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Sample records for acid esterase fae

  1. A feruloyl esterase (FAE) characterized by relatively high thermostability from the edible mushroom Russula virescens.

    PubMed

    Wang, Li; Zhang, Rui; Ma, Zengqiang; Wang, Hexiang; Ng, Tzibun

    2014-01-01

    A monomeric feruloyl esterase (FAE) with a molecular mass of 62 kDa was acquired from fresh fruiting bodies of the edible mushroom Russula virescens. The isolation procedure involved ion exchange chromatography on CM-cellulose, Q-Sepharose, and SP-Sepharose and finally fast protein liquid chromatography-gel filtration on Superdex 75. Two amino acid sequences were obtained after tryptic digestion, and they both showed some homology with the esterase of some fungi. Maximal activity was observed at pH 5.0 and at 50 °C. The enzyme displayed relatively high thermostability as evidenced by over 70 % residual activity at 70 °C and about 34 % residual activity at 80 °C. The K m and V max for this enzyme on methyl ferulate were 0.19 mM and 1.65 U/mg proteins, respectively. The purified FAE prefers methyl ferulate over methyl caffeate and is least active on methyl p-coumarate. The FAE activity was not significantly affected by the presence of cations such as Mn(2+), Ca(2+), Cd(2+), Zn(2+), Mg(2+), Cu(2+), and K(+) ions but inhibited by Al(3+), Hg(2+), Fe(2+), and Pb(2+) ions at a tested concentration of 2. 5 mM.

  2. Heterologous Expression of Two Ferulic Acid Esterases from Penicillium funiculosum

    NASA Astrophysics Data System (ADS)

    Knoshaug, Eric P.; Selig, Michael J.; Baker, John O.; Decker, Stephen R.; Himmel, Michael E.; Adney, William S.

    Two recombinant ferulic acid esterases from Penicillium funiculosum produced in Aspergillus awamori were evaluated for their ability to improve the digestibility of pretreated corn stover. The genes, faeA and faeB, were cloned from P. funiculosum and expressed in A. awamori using their native signal sequences. Both enzymes contain a catalytic domain connected to a family 1 carbohydrate-binding module by a threonine-rich linker peptide. Interestingly, the carbohydrate binding-module is N-terminal in FaeA and C-terminal in FaeB. The enzymes were purified to homogeneity using column chromatography, and their thermal stability was characterized by differential scanning microcalorimetry. We evaluated both enzymes for their potential to enhance the cellulolytic activity of purified Trichoderma reesei Cel7A on pretreated corn stover.

  3. Heterologous Expression of Two Ferulic Acid Esterases from Penicillium Funiculosum

    SciTech Connect

    Knoshaug, E. P.; Selig, M. J.; Baker, J. O.; Decker, S. R.; Himmel, M. E.; Adney, W. S.

    2008-01-01

    Two recombinant ferulic acid esterases from Penicillium funiculosum produced in Aspergillus awamori were evaluated for their ability to improve the digestibility of pretreated corn stover. The genes, faeA and faeB, were cloned from P. funiculosum and expressed in A. awamori using their native signal sequences. Both enzymes contain a catalytic domain connected to a family 1 carbohydrate-binding module by a threonine-rich linker peptide. Interestingly, the carbohydrate binding-module is N-terminal in FaeA and C-terminal in FaeB. The enzymes were purified to homogeneity using column chromatography, and their thermal stability was characterized by differential scanning microcalorimetry. We evaluated both enzymes for their potential to enhance the cellulolytic activity of purified Trichoderma reesei Cel7A on pretreated corn stover.

  4. The ferulic acid esterases of Chrysosporium lucknowense C1: purification, characterization and their potential application in biorefinery.

    PubMed

    Kühnel, S; Pouvreau, L; Appeldoorn, M M; Hinz, S W A; Schols, H A; Gruppen, H

    2012-01-01

    Three ferulic acid esterases from the filamentous fungus Chrysosporium lucknowense C1 were purified and characterized. The enzymes were most active at neutral pH and temperatures up to 45 °C. All enzymes released ferulic acid and p-coumaric acid from a soluble corn fibre fraction. Ferulic acid esterases FaeA1 and FaeA2 could also release complex dehydrodiferulic acids and dehydrotriferulic acids from corn fibre oligomers, but released only 20% of all ferulic acid present in sugar beet pectin oligomers. Ferulic acid esterase FaeB2 released almost no complex ferulic acid oligomers from corn fibre oligomers, but 60% of all ferulic acid from sugar beet pectin oligomers. The ferulic acid esterases were classified based on both, sequence similarity and their activities toward synthetic substrates. The type A ferulic acid esterases FaeA1 and FaeA2 are the first members of the phylogenetic subfamily 5 to be biochemically characterized. Type B ferulic acid esterase FaeB2 is a member of subfamily 6.

  5. Engineering Saccharomyces cerevisiae to produce feruloyl esterase for the release of ferulic acid from switchgrass

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Aspergillus niger ferulic acid esterase gene (faeA) was cloned into Saccharomyces cerevisiae via a yeast expression vector, resulting in efficient expression and secretion of the enzyme in the medium. The recombinant enzyme was purified to homogeneity by anion-exchange and hydrophobic interactio...

  6. Overexpression of Aspergillus tubingensis faeA in protease-deficient Aspergillus niger enables ferulic acid production from plant material.

    PubMed

    Zwane, Eunice N; Rose, Shaunita H; van Zyl, Willem H; Rumbold, Karl; Viljoen-Bloom, Marinda

    2014-06-01

    The production of ferulic acid esterase involved in the release of ferulic acid side groups from xylan was investigated in strains of Aspergillus tubingensis, Aspergillus carneus, Aspergillus niger and Rhizopus oryzae. The highest activity on triticale bran as sole carbon source was observed with the A. tubingensis T8.4 strain, which produced a type A ferulic acid esterase active against methyl p-coumarate, methyl ferulate and methyl sinapate. The activity of the A. tubingensis ferulic acid esterase (AtFAEA) was inhibited twofold by glucose and induced twofold in the presence of maize bran. An initial accumulation of endoglucanase was followed by the production of endoxylanase, suggesting a combined action with ferulic acid esterase on maize bran. A genomic copy of the A. tubingensis faeA gene was cloned and expressed in A. niger D15#26 under the control of the A. niger gpd promoter. The recombinant strain has reduced protease activity and does not acidify the media, therefore promoting high-level expression of recombinant enzymes. It produced 13.5 U/ml FAEA after 5 days on autoclaved maize bran as sole carbon source, which was threefold higher than for the A. tubingensis donor strain. The recombinant AtFAEA was able to extract 50 % of the available ferulic acid from non-pretreated maize bran, making this enzyme suitable for the biological production of ferulic acid from lignocellulosic plant material. PMID:24664515

  7. Overexpression of Aspergillus tubingensis faeA in protease-deficient Aspergillus niger enables ferulic acid production from plant material.

    PubMed

    Zwane, Eunice N; Rose, Shaunita H; van Zyl, Willem H; Rumbold, Karl; Viljoen-Bloom, Marinda

    2014-06-01

    The production of ferulic acid esterase involved in the release of ferulic acid side groups from xylan was investigated in strains of Aspergillus tubingensis, Aspergillus carneus, Aspergillus niger and Rhizopus oryzae. The highest activity on triticale bran as sole carbon source was observed with the A. tubingensis T8.4 strain, which produced a type A ferulic acid esterase active against methyl p-coumarate, methyl ferulate and methyl sinapate. The activity of the A. tubingensis ferulic acid esterase (AtFAEA) was inhibited twofold by glucose and induced twofold in the presence of maize bran. An initial accumulation of endoglucanase was followed by the production of endoxylanase, suggesting a combined action with ferulic acid esterase on maize bran. A genomic copy of the A. tubingensis faeA gene was cloned and expressed in A. niger D15#26 under the control of the A. niger gpd promoter. The recombinant strain has reduced protease activity and does not acidify the media, therefore promoting high-level expression of recombinant enzymes. It produced 13.5 U/ml FAEA after 5 days on autoclaved maize bran as sole carbon source, which was threefold higher than for the A. tubingensis donor strain. The recombinant AtFAEA was able to extract 50 % of the available ferulic acid from non-pretreated maize bran, making this enzyme suitable for the biological production of ferulic acid from lignocellulosic plant material.

  8. Characterization of FAE1 in the zero erucic acid germplasm of Brassica rapa L.

    PubMed

    Yan, Guixin; Li, Dan; Cai, Mengxian; Gao, Guizhen; Chen, Biyun; Xu, Kun; Li, Jun; Li, Feng; Wang, Nian; Qiao, Jiangwei; Li, Hao; Zhang, Tianyao; Wu, Xiaoming

    2015-06-01

    The modification of erucic acid content in seeds is one of the major goals for quality breeding in oil-yielding Brassica species. However, few low erucic acid (LEA) resources are available, and novel LEA genetic resources are being sought. Fatty acid elongase 1 (FAE1) is the key gene that controls erucic acid synthesis. However, the mechanism for erucic acid synthesis in B. rapa lacks systematic study. Here, we isolated zero erucic acid lines from 1981 Chinese landraces of B. rapa and found that the formation of LEA is not attributable to variations in FAE1 coding sequences, as reported for B. napus, but may be attributable to the decrease in FAE1 expression. Moreover, the FAE1 promoter sequences of LEA and high erucic acid materials shared 95% similarity. Twenty-eight bases deletions (containing a 24-base AT-rich region) were identified approximately 1300 bp upstream from the FAE1 start codon in the LEA accessions. The genotype with the deletions co-segregated with the LEA trait in the segregating population. This study isolated an LEA B. rapa resource that can be exploited in Brassica cultivation. The promoter variations might modify the expression level of FAE1, and the results shed light on novel regulation mechanisms for erucic acid synthesis.

  9. Characterization of FAE1 in the zero erucic acid germplasm of Brassica rapa L.

    PubMed Central

    Yan, Guixin; Li, Dan; Cai, Mengxian; Gao, Guizhen; Chen, Biyun; Xu, Kun; Li, Jun; Li, Feng; Wang, Nian; Qiao, Jiangwei; Li, Hao; Zhang, Tianyao; Wu, Xiaoming

    2015-01-01

    The modification of erucic acid content in seeds is one of the major goals for quality breeding in oil-yielding Brassica species. However, few low erucic acid (LEA) resources are available, and novel LEA genetic resources are being sought. Fatty acid elongase 1 (FAE1) is the key gene that controls erucic acid synthesis. However, the mechanism for erucic acid synthesis in B. rapa lacks systematic study. Here, we isolated zero erucic acid lines from 1981 Chinese landraces of B. rapa and found that the formation of LEA is not attributable to variations in FAE1 coding sequences, as reported for B. napus, but may be attributable to the decrease in FAE1 expression. Moreover, the FAE1 promoter sequences of LEA and high erucic acid materials shared 95% similarity. Twenty-eight bases deletions (containing a 24-base AT-rich region) were identified approximately 1300 bp upstream from the FAE1 start codon in the LEA accessions. The genotype with the deletions co-segregated with the LEA trait in the segregating population. This study isolated an LEA B. rapa resource that can be exploited in Brassica cultivation. The promoter variations might modify the expression level of FAE1, and the results shed light on novel regulation mechanisms for erucic acid synthesis. PMID:26175623

  10. Directed tagging of the Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene with the maize transposon activator.

    PubMed Central

    James, D W; Lim, E; Keller, J; Plooy, I; Ralston, E; Dooner, H K

    1995-01-01

    The FATTY ACID ELONGATION1 (FAE1) gene of Arabidopsis is required for the synthesis of very long chain fatty acids in the seed. The product of the FAE1 gene is presumed to be a condensing enzyme that extends the chain length of fatty acids from C18 to C20 and C22. We report here the cloning of FAE1 by directed transposon tagging with the maize element Activator (Ac). An unstable fae1 mutant was isolated in a line carrying Ac linked to the FAE1 locus on chromosome 4. Cosegregation and reversion analyses established that the new mutant was tagged by Ac. A DNA fragment flanking Ac was cloned by inverse polymerase chain reaction and used to isolate FAE1 genomic clones and a cDNA clone from a library made from immature siliques. The predicted amino acid sequence of the FAE1 protein shares homology with those of other condensing enzymes (chalcone synthase, stilbene synthases, and beta-ketoacyl-acyl carrier protein synthase III), supporting the notion that FAE1 is the structural gene for a synthase or condensing enzyme. FAE1 is expressed in developing seed, but not in leaves, as expected from the effect of the fae1 mutation on the fatty acid compositions of those tissues. PMID:7734965

  11. Contributions of a unique β-clamp to substrate recognition illuminates the molecular basis of exolysis in ferulic acid esterases.

    PubMed

    Gruninger, Robert J; Cote, Chris; McAllister, Tim A; Abbott, D Wade

    2016-04-01

    Lignocellulosic biomass is a promising renewable resource; however, deconstruction of this material is still the rate-limiting step. Major obstacles in the biocatalytic turnover of lignocellulose are ester-linked decorations that prevent access to primary structural polysaccharides. Enzymes targeting these esters represent promising biotools for increasing bioconversion efficiency. Ruminant livestock are unique in their ability to degrade lignocellulose through the action of their gut microbiome. The anaerobic fungi (phylum Neocallimastigomycota) are key members of this ecosystem that express a large repertoire of carbohydrate-active enzymes (CAZymes) with little sequence identity with characterized CAZymes [Lombard, Golaconda, Drula, Coutinho and Henrissat (2014) Nucleic Acids Res. 42: , D490-D495]. We have identified a carbohydrate esterase family 1 (CE1) ferulic acid esterase (FAE) belonging to Anaeromyces mucronatus(AmCE1/Fae1a), and determined its X-ray structure in both the presence [1.55 Å (1 Å=0.1 nm)] and absence (1.60 Å) of ferulic acid. AmCE1 adopts an α/β-hydrolase fold that is structurally conserved with bacterial FAEs, and possesses a unique loop, termed the β-clamp, that encloses the ligand. Isothermal titration calorimetry reveals that substrate binding is driven by enthalpic contributions, which overcomes a large entropic penalty. A comparative analysis of AmCE1 with related enzymes has uncovered the apparent structural basis for differential FAE activities targeting cross-linking ferulic acid conjugates compared with terminal decorations. Based on comparisons to structurally characterized FAEs, we propose that the β-clamp may define the structural basis of exolytic activities in FAEs. This provides a structure-based tool for predicting exolysis and endolysis in CE1. These insights hold promise for rationally identifying enzymes tailored for bioconversion of biomass with variations in cell wall composition.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  13. Feruloyl esterases from Schizophyllum commune to treat food industry side-streams.

    PubMed

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

    2016-11-01

    Agro-industrial side-streams are abundant and renewable resources of hydroxycinnamic acids with potential applications as antioxidants and preservatives in the food, health, cosmetic, and pharmaceutical industries. Feruloyl esterases (FAEs) from Schizophyllum commune were functionally expressed in Pichia pastoris with extracellular activities of 6000UL(-1). The recombinant enzymes, ScFaeD1 and ScFaeD2, released ferulic acid from destarched wheat bran and sugar beet pectin. Overnight incubation of coffee pulp released caffeic (>60%), ferulic (>80%) and p-coumaric acid (100%) indicating applicability for the valorization of food processing wastes and enhanced biomass degradation. Based on substrate specificity profiling and the release of diferulates from destarched wheat bran, the recombinant FAEs were characterized as type D FAEs. ScFaeD1 and ScFaeD2 preferably hydrolyzed feruloylated saccharides with ferulic acid esterified to the O-5 position of arabinose residues and showed an unprecedented ability to hydrolyze benzoic acid esters.

  14. Evolutionary Pattern of the FAE1 Gene in Brassicaceae and Its Correlation with the Erucic Acid Trait

    PubMed Central

    Li, Mimi; Peng, Bin; Guo, Haisong; Yan, Qinqin; Hang, Yueyu

    2013-01-01

    The fatty acid elongase 1 (FAE1) gene catalyzes the initial condensation step in the elongation pathway of VLCFA (very long chain fatty acid) biosynthesis and is thus a key gene in erucic acid biosynthesis. Based on a worldwide collection of 62 accessions representing 14 tribes, 31 genera, 51 species, 4 subspecies and 7 varieties, we conducted a phylogenetic reconstruction and correlation analysis between genetic variations in the FAE1 gene and the erucic acid trait, attempting to gain insight into the evolutionary patterns and the correlations between genetic variations in FAE1 and trait variations. The five clear, deeply diverged clades detected in the phylogenetic reconstruction are largely congruent with a previous multiple gene-derived phylogeny. The Ka/Ks ratio (<1) and overall low level of nucleotide diversity in the FAE1 gene suggest that purifying selection is the major evolutionary force acting on this gene. Sequence variations in FAE1 show a strong correlation with the content of erucic acid in seeds, suggesting a causal link between the two. Furthermore, we detected 16 mutations that were fixed between the low and high phenotypes of the FAE1 gene, which constitute candidate active sites in this gene for altering the content of erucic acid in seeds. Our findings begin to shed light on the evolutionary pattern of this important gene and represent the first step in elucidating how the sequence variations impact the production of erucic acid in plants. PMID:24358289

  15. Phenolic acid esterases, coding sequences and methods

    DOEpatents

    Blum, David L.; Kataeva, Irina; Li, Xin-Liang; Ljungdahl, Lars G.

    2002-01-01

    Described herein are four phenolic acid esterases, three of which correspond to domains of previously unknown function within bacterial xylanases, from XynY and XynZ of Clostridium thermocellum and from a xylanase of Ruminococcus. The fourth specifically exemplified xylanase is a protein encoded within the genome of Orpinomyces PC-2. The amino acids of these polypeptides and nucleotide sequences encoding them are provided. Recombinant host cells, expression vectors and methods for the recombinant production of phenolic acid esterases are also provided.

  16. Comparison of mesophilic and thermophilic feruloyl esterases: characterization of their substrate specificity for methyl phenylalkanoates.

    PubMed

    Topakas, Evangelos; Christakopoulos, Paul; Faulds, Craig B

    2005-02-23

    The active sites of feruloyl esterases from mesophilic and thermophilic sources were probed using methyl esters of phenylalkanoic acids. Only 13 out of 26 substrates tested were significant substrates for all the enzymes. Lengthening or shortening the aliphatic side chain while maintaining the same aromatic substitutions completely abolished activity for both enzymes, which demonstrates the importance of the correct distance between the aromatic group and the ester bond. Maintaining the phenylpropanoate structure but altering the substitutions of the aromatic ring demonstrated that the type-A esterase from the mesophilic fungus Fusarium oxysporum (FoFaeA) showed a preference for methoxylated substrates, in contrast to the type-B esterase from the same source (FoFaeB) and the thermophilic type-B (StFaeB) and type-C (StFaeC) from Sporotrichum thermophile, which preferred hydroxylated substrates. All four esterases hydrolyzed short chain aliphatic acid (C2-C4) esters of p-nitrophenol, but not the C12 ester of laurate. All the feruloyl esterases were able to release ferulic acid from the plant cell wall material in conjunction with a xylanase, but only the type-A esterase FoFaeA was effective in releasing the 5,5' form of diferulic acid. The thermophilic type-B esterase had a lower catalytic efficiency than its mesophilic counterpart, but released more ferulic acid from plant cell walls.

  17. Novel ferulate esterase from Gram-positive lactic acid bacteria and analyses of the recombinant enzyme produced in E. coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using a plate containing ethyl ferulate as sole carbon source, various bacteria cultures were screened for ferulate esterase (FAE). Among a dozen of species showing positive FAE, one Lactobacillus fermentum strain NRRL 1932 demonstrated the strongest activity. Using a published sequence of ferulate ...

  18. A halotolerant type A feruloyl esterase from Pleurotus eryngii.

    PubMed

    Nieter, Annabel; Haase-Aschoff, Paul; Linke, Diana; Nimtz, Manfred; Berger, Ralf G

    2014-03-01

    An extracellular feruloyl esterase (PeFaeA) from the culture supernatant of Pleurotus eryngii was purified to homogeneity using cation exchange, hydrophobic interaction, and size exclusion chromatography. The length of the complete coding sequence of PeFaeA was determined to 1668 bp corresponding to a protein of 555 amino acids. The catalytic triad of Ser-Glu-His demonstrated the uniqueness of the enzyme compared to previously published FAEs. The purified PeFaeA was a monomer with an estimated molecular mass of 67 kDa. Maximum feruloyl esterase (FAE) activity was observed at pH 5.0 and 50 °C, respectively. Metal ions (5 mM), except Hg(2+), had no significant influence on the enzyme activity. Substrate specificity profiling characterized the enzyme as a type A FAE preferring bulky natural substrates, such as feruloylated saccharides, rather than small synthetic ones. Km and kcat of the purified enzyme for methyl ferulate were 0.15 mM and 0.85 s(-1). In the presence of 3 M NaCl activity of the enzyme increased by 28 %. PeFaeA alone released only little ferulic acid from destarched wheat bran (DSWB), whereas after addition of Trichoderma viride xylanase the concentration increased more than 20 fold. PMID:24607359

  19. Cloning and functional characterization of the fatty acid elongase 1 (FAE1) gene from high erucic Crambe abyssinica cv. Prophet.

    PubMed

    Mietkiewska, Elzbieta; Brost, Jennifer M; Giblin, E Michael; Barton, Dennis L; Taylor, David C

    2007-09-01

    A genomic fatty acid elongation 1 (FAE1) clone was isolated from Crambe abyssinica. The genomic clone corresponds to a 1521-bp open reading frame, which encodes a protein of 507 amino acids. In yeast cells expression of CrFAE led to production of new very long chain monounsaturated fatty acids such as eicosenoic (20:1(delta11)) and erucic (22:1(delta13)) acids. Seed-specific expression in Arabidopsis thaliana resulted in up to a 12-fold increase in the proportion of erucic acid. On the other hand, in transgenic high-erucic Brassica carinata plants, the proportion of erucic acid was as high as 51.9% in the best transgenic line, a net increase of 40% compared to wild type. These results indicate that the CrFAE gene encodes a condensing enzyme involved in the biosynthesis of very long-chain fatty acids utilizing monounsaturated and saturated acyl substrates, with a strong capability for improving the erucic acid content.

  20. Lerisetron. FAES.

    PubMed

    Huckle, Richard

    2003-07-01

    Lerisetron is a 5-hydroxytryptamine3 receptor antagonist under development by FAES Farma for the potential treatment of emesis resulting from chemotherapy. Phase I trials of lerisetron were underway in Spain by 1994, and as of June 2000, the compound was in phase II trials in the UK. By the end of 2001, phase II trials had been completed and phase III trials had commenced. PMID:14619411

  1. Lerisetron. FAES.

    PubMed

    Huckle, Richard

    2003-07-01

    Lerisetron is a 5-hydroxytryptamine3 receptor antagonist under development by FAES Farma for the potential treatment of emesis resulting from chemotherapy. Phase I trials of lerisetron were underway in Spain by 1994, and as of June 2000, the compound was in phase II trials in the UK. By the end of 2001, phase II trials had been completed and phase III trials had commenced.

  2. A protease-insensitive feruloyl esterase from China Holstein cow rumen metagenomic library: expression, characterization, and utilization in ferulic acid release from wheat straw.

