones to DNA. After irradiation of the nuclei, the covalently linked protein-DNA complexes were isolated and the presence of histones in them demonstrated immunochemically. When chromatin from irradiated nuclei was treated with clostripain, which selectively cleaved the N-terminal tails of core histones, no one of them was found covalently linked to DNA, thus showing that crosslinking proceeded solely via the N-terminal regions. However, the crosslinking ability of the laser was preserved both upon physiological acetylation of histones, known to be restricted to the N-terminal tails, and with chemically acetylated chromatin. This finding is direct evidence that the postsynthetic histone acetylation does not release the N-terminal tails from interaction with DNA

  19. Monitoring the effect of belinostat in solid tumors by H4 acetylation

    DEFF Research Database (Denmark)

    Marquard, L.; Petersen, K.D.; Persson, M.

    2008-01-01

    after treatment with HDAC inhibitors, and could thus be used as a marker for monitoring cellular response to HDAC inhibitor treatment. Here we describe the utility of a newly described monoclonal antibody against acetylated H4 for immunohistochemistry on paraffin-embedded fine needle biopsies from nude...... acetylation in fine needle biopsies using the T25 antibody may prove useful in monitoring HDAC inhibitor efficacy in clinical trials involving humans with solid tumors Udgivelsesdato: 2008/5...

  20. Peripheral effects of FAAH deficiency on fuel and energy homeostasis: role of dysregulated lysine acetylation.

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    Bhavapriya Vaitheesvaran

    Full Text Available FAAH (fatty acid amide hydrolase, primarily expressed in the liver, hydrolyzes the endocannabinoids fatty acid ethanolamides (FAA. Human FAAH gene mutations are associated with increased body weight and obesity. In our present study, using targeted metabolite and lipid profiling, and new global acetylome profiling methodologies, we examined the role of the liver on fuel and energy homeostasis in whole body FAAH(-/- mice.FAAH(-/- mice exhibit altered energy homeostasis demonstrated by decreased oxygen consumption (Indirect calorimetry. FAAH(-/- mice are hyperinsulinemic and have adipose, skeletal and hepatic insulin resistance as indicated by stable isotope phenotyping (SIPHEN. Fed state skeletal muscle and liver triglyceride levels was increased 2-3 fold, while glycogen was decreased 42% and 57% respectively. Hepatic cholesterol synthesis was decreased 22% in FAAH(-/- mice. Dysregulated hepatic FAAH(-/- lysine acetylation was consistent with their metabolite profiling. Fasted to fed increases in hepatic FAAH(-/- acetyl-CoA (85%, p<0.01 corresponded to similar increases in citrate levels (45%. Altered FAAH(-/- mitochondrial malate dehydrogenase (MDH2 acetylation, which can affect the malate aspartate shuttle, was consistent with our observation of a 25% decrease in fed malate and aspartate levels. Decreased fasted but not fed dihydroxyacetone-P and glycerol-3-P levels in FAAH(-/- mice was consistent with a compensating contribution from decreased acetylation of fed FAAH(-/- aldolase B. Fed FAAH(-/- alcohol dehydrogenase (ADH acetylation was also decreased.Whole body FAAH deletion contributes to a pre-diabetic phenotype by mechanisms resulting in impairment of hepatic glucose and lipid metabolism. FAAH(-/- mice had altered hepatic lysine acetylation, the pattern sharing similarities with acetylation changes reported with chronic alcohol treatment. Dysregulated hepatic lysine acetylation seen with impaired FAA hydrolysis could support the liver