    PubMed

    Cheng, Fansheng; Sheng, Jiping; Cai, Ting; Jin, Jian; Liu, Wanzhen; Lin, Yanmei; Du, Yongxin; Zhang, Maoqiu; Shen, Lin

    2012-03-14

    A metagenomic library of China Holstein cow rumen microbes was constructed and screened for novel gene cluster. A novel feruloyl esterase (FAE) gene was identified with a length of 789 bp and encoded a protein displaying 56% identity to known esterase sequences. The gene was functionally expressed in Escherichia coli BL21 (DE3), and the total molecular weight of the recombined protein was 32.4 kDa. The purified enzyme showed a broad specificity against the four methyl esters of hydroxycinnamic acids and high activity (259.5 U/mg) to methyl ferulate at optimum conditions (pH 8.0, 40 °C). High thermal and pH stability were also observed. Moreover, the enzyme showed broad resistance to proteases. FAE-SH1 can enhance the release of ferulic acid from wheat straw with cellulase, β-1,4-endoxylanase, β-1,3-glucanase, and pectase. These features suggest FAE-SH1 as a good candidate to enhance biomass degradation and improve the health effects of food and forage.

  3. Seed-specific heterologous expression of a nasturtium FAE gene in Arabidopsis results in a dramatic increase in the proportion of erucic acid.

    PubMed

    Mietkiewska, Elzbieta; Giblin, E Michael; Wang, Song; Barton, Dennis L; Dirpaul, Joan; Brost, Jennifer M; Katavic, Vesna; Taylor, David C

    2004-09-01

    The fatty acid elongase [often designated FAE or beta-(or 3-) ketoacyl-CoA synthase] is a condensing enzyme and is the first component of the elongation complex involved in synthesis of erucic acid (22:1) in seeds of garden nasturtium (Tropaeolum majus). Using a degenerate primers approach, a cDNA of a putative embryo FAE was obtained showing high homology to known plant elongases. This cDNA contains a 1,512-bp open reading frame that encodes a protein of 504 amino acids. A genomic clone of the nasturtium FAE was isolated and sequence analyses indicated the absence of introns. Northern hybridization showed the expression of this nasturtium FAE gene to be restricted to the embryo. Southern hybridization revealed the nasturtium beta-ketoacyl-CoA synthase to be encoded by a small multigene family. To establish the function of the elongase homolog, the cDNA was introduced into two different heterologous chromosomal backgrounds (Arabidopsis and tobacco [Nicotiana tabacum]) under the control of a seed-specific (napin) promoter and the tandem 35S promoter, respectively. Seed-specific expression resulted in up to an 8-fold increase in erucic acid proportions in Arabidopsis seed oil, while constitutive expression in transgenic tobacco tissue resulted in increased proportions of very long chain saturated fatty acids. These results indicate that the nasturtium FAE gene encodes a condensing enzyme involved in the biosynthesis of very long chain fatty acids, utilizing monounsaturated and saturated acyl substrates. Given its strong and unique preference for elongating 20:1-CoA, the utility of the FAE gene product for directing or engineering increased synthesis of erucic acid is discussed.

  4. Probiotic Ferulic Acid Esterase Active Lactobacillus fermentum NCIMB 5221 APA Microcapsules for Oral Delivery: Preparation and in Vitro Characterization.

    PubMed

    Tomaro-Duchesneau, Catherine; Saha, Shyamali; Malhotra, Meenakshi; Coussa-Charley, Michael; Kahouli, Imen; Jones, Mitchell L; Labbé, Alain; Prakash, Satya

    2012-02-16

    Probiotics possess potential therapeutic and preventative effects for various diseases and metabolic disorders. One important limitation for the oral delivery of probiotics is the harsh conditions of the upper gastrointestinal tract (GIT) which challenge bacterial viability and activity. One proposed method to surpass this obstacle is the use of microencapsulation to improve the delivery of bacterial cells to the lower GIT. The aim of this study is to use alginate-poly-L-lysine-alginate (APA) microcapsules to encapsulate Lactobacillus fermentum NCIMB 5221 and characterize its enzymatic activity and viability through a simulated GIT. This specific strain, in previous research, was characterized for its inherent ferulic acid esterase (FAE) activity which could prove beneficial in the development of a therapeutic for the treatment and prevention of cancers and metabolic disorders. Our findings demonstrate that the APA microcapsule does not slow the mass transfer of substrate into and that of the FA product out of the microcapsule, while also not impairing bacterial cell viability. The use of simulated gastrointestinal conditions led to a significant 2.5 log difference in viability between the free (1.10 × 104 ± 1.00 × 103 cfu/mL) and the microencapsulated (5.50 × 106 ± 1.00 × 105 cfu/mL) L. fermentum NCIMB 5221 following exposure. The work presented here suggests that APA microencapsulation can be used as an effective oral delivery method for L. fermentum NCIMB 5221, a FAE-active probiotic strain.

  5. Feruloyl esterases from Schizophyllum commune to treat food industry side-streams.

    PubMed

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

    2016-11-01

    Agro-industrial side-streams are abundant and renewable resources of hydroxycinnamic acids with potential applications as antioxidants and preservatives in the food, health, cosmetic, and pharmaceutical industries. Feruloyl esterases (FAEs) from Schizophyllum commune were functionally expressed in Pichia pastoris with extracellular activities of 6000UL(-1). The recombinant enzymes, ScFaeD1 and ScFaeD2, released ferulic acid from destarched wheat bran and sugar beet pectin. Overnight incubation of coffee pulp released caffeic (>60%), ferulic (>80%) and p-coumaric acid (100%) indicating applicability for the valorization of food processing wastes and enhanced biomass degradation. Based on substrate specificity profiling and the release of diferulates from destarched wheat bran, the recombinant FAEs were characterized as type D FAEs. ScFaeD1 and ScFaeD2 preferably hydrolyzed feruloylated saccharides with ferulic acid esterified to the O-5 position of arabinose residues and showed an unprecedented ability to hydrolyze benzoic acid esters. PMID:27566510

  6. Characterization of a chimeric enzyme comprising feruloyl esterase and family 42 carbohydrate-binding module.

    PubMed

    Koseki, Takuya; Mochizuki, Keiji; Kisara, Hiroe; Miyanaga, Akimasa; Fushinobu, Shinya; Murayama, Tetsuya; Shiono, Yoshihito

    2010-03-01

    We engineered a chimeric enzyme (AwFaeA-CBM42) comprising of type-A feruloyl esterase from Aspergillus awamori (AwFaeA) and family 42 carbohydrate-binding module (AkCBM42) from glycoside hydrolase family 54 alpha-L-arabinofuranosidase of Aspergillus kawachii. The chimeric enzyme was successfully produced in Pichia pastoris and accumulated in the culture broth. The purified chimeric enzyme had an apparent relative molecular mass (M(r)) of 53,000. The chimeric enzyme binds to arabinoxylan; this indicates that the AkCBM42 in AwFaeA-CBM42 binds to arabinofuranose side chain moiety of arabinoxylan. The thermostability of the chimeric enzyme was greater than that of AwFaeA. No significant difference of the specific activity toward methyl ferulate was observed between the AwFaeA and chimeric enzyme, but the release of ferulic acid from insoluble arabinoxylan by the chimeric enzyme was approximately 4-fold higher than that achieved by AwFaeA alone. In addition, the chimeric enzyme and xylanase acted synergistically for the degradation of arabinoxylan. In conclusion, the findings of our study demonstrated that the components of the AwFaeA-CBM42 chimeric enzyme act synergistically to bring about the degradation of complex substrates and that the family 42 carbohydrate-binding module has potential for application in the degradation of polysaccharides.

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

    PubMed Central

    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 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 short listing the

  8. Feruloyl esterase production by Aspergillus terreus CECT 2808 and subsequent application to enzymatic hydrolysis.

    PubMed

    Pérez-Rodríguez, N; Moreira, C D; Torrado Agrasar, A; Domínguez, J M

    2016-09-01

    Ferulic acid esterases (FAE) were produced by Aspergillus terreus CECT 2808 from vine trimming shoots (VTS) and corn cob. Later, the fungal extracts thus obtained were used to enzymatically release ferulic acid (FA) from both substrates. Our findings showed a higher FAE activity in the enzymatic extracts produced on corn cob (0.070±0.004U/mL). Nevertheless, the enzymatic extracts produced on VTS demonstrated a better performance for FA release from both corn cob (2.05±0.01mg/g) and VTS (0.19±0.003mg/g). This result was probably because of the higher xylanase/FAE ratio determined in VTS extract. Therefore, an additional assay was carried out by supplementing corn cob extract with a commercial xylanase to test the influence of FAE/xylanase ratio in FA release. The results revealed the relevance of the FAE/xylanase ratio for an optimal FA release. PMID:27444329

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

    PubMed Central

    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

  10. Biomass-to-bio-products application of feruloyl esterase from Aspergillus clavatus.

    PubMed

    Damásio, André R L; Braga, Cleiton Márcio Pinto; Brenelli, Lívia B; Citadini, Ana Paula; Mandelli, Fernanda; Cota, Junio; de Almeida, Rodrigo Ferreira; Salvador, Victor Hugo; Paixao, Douglas Antonio Alvaredo; Segato, Fernando; Mercadante, Adriana Zerlotti; de Oliveira Neto, Mario; do Santos, Wanderley Dantas; Squina, Fabio M

    2013-08-01

    The structural polysaccharides contained in plant cell walls have been pointed to as a promising renewable alternative to petroleum and natural gas. Ferulic acid is a ubiquitous component of plant polysaccharides, which is found in either monomeric or dimeric forms and is covalently linked to arabinosyl residues. Ferulic acid has several commercial applications in food and pharmaceutical industries. The study herein introduces a novel feruloyl esterase from Aspergillus clavatus (AcFAE). Along with a comprehensive functional and biophysical characterization, the low-resolution structure of this enzyme was also determined by small-angle X-ray scattering. In addition, we described the production of phenolic compounds with antioxidant capacity from wheat arabinoxylan and sugarcane bagasse using AcFAE. The ability to specifically cleave ester linkages in hemicellulose is useful in several biotechnological applications, including improved accessibility to lignocellulosic enzymes for biofuel production.

  11. A Chlorogenic Acid Esterase with a Unique Substrate Specificity from Ustilago maydis

    PubMed Central

    Haase-Aschoff, Paul; Kelle, Sebastian; Linke, Diana; Krings, Ulrich; Popper, Lutz; Berger, Ralf G.

    2014-01-01

    An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83 mM−1 s−1, 7.63 mM−1 s−1, 3.83 mM−1 s−1 and 3.75 mM−1 s−1, respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme. PMID:25548041

  12. A chlorogenic acid esterase with a unique substrate specificity from Ustilago maydis.

    PubMed

    Nieter, Annabel; Haase-Aschoff, Paul; Kelle, Sebastian; Linke, Diana; Krings, Ulrich; Popper, Lutz; Berger, Ralf G

    2015-03-01

    An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83 mM(-1) s(-1), 7.63 mM(-1) s(-1), 3.83 mM(-1) s(-1) and 3.75 mM(-1) s(-1), respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme.

  13. 3-d structure-based amino acid sequence alignment of esterases, lipases and related proteins

    SciTech Connect

    Gentry, M.K.; Doctor, B.P.; Cygler, M.; Schrag, J.D.; Sussman, J.L.

    1993-05-13

    Acetylcholinesterase and butyrylcholinesterase, enzymes with potential as pretreatment drugs for organophosphate toxicity, are members of a larger family of homologous proteins that includes carboxylesterases, cholesterol esterases, lipases, and several nonhydrolytic proteins. A computer-generated alignment of 18 of the proteins, the acetylcholinesases, butyrylcholinesterases, carboxylesterases, some esterases, and the nonenzymatic proteins has been previously presented. More recently, the three-dimensional structures of two enzymes enzymes in this group, acetylcholinesterase from Torpedo californica and lipase from Geotrichum candidum, have been determined. Based on the x-ray structures and the superposition of these two enzymes, it was possible to obtain an improved amino acid sequence alignment of 32 members of this family of proteins. Examination of this alignment reveals that 24 amino acids are invariant in all of the hydrolytic proteins, and an additional 49 are well conserved. Conserved amino acids include those of the active site, the disulfide bridges, the salt bridges, in the core of the proteins, and at the edges of secondary structural elements. Comparison of the three-dimensional structures makes it possible to find a well-defined structural basis for the conservation of many of these amino acids.

  14. A non-modular type B feruloyl esterase from Neurospora crassa exhibits concentration-dependent substrate inhibition.

    PubMed Central

    Crepin, Valerie F; Faulds, Craig B; Connerton, Ian F

    2003-01-01

    Feruloyl esterases, a subclass of the carboxylic acid esterases (EC 3.1.1.1), are able to hydrolyse the ester bond between the hydroxycinnamic acids and sugars present in the plant cell wall. The enzymes have been classified as type A or type B, based on their substrate specificity for aromatic moieties. We show that Neurospora crassa has the ability to produce multiple ferulic acid esterase activities depending upon the length of fermentation with either sugar beet pulp or wheat bran substrates. A gene identified on the basis of its expression on sugar beet pulp has been cloned and overexpressed in Pichia pastoris. The gene encodes a single-domain ferulic acid esterase, which represents the first report of a non-modular type B enzyme (fae-1 gene; GenBank accession no. AJ293029). The purified recombinant protein has been shown to exhibit concentration-dependent substrate inhibition (K(m) 0.048 mM, K (i) 2.5 mM and V(max) 8.2 units/mg against methyl 3,4-dihydroxycinnamate). The kinetic behaviour of the non-modular enzyme is discussed in terms of the diversity in the roles of the feruloyl esterases in the mobilization of plant cell wall materials and their respective modes of action. PMID:12435269

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

    SciTech Connect

    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.

  16. Dehydrogenases, Acid and Alkaline Phosphatases, and Esterases for Chemotaxonomy of Selected Meloidogyne, Ditylenchus, Heterodera and Aphelenchus spp.

    PubMed Central

    Dickson, D. W.; Huisingh, D.; Sasser, J. N.

    1971-01-01

    Various taxonomically useful profiles of four dehydrogenases (lactate, malate, glucose-6-phosphate, and a-glycerophosphate) and three hydrolases (acid and alkaline phosphatase and esterase) were detected in whole nematode homogenates of Meloidogyne javanica, M. hapla, M. incognita, M. arenaria, Ditylenchus dipsaci, D. triformis, Heterodera glycines, and Aphelenchus avenae. The enzyme profiles were stable in populations cultured on several different hosts. A tentative enzymically-determined phylogeny of Meloidogyne is given. PMID:19322334

  17. Biochemical characterization of a halotolerant feruloyl esterase from Actinomyces spp.: refolding and activity following thermal deactivation.

    PubMed

    Hunt, Cameron J; Tanksale, Akshat; Haritos, Victoria S

    2016-02-01

    Ferulic acid esterases (FAE, EC. 3.1.1.73) hydrolyse the linkage between hemicellulose and lignin and thus have potential for use in mild enzymatic pretreatment of biomass as an alternative to thermochemical approaches. Here, we report the characterization of a novel FAE (ActOFaeI) obtained from the bacterium, Actinomyces sp. oral which was recombinantly expressed in Escherichia coli BL21 in two forms: with and without its putative signal peptide. The truncated form was found to have <10 % relative activity compared to the full length and was more prone to aggregation after purification. The enzyme with retained peptide demonstrated 2 to 4-fold higher activity against methyl caffeate and methyl p-coumarate, with specific activities of 477.6 and 174.4 U mg(-1) respectively, than the equivalent activities of the benchmark FAE from Aspergillus niger A and B. ActOFaeI retained activity over a broad pH range with a maximum at 9 but >90 % relative activity at pH 6.5 and an optimum reaction temperature of 30 °C. ActOFaeI increased activity by 15% in high salt conditions (1000 mMNaCl) and its thermal unfolding temperature improved from 41.5 °C in standard buffer to 74 °C in the presence of 2500 mM sodium malonate. ActOFaeI also released ferulic acid from destarched wheat bran when combined with a xylanase preparation. After treatment above the thermal denaturation temperature followed by cooling to room temperature, ActOFaeI demonstrated spontaneous refolding into an active state. ActOFaeI displays many useful characteristics for enzymatic pretreatment of lignocellulose and contributes to our understanding of this important family.

  18. Heterologous production and characterization of a chlorogenic acid esterase from Ustilago maydis with a potential use in baking.

    PubMed

    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. PMID:27173527

  19. Heterologous production and characterization of a chlorogenic acid esterase from Ustilago maydis with a potential use in baking.

    PubMed

    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.

  20. The Staphylococcus aureus Methicillin Resistance Factor FmtA Is a d-Amino Esterase That Acts on Teichoic Acids

    PubMed Central

    Rahman, Muhammad M.; Hunter, Howard N.; Prova, Shamina; Verma, Vidhu; Qamar, Aneela

    2016-01-01

    ABSTRACT The methicillin resistance factor encoded by fmtA is a core member of the Staphylococcus aureus cell wall stimulon, but its function has remained elusive for the past two decades. First identified as a factor that affects methicillin resistance in S. aureus strains, FmtA was later shown to interact with teichoic acids and to localize to the cell division septum. We have made a breakthrough in understanding FmtA function. We show that FmtA hydrolyzes the ester bond between d-Ala and the backbone of teichoic acids, which are polyglycerol-phosphate or polyribitol-phosphate polymers found in the S. aureus cell envelope. FmtA contains two conserved motifs found in serine active-site penicillin-binding proteins (PBPs) and β-lactamases. The conserved SXXK motif was found to be important for the d-amino esterase activity of FmtA. Moreover, we show that deletion of fmtA (ΔfmtA) led to higher levels of d-Ala in teichoic acids, and this effect was reversed by complementation of ΔfmtA with fmtA. The positive charge on d-Ala partially masks the negative charge of the polyol-phosphate backbone of teichoic acids; hence, a change in the d-Ala content will result in modulation of their charge. Cell division, biofilm formation, autolysis, and colonization are among the many processes in S. aureus affected by the d-Ala content and overall charge of the cell surface teichoic acids. The esterase activity of FmtA and the regulation of fmtA suggest that FmtA functions as a modulator of teichoic acid charge, thus FmtA may be involved in S. aureus cell division, biofilm formation, autolysis, and colonization. PMID:26861022

  1. Synergistic Enhancement of Cellobiohydrolase Performance on Pretreated Corn Stover by Addition of Xylanase and Esterase Activities

    SciTech Connect

    Selig, M. J.; Knoshaug E. P.; Adney, W. S.; Himmel, M. E.; Decker, S. R.

    2007-11-01

    Significant increases in the depolymerization of corn stover cellulose by cellobiohydrolase I (Cel7A) from Trichoderma reesei were observed using small quantities of non-cellulolytic cell wall-degrading enzymes. Purified endoxylanase (XynA), ferulic acid esterase (FaeA), and acetyl xylan esterase (Axe1) all enhanced Cel7A performance on corn stover subjected to hot water pretreatment. In all cases, the addition of these activities improved the effectiveness of the enzymatic hydrolysis in terms of the quantity of cellulose converted per milligram of total protein. Improvement in cellobiose release by the addition of the non-cellulolytic enzymes ranged from a 13-84% increase over Cel7A alone. The most effective combinations included the addition of both XynA and Axe1, which synergistically enhance xylan conversions resulting in additional synergistic improvements in glucan conversion. Additionally, we note a direct relationship between enzymatic xylan removal in the presence of XynA and the enhancement of cellulose hydrolysis by Cel7A.

  2. Hydrolysis of chicoric and caftaric acids with esterases and Lactobacillus johnsonii in Vitro and in a gastrointestinal model.

    PubMed

    Bel-Rhlid, Rachid; Pagé-Zoerkler, Nicole; Fumeaux, René; Ho-Dac, Thang; Chuat, Jean-Yves; Sauvageat, Jean Luc; Raab, Thomas

    2012-09-12

    Chicoric acid (ChA) and caftaric acid (CafA) were identified as bioactive components of chicory and have been ascribed a number of health benefits. This study investigated the hydrolysis of ChA and CafA with enzymes and a probiotic bacterium Lactobacillus johnsonii (La1). Esterase from Aspergillus japonicus (24 U/mg) hydrolyzed 100% of ChA (5 mM) and CafA (5 mM) after 3 h, at pH 7.0 and 37 °C. Under the same reaction conditions, 100% hydrolysis of ChA and CafA was achieved with a spray-dried preparation of La1. The addition of La1 (100 mg/mL, 3.3 E9 cfu/g) to CafA solution in a gastrointestinal model (GI model) resulted in 65% hydrolysis of CafA. This model simulates the physicochemical conditions of the human gastrointestinal tract. No hydrolysis of CafA was observed after passage through the GI model in the absence of La1. The results of this study support the hypothesis that ChA and CafA are degraded by gut microflora before absorption and metabolization. PMID:22920606

  3. A novel thermoalkalostable esterase from Acidicaldus sp. strain USBA-GBX-499 with enantioselectivity isolated from an acidic hot springs of Colombian Andes.

    PubMed

    López, Gina; Chow, Jennifer; Bongen, Patrick; Lauinger, Benjamin; Pietruszka, Jörg; Streit, Wolfgang R; Baena, Sandra

    2014-10-01

    Several thermo- and mesoacidophilic bacterial strains that revealed high lipolytic activity were isolated from water samples derived from acidic hot springs in Los Nevados National Natural Park (Colombia). A novel lipolytic enzyme named 499EST was obtained from the thermoacidophilic alpha-Proteobacterium Acidicaldus USBA-GBX-499. The gene estA encoded a 313-amino-acid protein named 499EST. The deduced amino acid sequence showed the highest identity (58 %) with a putative α/β hydrolase from Acidiphilium sp. (ZP_08632277.1). Sequence alignments and phylogenetic analysis indicated that 499EST is a new member of the bacterial esterase/lipase family IV. The esterase reveals its optimum catalytic activity at 55 °C and pH 9.0. Kinetic studies showed that 499EST preferentially hydrolyzed middle-length acyl chains (C6-C8), especially p-nitrophenyl (p-NP) caproate (C6). Its thermostability and activity were strongly enhanced by adding 6 mM FeCl3. High stability in the presence of water-miscible solvents such as dimethyl sulfoxide and glycerol was observed. This enzyme also exhibits stability under harsh environmental conditions and enantioselectivity towards naproxen and ibuprofen esters, yielding the medically relevant (S)-enantiomers. In conclusion, according to our knowledge, 499EST is the first thermoalkalostable esterase derived from a Gram-negative thermoacidophilic bacterium.

  4. A novel thermoalkalostable esterase from Acidicaldus sp. strain USBA-GBX-499 with enantioselectivity isolated from an acidic hot springs of Colombian Andes.

    PubMed

    López, Gina; Chow, Jennifer; Bongen, Patrick; Lauinger, Benjamin; Pietruszka, Jörg; Streit, Wolfgang R; Baena, Sandra

    2014-10-01

    Several thermo- and mesoacidophilic bacterial strains that revealed high lipolytic activity were isolated from water samples derived from acidic hot springs in Los Nevados National Natural Park (Colombia). A novel lipolytic enzyme named 499EST was obtained from the thermoacidophilic alpha-Proteobacterium Acidicaldus USBA-GBX-499. The gene estA encoded a 313-amino-acid protein named 499EST. The deduced amino acid sequence showed the highest identity (58 %) with a putative α/β hydrolase from Acidiphilium sp. (ZP_08632277.1). Sequence alignments and phylogenetic analysis indicated that 499EST is a new member of the bacterial esterase/lipase family IV. The esterase reveals its optimum catalytic activity at 55 °C and pH 9.0. Kinetic studies showed that 499EST preferentially hydrolyzed middle-length acyl chains (C6-C8), especially p-nitrophenyl (p-NP) caproate (C6). Its thermostability and activity were strongly enhanced by adding 6 mM FeCl3. High stability in the presence of water-miscible solvents such as dimethyl sulfoxide and glycerol was observed. This enzyme also exhibits stability under harsh environmental conditions and enantioselectivity towards naproxen and ibuprofen esters, yielding the medically relevant (S)-enantiomers. In conclusion, according to our knowledge, 499EST is the first thermoalkalostable esterase derived from a Gram-negative thermoacidophilic bacterium. PMID:24818691

  5. Characterization of Yersinia enterocolitica, Y. intermedia, Y. aldovae, Y. frederiksenii, Y. kristensenii and Y. pseudotuberculosis by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate and malate dehydrogenases.

    PubMed

    Goullet, P; Picard, B

    1988-02-01

    Acid phosphatase, esterases, and glutamate and malate dehydrogenases of 192 strains of Yersinia enterocolitica, Y. intermedia, Y. aldovae, Y. frederiksenii, Y. kristensenii and Y. pseudotuberculosis were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gels. The six species were clearly separated from each other by their distinct enzyme electrophoretic polymorphism. For Y. enterocolitica, the strains of biotype 5 were differentiated from the other biotypes by the mobility of glutamate dehydrogenase. For Y. frederiksenii, six zymotypes were delineated by pI and by the mobility of the enzymes. Variation in number or mobility of esterases within each species could represent a marker for epidemiological and ecological analyses. A linear relationship was obtained between the mean genetic diversity coefficient of enzymes and the mean percentage DNA-DNA relatedness of Y. intermedia, Y. aldovae, Y. enterocolitica and Y. frederiksenii.

  6. Genetic studies of water buffalo blood markers. I. Red cell acid phosphatase, albumin, catalase, red cell alpha-esterase-3, group-specific component, and protease inhibitor.

    PubMed

    Tan, S G; Barker, J S; Selvaraj, O S; Mukherjee, T K; Wong, Y F

    1993-06-01

    We have developed the methodologies for typing and family studies to establish the modes of inheritance of water buffalo red cell acid phosphatase (Acp), protease inhibitor (Pi), and group-specific component (Gc) on isoelectric focusing and albumin (Alb), red cell alpha-esterase-3 (Est-3), and catalase (Cat) on polyacrylamide gel electrophoresis. Family studies showed that Pi, Gc, Alb, and Cat are coded by autosomal genes with two codominant alleles, while Est-3 is autosomal with two codominant alleles and a recessive null allele and Acp exhibits three codominant alleles.

  7. The enzymatic nature of C'1r. Conversion of C'1s to C'1 esterase and digestion of amino acid esters by C'1r.

    PubMed

    Naff, G B; Ratnoff, O S

    1968-10-01

    Human C'1, a macromolecular complex composed of three subunits, is the zymogen for at least two distinct enzymes. Preparations of one subunit, C'1r, functioned as a protease which converted another subunit, C'1s, to C'1 esterase. The conversion of C'1s to C'1 esterase by C'1r was blocked by Liquoid, phenyl methylsulfonyl fluoride, and calcium ions, but not by soybean trypsin inhibitor, hirudin, or heparin. Preparations of C'1r also possessed two additional functions, i.e., the ability to hydrolyze certain synthetic amino acid esters and to participate in immune hemolysis. Evidence was presented which indicates that these three functions are properties of a single entity, C'1r, but not of the same portion of its molecular structure. These observations suggest that C'1r has at least two active sites, one for its reaction with C'1q, an additional subunit of C'1, and one for its reaction with C'1s; together, the three subcomponents, C'1q, C'1r, and C'1s, form a single functional unit, the first component of complement.

  8. Addition of feruloyl esterase and xylanase produced on-site improves sugarcane bagasse hydrolysis.

    PubMed

    Braga, Cleiton Márcio Pinto; Delabona, Priscila da Silva; Lima, Deise Juliana da Silva; Paixão, Douglas Antônio Alvaredo; Pradella, José Geraldo da Cruz; Farinas, Cristiane Sanchez

    2014-10-01

    Accessory enzymes that assist biomass degradation could be used to improve the recovery of fermentable sugar for use in biorefineries. In this study, different fungal strains isolated from the Amazon rainforest were evaluated in terms of their ability to produce feruloyl esterase (FAE) and xylanase enzymes, and an assessment was made of the contributions of the enzymes in the hydrolysis of pretreated sugarcane bagasse. In the selection step, screening using plate assays was followed by shake flask submerged cultivations. After carbon source selection and cultivation in a stirred-tank bioreactor, Aspergillusoryzae P21C3 proved to be a promising strain for production of the enzymes. Supplementation of a commercial enzyme preparation with 30% (v/v) crude enzymatic complex from A. oryzae P21C3 increased the conversion of cellulose derived from pretreated sugarcane bagasse by 36%. Supplementation with FAE and xylanase enzymes produced on-site can therefore be used to improve the hydrolysis of sugarcane bagasse.

  9. Determination of acid alpha-naphthyl acetate esterase enzyme activity in peripheral blood leukocytes of gazelles (Gazella subgutturosa).

    PubMed

    Altunay, H; Harem, I S; Harem, M K; Asti, R N; Kurtdede, N

    2008-12-01

    We examined gazelle peripheral blood leucocytes using the alpha-Naphthyl acetate esterase (ANAE) staining technique (pH 5.8). Our purpose was to determine the percentage of ANAE positive lymphocytes. The proportion of ANAE positive T-lymphocytes was 72%. T-lymphocytes showed an ANAE positive reaction, but eosinophilic granulocytes and monocytes also showed a positive reaction. By contrast, no reaction was detected in B-lymphocytes, neutrophil granulocytes or platelets. The reaction observed in T-lymphocytes was a red-brown coloration, usually 1-2 granules, but enough granules to fill the cytoplasm were detected rarely. As a result of ANAE enzyme staining, we concluded that the staining technique can be used as a cytochemical marker for gazelle T-lymphocytes. PMID:19085516

  10. Inter-conversion of catalytic abilities in a bifunctional carboxyl/feruloyl-esterase from earthworm gut metagenome.

    PubMed

    Vieites, José María; Ghazi, Azam; Beloqui, Ana; Polaina, Julio; Andreu, José M; Golyshina, Olga V; Nechitaylo, Taras Y; Waliczek, Agnes; Yakimov, Michail M; Golyshin, Peter N; Ferrer, Manuel

    2010-01-01

    Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structural similarity. In present study we have exploited functional metagenomics, saturation mutagenesis and experimental protein evolution to explore residues that have a significant role in substrate discrimination. We used an enzyme, designated 3A6, derived from the earthworm gut metagenome that exhibits CE and feruloyl esterase (FAE) activities with p-nitrophenyl and cinnamate esters, respectively, with a [(k(cat)/K(m))](CE)/[(k(cat)/K(m))](FAE) factor of 17. Modelling-guided saturation mutagenesis at specific hotspots (Lys(281), Asp(282), Asn(316) and Lys(317)) situated close to the catalytic core (Ser(143)/Asp(273)/His(305)) and a deletion of a 34-AA-long peptide fragment yielded mutants with the highest CE activity, while cinnamate ester bond hydrolysis was effectively abolished. Although, single to triple mutants with both improved activities (up to 180-fold in k(cat)/K(m) values) and enzymes with inverted specificity ((k(cat)/K(m))(CE)/(k(cat)/K(m))(FAE) ratio of ∼0.4) were identified, no CE inactive variant was found. Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination. We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to -5.6 J mol(-1)), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to -13.7 J mol(-1)) was found. Results suggest that the FAE activity in 3A6 may have evolved via introduction of a limited number of 'hot spot' mutations in a common CE ancestor, which may retain the original hydrolytic activity due to lower restrictive energy barriers but conveys a dynamic energetically favourable switch of a second hydrolytic reaction. PMID:21255305

  11. Phenol esterase activity of porcine skin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The alkyl esters of plant-derived phenols may serve as slow-release sources for cutaneous delivery of antioxidants. The ability of skin esterases to hydrolyze phenolic esters was examined. Esters of tyrosol and hydroxytyrosol were prepared from decanoic and lipoic acids. Ferulic acid was esterified ...

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

    SciTech Connect

    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.

  13. Baby Fae: the "anything goes" school of human experimentation.

    PubMed

    Annas, G J

    1985-02-01

    The case of Baby Fae is likened to previous cases of high-risk xenograft experiments in which members of groups at particular risk for exploitation were sacrificed to scientific progress in violation of major precepts of the Nuremberg Code. Annas finds fault with the scientific justification for performing the experiment on an infant, the adequacy of the Institutional Review Board review, and the quality of the parents' consent. He concludes that, if current federal regulations cannot prevent such gross exploitation of the terminally ill, they must be revised. In addition, a "national review board" should perhaps be established to deal with complex new technologies.

  14. Efficient production of lignocellulolytic enzymes xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by the mutant strain Aspergillus awamori 2B.361 U2/1

    PubMed Central

    Gottschalk, Leda Maria Fortes; de Sousa Paredes, Raquel; Teixeira, Ricardo Sposina Sobral; da Silva, Ayla Sant’Ana; da Silva Bon, Elba Pinto

    2013-01-01

    The production of xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by Aspergillus awamori 2B.361 U2/1, a hyper producer of glucoamylase and pectinase, was evaluated using selected conditions regarding nitrogen nutrition. Submerged cultivations were carried out at 30 °C and 200 rpm in growth media containing 30 g wheat bran/L as main carbon source and either yeast extract, ammonium sulfate, sodium nitrate or urea, as nitrogen sources; in all cases it was used a fixed molar carbon to molar nitrogen concentration of 10.3. The use of poor nitrogen sources favored the accumulation of xylanase, β-xylosidase and ferulic acid esterase to a peak concentrations of 44,880; 640 and 118 U/L, respectively, for sodium nitrate and of 34,580, 685 and 170 U/L, respectively, for urea. However, the highest β-glucosidase accumulation of 10,470 U/L was observed when the rich organic nitrogen source yeast extract was used. The maxima accumulation of filter paper activity, xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by A. awamori 2B.361 U2/1 was compared to that produced by Trichoderma reesei Rut-C30. The level of β-glucosidase was over 17-fold higher for the Aspergillus strain, whereas the levels of xylanase and β-xylosidase were over 2-fold higher. This strain also produced ferulic acid esterase (170 U/L), which was not detected in the T. reesei culture. PMID:24294256

  15. The ultrastructure of bovine ileal follicle-associated epithelial (FAE) cells during the perinatal period.

    PubMed Central

    Asari, M; Kano, Y; Wakui, S; Nishita, T; Matsushita, H; Oshige, H

    1989-01-01

    The ileal follicle-associated epithelial (FAE) cells in bovine fetuses and neonates were examined by light and electron microscopy. In 7-9 months old fetuses (68, 82 and 86 cm CRL) the dome epithelium was usually a little thinner than elsewhere and contained more intra-epithelial leucocytes. FAE cells were already distinguishable by their being more cuboidal and eosinophilic than the other epithelial cells. The cytoplasm of the FAE cells bulged noticeably into the lumen and contained numerous mitochondria and vacuoles. At 18 hours and 21 hours after birth, the dome epithelium was more columnar and eosinophilic than previously and contained more intra-epithelial leucocytes. The FAE cells showed characteristic bulging of large cytoplasmic processes into the lumen, as seen in the previous stage. In the cytoplasm, moderate numbers of mitochondria, numerous vesicles and microtubules could be seen. Frequently degenerated FAE cells could also be found among normal FAE cells in the epithelium. After this stage the cytoplasmic processes almost disappeared but distribution of the other organelles was similar to that seen at the previous stage except that multivesicular bodies were frequently seen in the apical cytoplasm. These histological results suggest that bovine ileal FAE cells are histologically and functionally mature by birth and that at birth they seem to be able to react against the penetration of pathogenic substances from the extrauterine environment. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:2606783

  16. The Wood Rot Ascomycete Xylaria polymorpha Produces a Novel GH78 Glycoside Hydrolase That Exhibits α-l-Rhamnosidase and Feruloyl Esterase Activities and Releases Hydroxycinnamic Acids from Lignocelluloses

    PubMed Central

    Nghi, Do Huu; Bittner, Britta; Kellner, Harald; Jehmlich, Nico; Ullrich, René; Pecyna, Marek J.; Nousiainen, Paula; Sipilä, Jussi; Huong, Le Mai; Hofrichter, Martin

    2012-01-01

    Soft rot (type II) fungi belonging to the family Xylariaceae are known to substantially degrade hardwood by means of their poorly understood lignocellulolytic system, which comprises various hydrolases, including feruloyl esterases and laccase. In the present study, several members of the Xylariaceae were found to exhibit high feruloyl esterase activity during growth on lignocellulosic materials such as wheat straw (up to 1,675 mU g−1) or beech wood (up to 80 mU g−1). Following the ester-cleaving activity toward methyl ferulate, a hydrolase of Xylaria polymorpha was produced in solid-state culture on wheat straw and purified by different steps of anion-exchange and size-exclusion chromatography to apparent homogeneity (specific activity, 2.2 U mg−1). The peptide sequence of the purified protein deduced from the gene sequence and verified by de novo peptide sequencing shows high similarity to putative α-l-rhamnosidase sequences belonging to the glycoside hydrolase family 78 (GH78; classified under EC 3.2.1.40). The purified enzyme (98 kDa by SDS-PAGE, 103 kDa by size-exclusion chromatography; pI 3.7) converted diverse glycosides (e.g., α-l-rhamnopyranoside and α-l-arabinofuranoside) but also natural and synthetic esters (e.g., chlorogenic acid, hydroxycinnamic acid glycoside esters, veratric acid esters, or p-nitrophenyl acetate) and released free hydroxycinnamic acids (ferulic and coumaric acid) from arabinoxylan and milled wheat straw. These catalytic properties strongly suggest that X. polymorpha GH78 is a multifunctional enzyme. It is the first fungal enzyme that combines glycosyl hydrolase with esterase activities and may help this soft rot fungus to degrade lignocelluloses. PMID:22544251

  17. Cytogenetic characterization and fae1 gene variation in progenies from asymmetric somatic hybrids between Brassica napus and Crambe abyssinica.

    PubMed

    Wang, Y P; Snowdon, R J; Rudloff, E; Wehling, P; Friedt, W; Sonntag, K

    2004-08-01

    Sexual progenies of asymmetric somatic hybrids between Brassica napus and Crambe abyssinica were analyzed with respect to chromosomal behavior, fae1 gene introgression, fertility, and fatty-acid composition of the seed. Among 24 progeny plants investigated, 11 plants had 38 chromosomes and were characterized by the occurrence of normal meiosis with 19 bivalents. The other 13 plants had more than 38 chromosomes, constituting a complete chromosomal set from B. napus plus different numbers of additional chromosomes from C. abyssinica. The chromosomes of B. napus and C. abyssinica origin could be clearly discriminated by genomic in situ hybridization (GISH) in mitotic and meiotic cells. Furthermore, meiotic GISH enabled identification of intergenomic chromatin bridges and of asynchrony between the B. napus and C. abyssinca meiotic cycles. Lagging, bridging and late disjunction of univalents derived from C. abyssinica were observed. Analysis of cleaved amplified polymorphic sequence (CAPS) markers derived from the fae1 gene showed novel patterns different from the B. napus recipient in some hybrid offspring. Most of the progeny plants had a high pollen fertility and seed set, and some contained significantly greater amounts of seed erucic acid than the B. napus parent. This study demonstrates that a part of the C. abyssinica genome can be transferred into B. napus via asymmetric hybridization and maintained in sexual progenies of the hybrids. Furthermore, it confirms that UV irradiation improves the fertility of the hybrid and of its sexual progeny via chromosomal elimination and facilitates the introgression of exotic genetic material into crop species.

  18. [Role of Human Orphan Esterases in Drug-induced Toxicity].

    PubMed

    Fukami, Tatsuki

    2015-01-01

    Esterases hydrolyze compounds containing ester, amide, and thioester bonds, causing prodrug activation or detoxification. Among esterases, carboxylesterases have been studied in depth due to their ability to hydrolyze a variety of drugs. However, there are several drugs for which the involved esterase(s) is unknown. We found that flutamide, phenacetin, rifamycins (rifampicin, rifabutin, and rifapentine), and indiplon are hydrolyzed by arylacetamide deacetylase (AADAC), which is highly expressed in human liver and gastrointestinal tissues. Flutamide hydrolysis is considered associated with hepatotoxicity. Phenacetin, a prodrug of acetaminophen, was withdrawn due to side effects such as methemoglobinemia and renal failure. It was demonstrated in vitro and in vivo using mice that AADAC is responsible for phenacetin hydrolysis, which leads to methemoglobinemia. In addition, it was shown that AADAC-mediated hydrolysis attenuates the cytotoxicity of rifamycins. Thus AADAC plays critical roles in drug-induced toxicity. Another orphan esterase, α/β hydrolase domain containing 10 (ABHD10), was found responsible for deglucuronidation of acyl-glucuronides including mycophenolic acid acyl-glucuronide and probenecid acyl-glucuronide. Because acyl-glucuronides appear associated with toxicity, ABHD10 would function as a detoxification enzyme. The roles of orphan esterases are becoming increasingly understood. Further studies will facilitate our knowledge of the pharmacologic and toxicological significance of orphan esterases in drug therapy. PMID:26521872

  19. Immunogenicity of recombinant F4 (K88) fimbrial adhesin FaeG expressed in tobacco chloroplast.

    PubMed

    Shen, Huifeng; Qian, Bingjun; Chen, Weiwei; Liu, Zhenhua; Yang, Litao; Zhang, Dabing; Liang, Wanqi

    2010-08-01

    To test the possibility of producing the novel vaccine in plants against diarrhea normally found in neonatal and newly weaned piglets, the faeG gene, encoding a major F4ac fimbrial subunit protein, was introduced into the tobacco chloroplast genome. After two rounds of selection under spectinomycin, we obtained the transgenic plants nearly homoplasmic. RNA gel blot analysis indicated that faeG and the antibiotic selective gene aminoglycoside 3' adenylyltransferase (aadA) were highly transcribed as a dicistron, while the translational level of recombinant FaeG in transplastomic tobacco was about 0.15% of total soluble protein. The immunogenicity of recombinant FaeG produced in tobacco chloroplasts was confirmed by the observation that FaeG-specific antibodies were elicited in mice immunized with total soluble protein of transgenic plants, as well as the result that mouse sera stimulated by chloroplast-derived recombinant FaeG could neutralize F4ac enterotoxigenic Escherichia coli (ETEC) in vivo. This study provides a new alternative for producing the ETEC vaccine using the chloroplast expression system.

  20. F4+ enterotoxigenic Escherichia coli (ETEC) adhesion mediated by the major fimbrial subunit FaeG.

    PubMed

    Xia, Pengpeng; Song, Yujie; Zou, Yajie; Yang, Ying; Zhu, Guoqiang

    2015-09-01

    The FaeG subunit is the major constituent of F4(+) fimbriae, associated with glycoprotein and/or glycolipid receptor recognition and majorly contributes to the pathogen attachment to the host cells. To investigate the key factor involved in the fimbrial binding of F4(+) Escherichia coli, both the recombinant E. coli SE5000 strains carrying the fae operon gene clusters that express the different types of fimbriae in vitro, named as rF4ab, rF4ac, and rF4ad, respectively, corresponding to the fimbrial types F4ab, F4ac, and F4ad, and the three isogenic in-frame faeG gene deletion mutants were constructed. The adhesion assays and adhesion inhibition assays showed that ΔfaeG mutants had a significant reduction in the binding to porcine brush border as well as the intestinal epithelial cell lines, while the complemented strain ΔfaeG/pfaeG restored the adhesion function. The recombinant bacterial strains rF4ab, rF4ac, and rF4ad have the same binding property as wild-type F4(+) E. coli strains do and improvement in terms of binding to porcine brush border and the intestinal epithelial cells, and the adherence was blocked by the monoclonal antibody anti-F4 fimbriae. These data demonstrate that the fimbrial binding of F4(+) E. coli is directly mediated by the major FaeG subunit. PMID:25847483

  1. Immunogenicity of recombinant F4 (K88) fimbrial adhesin FaeG expressed in tobacco chloroplast.

    PubMed

    Shen, Huifeng; Qian, Bingjun; Chen, Weiwei; Liu, Zhenhua; Yang, Litao; Zhang, Dabing; Liang, Wanqi

    2010-08-01

    To test the possibility of producing the novel vaccine in plants against diarrhea normally found in neonatal and newly weaned piglets, the faeG gene, encoding a major F4ac fimbrial subunit protein, was introduced into the tobacco chloroplast genome. After two rounds of selection under spectinomycin, we obtained the transgenic plants nearly homoplasmic. RNA gel blot analysis indicated that faeG and the antibiotic selective gene aminoglycoside 3' adenylyltransferase (aadA) were highly transcribed as a dicistron, while the translational level of recombinant FaeG in transplastomic tobacco was about 0.15% of total soluble protein. The immunogenicity of recombinant FaeG produced in tobacco chloroplasts was confirmed by the observation that FaeG-specific antibodies were elicited in mice immunized with total soluble protein of transgenic plants, as well as the result that mouse sera stimulated by chloroplast-derived recombinant FaeG could neutralize F4ac enterotoxigenic Escherichia coli (ETEC) in vivo. This study provides a new alternative for producing the ETEC vaccine using the chloroplast expression system. PMID:20705597

  2. Restoring enzyme activity in nonfunctional low erucic acid Brassica napus fatty acid elongase 1 by a single amino acid substitution.

    PubMed

    Katavic, Vesna; Mietkiewska, Elzbieta; Barton, Dennis L; Giblin, E Michael; Reed, Darwin W; Taylor, David C

    2002-11-01

    Genomic fatty acid elongation 1 (FAE1) clones from high erucic acid (HEA) Brassica napus, Brassica rapa and Brassica oleracea, and low erucic acid (LEA) B. napus cv. Westar, were amplified by PCR and expressed in yeast cells under the control of the strong galactose-inducible promoter. As expected, yeast cells expressing the FAE1 genes from HEA Brassica spp. synthesized very long chain monounsaturated fatty acids that are not normally found in yeast, while fatty acid profiles of yeast cells expressing the FAE1 gene from LEA B. napus were identical to control yeast samples. In agreement with published findings regarding different HEA and LEA B. napus cultivars, comparison of FAE1 protein sequences from HEA and LEA Brassicaceae revealed one crucial amino acid difference: the serine residue at position 282 of the HEA FAE1 sequences is substituted by phenylalanine in LEA B. napus cv. Westar. Using site directed mutagenesis, the phenylalanine 282 residue was substituted with a serine residue in the FAE1 polypeptide from B. napus cv. Westar, the mutated gene was expressed in yeast and GC analysis revealed the presence of very long chain monounsaturated fatty acids (VLCMFAs), indicating that the elongase activity was restored in the LEA FAE1 enzyme by the single amino acid substitution. Thus, for the first time, the low erucic acid trait in canola B. napus can be attributed to a single amino acid substitution which prevents the biosynthesis of the eicosenoic and erucic acids.

  3. [Purification and characterization of esterase from Morganella morganii ZJB-09203].

    PubMed

    Zheng, Renchao; Wang, Tianzhen; Li, Xiaojun; Zheng, Yuguo

    2014-01-01

    Enantioselective hydrolysis of 2-carboxyethyl-3-cyano-5-methylhexanoic acid (CNDE) is the key step in chemoenzymatic synthesis of pregabalin. We purified an intracellular carboxyl esterase from Morganella morganii ZJB-09203, which exhibited high enantioselectivity and activity towards CNDE. The carboxyl esterase was purified to electrophoretic homogeneity by ammonium sulfate fraction precipitation, Phenyl Sepharose 6 FF hydrophobic interaction chromatography, anion exchange with DEAE Sephadex A-50 and Bio-Scale CHT column. The purified enzyme was a monomer with molecular mass of 68 kDa determined by SDS-PAGE and gel chromatography. Substrate specificity of the enzyme towards p-nitrophenyl esters suggested that the purified enzyme was an esterase. The optimal reaction pH for CNDE hydrolysis was 9.0, and optimal temperature was 45 degrees C. The esterase was stable between pH 7.0 and 9.0, and at 40 degrees C. The enzyme activity was enhanced by Ca2+, Cu2+ and Mn2+, whereas strongly inhibited by Co2+, Fe3+, Ni2+ and EDTA. Meanwhile, we investigated the kinetic parameters of the esterase towards p-nitrophenyl esters and effect of CNDE concentration on conversion. The present study reported the esterase capable of stereospecific hydrolysis of CNDE for the first time. Our research will provide foundations for industrial production of Pregabalin using the new biocatalyst.

  4. Immune responses elicited in mice with recombinant Lactococcus lactis expressing F4 fimbrial adhesin FaeG by oral immunization.

    PubMed

    Liu, Shujie; Li, Yongming; Xu, Ziwei; Wang, Yicheng

    2010-08-01

    Enterotoxigenic Escherichia coli (ETEC) is a major pathogenic agent causing piglet diarrhea. The major subunit and adhesin FaeG of F4(+) ETEC is an important virulence factor with strong immunogenicity. To determine whether Lactococcus lactis (L. lactis) could effectively deliver FaeG to the mucosal immune system, recombinant L. lactis expressing FaeG was constructed, and immune responses in mice following oral route delivery of recombinant L. lactis were explored. The production of FaeG expressed in L. lactis was up to approximately 10% of soluble whole-cell proteins, and recombinant FaeG (rFaeG) possessed good immunoreactivity by Western blot analysis. Oral immunization with recombinant L. lactis expressing FaeG induced F4-specific mucosal and systemic immune responses in the mice. In addition, high dose recombinant L. lactis or co-administration of high dose recombinant L. lactis with CTB enhanced the immune responses. These results suggested that L. lactis expressing FaeG was a promising candidate vaccine against ETEC. PMID:20532816

  5. Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media

    DOEpatents

    Arnold, Frances H.; Moore, Jeffrey C.

    1998-01-01

    A method for isolating and identifying modified para-nitrobenzyl esterases which exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase.

  6. Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media

    DOEpatents

    Arnold, Frances H.; Moore, Jeffrey C.

    1999-01-01

    A method for isolating and identifying modified para-nitrobenzyl esterases which exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase.

  7. Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media

    DOEpatents

    Arnold, F.H.; Moore, J.C.

    1998-04-21

    A method is disclosed for isolating and identifying modified para-nitrobenzyl esterases. These enzymes exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase. 43 figs.

  8. Para-nitrobenzyl esterases with enhanced activity in aqueous and nonaqueous media

    DOEpatents

    Arnold, F.H.; Moore, J.C.

    1999-05-25

    A method is disclosed for isolating and identifying modified para-nitrobenzyl esterases which exhibit improved stability and/or esterase hydrolysis activity toward selected substrates and under selected reaction conditions relative to the unmodified para-nitrobenzyl esterase. The method involves preparing a library of modified para-nitrobenzyl esterase nucleic acid segments (genes) which have nucleotide sequences that differ from the nucleic acid segment which encodes for unmodified para-nitrobenzyl esterase. The library of modified para-nitrobenzyl nucleic acid segments is expressed to provide a plurality of modified enzymes. The clones expressing modified enzymes are then screened to identify which enzymes have improved esterase activity by measuring the ability of the enzymes to hydrolyze the selected substrate under the selected reaction conditions. Specific modified para-nitrobenzyl esterases are disclosed which have improved stability and/or ester hydrolysis activity in aqueous or aqueous-organic media relative to the stability and/or ester hydrolysis activity of unmodified naturally occurring para-nitrobenzyl esterase. 43 figs.

  9. RNAi knockdown of fatty acid elongase1 alters fatty acid composition in Brassica napus.

    PubMed

    Shi, Jianghua; Lang, Chunxiu; Wu, Xuelong; Liu, Renhu; Zheng, Tao; Zhang, Dongqing; Chen, Jinqing; Wu, Guanting

    2015-10-23

    The quality and end-use of oil from oilseed crops is determined by its fatty acid composition. In particular, the relative proportions of erucic and oleic acids are key selection traits for breeders. The goal of our research is to genetically improve the nutritional quality of Brassica napus cultivar CY2, the oil of which is high in erucic acid (about 40%) and low in oleic acid (about 20%). Here, we report the use of a seed-specific napin A promoter to drive the knockdown of BnFAE1 in transgenic CY2. Southern blotting results confirmed the presence of the transgene. RT-PCR analysis showed that the levels of BnFAE1 were greatly decreased in BnFAE1-Ri lines compared with the CY2 cultivar. Knockdown of BnFAE1 sharply decreased the levels of erucic acid (less than 3%), largely increased the contents of oleic acid (more than 60%) and slightly increased the polyunsaturated chain fatty acids. Compared with high erucic acid parents, expression of BnFAE1 was dramatically decreased in developing F1 seeds derived from reciprocally crossed BnFAE1-Ri lines and high erucic acid cultivars. In addition, F1 seeds derived from reciprocal crosses between BnFAE1-Ri lines and high erucic acid cultivars showed significantly increased oleic acid (more than 52%) and sharply decreased erucic acid (less than 4%), demonstrating that the RNAi construct of BnFAE1 can effectively interfere with the target gene in F1 seeds. Taken together, our results demonstrate that BnFAE1 is a reliable target for genetic improvement of rapeseed in seed oil quality promotion.

  10. Fumaric acid esters: an alternative systemic treatment for psoriasis.

    PubMed

    Ameen, M; Russell-Jones, R

    1999-09-01

    We report the successful clearance of severe chronic plaque psoriasis following treatment with fumaric acid esters (FAE) in two patients who had failed previous systemic therapy. FAE is gaining increasing acceptance for the treatment of psoriasis in countries such as Germany and the Netherlands, but at present remains unlicensed in Britain.

  11. In situ localization of the genetic locus encoding the lysosomal acid lipase/cholesteryl esterase (LIPA) deficient in wolman disease to chromosome 10q23. 2-q23. 3

    SciTech Connect

    Anderson, R.A.; Rao, N.; Byrum, R.S.; Rothschild, C.B.; Bowden, D.W.; Hayworth, R.; Pettenati, M. )

    1993-01-01

    Human acid lipase/cholesteryl esterase (EC 3.1.1.13) is a 46-kDa glycoprotein required for the lysosomal hydrolysis of cholesteryl esters and triglycerides that cells acquire through the receptor-mediated endocytosis of low-density lipoproteins. This activity is essential in the provision of free cholesterol for cell metabolism as well as for the feedback signal that modulates endogenous cellular cholesterol production. The extremely low level of lysosomal acid lipase in patients afflicted with the hereditary, allelic lysosomal storage disorders Woman disease (WD) and cholesteryl ester storage disease (CESD) (MIM Number 278000 (6)) is associated with the massive intralysosomal lipid storage and derangements in the regulation of cellular cholesterol production (10). Both WD and CESD cells lack a specific acid lipase isoenzyme and it is thought that the different mutations associated with WD and CESD are in the structural gene for this isoenzyme, LIPA. Analysis of the activity of the acid lipase isoenzyme in cell extracts from human-Chinese hamster somatic cell hybrids (4, 11) demonstrated the concordant segregation of the gene locus for lysosomal acid lipase with the glutamate oxaloacetate transaminase-1 (GOT1) enzyme marker for human chromosome 10 which was subsequently localized to 10q24.1 q25.1 (8). 11 refs., 1 figs.

  12. Glucuronoyl esterases are active on polymeric substrate, methyl esterified glucuronoxylan

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Alkali extracted beechwood glucuronoxylan methyl ester prepared by esterification of 4-O-methyl-D-glucuronic acid side residues by methanol was found to serve as substrate of microbial glucuronoyl esterases from Ruminococcus flavefaciens, Schizophyllum commune and Trichoderma reesei. The enzymatic d...

  13. Lipases or esterases: does it really matter? Toward a new bio-physico-chemical classification.

    PubMed

    Ali, Yassine Ben; Verger, Robert; Abousalham, Abdelkarim

    2012-01-01

    Carboxylester hydrolases, commonly named esterases, consist of a large spectrum of enzymes defined by their ability to catalyze the hydrolysis of carboxylic ester bonds and are widely distributed among animals, plants, and microorganisms. Lipases are lipolytic enzymes which constitute a special class of carboxylic esterases capable of releasing long-chain fatty acids from natural water-insoluble carboxylic esters. However, up to now, several unsuccessful attempts aimed at differentiating "lipases" from "esterases" by using various criteria. These criteria were based on the first substrate used chronologically, primary sequence comparisons, some kinetic parameters, or some structural features.Lipids are biological compounds which, by definition, are insoluble in water. Taking into account this basic physico-chemical criterion, we primarily distinguish lipolytic esterases (L, acting on lipids) from nonlipolytic esterases (NL, not acting on lipids). In view of the biochemical data accumulated up to now, we proposed a new classification of esterases based on various criteria of physico-chemical, chemical, anatomical, or cellular nature. We believe that the present attempt matters scientifically for several reasons: (1) to help newcomers in the field, performing a few key experiments to figure out if a newly isolated esterase is lipolytic or not; (2) to clarify a debate between scientists in the field; and (3) to formulate questions which are relevant to the still unsolved problem of the structure-function relationships of esterases. PMID:22426710

  14. Lipases or esterases: does it really matter? Toward a new bio-physico-chemical classification.

    PubMed

    Ali, Yassine Ben; Verger, Robert; Abousalham, Abdelkarim

    2012-01-01

    Carboxylester hydrolases, commonly named esterases, consist of a large spectrum of enzymes defined by their ability to catalyze the hydrolysis of carboxylic ester bonds and are widely distributed among animals, plants, and microorganisms. Lipases are lipolytic enzymes which constitute a special class of carboxylic esterases capable of releasing long-chain fatty acids from natural water-insoluble carboxylic esters. However, up to now, several unsuccessful attempts aimed at differentiating "lipases" from "esterases" by using various criteria. These criteria were based on the first substrate used chronologically, primary sequence comparisons, some kinetic parameters, or some structural features.Lipids are biological compounds which, by definition, are insoluble in water. Taking into account this basic physico-chemical criterion, we primarily distinguish lipolytic esterases (L, acting on lipids) from nonlipolytic esterases (NL, not acting on lipids). In view of the biochemical data accumulated up to now, we proposed a new classification of esterases based on various criteria of physico-chemical, chemical, anatomical, or cellular nature. We believe that the present attempt matters scientifically for several reasons: (1) to help newcomers in the field, performing a few key experiments to figure out if a newly isolated esterase is lipolytic or not; (2) to clarify a debate between scientists in the field; and (3) to formulate questions which are relevant to the still unsolved problem of the structure-function relationships of esterases.

  15. Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification.

    PubMed

    Berlemont, Renaud; Spee, Olivier; Delsaute, Maud; Lara, Yannick; Schuldes, Jörg; Simon, Carola; Power, Pablo; Daniel, Rolf; Galleni, Moreno

    2013-01-01

    in order to isolate novel organic solvent-tolerant (OST) lipases, a metagenomic library was built using DNA derived from a temperate forest soil sample. A two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. The deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described OST lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 α/β hydrolase subgroup (abH04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (C4) compounds, behaving as an esterase rather than a lipase. The RBest1 esterase was purified and biochemically characterized. The optimal esterase activity was observed at pH 6.5 and at temperatures ranging from 38 to 45 °C. Enzymatic activity, determined by hydrolysis of p-nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. Noteworthy, RBest1 remains significantly active at high ionic strength. These findings suggest that RBest1 possesses the ability of OST enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins.

  16. Regiospecific Ester Hydrolysis by Orange Peel Esterase - An Undergraduate Experiment.

    NASA Astrophysics Data System (ADS)

    Bugg, Timothy D. H.; Lewin, Andrew M.; Catlin, Eric R.

    1997-01-01

    A simple but effective experiment has been developed to demonstrate the regiospecificity of enzyme catalysis using an esterase activity easily isolated from orange peel. The experiment involves the preparation of diester derivatives of para-, meta- and ortho-hydroxybenzoic acid (e.g. methyl 4-acetoxy-benzoic acid). The derivatives are incubated with orange peel esterase, as a crude extract, and with commercially available pig liver esterase and porcine pancreatic lipase. The enzymatic hydrolysis reactions are monitored by thin layer chromatography, revealing which of the two ester groups is hydrolysed, and the rate of the enzyme-catalysed reaction. The results of a group experiment revealed that in all cases hydrolysis was observed with at least one enzyme, and in most cases the enzymatic hydrolysis was specific for production of either the hydroxy-ester or acyl-acid product. Specificity towards the ortho-substituted series was markedly different to that of the para-substituted series, which could be rationalised in the case of pig liver esterase by a published active site model.

  17. Structural features determining thermal adaptation of esterases.

    PubMed

    Kovacic, Filip; Mandrysch, Agathe; Poojari, Chetan; Strodel, Birgit; Jaeger, Karl-Erich

    2016-02-01

    The adaptation of microorganisms to extreme living temperatures requires the evolution of enzymes with a high catalytic efficiency under these conditions. Such extremophilic enzymes represent valuable tools to study the relationship between protein stability, dynamics and function. Nevertheless, the multiple effects of temperature on the structure and function of enzymes are still poorly understood at the molecular level. Our analysis of four homologous esterases isolated from bacteria living at temperatures ranging from 10°C to 70°C suggested an adaptation route for the modulation of protein thermal properties through the optimization of local flexibility at the protein surface. While the biochemical properties of the recombinant esterases are conserved, their thermal properties have evolved to resemble those of the respective bacterial habitats. Molecular dynamics simulations at temperatures around the optimal temperatures for enzyme catalysis revealed temperature-dependent flexibility of four surface-exposed loops. While the flexibility of some loops increased with raising the temperature and decreased with lowering the temperature, as expected for those loops contributing to the protein stability, other loops showed an increment of flexibility upon lowering and raising the temperature. Preserved flexibility in these regions seems to be important for proper enzyme function. The structural differences of these four loops, distant from the active site, are substantially larger than for the overall protein structure, indicating that amino acid exchanges within these loops occurred more frequently thereby allowing the bacteria to tune atomic interactions for different temperature requirements without interfering with the overall enzyme function.

  18. Subcutaneous or oral immunization of mice with Lactococcus lactis expressing F4 fimbrial adhesin FaeG.

    PubMed

    Liu, Shujie; Li, Yongming; Xu, Ziwei; Wang, Yicheng

    2013-01-01

    Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of diarrhea in neonatal and postweaning piglets. Fimbrial adhesion of ETEC has been considered an important colonization factor with antigenicity. To safely and effectively deliver the F4 (K88) fimbrial adhesin FaeG to the immune system, we have previously constructed the secretory expression vector pNZ8112-faeG, and FaeG was produced in cytoplasmic form in Lactococcus lactis. In this work, BALB/c mice were immunized with recombinant L. lactis to further determine the immunogenicity of recombinant FaeG (rFaeG) via the subcutaneous or oral route. Subcutaneous immunization in mice with recombinant L. lactis induced a significant increase in the F4-specific serum IgG titer and the number of antibody-secreting cells (ASCs) in the spleen. Oral immunization of mice with recombinant L. lactis induced mucosal and systemic F4-specific immune responses and increased the number of ASCs in the spleen, mesenteric lymph nodes and Peyer's patches. High-dose (2.8 × 10(11) CFU) recombinant strains and adjuvant cholera toxin B subunit enhanced specific mucosal immune responses. The results suggest the feasibility of delivering rFaeG expressed in L. lactis to the immune system in order to induce an F4-specific immune response. PMID:23386358

  19. Influenza C virus esterase: analysis of catalytic site, inhibition, and possible function.

    PubMed Central

    Vlasak, R; Muster, T; Lauro, A M; Powers, J C; Palese, P

    1989-01-01

    The active site serine of the acetylesterase of influenza C virus was localized to amino acid 71 of the hemagglutinin-esterase protein by affinity labeling with 3H-labeled diisopropylfluorophosphate. This serine and the adjacent amino acids (Phe-Gly-Asp-Ser) are part of a consensus sequence motif found in serine hydrolases. Since comparative analysis failed to reveal esterase sequence similarities with other serine hydrolases, we suggest that this viral enzyme is a serine hydrolase constituting a new family of serine esterases. Furthermore, we found that the influenza C virus esterase was inhibited by isocoumarin derivatives, with 3,4-dichloroisocoumarin being the most potent inhibitor. Addition of this compound prevented elution of influenza C virus from erythrocytes and inhibited virus infectivity, possibly through inhibition of virus entry into cells. Images PMID:2495370

  20. Influenza C virus esterase: analysis of catalytic site, inhibition, and possible function

    SciTech Connect

    Vlasak, R.; Muster, T.; Lauro, A.M.; Powers, J.C.; Palese, P.

    1989-05-01

    The active site serine of the acetylesterase of influenza C virus was localized to amino acid 71 of the hemagglutinin-esterase protein by affinity labeling with /sup 3/H-labeled diisopropylfluorophosphate. This serine and the adjacent amino acids (Phe-Gly-Asp-Ser) are part of a consensus sequence motif found in serine hydrolases. Since comparative analysis failed to reveal esterase sequence similarities with other serine hydrolases, the authors suggest that this viral enzyme is a serine hydrolase constituting a new family of serine esterases. Furthermore, they found that the influenza C virus esterase was inhibited by isocoumarin derivatives, with 3,4-dichloroisocoumarin being the most potent inhibitor. Addition of this compound prevented elution of influenza C virus from erythrocytes and inhibited virus infectivity, possibly through inhibition of virus entry into cells.

  1. Esterase and lipase in camel tick Hyalomma dromedarii (Acari: Ixodidae) during embryogenesis.

    PubMed

    Fahmy, Afaf S; Abdel-Gany, Somia S; Mohamed, Tarek M; Mohamed, Saleh A

    2004-02-01

    Esterase and lipase activity showed significant changes during embryogenesis of camel tick Hyalomma dromedarii. From the elution profile of chromatography on DEAE-cellulose, six forms of H. dromedarii esterase (El to EVI) can be distinguished. Esterase EIII was purified to homogeneity after chromatography on Sepharose 6B. The molecular mass of esterase EIII was 45 kDa for the native enzyme and represented a monomer of 45 kDa by SDS-PAGE. Esterase EIII had an acidic pI at 5.3. Lipase activity was detected in the same DEAE-cellulose peaks (LI to LVI) of H. dromedarii esterases. The highest lipase activity was exhibited by lipase LIII. Esterase EIII and lipase LIII were compared with respect to Michaelis constant, substrate specificity, temperature optimum, heat stability, pH optimum, effect of metal ions and inhibitors. This study suggests that H. dromedarii lipolytic enzymes may play a central role in the interconversion of lipovitellins during embryogenesis. PMID:14990212

  2. 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

    PubMed Central

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

    2015-01-01

    ABSTRACT 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. IMPORTANCE 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

  3. High-throughput screening method for lipases/esterases.

    PubMed

    Mateos-Díaz, Eduardo; Rodríguez, Jorge Alberto; de Los Ángeles Camacho-Ruiz, María; Mateos-Díaz, Juan Carlos

    2012-01-01

    High-throughput screening (HTS) methods for lipases and esterases are generally performed by using synthetic chromogenic substrates (e.g., p-nitrophenyl, resorufin, and umbelliferyl esters) which may be misleading since they are not their natural substrates (e.g., partially or insoluble triglycerides). In previous works, we have shown that soluble nonchromogenic substrates and p-nitrophenol (as a pH indicator) can be used to quantify the hydrolysis and estimate the substrate selectivity of lipases and esterases from several sources. However, in order to implement a spectrophotometric HTS method using partially or insoluble triglycerides, it is necessary to find particular conditions which allow a quantitative detection of the enzymatic activity. In this work, we used Triton X-100, CHAPS, and N-lauroyl sarcosine as emulsifiers, β-cyclodextrin as a fatty acid captor, and two substrate concentrations, 1 mM of tributyrin (TC4) and 5 mM of trioctanoin (TC8), to improve the test conditions. To demonstrate the utility of this method, we screened 12 enzymes (commercial preparations and culture broth extracts) for the hydrolysis of TC4 and TC8, which are both classical substrates for lipases and esterases (for esterases, only TC4 may be hydrolyzed). Subsequent pH-stat experiments were performed to confirm the preference of substrate hydrolysis with the hydrolases tested. We have shown that this method is very useful for screening a high number of lipases (hydrolysis of TC4 and TC8) or esterases (only hydrolysis of TC4) from wild isolates or variants generated by directed evolution using nonchromogenic triglycerides directly in the test.

  4. High-throughput screening method for lipases/esterases.

    PubMed

    Mateos-Díaz, Eduardo; Rodríguez, Jorge Alberto; de Los Ángeles Camacho-Ruiz, María; Mateos-Díaz, Juan Carlos

    2012-01-01

    High-throughput screening (HTS) methods for lipases and esterases are generally performed by using synthetic chromogenic substrates (e.g., p-nitrophenyl, resorufin, and umbelliferyl esters) which may be misleading since they are not their natural substrates (e.g., partially or insoluble triglycerides). In previous works, we have shown that soluble nonchromogenic substrates and p-nitrophenol (as a pH indicator) can be used to quantify the hydrolysis and estimate the substrate selectivity of lipases and esterases from several sources. However, in order to implement a spectrophotometric HTS method using partially or insoluble triglycerides, it is necessary to find particular conditions which allow a quantitative detection of the enzymatic activity. In this work, we used Triton X-100, CHAPS, and N-lauroyl sarcosine as emulsifiers, β-cyclodextrin as a fatty acid captor, and two substrate concentrations, 1 mM of tributyrin (TC4) and 5 mM of trioctanoin (TC8), to improve the test conditions. To demonstrate the utility of this method, we screened 12 enzymes (commercial preparations and culture broth extracts) for the hydrolysis of TC4 and TC8, which are both classical substrates for lipases and esterases (for esterases, only TC4 may be hydrolyzed). Subsequent pH-stat experiments were performed to confirm the preference of substrate hydrolysis with the hydrolases tested. We have shown that this method is very useful for screening a high number of lipases (hydrolysis of TC4 and TC8) or esterases (only hydrolysis of TC4) from wild isolates or variants generated by directed evolution using nonchromogenic triglycerides directly in the test. PMID:22426713

  5. A food-grade fimbrial adhesin FaeG expression system in Lactococcus lactis and Lactobacillus casei.

    PubMed

    Lu, W W; Wang, T; Wang, Y; Xin, M; Kong, J

    2016-03-01

    Enterotoxigenic Escherichia coli (ETEC) infection is the major cause of diarrhea in neonatal piglets. The fimbriae as colonizing factor in the pathogenesis of ETEC constitute a primary target for vaccination against ETEC. Lactic acid bacteria (LAB) are attractive tools to deliver antigens at the mucosal level. With the safety of genetically modified LAB in mind, a food-grade secretion vector (pALRc or pALRb) was constructed with DNA entirely from LAB, including the replicon, promoter, signal peptide, and selection marker alanine racemase gene (alr). To evaluate the feasibility of the system, the nuclease gene (nuc) from Staphylococcus aureus was used as a reporter to be expressed in both Lactococcus lactis and Lactobacillus casei. Subsequently, the extracellular secretion of the fimbrial adhesin FaeG of ETEC was confirmed by Western blot analysis. These results showed that this food-grade expression system has potential as the delivery vehicle for the safe use of genetically modified LAB for the development of vaccines against ETEC infection.

  6. A food-grade fimbrial adhesin FaeG expression system in Lactococcus lactis and Lactobacillus casei.

    PubMed

    Lu, W W; Wang, T; Wang, Y; Xin, M; Kong, J

    2016-03-01

    Enterotoxigenic Escherichia coli (ETEC) infection is the major cause of diarrhea in neonatal piglets. The fimbriae as colonizing factor in the pathogenesis of ETEC constitute a primary target for vaccination against ETEC. Lactic acid bacteria (LAB) are attractive tools to deliver antigens at the mucosal level. With the safety of genetically modified LAB in mind, a food-grade secretion vector (pALRc or pALRb) was constructed with DNA entirely from LAB, including the replicon, promoter, signal peptide, and selection marker alanine racemase gene (alr). To evaluate the feasibility of the system, the nuclease gene (nuc) from Staphylococcus aureus was used as a reporter to be expressed in both Lactococcus lactis and Lactobacillus casei. Subsequently, the extracellular secretion of the fimbrial adhesin FaeG of ETEC was confirmed by Western blot analysis. These results showed that this food-grade expression system has potential as the delivery vehicle for the safe use of genetically modified LAB for the development of vaccines against ETEC infection. PMID:26825016

  7. Recent advances in the biomedical applications of fumaric acid and its ester derivatives: The multifaceted alternative therapeutics.

    PubMed

    Das, Ratul Kumar; Brar, Satinder Kaur; Verma, Mausam

    2016-04-01

    Several lines of evidence have demonstrated the potential biomedical applications of fumaric acid (FA) and its ester derivatives against many human disease conditions. Fumaric acid esters (FAEs) have been licensed for the systemic treatment of the immune-mediated disease psoriasis. Biogen Idec Inc. announced about the safety and efficacy of the formulation FAE (BG-12) for treating RRMS (relapsing-remitting multiple sclerosis). Another FAE formulation DMF (dimethyl fumarate) was found to be capable of reduction in inflammatory cardiac conditions, such as autoimmune myocarditis and ischemia and reperfusion. DMF has also been reported to be effective as a potential neuroprotectant against the HIV-associated neurocognitive disorders (HAND). Many in vivo studies carried out on rat and mice models indicated inhibitory effects of fumaric acid on carcinogenesis of different origins. Moreover, FAEs has emerged as an important matrix ingredient in the fabrication of biodegradable scaffolds for tissue engineering applications. Drug delivery vehicles composed of FAEs have shown promising results in delivering some leading drug molecules. Apart from these specific applications and findings, many more studies on FAEs have revealed new therapeutic potentials with the scope of clinical applications. However, until now, this scattered vital information has not been written into a collective account and analyzed for minute details. The aim of this paper is to review the advancement made in the biomedical application of FA and FAEs and to focus on the clinical investigation and molecular interpretation of the beneficial effects of FA and FAEs. PMID:26922546

  8. Exposure to a Cutinase-like Serine Esterase Triggers Rapid Lysis of Multiple Mycobacterial Species*

    PubMed Central

    Yang, Yong; Bhatti, Alexandra; Ke, Danxia; Gonzalez-Juarrero, Mercedes; Lenaerts, Anne; Kremer, Laurent; Guerardel, Yann; Zhang, Peijun; Ojha, Anil K.

    2013-01-01

    Mycobacteria are shaped by a thick envelope made of an array of uniquely structured lipids and polysaccharides. However, the spatial organization of these molecules remains unclear. Here, we show that exposure to an esterase from Mycobacterium smegmatis (Msmeg_1529), hydrolyzing the ester linkage of trehalose dimycolate in vitro, triggers rapid and efficient lysis of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium marinum. Exposure to the esterase immediately releases free mycolic acids, while concomitantly depleting trehalose mycolates. Moreover, lysis could be competitively inhibited by an excess of purified trehalose dimycolate and was abolished by a S124A mutation affecting the catalytic activity of the esterase. These findings are consistent with an indispensable structural role of trehalose mycolates in the architectural design of the exposed surface of the mycobacterial envelope. Importantly, we also demonstrate that the esterase-mediated rapid lysis of M. tuberculosis significantly improves its detection in paucibacillary samples. PMID:23155047

  9. Cloning, sequencing, and regulation of expression of an extracellular esterase gene from the plant pathogen Streptomyces scabies.

    PubMed Central

    Raymer, G; Willard, J M; Schottel, J L

    1990-01-01

    The gene that encodes the extracellular esterase produced by Streptomyces scabies has been cloned and sequenced. The gene was identified by hybridization to a synthetic oligonucleotide that corresponds to the amino-terminal amino acid sequence determined for the secreted form of the esterase. Nucleotide sequence analysis predicted a 345-amino-acid open reading frame, a putative ribosome-binding site, and 39 amino acids at the amino terminus of the sequence that is not found in the secreted protein. This 39-amino-acid sequence has many of the characteristics common to known signal peptides. End mapping the esterase transcript revealed a single 5' end of the mRNA located 51 nucleotides upstream from the start point for translation. Northern (RNA) hybridization analysis of the esterase message by using the cloned esterase gene as a probe indicated that the esterase mRNA is about 1,440 nucleotides in length and was detected only when the cells were grown in the presence of zinc. These results suggest that the level of esterase mRNA detected in the cells is regulated by zinc. Images PMID:2254271

  10. Selective induction of xenobiotic metabolizing esterases/amidases of liver by methaqualone consumption.

    PubMed

    Kaur, S; Ali, B

    1983-08-01

    The present investigation reports the influence of po and ip methaqualone administration on the hydrolytic metabolism of acetylsalicylic acid, procaine, p-nitrophenylacetate, acetanilid, and butyrylcholine in the liver, kidney, and brain of male rats. Oral administration of methaqualone (60 mg/kg/day) to rats for 20 days caused 41.0, 46.5, and 55.0% stimulation of acetylsalicyclic acid esterase I, acetylsalicyclic acid esterase II, and acetanilid N-deacetylase, respectively, in the liver. Under such conditions, the activities of other esterases remained unaffected. The responses of tissue esterases to ip methaqualone treatment (40 mg/kg/day for 6 days) were similar to those observed after po methaqualone administration. Since a single po dose of methaqualone failed to produce any alteration in the rate of metabolism of acetylsalicylic acid, procaine, p-nitrophenylacetate, acetanilid, and butyrylcholine within 20 hr, it may be interpreted that the stimulation of acetylsalicylic acid and acetanilid metabolism is possibly due to selective enhanced de novo synthesis of the enzymes/isozymes necessary for the hydrolysis of the two drugs. The ability of the kidney and brain to metabolize the esters/amides was not modified by po or ip methaqualone pretreatment suggesting the possibility of noninducible forms of renal and neuronal esterases/amidases.

  11. The Serratia marcescens bioH gene encodes an esterase.

    PubMed

    Akatsuka, Hiroyuki; Kawai, Eri; Sakurai, Naoki; Omori, Kenji

    2003-01-01

    The 3.9 kb chromosomal DNA was cloned from Serratia marcescens Sr41, which confers on Escherichia coli cells a phenotype of clear halo formation on tributyrin agar plates. Three complete open reading frames (ORFs) were identified in the inserted DNA, and one ORF was demonstrated to encode a 28 kDa protein of 255 amino acids related to esterase activity. Interestingly, the ORF was 70% identical to a product of the E. coli bioH gene, which lies at a locus separated from the bioABFCD operon and acts in the early steps of the biotin synthetic pathway before pimeloyl-CoA synthesis. This gene complemented a bioH-deficient mutation of E. coli. From the sequence analysis, BioH is presumed to be a serine hydrolase, which belongs to the alpha/beta hydrolase-fold family comprising a wide variety of hydrolases including esterases. A catalytic triad composed of a nucleophilic residue (Ser80), an acidic residue (Asp206), and histidine (His234) was conserved in BioH, and the nucleophilic residue Ser, a catalytic center, was situated in the consensus sequence of G-X-S-X-G-G, a nucleophile elbow. Although the enzymatic function of BioH is not yet elucidated, the bioH gene products from S. marcescens and E. coli show esterase activity, which may imply the hydrolysis of a precursor leading to pimeloyl-CoA ester. The esterase activity of BioH and its CoA binding activity recently reported agree with a current hypothesis of pimeloyl-CoA ester synthesis from CoA and acylester derivatives including an acyl-carrier protein.

  12. Differences in Esterase Activity to Aspirin and p-Nitrophenyl Acetate among Human Serum Albumin Preparations.

    PubMed

    Tatsumi, Akitoshi; Okada, Masaya; Inagaki, Yoshihiro; Inoue, Sachiyo; Hamaguchi, Tsuneo; Iwakawa, Seigo

    2016-01-01

    Human serum albumin (HSA) has two major ligand-binding sites, sites I and II, and also hydrolyzes some compounds at both sites. In the present study, we investigated differences in esterase activity among HSA preparations, and also the effects of warfarin, indomethacin, and naproxen on the hydrolytic activities of HSA to aspirin and p-nitrophenyl acetate. The esterase activities of HSA to aspirin or p-nitrophenyl acetate were measured from the pseudo-first-order formation rate constant (kobs) of salicylic acid or p-nitrophenol by HSA. Inter-lot variations were observed in the esterase activities of HSA to aspirin and p-nitrophenyl acetate; however, the esterase activity of HSA to aspirin did not correlate with that to p-nitrophenyl acetate. The inhibitory effects of warfarin and indomethacin on the esterase activity of HSA to aspirin were stronger than that of naproxen. In contrast, the inhibitory effect of naproxen on the esterase activity of HSA to p-nitrophenyl acetate was stronger than those of warfarin and indomethacin. These results suggest that the administration of different commercial HSA preparations and the co-administration with site I or II high-affinity binding drugs may change the pharmacokinetic profiles of drugs that are hydrolyzed by HSA. PMID:27476944

  13. Evolution of a new function in an esterase: simple amino acid substitutions enable the activity present in the larger paralog, BioH.

    PubMed

    Flores, Humberto; Lin, Steven; Contreras-Ferrat, Gabriel; Cronan, John E; Morett, Enrique

    2012-08-01

    Gene duplication and divergence are essential processes for the evolution of new activities. Divergence may be gradual, involving simple amino acid residue substitutions, or drastic, such that larger structural elements are inserted, deleted or rearranged. Vast protein sequence comparisons, supported by some experimental evidence, argue that large structural modifications have been necessary for certain catalytic activities to evolve. However, it is not clear whether these activities could not have been attained by gradual changes. Interestingly, catalytic promiscuity could play a fundamental evolutionary role: a preexistent secondary activity could be increased by simple amino acid residue substitutions that do not affect the enzyme's primary activity. The promiscuous profile of the enzyme may be modified gradually by genetic drift, making a pool of potentially useful activities that can be selected before duplication. In this work, we used random mutagenesis and in vivo selection to evolve the Pseudomonas aeruginosa PAO1 carboxylesterase PA3859, a small protein, to attain the function of BioH, a much larger paralog involved in biotin biosynthesis. BioH was chosen as a target activity because it provides a highly sensitive selection for evolved enzymatic activities by auxotrophy complementation. After only two cycles of directed evolution, mutants with the ability to efficiently complement biotin auxotrophy were selected. The in vivo and in vitro characterization showed that the activity of one of our mutant proteins was similar to that of the wild-type BioH enzyme. Our results demonstrate that it is possible to evolve enzymatic activities present in larger proteins by discrete amino acid substitutions.

  14. Comparative analysis for the production of fatty acid alkyl esterase using whole cell biocatalyst and purified enzyme from Rhizopus oryzae on waste cooking oil (sunflower oil).

    PubMed

    Balasubramaniam, Bharathiraja; Sudalaiyadum Perumal, Ayyappasamy; Jayaraman, Jayamuthunagai; Mani, Jayakumar; Ramanujam, Praveenkumar

    2012-08-01

    The petroleum fuel is nearing the line of extinction. Recent research and technology have provided promising outcomes to rely on biodiesel as the alternative and conventional source of fuel. The use of renewable source - vegetable oil constitutes the main stream of research. In this preliminary study, Waste Cooking Oil (WCO) was used as the substrate for biodiesel production. Lipase enzyme producing fungi Rhizopus oryzae 262 and commercially available pure lipase enzyme were used for comparative study in the production of Fatty Acid Alkyl Esters (FAAE). The whole cell (RO 262) and pure lipase enzyme (PE) were immobilized using calcium alginate beads. Calcium alginate was prepared by optimizing with different molar ratios of calcium chloride and different per cent sodium alginate. Entrapment immobilization was done for whole cell biocatalyst (WCB). PE was also immobilized by entrapment for the transesterification reaction. Seven different solvents - methanol, ethanol, n-propanol, n-butanol, iso-propanol, iso-butanol and iso-amyl alcohol were used as the acyl acceptors. The reaction parameters like temperature (30°C), molar ratio (1:3 - oil:solvent), reaction time (24 h), and amount of enzyme (10% mass ratio to oil) were also optimized for methanol alone. The same parameters were adopted for the other acyl acceptors too. Among the different acyl acceptors - methanol, whose reaction parameters were optimized showed maximum conversion of triglycerides to FAAE-94% with PE and 84% with WCB. On the whole, PE showed better catalytic converting ability with all the acyl acceptor compared to WCB. Gas chromatography analysis (GC) was done to determine the fatty acid composition of WCO (sunflower oil) and FAAE production with different acyl acceptors.

  15. A standardised challenge model with an enterotoxigenic F4+ Escherichia coli strain in piglets assessing clinical traits and faecal shedding of fae and est-II toxin genes.

    PubMed

    Spitzer, Franz; Vahjen, Wilfried; Pieper, Robert; Martinez-Vallespin, Beatriz; Zentek, Jürgen

    2014-12-01

    This study evaluated the effect of five feed additives on post weaning diarrhoea (PWD) in piglets challenged 3 d after weaning with an enterotoxigenic Escherichia coli strain (ETEC). In three experimental runs, a total of 84 piglets was weaned at 21 days of age and randomly assigned to seven treatments. As dietary treatment, piglets were fed a basal diet or diets with addition of bovine colostrum (0.2%), pineapple stem extract containing bromelain (0.2%), an autolysed yeast preparation (Saccharomyces cerevisiae) (0.1%), a combination of organic acids (0.7%) and a phytogenic product with thyme essential oil (0.015%). A porcine ETEC, serotype O149:K91:K88ac was given twice via oral infection on day 3 after weaning at 10(10) colony forming units/animal. One group of piglets was fed the basal diet without ETEC challenge. Traits included clinical sores, body temperature, faecal scoring and determination of faecal dry matter and the shedding of fae and est-II ETEC toxin genes. After weaning, non-challenged control piglets did not show signs of diarrhoea or impaired health, while the majority of infected piglets had a drop in body temperature, signs of diarrhoea and impaired general health. Mortality, the decrease of faecal dry matter and shedding of the toxin genes fae and est-II were not affected by the different additives. In conclusion, the ETEC challenge model induced distinct clinical signs of PWD in piglets, but the tested feed additives had no preventive effect under these conditions.

  16. A standardised challenge model with an enterotoxigenic F4+ Escherichia coli strain in piglets assessing clinical traits and faecal shedding of fae and est-II toxin genes.

    PubMed

    Spitzer, Franz; Vahjen, Wilfried; Pieper, Robert; Martinez-Vallespin, Beatriz; Zentek, Jürgen

    2014-12-01

    This study evaluated the effect of five feed additives on post weaning diarrhoea (PWD) in piglets challenged 3 d after weaning with an enterotoxigenic Escherichia coli strain (ETEC). In three experimental runs, a total of 84 piglets was weaned at 21 days of age and randomly assigned to seven treatments. As dietary treatment, piglets were fed a basal diet or diets with addition of bovine colostrum (0.2%), pineapple stem extract containing bromelain (0.2%), an autolysed yeast preparation (Saccharomyces cerevisiae) (0.1%), a combination of organic acids (0.7%) and a phytogenic product with thyme essential oil (0.015%). A porcine ETEC, serotype O149:K91:K88ac was given twice via oral infection on day 3 after weaning at 10(10) colony forming units/animal. One group of piglets was fed the basal diet without ETEC challenge. Traits included clinical sores, body temperature, faecal scoring and determination of faecal dry matter and the shedding of fae and est-II ETEC toxin genes. After weaning, non-challenged control piglets did not show signs of diarrhoea or impaired health, while the majority of infected piglets had a drop in body temperature, signs of diarrhoea and impaired general health. Mortality, the decrease of faecal dry matter and shedding of the toxin genes fae and est-II were not affected by the different additives. In conclusion, the ETEC challenge model induced distinct clinical signs of PWD in piglets, but the tested feed additives had no preventive effect under these conditions. PMID:25313936

  17. Preparation and Properties of Novel Dentin Adhesives with Esterase Resistance

    PubMed Central

    Park, Jong-Gu; Ye, Qiang; Topp, Elizabeth M.; Kostoryz, Elisabet L.; Wang, Yong; Kieweg, Sarah L.; Spencer, Paulette

    2012-01-01

    A new methacrylate monomer, trimethylolpropane mono allyl ether dimethacrylate (TMPEDMA), was synthesized and evaluated. This branched methacrylate was designed to increase esterase-resistance when incorporated into conventional HEMA (2-hydroxyethyl methacrylate)/BisGMA (2,2-bis[4(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl] propane) dental adhesives. The new adhesives, HEMA/BisGMA/TMPEDMA in a 45/30/25 (w/w) ratio were formulated with H2O at 0 (A0T) and 8 wt % water (A8T) and compared with control adhesives (HEMA/BisGMA, 45/55 (w/w), at 0 (A0) and 8 wt % (A8) water). Camphoroquinone (CQ), 2-(dimethylamino) ethyl methacrylate and diphenyliodonium hexafluorophosphate were used as photoinitiators. The new adhesives showed a degree of conversion comparable with the control and improved modulus and glass transition temperature (Tg). Exposure of photopolymerized discs to porcine liver esterase for up to eight days showed that the net cumulative methacrylic acid (MAA) release in adhesives formulated with the new monomer and 8% water (A8T: 182 μg/mL) was dramatically (P < 0.05) decreased in comparison to the control (A8: 361.6 μg/mL). The results demonstrate that adhesives made with the new monomer and cured in water to simulate wet bonding are more resistant to esterase than conventional HEMA/BisGMA adhesive. PMID:22919119

  18. Induction of specific immune responses in piglets by intramuscular immunization with fimbrial adhesin FaeG expressed in Lactococcus lactis.

    PubMed

    Liu, Shujie; Li, Yongming; Xu, Ziwei

    2013-08-01

    Fimbrial adhesin plays a critical role in the pathogenesis of enterotoxigenic Escherichia coli (ETEC)-induced piglet diarrhoea. Lactococcus lactis is an attractive food-grade host for the production of heterologous antigens. We previously demonstrated that fimbrial adhesin FaeG was expressed in L. lactis and that oral immunization in mice with recombinant L. lactis expressing FaeG induced F4-specific mucosal and systemic immune responses. In the present study, we explored the immune responses of piglets induced by intramuscular vaccination with recombinant L. lactis expressing rFaeG. Intramuscular vaccination resulted in significantly elevated serum IgG level and modest increases in serum IgA and IgM levels. In addition, IgG, IgA, and IgM antibody secreting cells were induced in the spleen, mesenteric lymph nodes, and jejunum. The growth performance of piglets was not influenced by intramuscular vaccination. The results suggest that L. lactis expressing FaeG is a promising candidate vaccine against ETEC. PMID:23540979

  19. Literacy-Based Supports for Young Adults with FAS/FAE [Fetal Alcohol Syndrome/Fetal Alcohol Effects].

    ERIC Educational Resources Information Center

    Raymond, Margaret; Belanger, Joe

    During a 1-year period, a study investigated the contributions made by 3 literacy-based supports (support circles, cognitive compensatory tools, and cognitive enhancement tools) to the lives of 5 young adults, aged 16-34, with FAS/FAE (Fetal Alcohol Syndrome/Fetal Alcohol Effects). Four of the five subjects had IQs (intelligence quotients) above…

  20. The Secreted Esterase of Propionibacterium freudenreichii Has a Major Role in Cheese Lipolysis

    PubMed Central

    Abeijón Mukdsi, María Claudia; Falentin, Hélène; Maillard, Marie-Bernadette; Chuat, Victoria; Medina, Roxana Beatriz; Parayre, Sandrine

    2014-01-01

    Free fatty acids are important flavor compounds in cheese. Propionibacterium freudenreichii is the main agent of their release through lipolysis in Swiss cheese. Our aim was to identify the esterase(s) involved in lipolysis by P. freudenreichii. We targeted two previously identified esterases: one secreted esterase, PF#279, and one putative cell wall-anchored esterase, PF#774. To evaluate their role in lipolysis, we constructed overexpression and knockout mutants of P. freudenreichii CIRM-BIA1T for each corresponding gene. The sequences of both genes were also compared in 21 wild-type strains. All strains were assessed for their lipolytic activity on milk fat. The lipolytic activity observed matched data previously reported in cheese, thus validating the relevance of the method used. The mutants overexpressing PF#279 or PF#774 released four times more fatty acids than the wild-type strain, demonstrating that both enzymes are lipolytic esterases. However, inactivation of the pf279 gene induced a 75% reduction in the lipolytic activity compared to that of the wild-type strain, whereas inactivation of the pf774 gene did not modify the phenotype. Two of the 21 wild-type strains tested did not display any detectable lipolytic activity. Interestingly, these two strains exhibited the same single-nucleotide deletion at the beginning of the pf279 gene sequence, leading to a premature stop codon, whereas they harbored a pf774 gene highly similar to that of the other strains. Taken together, these results clearly demonstrate that PF#279 is the main lipolytic esterase in P. freudenreichii and a key agent of Swiss cheese lipolysis. PMID:24242250

  1. Isolation of acetyl esterase mutants of Bacillus subtilis 168.

    PubMed Central

    Higerd, T B

    1977-01-01

    Five mutants of Bacillus subtilis 168 defective in an intracellular esterase activity were identified. By polyacrylamide gel electrophoresis, four of the mutants were shown to lack esterase B activity, and the fifth lacked esterase A activity. All of the back-crossed esterase mutants were able to sporulate at wild-type frequency and produce exoprotease(s) and antibiotic(s). No difference in motility could be attributed to the esterase mutation. PBS1 transduction analysis showed all the esterase B mutations to be linked to the hisA marker. Images PMID:402361

  2. Cloning of fatty acid elongase1 gene and molecular identification of A and C genome in Brassica species.

    PubMed

    Wu, YuHua; Xiao, Ling; Wu, Gang; Lu, ChangMing

    2007-06-01

    The fatty acid elongase 1 (FAE1) genes of Brassic napus were cloned from two cultivars, i.e. Zhongshuan No. 9 with low erucic acid content, and Zhongyou 821 with high erucic acid content, using the degenerate PCR primers. The sequence analysis showed that there was no intron within the FAE1 genes. The FAE1 genes from Zhongyou 821 contained a coding sequence of 1521 nucleotides, and those cloned from Zhongshuan No. 9 contained a 1517 bp coding sequence. Alignment of the FAE1 sequences from Brassica rapa, B. oleracea and B. napus detected 31 single nucleotide polymorphic sites (2.03%), which resulted in 7 amino-acid substitutions. Further analysis indicated that 19 SNPs were genome-specific, of which, 95% were synonymous mutations. The nucleotide substitution at position 1217 in the FAE1 genes led to a specific site of restricted cleavage. An AvrII cleavage site was present only in the C genome genes and absent in the A genome FAE1 genes. Digestion profile of the FAE1 sequences from B. rapa, B. oleracea and B. napus produced with AvrII confirmed that the FAE1 genes of B. oleracea origin was recognized and digested, while that of B. rapa origin could not. The results indicated that by AvrII cleavage it was possible to distinguish B. rapa from B. oleracea and between the A and C genome of B. napus. In addition, the FAE1 genes could be used as marker genes to detect the pollen flow of B. napus, thus providing an alternative method for risk assessment of gene flow.

  3. Biochemical Characterization of a First Fungal Esterase from Rhizomucor miehei Showing High Efficiency of Ester Synthesis

    PubMed Central

    Liu, Yu; Xu, Haibo; Yan, Qiaojuan; Yang, Shaoqing; Duan, Xiaojie; Jiang, Zhengqiang

    2013-01-01

    Background Esterases with excellent merits suitable for commercial use in ester production field are still insufficient. The aim of this research is to advance our understanding by seeking for more unusual esterases and revealing their characterizations for ester synthesis. Methodology/Principal Findings A novel esterase-encoding gene from Rhizomucor miehei (RmEstA) was cloned and expressed in Escherichia coli. Sequence analysis revealed a 975-bp ORF encoding a 324-amino-acid polypeptide belonging to the hormone-sensitive lipase (HSL) family IV and showing highest similarity (44%) to the Paenibacillus mucilaginosus esterase/lipase. Recombinant RmEstA was purified to homogeneity: it was 34 kDa by SDS-PAGE and showed optimal pH and temperature of 6.5 and 45°C, respectively. The enzyme was stable to 50°C, under a broad pH range (5.0–10.6). RmEstA exhibited broad substrate specificity toward p-nitrophenol esters and short-acyl-chain triglycerols, with highest activities (1,480 U mg−1 and 228 U mg−1) for p-nitrophenyl hexanoate and tributyrin, respectively. RmEstA efficiently synthesized butyl butyrate (92% conversion yield) when immobilized on AOT-based organogel. Conclusion RmEstA has great potential for industrial applications. RmEstA is the first reported esterase from Rhizomucor miehei. PMID:24204998

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

    PubMed

    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. PMID:25575887

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

    PubMed

    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.

  6. Molecular cloning and characterization of a new and highly thermostable esterase from Geobacillus sp. JM6.

    PubMed

    Zhu, Yanbing; Zheng, Wenguang; Ni, Hui; Liu, Han; Xiao, Anfeng; Cai, Huinong

    2015-10-01

    A new lipolytic enzyme gene was cloned from a thermophile Geobacillus sp. JM6. The gene contained 750 bp and encoded a 249-amino acid protein. The recombinant enzyme was expressed and purified from Escherichia coli BL21 (DE3) with a molecular mass of 33.6 kDa. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding the highest activity with p-nitrophenyl butyrate. When p-nitrophenyl butyrate was used as a substrate, the optimum reaction temperature and pH for the enzyme were 60 °C and pH 7.5, respectively. Geobacillus sp. JM6 esterase showed excellent thermostability with 68% residual activity after incubation at 100 °C for 18 h. A theoretical structural model of strain JM6 esterase was developed with a monoacylglycerol lipase from Bacillus sp. H-257 as a template. The predicted core structure exhibits an α/β hydrolase fold, and a putative catalytic triad (Ser97, Asp196, and His226) was identified. Inhibition assays with PMSF indicated that serine residue is involved in the catalytic activity of strain JM6 esterase. The recombinant esterase showed a relatively good tolerance to the detected detergents and denaturants, such as SDS, Chaps, Tween 20, Tween 80, Triton X-100, sodium deoxycholate, urea, and guanidine hydrochloride.

  7. A novel esterase from a marine metagenomic library exhibiting salt tolerance ability.

    PubMed

    Fang, Zemin; Li, Jingjing; Wang, Quan; Fang, Wei; Peng, Hui; Zhang, Xuecheng; Xiao, Yazhong

    2014-06-28

    A putative lipolytic enzyme gene, named as est9x, was obtained from a marine microbial metagenome of the South China Sea. Sequence analysis showed that Est9X shares lower than 27% sequence identities with the characterized lipolytic enzymes, but possesses a catalytic triad highly conserved in lipolytic enzymes of the α/β hydrolase superfamily. By phylogenetic tree construction, Est9X was grouped into a new lipase/esterase family. To understand Est9X protein in depth, it was recombinantly expressed, purified, and biochemically characterized. Within potential hydrolytic activities, only lipase/esterase activity was detected for Est9X, confirming its identity as a lipolytic enzyme. When using p-nitrophenol esters with varying lengths of fatty acid as substrates, Est9X exhibited the highest activity to the C2 substrate, indicating it is an esterase. The optimal activity of Est9X occurred at a temperature of 65°C, and Est9X was pretty stable below the optimum temperature. Distinguished from other salttolerant esterases, Est9X's activity was tolerant to and even promoted by as high as 4 M NaCl. Our results imply that Est9X is a unique esterase and could be a potential candidate for industrial application under extreme conditions.

  8. Gene Cloning and Nucleotide Sequencing and Properties of a Cocaine Esterase from Rhodococcus sp. Strain MB1

    PubMed Central

    Bresler, Matthew M.; Rosser, Susan J.; Basran, Amrik; Bruce, Neil C.

    2000-01-01

    A strain of Rhodococcus designated MB1, which was capable of utilizing cocaine as a sole source of carbon and nitrogen for growth, was isolated from rhizosphere soil of the tropane alkaloid-producing plant Erythroxylum coca. A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized by Rhodococcus sp. strain MB1. The structural gene encoding a cocaine esterase, designated cocE, was cloned from Rhodococcus sp. strain MB1 genomic libraries by screening recombinant strains of Rhodococcus erythropolis CW25 for growth on cocaine. The nucleotide sequence of cocE corresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids. The amino acid sequence of cocaine esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serine esterase. The cocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed in Escherichia coli. The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with an Mr of approximately 65,000. The apparent Km of the enzyme (mean ± standard deviation) for cocaine was measured as 1.33 ± 0.085 mM. These findings are of potential use in the development of a linked assay for the detection of illicit cocaine. PMID:10698749

  9. Ectopic expression of FaesAP3, a Fagopyrum esculentum (Polygonaceae) AP3 orthologous gene rescues stamen development in an Arabidopsis ap3 mutant.

    PubMed

    Fang, Zheng-wu; Qi, Rui; Li, Xiao-fang; Liu, Zhi-xiong

    2014-10-25

    Arabidopsis thaliana APETALA3 (AP3) and Antirrhinum majus DEFICIENS (DEF) MADS box genes are required to specify petal and stamen identity. AP3 and DEF are members of the euAP3 lineage, which arose by gene duplication coincident with radiation of the core eudicots. In order to investigate the molecular mechanisms underlying organ development in early diverging clades of core eudicots, we isolated and identified an AP3 homolog, FaesAP3, from Fagopyrum esculentum (buckwheat, Polygonaceae), a multi-food-use pseudocereal with healing benefits. Protein sequence alignment and phylogenetic analyses revealed that FaesAP3 grouped into the euAP3 lineage. Expression analysis showed that FaesAP3 was transcribed only in developing stamens, and differed from AP3 and DEF, which expressed in developing petals and stamens. Moreover, ectopic expression of FaesAP3 rescued stamen development without complementation of petal development in an Arabidopsis ap3 mutant. Our results suggest that FaesAP3 is involved in the development of stamens in buckwheat. These results also suggest that FaesAP3 holds some potential for biotechnical engineering to create a male sterile line of F. esculentum. PMID:25149019

  10. Comparison of fungal carbohydrate esterases of family CE16 on artificial and natural substrates.

    PubMed

    Puchart, Vladimír; Agger, Jane W; Berrin, Jean-Guy; Várnai, Anikó; Westereng, Bjørge; Biely, Peter

    2016-09-10

    The enzymatic conversion of acetylated hardwood glucuronoxylan to functional food oligomers, biochemicals or fermentable monomers requires besides glycoside hydrolases enzymes liberating acetic acid esterifying position 2 and/or 3 in xylopyranosyl (Xylp) residues. The 3-O-acetyl group at internal Xylp residues substituted by MeGlcA is the only acetyl group of hardwood acetylglucuronoxylan and its fragments not attacked by acetylxylan esterases of carbohydrate esterase (CE) families 1, 4, 5 and 6 and by hemicellulolytic acetyl esterases classified in CE family 16. Monoacetylated aldotetraouronic acid 3″-Ac(3)MeGlcA(3)Xyl3, generated from the polysaccharide by GH10 endoxylanases, appears to be one of the most resistant fragments. The presence of the two substituents on the non-reducing-end Xylp residue prevents liberation of MeGlcA by α-glucuronidase of family GH67 and blocks the action of acetylxylan esterases. The Ac(3)MeGlcA(3)Xyl3 was isolated from an enzymatic hydrolysate of birchwood acetylglucuronoxylan and characterized by (1)H NMR spectroscopy as a mixture of two positional isomers, 3″-Ac(3)MeGlcA(3)Xyl3 and 4″-Ac(3)MeGlcA(3)Xyl3, the latter being the result of acetyl group migration. The mixture was used as a substrate for three members of CE16 family of fungal origin. Trichoderma reesei CE16 esterase, inactive on polymeric substrate, deacetylated both isomers. Podospora anserina and Aspergillus niger esterases, active on acetylglucuronoxylan, deesterified effectively only the 4″-isomer. The results indicate catalytic diversity among CE16 enzymes, but also their common and unifying catalytic ability to exo-deacetylate positions 3 and 4 on non-reducing-end Xylp residues, which is an important step in plant hemicellulose saccharification. PMID:27439201

  11. Differences in distribution of esterase between cell fractions of rat liver homogenates prepared in various media. Relevance to the lysosomal location of the enzyme in the intact cell

    PubMed Central

    Barrow, Patience C.; Holt, S. J.

    1971-01-01

    The distribution of esterase in subcellular fractions of rat liver homogenates was compared with that of the lysosomal enzyme acid phosphatase and the microsomal enzyme glucose 6-phosphatase. Most of the esterase from sucrose homogenate sediments with glucose 6-phosphatase and about 8% is recovered in the supernatant. However, up to 53% of the esterase can be washed from microtome sections of unfixed liver, in which less cellular damage would be expected than that caused by homogenization. About 40% of both esterase and acid phosphatase are recovered in the soluble fraction after homogenization in aqueous glycerol or in a two-phase system (Arcton 113–0.25m-sucrose), although glucose 6-phosphatase is still recovered in the microsomal fraction of such homogenates. The esterase of the microsomal fraction prepared from a sucrose homogenate is much more readily released by treatment with 0.26% deoxycholate than are other constituents of this fraction. The release of esterase from the microsomal fraction by the detergent and its concomitant release with acid phosphatase after homogenization in glycerol or the two-phase system suggests that a greater proportion of esterase may be present in lysosomes of the intact cell than is indicated by the results of standard fractionation procedures. PMID:4335692

  12. The role of calcium in the hydrolysis of the organophosphate paraoxon by human serum A-esterase.

    PubMed

    Vitarius, J A; Sultatos, L G

    1995-01-01

    Human serum A-esterase is a calcium-dependent enzyme that hydrolyzes the organophosphate paraoxon by an Ordered Uni Bi kinetic mechanism. Incubation of various concentrations of calcium chloride with human serum A-esterase resulted in corresponding changes in appk3 and appE for the reaction, while appk2 was unaffected. Carboxyglutamic acid (CAG) prevented calcium chloride from altering appk3, but not appE. Similarly CAG reduced the calcium-stimulated nonenzymatic hydrolysis of paraoxon, as well as the calcium-stimulated de-phosphorylation of chymotrypsin phosphorylated by paraoxon. These results suggest that calcium plays two roles in the hydrolysis of paraoxon by A-esterase. Firstly, calcium is required in order to maintain an active site. In this capacity calcium might participate directly in the catalytic reaction, or it might be required in order to maintain the appropriate confirmation of the active site. And secondly, free calcium (or calcium weakly associated with A-esterase) facilitates the removal of diethyl phosphate from A-esterase, probably by polarizing the P = O bond of the diethyl phosphate-A-esterase intermediate, thereby rendering phosphorus more susceptible to nucleophilic attack by hydroxide ions. PMID:7823759

  13. New Extremophilic Lipases and Esterases from Metagenomics

    PubMed Central

    López-López, Olalla; Cerdán, Maria E; González Siso, Maria I

    2014-01-01

    Lipolytic enzymes catalyze the hydrolysis of ester bonds in the presence of water. In media with low water content or in organic solvents, they can catalyze synthetic reactions such as esterification and transesterification. Lipases and esterases, in particular those from extremophilic origin, are robust enzymes, functional under the harsh conditions of industrial processes owing to their inherent thermostability and resistance towards organic solvents, which combined with their high chemo-, regio- and enantioselectivity make them very attractive biocatalysts for a variety of industrial applications. Likewise, enzymes from extremophile sources can provide additional features such as activity at extreme temperatures, extreme pH values or high salinity levels, which could be interesting for certain purposes. New lipases and esterases have traditionally been discovered by the isolation of microbial strains producing lipolytic activity. The Genome Projects Era allowed genome mining, exploiting homology with known lipases and esterases, to be used in the search for new enzymes. The Metagenomic Era meant a step forward in this field with the study of the metagenome, the pool of genomes in an environmental microbial community. Current molecular biology techniques make it possible to construct total environmental DNA libraries, including the genomes of unculturable organisms, opening a new window to a vast field of unknown enzymes with new and unique properties. Here, we review the latest advances and findings from research into new extremophilic lipases and esterases, using metagenomic approaches, and their potential industrial and biotechnological applications. PMID:24588890

  14. Review on technological and scientific aspects of feruloyl esterases: A versatile enzyme for biorefining of biomass.

    PubMed

    Gopalan, Nishant; Rodríguez-Duran, L V; Saucedo-Castaneda, G; Nampoothiri, K Madhavan

    2015-10-01

    With increasing focus on sustainable energy, bio-refining from lignocellulosic biomass has become a thrust area of research. With most of the works being focused on biofuels, significant efforts are also being directed towards other value added products. Feruloyl esterases (EC. 3.1.1.73) can be used as a tool for bio-refining of lignocellulosic material for the recovery and purification of ferulic acid and related hydroxycinnamic acids ubiquitously found in the plant cell wall. More and more genes coding for feruloyl esterases have been mined out from various sources to allow efficient enzymatic release of ferulic acid and allied hydroxycinnamic acids (HCAs) from plant-based biomass. A sum up on enzymatic extraction of HCAs and its recovery from less explored agro residual by-products is still a missing link and this review brushes up the achieved landmarks so far in this direction and also covers a detailed patent search on this biomass refining enzyme.

  15. Review on technological and scientific aspects of feruloyl esterases: A versatile enzyme for biorefining of biomass.

    PubMed

    Gopalan, Nishant; Rodríguez-Duran, L V; Saucedo-Castaneda, G; Nampoothiri, K Madhavan

    2015-10-01

    With increasing focus on sustainable energy, bio-refining from lignocellulosic biomass has become a thrust area of research. With most of the works being focused on biofuels, significant efforts are also being directed towards other value added products. Feruloyl esterases (EC. 3.1.1.73) can be used as a tool for bio-refining of lignocellulosic material for the recovery and purification of ferulic acid and related hydroxycinnamic acids ubiquitously found in the plant cell wall. More and more genes coding for feruloyl esterases have been mined out from various sources to allow efficient enzymatic release of ferulic acid and allied hydroxycinnamic acids (HCAs) from plant-based biomass. A sum up on enzymatic extraction of HCAs and its recovery from less explored agro residual by-products is still a missing link and this review brushes up the achieved landmarks so far in this direction and also covers a detailed patent search on this biomass refining enzyme. PMID:26159377

  16. Transposable Element Insertion and Epigenetic Modification Cause the Multiallelic Variation in the Expression of FAE1 in Sinapis alba[W][OPEN

    PubMed Central

    Zeng, Fangqin; Cheng, Bifang

    2014-01-01

    Naturally occurring heritable variation provides a fundamental resource to reveal the genetic and molecular bases of traits in forward genetic studies. Here, we report the molecular basis of the differences in the four alleles E1, E2, E3, and e of the FATTY ACID ELONGATION1 (FAE1) gene controlling high, medium, low, and zero erucic content in yellow mustard (Sinapis alba). E1 represents a fully functional allele with a coding DNA sequence (CDS) of 1521 bp and a promoter adjacent to the CDS. The null allele e resulted from an insertional disruption in the CDS by Sal-PIF, a 3100-bp PIF/Harbinger-like DNA transposon, whereas E2 and E3 originated from the insertion of Sal-T1, a 4863-bp Copia-like retrotransposon, in the 5′ untranslated region. E3 was identical to E2 but showed cytosine methylation in the promoter region and was thus an epiallele having a further reduction in expression. The coding regions of E2 and E3 also contained five single-nucleotide polymorphisms (SNPs) not present in E1, but expression studies in Saccharomyces cerevisiae indicated that these SNPs did not affect enzyme functionality. These results demonstrate a comprehensive molecular framework for the interplay of transposon insertion, SNP/indel mutation, and epigenetic modification influencing the broad range of natural genetic variation in plants. PMID:24934174

  17. Characterization of esterases from Cucurbita pepo cv. "Eskandrani".

    PubMed

    Fahmy, Afaf S; Abo-Zeid, Amal Z; Mohamed, Tarek M; Ghanem, Hala M; Borai, Ibrahim H; Mohamed, Saleh A

    2008-01-01

    Two of the six esterases identified in Cucurbita pepo cv. "Eskandrani" were purified to homogeneity using two chromatography steps: anion exchange and gel filtration. The molecular weights of C. pepo esterases EIc and EII were 50,000 +/- 1500 and 68,000 +/- 1900 Da from gel filtration and 47,000 and 66,000 Da from SDS/PAGE, respectively, suggesting a monomeric structure for both enzymes. Esterases EIc and EII had K(m) values of 1.22 and 1.56 mM and pH optima at 9.0 and 8.0, respectively. The substrate specificity of C. pepo esterases EIc and EII were determined for a number of p-nitrophenyl esters, where their affinity toward these substrates were decreased as carbon atom number increased. Esterases EIc and EII had the same temperature optima, 40 degrees C. Thermal stability studies of esterases EIc and EII indicated that half maximal activities of EIc and EII esterases were reached at 55 degrees C and 50 degrees C, while they lost 45%, 51% and 70%, 77% of their activities after 30 and 90 min of incubation at 40 degrees C, respectively. The effect of different metal cations and inhibitors were examined. The inhibition studies revealed that the active sites of the two esterases contain serine and cysteine residues. The characteristics of C. pepo esterases are closely similar to those of microbial esterases used in food processing and food industry. PMID:17321740

  18. A thermoactive uropygial esterase from chicken: purification, characterisation and synthesis of flavour esters.

    PubMed

    Fendri, Ahmed; Louati, Hanen; Sellami, Mohamed; Gargouri, Héla; Smichi, Nabil; Zarai, Zied; Aissa, Imen; Miled, Nabil; Gargouri, Youssef

    2012-06-01

    A lipolytic activity was located in the chicken uropygial glands, from which a carboxylesterase (CUE) was purified. Pure CUE has an apparent molecular mass of 50 kDa. The purified esterase displayed its maximal activity (200 U/mg) on short-chain triacylglycerols (tributyrin) at a temperature of 50°C. No significant lipolytic activity was found when medium chain (trioctanoin) or long chain (olive oil) triacylglycerols were used as substrates. The enzyme retained 75% of its maximal activity when incubated during 2h at 50°C. The NH(2)-terminal amino acid sequence showed similarities with the esterase purified recently from turkey pharyngeal tissue. Esterase activity remains stable after its incubation during 30 min in presence of organic solvents such as hexane or butanol. CUE is a serine enzyme since it was inactivated by phenylmethanesulphonyl fluoride (PMSF), a serine-specific inhibitor. The purified enzyme, which tolerates the presence of some organic solvent and a high temperature, can be used in non-aqueous synthesis reactions. Hence, the uropygial esterase immobilised onto CaCO(3) was tested to produce the isoamyl and the butyl acetate (flavour esters). Reactions were performed at 50°C in presence of hexane. High synthesis yields of 91 and 67.8% were obtained for isoamyl and butyl acetate, respectively. PMID:22531158

  19. Xylella fastidiosa esterase rather than hydroxynitrile lyase.

    PubMed

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

    2015-03-01

    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. PMID:25684099

  20. Uncovering divergent evolution of α/β-hydrolases: a surprising residue substitution needed to convert Hevea brasiliensis hydroxynitrile lyase into an esterase.

    PubMed

    Nedrud, David M; Lin, Hui; Lopez, Gilsinia; Padhi, Santosh K; Legatt, Graig A; Kaz-Lauskas, Romas J

    2014-11-01

    Hevea brasiliensis hydroxynitrile lyase (HbHNL) and salicylic acid binding protein 2 (SABP2, an esterase) share 45% amino acid sequence identity, the same protein fold, and even the same catalytic triad of Ser-His-Asp. However, they catalyze different reactions: cleavage of hydroxynitriles and hydrolysis of esters, respectively. To understand how other active site differences in the two enzymes enable the same catalytic triad to catalyze different reactions, we substituted amino acid residues in HbHNL with the corresponding residues from SABP2, expecting hydroxynitrile lyase activity to decrease and esterase activity to increase. Previous mechanistic studies and x-ray crystallography suggested that esterase activity requires removal of an active site lysine and threonine from the hydroxynitrile lyase. The Thr11Gly Lys236Gly substitutions in HbHNL reduced hydroxynitrile lyase activity for cleavage of mandelonitrile 100-fold, but increased esterase activity only threefold to kcat ~ 0.1 min(-1) for hydrolysis of p-nitrophenyl acetate. Adding a third substitution - Glu79His - increased esterase activity more than tenfold to kcat ~ 1.6 min(-1). The specificity constant (kcat/KM) for this triple substitution variant versus wild type HbHNL shifted more than one million-fold from hydroxynitrile lyase activity (acetone cyanohydrin substrate) to esterase activity (p-nitrophenyl acetate substrate). The contribution of Glu79His to esterase activity was surprising since esterases and lipases contain many different amino acids at this position, including glutamate. Saturation mutagenesis at position 79 showed that 13 of 19 possible amino acid substitutions increased esterase activity, suggesting that removal of glutamate, not addition of histidine, increased esterase activity. Molecular modeling indicates that Glu79 disrupts esterase activity in HbHNL when its negatively charged side chain distorts the orientation of the catalytic histidine. Naturally occurring glutamate at the

  1. Uncovering divergent evolution of α/β-hydrolases: a surprising residue substitution needed to convert Hevea brasiliensis hydroxynitrile lyase into an esterase

    PubMed Central

    Nedrud, David M.; Lin, Hui; Lopez, Gilsinia; Padhi, Santosh K.; Legatt, Graig A.

    2014-01-01

    Hevea brasiliensis hydroxynitrile lyase (HbHNL) and salicylic acid binding protein 2 (SABP2, an esterase) share 45% amino acid sequence identity, the same protein fold, and even the same catalytic triad of Ser-His-Asp. However, they catalyze different reactions: cleavage of hydroxynitriles and hydrolysis of esters, respectively. To understand how other active site differences in the two enzymes enable the same catalytic triad to catalyze different reactions, we substituted amino acid residues in HbHNL with the corresponding residues from SABP2, expecting hydroxynitrile lyase activity to decrease and esterase activity to increase. Previous mechanistic studies and x-ray crystallography suggested that esterase activity requires removal of an active site lysine and threonine from the hydroxynitrile lyase. The Thr11Gly Lys236Gly substitutions in HbHNL reduced hydroxynitrile lyase activity for cleavage of mandelonitrile 100-fold, but increased esterase activity only threefold to kcat ~ 0.1 min−1 for hydrolysis of p-nitrophenyl acetate. Adding a third substitution – Glu79His – increased esterase activity more than tenfold to kcat ~ 1.6 min−1. The specificity constant (kcat/KM) for this triple substitution variant versus wild type HbHNL shifted more than one million-fold from hydroxynitrile lyase activity (acetone cyanohydrin substrate) to esterase activity (p-nitrophenyl acetate substrate). The contribution of Glu79His to esterase activity was surprising since esterases and lipases contain many different amino acids at this position, including glutamate. Saturation mutagenesis at position 79 showed that 13 of 19 possible amino acid substitutions increased esterase activity, suggesting that removal of glutamate, not addition of histidine, increased esterase activity. Molecular modeling indicates that Glu79 disrupts esterase activity in HbHNL when its negatively charged side chain distorts the orientation of the catalytic histidine. Naturally occurring glutamate at

  2. Uncovering divergent evolution of α/β-hydrolases: a surprising residue substitution needed to convert Hevea brasiliensis hydroxynitrile lyase into an esterase.

    PubMed

    Nedrud, David M; Lin, Hui; Lopez, Gilsinia; Padhi, Santosh K; Legatt, Graig A; Kaz-Lauskas, Romas J

    2014-11-01

    Hevea brasiliensis hydroxynitrile lyase (HbHNL) and salicylic acid binding protein 2 (SABP2, an esterase) share 45% amino acid sequence identity, the same protein fold, and even the same catalytic triad of Ser-His-Asp. However, they catalyze different reactions: cleavage of hydroxynitriles and hydrolysis of esters, respectively. To understand how other active site differences in the two enzymes enable the same catalytic triad to catalyze different reactions, we substituted amino acid residues in HbHNL with the corresponding residues from SABP2, expecting hydroxynitrile lyase activity to decrease and esterase activity to increase. Previous mechanistic studies and x-ray crystallography suggested that esterase activity requires removal of an active site lysine and threonine from the hydroxynitrile lyase. The Thr11Gly Lys236Gly substitutions in HbHNL reduced hydroxynitrile lyase activity for cleavage of mandelonitrile 100-fold, but increased esterase activity only threefold to kcat ~ 0.1 min(-1) for hydrolysis of p-nitrophenyl acetate. Adding a third substitution - Glu79His - increased esterase activity more than tenfold to kcat ~ 1.6 min(-1). The specificity constant (kcat/KM) for this triple substitution variant versus wild type HbHNL shifted more than one million-fold from hydroxynitrile lyase activity (acetone cyanohydrin substrate) to esterase activity (p-nitrophenyl acetate substrate). The contribution of Glu79His to esterase activity was surprising since esterases and lipases contain many different amino acids at this position, including glutamate. Saturation mutagenesis at position 79 showed that 13 of 19 possible amino acid substitutions increased esterase activity, suggesting that removal of glutamate, not addition of histidine, increased esterase activity. Molecular modeling indicates that Glu79 disrupts esterase activity in HbHNL when its negatively charged side chain distorts the orientation of the catalytic histidine. Naturally occurring glutamate at the

  3. β-Glucuronidase-coupled assays of glucuronoyl esterases.

    PubMed

    Fraňová, Lucia; Puchart, Vladimír; Biely, Peter

    2016-10-01

    Glucuronoyl esterases (GEs) are microbial enzymes with potential to cleave the ester bonds between lignin alcohols and xylan-bound 4-O-methyl-d-glucuronic acid in plant cell walls. This activity renders GEs attractive research targets for biotechnological applications. One of the factors impeding the progress in GE research is the lack of suitable substrates. In this work, we report a facile preparation of methyl esters of chromogenic 4-nitrophenyl and 5-bromo-4-chloro-3-indolyl β-D-glucuronides for qualitative and quantitative GE assay coupled with β-glucuronidase as the auxiliary enzyme. The indolyl derivative affording a blue indigo-type product is suitable for rapid and sensitive assay of GE in commercial preparations as well as for high throughput screening of microorganisms and genomic and metagenomic libraries. PMID:27452816

  4. Novel choline esterase based sensor for monitoring of organophosphorus pollutants

    SciTech Connect

    Wilkins, E.S.; Ghindilis, A.L.; Atanasov, P.

    1996-12-31

    Organophosphorus compounds are significant major environmental pollutants due to their intensive use as pesticides. The modern techniques based on inhibition of choline esterase enzyme activity are discussed. Potentiometric electrodes based on detection of choline esterase inhibition by analytes has been developed. The detection of choline esterase activity is based on the novel principle of molecular transduction. Immobilized peroxidase acting as the molecular transducer, catalyzes the electroreduction of hydrogen peroxide by direct (mediatorless) electron transfer. The sensing element consists of a carbon based electrode containing an assembly of co-immobilized enzymes: choline esterase, choline oxidase and peroxidase.

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

    SciTech Connect

    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

  6. Improved biomass degradation using fungal glucuronoyl-esterases-hydrolysis of natural corn fiber substrate.

    PubMed

    d'Errico, Clotilde; Börjesson, Johan; Ding, Hanshu; Krogh, Kristian B R M; Spodsberg, Nikolaj; Madsen, Robert; Monrad, Rune Nygaard

    2016-02-10

    Lignin-carbohydrate complexes (LCCs) are in part responsible for the recalcitrance of lignocellulosics in relation to industrial utilization of biomass for biofuels. Glucuronoyl esterases (GEs) belonging to the carbohydrate esterase family 15 have been proposed to be able to degrade ester LCCs between glucuronic acids in xylans and lignin alcohols. By means of synthesized complex LCC model substrates we provide kinetic data suggesting a preference of fungal GEs for esters of bulky arylalkyl alcohols such as ester LCCs. Furthermore, using natural corn fiber substrate we report the first examples of improved degradation of lignocellulosic biomass by the use of GEs. Improved C5 sugar, glucose and glucuronic acid release was observed when heat pretreated corn fiber was incubated in the presence of GEs from Cerrena unicolor and Trichoderma reesei on top of different commercial cellulase/hemicellulase preparations. These results emphasize the potential of GEs for delignification of biomass thereby improving the overall yield of fermentable sugars for biofuel production. PMID:26712478

  7. Improved biomass degradation using fungal glucuronoyl-esterases-hydrolysis of natural corn fiber substrate.

    PubMed

    d'Errico, Clotilde; Börjesson, Johan; Ding, Hanshu; Krogh, Kristian B R M; Spodsberg, Nikolaj; Madsen, Robert; Monrad, Rune Nygaard

    2016-02-10

    Lignin-carbohydrate complexes (LCCs) are in part responsible for the recalcitrance of lignocellulosics in relation to industrial utilization of biomass for biofuels. Glucuronoyl esterases (GEs) belonging to the carbohydrate esterase family 15 have been proposed to be able to degrade ester LCCs between glucuronic acids in xylans and lignin alcohols. By means of synthesized complex LCC model substrates we provide kinetic data suggesting a preference of fungal GEs for esters of bulky arylalkyl alcohols such as ester LCCs. Furthermore, using natural corn fiber substrate we report the first examples of improved degradation of lignocellulosic biomass by the use of GEs. Improved C5 sugar, glucose and glucuronic acid release was observed when heat pretreated corn fiber was incubated in the presence of GEs from Cerrena unicolor and Trichoderma reesei on top of different commercial cellulase/hemicellulase preparations. These results emphasize the potential of GEs for delignification of biomass thereby improving the overall yield of fermentable sugars for biofuel production.

  8. Conserved tyrosine 182 residue in hyperthermophilic esterase EstE1 plays a critical role in stabilizing the active site.

    PubMed

    Truongvan, Ngoc; Chung, Hye-Shin; Jang, Sei-Heon; Lee, ChangWoo

    2016-03-01

    An aromatic amino acid, Tyr or Trp, located in the esterase active site wall, is highly conserved, with hyperthermophilic esterases showing preference for Tyr and lower temperature esterases showing preference for Trp. In this study, we investigated the role of Tyr(182) in the active site wall of hyperthermophilic esterase EstE1. Mutation of Tyr to Phe or Ala had a moderate effect on EstE1 thermal stability. However, a small-to-large mutation such as Tyr to His or Trp had a devastating effect on thermal stability. All mutant EstE1 enzymes showed reduced catalytic rates and enhanced substrate affinities as compared with wild-type EstE1. Hydrogen bond formation involving Tyr(182) was unimportant for maintaining EstE1 thermal stability, as the EstE1 structure is already adapted to high temperatures via increased intramolecular interactions. However, removal of hydrogen bond from Tyr(182) significantly decreased EstE1 catalytic activity, suggesting its role in stabilization of the active site. These results suggest that Tyr is preferred over a similarly sized Phe residue or bulky His or Trp residue in the active site walls of hyperthermophilic esterases for stabilizing the active site and regulating catalytic activity at high temperatures. PMID:26838013

  9. VvMJE1 of the grapevine (Vitis vinifera) VvMES methylesterase family encodes for methyl jasmonate esterase and has a role in stress response

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The known members of the plant methyl esterase (MES) family catalyze hydrolysis of a C-O ester linkage of methyl esters of several phytohormones including indole-3-acetic acid, salicylic acid, and jasmonic acid. The genome of grapevine (Vitis vinifera) was found to contain 15 MES genes, designated V...

  10. Novel xylanase from a holstein cattle rumen metagenomic library and its application in xylooligosaccharide and ferulic Acid production from wheat straw.

    PubMed

    Cheng, Fansheng; Sheng, Jiping; Dong, Rubo; Men, Yejun; Gan, Lin; Shen, Lin

    2012-12-26

    A novel gene fragment containing a xylanase was identified from a Holstein cattle rumen metagenomic library. The novel xylanase (Xyln-SH1) belonged to the glycoside hydrolase family 10 (GH10) and exhibited a maximum of 44% identity to the glycoside hydrolase from Clostridium thermocellum ATCC 27405. Xyln-SH1 was heterologously expressed, purified, and characterized. A high level of activity was obtained under the optimum conditions of pH 6.5 and 40 °C. A substrate utilization study indicated that Xyln-SH1 was cellulase-free and strictly specific to xylan from softwood. The synergistic effects of Xyln-SH1 and feruloyl esterase (FAE-SH1) were observed for the release of xylooligosaccharides (XOS) and ferulic acid (FA) from wheat straw. In addition, a high dose of Xyln-SH1 alone was observed to improve the release of FA from wheat straw. These features suggest that this enzyme has substantial potential to improve biomass degradation and industrial applications.

  11. Esterases and putative lipases from tropical isolates of Aureobasidium pullulans.

    PubMed

    Kudanga, Tukayi; Mwenje, Eddie; Mandivenga, Faith; Read, John S

    2007-04-01

    Esterases and lipases have been studied in a number of fungi, though very little is known about esterases from Aureobasidium pullulans especially from the African tropics. In this study, forty-two Zimbabwean isolates were screened for lipase activity on tributyrin agar. Extracellular esterase activities of seven selected isolates were studied under varying conditions using para-nitrophenol acetate as substrate. Twenty isolates (48%) showed lipolytic activity; sixteen showed negative results for lipase activity while the rest showed weak activities. Esterase activities in broth cultures ranged from 0.011-0.223 mmol/microg protein/min while activities ranged from 1.5-12.8 U/ml under solid state fermentation. The esterases were optimally active at pH 7.6-8.0, showed a temperature optimum of 35 degrees C and retained more than 50% activity at temperatures up to 60 degrees C and at pH 4.0-7.0 after 150 min. Enzyme production was optimal after 5-6 days with diammonium hydrogen phosphate as nitrogen source. Isolates showed variations in preference for carbon source for esterase production. The A. pullulans esterases differed from most fungal esterases in that they are optimally active in alkaline conditions and are active over a broad pH range. PMID:17440916

  12. Identification of petrogenic produced water components as acetylcholine esterase inhibitors.

    PubMed

    Froment, Jean; Langford, Katherine; Tollefsen, Knut Erik; Bråte, Inger Lise N; Brooks, Steven J; Thomas, Kevin V

    2016-08-01

    Effect-directed analysis (EDA) was applied to identify acetylcholine esterase (AChE) inhibitors in produced water. Common produced water components from oil production activities, such as polycyclic aromatic hydrocarbons (PAHs), alkylphenols, and naphthenic acids were tested for AChE inhibition using a simple mixture of PAHs and naphthenic acids. Produced water samples collected from two offshore platforms in the Norwegian sector of the North Sea were extracted by solid phase extraction and fractionated by open-column liquid solid chromatography and high-performance liquid chromatography (HPLC) before being tested using a high-throughput and automated AChE assay. The HPLC fractions causing the strongest AChE inhibition were analysed by gas chromatography coupled to a high-resolution time-of-flight mass spectrometry (GC-HR-ToF-MS). Butylated hydroxytoluene and 4-phenyl-1,2-dihydronaphthalene were identified as two produced water components capable of inhibiting AChE at low concentrations. In order to assess the potential presence of such compounds discharged into aquatic ecosystems, AChE activity in fish tissues was measured. Saithe (Pollachius virens) caught near two offshore platforms showed lower enzymatic activity than those collected from a reference location. Target analysis of saithe did not detected the presence of these two putative AChE inhibitors and suggest that additional compounds such as PAHs, naphthenic acids and yet un-identified compounds may also contribute to the purported AChE inhibition observed in saithe. PMID:27176761

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

    NASA Astrophysics Data System (ADS)

    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.

  14. A cold-adapted esterase of a novel marine isolate, Pseudoalteromonas arctica: gene cloning, enzyme purification and characterization.

    PubMed

    Al Khudary, Rami; Venkatachalam, Ramprasath; Katzer, Moritz; Elleuche, Skander; Antranikian, Garabed

    2010-05-01

    A gene encoding an esterase (estO) was identified and sequenced from a gene library screen of the psychrotolerant bacterium Pseudoalteromonas arctica. Analysis of the 1,203 bp coding region revealed that the deduced peptide sequence is composed of 400 amino acids with a predicted molecular mass of 44.1 kDa. EstO contains a N-terminal esterase domain and an additional OsmC domain at the C-terminus (osmotically induced family of proteins). The highly conserved five-residue motif typical for all alpha/beta hydrolases (G x S x G) was detected from position 104 to 108 together with a putative catalytic triad consisting of Ser(106), Asp(196), and His(225). Sequence comparison showed that EstO exhibits 90% amino acid identity with hypothetical proteins containing similar esterase and OsmC domains but only around 10% identity to the amino acid sequences of known esterases. EstO variants with and without the OsmC domain were produced and purified as His-tag fusion proteins in E. coli. EstO displayed an optimum pH of 7.5 and optimum temperature of 25 degrees C with more than 50% retained activity at the freezing point of water. The thermostability of EstO (50% activity after 5 h at 40 degrees C) dramatically increased in the truncated variant (50% activity after 2.5 h at 90 degrees C). Furthermore, the esterase displays broad substrate specificity for esters of short-chain fatty acids (C(2)-C(8)).

  15. Cloning, expression and characterization of a novel cold-active and organic solvent-tolerant esterase from Monascus ruber M7.

    PubMed

    Guo, Hailun; Zhang, Yan; Shao, Yanchun; Chen, Wanping; Chen, Fusheng; Li, Mu

    2016-07-01

    Cold active esterases are a class of important biocatalysts that exhibit high activity at low temperatures. In this study, a search for putative cold-active esterase encoding genes from Monascus ruber M7 was performed. A cold-active esterase, named Lip10, was isolated, cloned, purified, and characterized. Amino acid sequence analysis reveals that Lip10 contained a conserved sequence motif Gly(173)-Xaa-Ser(175)-Xaa-Gly(177) that is also present in the majority of esterases and lipases. Phylogenetic analysis indicated that Lip10 was a novel microbial esterase. The lip10 gene was cloned and heterologously expressed in Escherichia coli BL21(DE3), resulting in the expression of an active and soluble protein that constituted 40 % of the total cell protein content. Lip10 maintained almost 50 % of its maximal activity at 4-10 °C, with optimal activity at 40 °C. Furthermore, Lip10 retained 184-216 % of its original activity, after incubation in 50 % (v/v) hydrophobic organic solvents for 24 h. The enzyme also exhibited high activity under alkaline conditions and good tolerance to metal ions in the reaction mixture. These results indicate that Lip10 may have potential uses in chemical synthesis and food processing industrial applications as an esterase. PMID:27209523

  16. Est10: A Novel Alkaline Esterase Isolated from Bovine Rumen Belonging to the New Family XV of Lipolytic Enzymes

    PubMed Central

    Rodríguez, María Cecilia; Loaces, Inés; Amarelle, Vanesa; Senatore, Daniella; Iriarte, Andrés; Fabiano, Elena; Noya, Francisco

    2015-01-01

    A metagenomic fosmid library from bovine rumen was used to identify clones with lipolytic activity. One positive clone was isolated. The gene responsible for the observed phenotype was identified by in vitro transposon mutagenesis and sequencing and was named est10. The 367 amino acids sequence harbors a signal peptide, the conserved secondary structure arrangement of alpha/beta hydrolases, and a GHSQG pentapeptide which is characteristic of esterases and lipases. Homology based 3D-modelling confirmed the conserved spatial orientation of the serine in a nucleophilic elbow. By sequence comparison, Est10 is related to hydrolases that are grouped into the non-specific Pfam family DUF3089 and to other characterized esterases that were recently classified into the new family XV of lipolytic enzymes. Est10 was heterologously expressed in Escherichia coli as a His-tagged fusion protein, purified and biochemically characterized. Est10 showed maximum activity towards C4 aliphatic chains and undetectable activity towards C10 and longer chains which prompted its classification as an esterase. However, it was able to efficiently catalyze the hydrolysis of aryl esters such as methyl phenylacetate and phenyl acetate. The optimum pH of this enzyme is 9.0, which is uncommon for esterases, and it exhibits an optimal temperature at 40°C. The activity of Est10 was inhibited by metal ions, detergents, chelating agents and additives. We have characterized an alkaline esterase produced by a still unidentified bacterium belonging to a recently proposed new family of esterases. PMID:25973851

  17. Purification and characterization of an extracellular esterase with organic solvent tolerance from a halotolerant isolate, Salimicrobium sp. LY19

    PubMed Central

    2013-01-01

    Background Halotolerant bacteria are excellent sources for selecting novel enzymes. Being intrinsically stable and active under high salinities, enzymes from these prokaryotes have evolved to function optimally under extreme conditions, making them robust biocatalysts with potential applications in harsh industrial processes. Results A halotolerant strain LY19 showing lipolytic activity was isolated from saline soil of Yuncheng Salt Lake, China. It was identified as belonging to the genus of Salimicrobium by 16S rRNA gene sequence analysis. The extracellular enzyme was purified to homogeneity with molecular mass of 57 kDa by SDS-PAGE. Substrate specificity test revealed that the enzyme preferred short-chain p-nitrophenyl esters and exhibited maximum activity towards p-nitrophenyl butyrate (p-NPB), indicating an esterase activity. The esterase was highly active and stable over broad temperature (20°C-70°C), pH (7.0-10.0) and NaCl concentration (2.5%-25%) ranges, with an optimum at 50°C, pH 7.0 and 5% NaCl. Significant inhibition of the esterase was shown by ethylenediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF) and phenylarsine oxide (PAO), which indicated that it was a metalloenzyme with serine and cysteine residues essential for enzyme activity. Moreover, the esterase displayed high activity and stability in the presence of hydrophobic organic solvents with log Pow ≥ 0.88 than in the absence of an organic solvent or in the presence of hydrophilic solvents. Conclusions Results from the present study indicated the novel extracellular esterase from Salimicrobium sp. LY19 exhibited thermostable, alkali-stable, halotolerant and organic solvent-tolerant properties. These features led us to conclude that the esterase may have considerable potential for industrial applications in organic synthesis reactions. PMID:24325447

  18. Structure and activity of the cold-active and anion-activated carboxyl esterase OLEI01171 from the oil-degrading marine bacterium Oleispira antarctica

    PubMed Central

    Lemak, Sofia; Tchigvintsev, Anatoli; Petit, Pierre; Flick, Robert; Singer, Alexander U.; Brown, Greg; Evdokimova, Elena; Egorova, Olga; Gonzalez, Claudio F.; Chernikova, Tatyana N.; Yakimov, Michail M.; Kube, Michael; Reinhardt, Richard; Golyshin, Peter N.; Savchenko, Alexei; Yakunin, Alexander F.

    2014-01-01

    The uncharacterized α/β-hydrolase protein OLEI01171 from the psychrophilic marine bacterium Oleispira antarctica belongs to the PF00756 family of putative esterases, which also includes human esterase D. In the present paper we show that purified recombinant OLEI01171 exhibits high esterase activity against the model esterase substrate α-naphthyl acetate at 5 – 30°C with maximal activity at 15–20°C. The esterase activity of OLEI01171 was stimulated 3–8-fold by the addition of chloride or several other anions (0.1–1.0 M). Compared with mesophilic PF00756 esterases, OLEI01171 exhibited a lower overall protein thermostability. Two crystal structures ofOLEI01171 were solved at 1.75 and 2.1 Å resolution and revealed a classical serine hydrolase catalytic triad and the presence of a chloride or bromide ion bound in the active site close to the catalytic Ser148.Both anions were found to co-ordinate a potential catalytic water molecule located in the vicinity of the catalytic triad His257. The results of the present study suggest that the bound anion perhaps contributes to the polarization of the catalytic water molecule and increases the rate of the hydrolysis of an acyl-enzyme intermediate. Alanine replacement mutagenesis of OLEI01171 identified ten amino acid residues important for esterase activity. The replacement of Asn225 by lysine had no significant effect on the activity or thermostability of OLEI01171, but resulted in a detectable increase of activity at 35–45°C. The present study has provided insight into the molecular mechanisms of activity of a cold-active and anion-activated carboxyl esterase. PMID:22519667

  19. Esterase patterns of species in the Drosophila buzzatii cluster.

    PubMed

    Lapenta, A S; de Campos Bicudo, H E; Ceron, C R; Cordeiro, J A

    1995-01-01

    A comparative analysis was made of the esterase isoenzyme patterns of eight iso-female lines, four of Drosophila serido (B31 D1, A55, B59, Q1, B50Q3), two of D. koepferae (B20D2 and B25D7), one of D. seriema (A95) and one of D. buzzatii (Buz). In all, 43 bands in the spectrum of esterase isoenzymes were detected by electrophoresis in polyacrylamide gels. They showed variations in specific reactions with alpha and beta-naphthyl acetate, number of patterns yielded in their intra-isofemale line combinations, frequencies of such combinations and the thickness and staining degree of some bands, in different individuals, lines and species. Among bands detected exclusively in males, seven may be considered sex-specific (5 alpha-esterases and 2 beta-esterases). These male-specific alpha-esterases have in common the inability to cleave beta-naphthyl acetate in the absence of alpha-naphthyl, denoting a possible common function. The similarity index (SI) and analysis of dependence were calculated in an attempt to quantify the differentiation of the iso-female lines studied, on the basis of esterase bands. SI mean value allowed the separation of the isofemale lines into five classes. Each species had its own pattern of esterase bands, but some bands were shared. A divergence hypothesis for the isofemale lines and the species is discussed.

  20. Mycobacteriophage Lysin B is a novel mycolylarabinogalactan esterase

    SciTech Connect

    Payne, K.; Sun, Q.; Sacchettini, J.; Hatfull, G.F.

    2010-08-27

    Mycobacteriophages encounter a unique problem among phages of Gram-positive bacteria, in that lysis must not only degrade the peptidoglycan layer but also circumvent a mycolic acid-rich outer membrane covalently attached to the arabinogalactan-peptidoglycan complex. Mycobacteriophages accomplish this by producing two lysis enzymes, Lysin A (LysA) that hydrolyses peptidoglycan, and Lysin B (LysB), a novel mycolylarabinogalactan esterase, that cleaves the mycolylarabinogalactan bond to release free mycolic acids. The D29 LysB structure shows an {alpha}/{beta} hydrolase organization with a catalytic triad common to cutinases, but which contains an additional four-helix domain implicated in the binding of lipid substrates. Whereas LysA is essential for mycobacterial lysis, a Giles {Delta}lysB mutant mycobacteriophage is viable, but defective in the normal timing, progression and completion of host cell lysis. We propose that LysB facilitates lysis by compromising the integrity of the mycobacterial outer membrane linkage to the arabinogalactan-peptidoglycan layer.

  1. Mechanism of action of Neisseria gonorrhoeae O-acetylpeptidoglycan esterase, an SGNH serine esterase.

    PubMed

    Pfeffer, John M; Weadge, Joel T; Clarke, Anthony J

    2013-01-25

    O-Acetylpeptidoglycan esterase from Neisseria gonorrhoeae functions to release O-acetyl groups from the C-6 position of muramoyl residues in O-acetylated peptidoglycan, thereby permitting the continued metabolism of this essential cell wall heteropolymer. It has been demonstrated to be a serine esterase with sequence similarity to the family CE-3 carbohydrate esterases of the CAZy classification system. In the absence of a three-dimensional structure for any Ape, further knowledge of its structure and function relationship is dependent on modeling and kinetic studies. In this study, we predicted Neisseria gonorrhoeae Ape1a to be an SGNH hydrolase with an adopted α/β-hydrolase fold containing a central twisted four-stranded parallel β-sheet flanked by six α-helices with the putative catalytic triad, Asp-366, His-369, and Ser-80 appropriately aligned within a pocket. The role of eight invariant and highly conserved residues localized to the active site was investigated by site-directed replacements coupled with kinetic characterization and binding studies of the resultant engineered enzymes. Based on these data and theoretical considerations, Gly-236 and Asn-268 were identified as participating at the oxyanion hole to stabilize the tetrahedral species in the reaction mechanism, whereas Gly-78, Asp-79, His-81, Asn-235, Thr-267, and Val-368 are proposed to position appropriately the catalytic residues and participate in substrate binding. PMID:23209280

  2. Identification and characterization of a novel salt-tolerant esterase from a Tibetan glacier metagenomic library.

    PubMed

    De Santi, Concetta; Ambrosino, Luca; Tedesco, Pietro; Zhai, Lei; Zhou, Cheng; Xue, Yanfen; Ma, Yanhe; de Pascale, Donatella

    2015-01-01

    A salt-tolerant esterase, designated H9Est, was identified from a metagenomic library of the Karuola glacier. H9Est gene comprised 1071 bp and encoded a polypeptide of 357 amino acids with a molecular mass of 40 kDa. Sequence analysis revealed that H9Est belonged to the family IV of bacterial lypolitic enzyme. H9Est was overexpressed in Escherichia coli and the purified enzyme showed hydrolytic activity towards p-nitrophenyl esters with carbon chain from 2 to 8. The optimal esterase activity was at 40°C and pH 8.0 and the enzyme retained its activity towards some miscible organic solvents such as polyethylene glycol. A three-dimensional model of H9Est revealed that S200, D294, and H324 formed the H9Est catalytic triad. Circular Dichroism spectra and molecular dynamic simulation indicated that the esterase had a wide denaturation temperature range and flexible loops that would be beneficial for H9Est performance at low temperatures while retaining heat-resistant features. PMID:25920073

  3. Characterization of two metagenome-derived esterases that reactivate chloramphenicol by counteracting chloramphenicol acetyltransferase.

    PubMed

    Tao, Weixin; Lee, Myung Hwan; Yoon, Mi-Young; Kim, Jin-Cheol; Malhotra, Shweta; Wu, Jing; Hwang, Eul Chul; Lee, Seon-Woo

    2011-12-01

    Function-driven metagenomic analysis is a powerful approach to screening for novel biocatalysts. In this study, we investigated lipolytic enzymes selected from an alluvial soil metagenomic library, and identified two novel esterases, EstDL26 and EstDL136. EstDL26 and EstDL136 reactivated chloramphenicol from its acetyl derivates by counteracting the chloramphenicol acetyltransferase (CAT) activity in Escherichia coli. These two enzymes showed only 27% identity in amino acid sequence to each other; however both preferentially hydrolyzed short-chain p-nitrophenyl esters (< or =C5) and showed mesophilic properties. In vitro, EstDL136 catalyzed the deacetylation of 1- and 3- acetyl and 1,3-diacetyl derivates; in contrast, EstDL26 was not capable of the deacetylation at C1, indicating a potential regioselectivity. EstDL26 and EstDL136 were similar to microbial hormone-sensitive lipase (HSL), and since chloramphenicol acetate esterase (CAE) activity was detected from two other soil esterases in the HSL family, this suggests a distribution of CAE among the soil microorganisms. The isolation and characterization of EstDL26 and EstDL136 in this study may be helpful in understanding the diversity of CAE enzymes and their potential role in releasing active chloramphenicol in the producing bacteria. PMID:22210605

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

    PubMed

    Lopes, Jose L S; Yoneda, Juliana S; Martins, Julia M; DeMarco, Ricardo; Jameson, David M; Castro, Aline M; Bossolan, Nelma R S; Wallace, B A; Araujo, Ana P U

    2016-01-01

    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. PMID:27351338

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

    PubMed Central

    Martins, Julia M.; DeMarco, Ricardo; Jameson, David M.; Castro, Aline M.; Bossolan, Nelma R. S.; Wallace, B. A.; Araujo, Ana P. U.

    2016-01-01

    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. PMID:27351338

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

    PubMed Central

    De Santi, Concetta; Willassen, Nils Peder

    2016-01-01

    Background 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. Results 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. PMID:27433797

  7. Characterization of a Feruloyl Esterase from Lactobacillus plantarum

    PubMed Central

    Esteban-Torres, María; Reverón, Inés; Mancheño, José Miguel; de las Rivas, Blanca

    2013-01-01

    Lactobacillus plantarum is frequently found in the fermentation of plant-derived food products, where hydroxycinnamoyl esters are abundant. L. plantarum WCFS1 cultures were unable to hydrolyze hydroxycinnamoyl esters; however, cell extracts from the strain partially hydrolyze methyl ferulate and methyl p-coumarate. In order to discover whether the protein Lp_0796 is the enzyme responsible for this hydrolytic activity, it was recombinantly overproduced and enzymatically characterized. Lp_0796 is an esterase that, among other substrates, is able to efficiently hydrolyze the four model substrates for feruloyl esterases (methyl ferulate, methyl caffeate, methyl p-coumarate, and methyl sinapinate). A screening test for the detection of the gene encoding feruloyl esterase Lp_0796 revealed that it is generally present among L. plantarum strains. The present study constitutes the description of feruloyl esterase activity in L. plantarum and provides new insights into the metabolism of hydroxycinnamic compounds in this bacterial species. PMID:23793626

  8. Esterases of Varroa destructor (Acari: Varroidae), parasitic mite of the honeybee.

    PubMed

    Dmitryjuk, Małgorzata; Żołtowska, Krystyna; Frączek, Regina; Lipiński, Zbigniew

    2014-04-01

    Varroa destructor is an ectoparasite that causes serious damage to the population of the honeybee. Increasing resistance of the parasite to acaricides is related, among others, to metabolic adaptations of its esterases to facilitate decomposition of the chemicals used. Esterases are a large heterogeneous group of enzymes that metabolize a number of endogenous and exogenous substrates with ester binding. The aim of the present study was to determine the activity of esterases in the body extracts (BE) and excretion/secretion products (E/SP) of the mite. The enzymes contained in the E/SP should originate mainly from the salivary glands and the alimentary system and they may play a particularly important role in the first line of defence of the mite against acaricides. Activity of cholinesterases (ChEs) [acetylcholinesterase (AChE) and butyrylcholinesterase], carboxylesterases (CEs) and phosphatases [alkaline phosphatase (AP) and acid phosphatase (AcP)] was investigated. The activity of all the enzymes except AChE was higher in the E/SP than in the BE. ChEs from the BE and from the E/SP reacted differently on eserine, a ChE inhibitor. Eserine inhibited both enzymes from the BE, increased decomposition of acetylcholine, but did not influence hydrolysis of butyrylcholine by the E/SP. Activity of the CEs from the BE in relation to the esters of carboxylic acids can be presented in the following series: C10 > C12 > C14 > C8 > C2 > C4 = C16, while activity of the CEs from the E/SP was: C4 > C8 > C2 > C14 > C10 > C12 > C16. The inhibitor of CEs, triphenyl phosphate, reduced the activity of esterases C2–C8 and C14–C16; however, it acted in the opposite way to CEs C10 and C12. The activity of both phosphatases was higher in the E/SP than in the BE (AcP about twofold and AP about 2.6-fold); the activities of AP and AcP in the same material were similar. Given the role of esterases in resistance to pesticides, further studies are necessary to obtain complete biochemical

  9. Structure and properties of the esterase from non-LTR retrotransposons suggest a role for lipids in retrotransposition.

    PubMed

    Schneider, Anna M; Schmidt, Steffen; Jonas, Stefanie; Vollmer, Benjamin; Khazina, Elena; Weichenrieder, Oliver

    2013-12-01

    Non-LTR retrotransposons are mobile genetic elements and play a major role in eukaryotic genome evolution and disease. Similar to retroviruses they encode a reverse transcriptase, but their genomic integration mechanism is fundamentally different, and they lack homologs of the retroviral nucleocapsid-forming protein Gag. Instead, their first open reading frames encode distinct multi-domain proteins (ORF1ps) presumed to package the retrotransposon-encoded RNA into ribonucleoprotein particles (RNPs). The mechanistic roles of ORF1ps are poorly understood, particularly of ORF1ps that appear to harbor an enzymatic function in the form of an SGNH-type lipolytic acetylesterase. We determined the crystal structures of the coiled coil and esterase domains of the ORF1p from the Danio rerio ZfL2-1 element. We demonstrate a dimerization of the coiled coil and a hydrolytic activity of the esterase. Furthermore, the esterase binds negatively charged phospholipids and liposomes, but not oligo-(A) RNA. Unexpectedly, the esterase can split into two dynamic half-domains, suited to engulf long fatty acid substrates extending from the active site. These properties indicate a role for lipids and membranes in non-LTR retrotransposition. We speculate that Gag-like membrane targeting properties of ORF1ps could play a role in RNP assembly and in membrane-dependent transport or localization processes. PMID:24003030

  10. Functional Characterization of a Novel Marine Microbial Esterase and its Utilization in the Enantioselective Preparation of (R)-Methyl 2-Chloropropionate.

    PubMed

    Cao, Yingying; Deng, Dun; Sun, Aijun; Zhang, Yun; Hu, Yunfeng

    2016-09-01

    Chiral 2-chloropropanoic acids and their ester derivatives are crucial intermediates in the synthesis of many chemicals, especially herbicides. The enzymatic synthesis of chiral 2-chloropropanoic acids and their ester derivatives by esterases was not easily achieved, because the structural difference between the two enantiomers was too small to be recognized by esterases. Herein, we report the expression and functional characterization of one novel low temperature-resistant esterase EST12-7 identified from the genome of Pseudonocardia antitumoralis SCSIO 01299 isolated from the sediments of the South China Sea. Biocatalyst EST12-7 could hydrolyze racemic methyl 2-chloropropinate and generate optically pure (R)-methyl 2-chloropropinate with high enantiomeric excess (>99 %) and conversion (>49 %) after process optimization. Notably, the addition of different surfactants and using surfactants of different concentrations in the kinetic resolution catalyzed by EST12-7 could greatly affect the enantiomeric excess and conversion rate of product (R)-methyl 2-chloropropinate.

  11. Improved enantioselectivity of thermostable esterase from Archaeoglobus fulgidus toward (S)-ketoprofen ethyl ester by directed evolution and characterization of mutant esterases.

    PubMed

    Kim, Jinyeong; Kim, Seungbum; Yoon, Sangyoung; Hong, Eunsoo; Ryu, Yeonwoo

    2015-08-01

    Thermostable esterases have potential applications in various biotechnology industries because of their resistance to high temperature and organic solvents. In a previous study, we isolated an esterase from Archaeoglobus fulgidus DSM 4304 (Est-AF), which showed high thermostability but low enantioselectivity toward (S)-ketoprofen ethyl ester. (R)-ketoprofenor (S)-ketoprofenis produced by esterase hydrolysis of the ester bond of (R,S)-ketoprofen ethyl ester and (S)-ketoprofen has better pharmaceutical activity and lower side effects than (R)-ketoprofen. Therefore, we have generated mutants of Est-AF that retained high thermostability whilst improving enantioselectivity. A library of Est-AF mutants was created by error-prone polymerase chain reaction, and mutants with improved enantioselectivity were isolated by site-saturation mutagenesis. The regions of Est-AF containing amino acid mutations were analyzed by homology modeling of its three-dimensional structure, and structure-based explanations for the changes in enantioselectivity are proposed. Finally, we isolated two mutants showing improved enantioselectivity over Est-AF (ee% = -16.2 ± 0.2 and E = 0.7 ± 0.0): V138G (ee% = 35.9 ± 1.0 and E = 3.0 ± 0.1) and V138G/L200R (ee% = 89.2 ± 0.2 and E = 19.5 ± 0.5). We also investigated various characteristics of these mutants and found that the mutants showed similar thermostability and resistance to additives or organic solvents to Est-AF, without a significant trade-off between activity and stability.

  12. A glucuronoyl esterase from Acremonium alcalophilum cleaves native lignin-carbohydrate ester bonds.

    PubMed

    Arnling Bååth, Jenny; Giummarella, Nicola; Klaubauf, Sylvia; Lawoko, Martin; Olsson, Lisbeth

    2016-08-01

    The Glucuronoyl esterases (GE) have been proposed to target lignin-carbohydrate (LC) ester bonds between lignin moieties and glucuronic acid side groups of xylan, but to date, no direct observations of enzymatic cleavage on native LC ester bonds have been demonstrated. In the present investigation, LCC fractions from spruce and birch were treated with a recombinantly produced GE originating from Acremonium alcalophilum (AaGE1). A combination of size exclusion chromatography and (31) P NMR analyses of phosphitylated LCC samples, before and after AaGE1 treatment provided the first evidence for cleavage of the LC ester linkages existing in wood. PMID:27397104

  13. Characterization of EST3: a metagenome-derived esterase with suitable properties for biotechnological applications.

    PubMed

    Maester, Thaís Carvalho; Pereira, Mariana Rangel; Machado Sierra, E G; Balan, Andrea; de Macedo Lemos, Eliana Gertrudes

    2016-07-01

    Metagenomic libraries from diverse environments have been extensive sources of many lipases and esterases; nevertheless, most of these enzymes remain biochemically uncharacterized. We previously built a metagenomic fosmid library from a microbial consortium specialized for diesel oil degradation and tested it for lipolytic activity. In the present study, we identified the PL14.H10 clone that was subcloned and sequenced, which enabled the identification of the EST3 protein. This enzyme exhibited 74 % amino acid identity with the uncharacterized alpha/beta hydrolase from Parvibaculum lavamentivorans [GenBank: WP012110575.1] and was classified into lipolytic enzyme family IV. Biochemical characterization revealed that EST3 presents high activity in a wide range of temperature with highest activity from 41 to 45 °C. Also, this thermostable esterase acts from mild acidic to alkaline conditions with an optimum pH of 6.0. The enzyme exhibited activity against p-nitrophenyl esters of different chain lengths and highest catalytic efficiency against p-nitrophenyl caprylate. The activity of the protein was increased in the presence of 0.5 mM of Mn(+2), Li(+), EDTA, and 1 % of CTAB and exhibited half of the activity in the presence of 10 % methanol and ethanol. Moreover, the homology model of EST3 was built and compared to other esterases, revealing a substrate channel that should fit a wide range of substrates. Taken together, the data presented in this work reveal the unique and interesting characteristics of EST3 that might be explored for further use in biotechnological applications.

  14. High throughput genome-specific and gene-specific molecular markers for erucic acid genes in Brassica napus (L.) for marker-assisted selection in plant breeding.

    PubMed

    Rahman, Mukhlesur; Sun, Zudong; McVetty, Peter B E; Li, Genyi

    2008-10-01

    A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. A BAC clone anchoring Bn-FAE1.1 from a B. rapa BAC library and a BAC clone anchoring Bn-FAE1.2 from a B. oleracea BAC library were used in this research. After sequencing the gene flanking regions, it was found that the dissimilarity of the flanking sequences of these two FAE1 homologs facilitated the design of genome-specific primers that could amplify the corresponding genome in allotetraploid B. napus. The two-base deletion in the C genome gene was detected as a sequence-characterized amplified region (SCAR) marker. To increase the throughput, one genome-specific primer was labeled with four fluorescence dyes and combined with 20 different primers to produce PCR products with different fragment sizes. Eventually, a super pool of 80 samples was detected simultaneously. This dramatically reduces the cost of marker detection. The single base change in the Bn-FAE1.1 gene was detected as single nucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexing primer set was designed by adding a polyT to the 5' primer end to increase SNP detection throughput through sample pooling. Furthermore, the Bn-FAE1.1 and Bn-FAE1.2 were integrated into the N8 and N13 linkage groups of our previously reported high-density sequence-related amplified polymorphism (SRAP) map, respectively. There were 124 SRAP markers in a N8 bin in which the Bn-FAE1.1 gene-specific SCAR marker was located and 46 SRAP markers in a N13 bin into which the Bn-FAE1.2 SNP marker was integrated. These three kinds of high throughput molecular markers have been successfully implemented in our canola/rapeseed breeding programs.

  15. Penicillium brasilianum as an enzyme factory; the essential role of feruloyl esterases for the hydrolysis of the plant cell wall.

    PubMed

    Panagiotou, Gianni; Olavarria, Reyes; Olsson, Lisbeth

    2007-06-30

    The production of arabinoxylan-degrading enzymes by the fungus Penicillium brasilianum, grown on different carbon and nitrogen sources as well as different environmental conditions was investigated. Highest feruloyl esterase (225 mU/ml) and alpha-L-arabinofuranosidase (211 mU/ml) activities were obtained when P. brasilianum was grown on sugar beet pulp, whereas maximum xylanase (17 U/ml) activity was found during growth on oat spelt xylan. Yeast extract was the preferable nitrogen source for the production of all the three enzymes. Further optimization of the production of the crude enzyme mixture was examined by experimental design using a D-optimal quadratic model. Investigation of the microbial regulation of enzyme production showed that the presence of free ferulic acid further stimulated the production and pointing to that the fungal regulatory mechanism involved a coordinated production and secretion of feruloyl esterase, xylanase and alpha-L-arabinofuranosidase. Since agroindustrial by-products are a potential source of phenolic acids, crude enzyme mixtures of P. brasilianum were tested for their hydrolysis abilities against eight complex or model substrates. While total release of phenolic acids and pentoses was not observed, the synergistic enhancement of hydrolysis in the presence of feruloyl esterase was clearly demonstrated.

  16. Interactions between dietary oil treatments and genetic variants modulate fatty acid ethanolamides in plasma and body weight composition.

    PubMed

    Pu, Shuaihua; Eck, Peter; Jenkins, David J A; Connelly, Philip W; Lamarche, Benoît; Kris-Etherton, Penny M; West, Sheila G; Liu, Xiaoran; Jones, Peter J H

    2016-03-28

    Fatty acid ethanolamides (FAE), a group of lipid mediators derived from long-chain fatty acids (FA), mediate biological activities including activation of cannabinoid receptors, stimulation of fat oxidation and regulation of satiety. However, how circulating FAE levels are influenced by FA intake in humans remains unclear. The objective of the present study was to investigate the response of six major circulating FAE to various dietary oil treatments in a five-period, cross-over, randomised, double-blind, clinical study in volunteers with abdominal obesity. The treatment oils (60 g/12 552 kJ per d (60 g/3000 kcal per d)) provided for 30 d were as follows: conventional canola oil, high oleic canola oil, high oleic canola oil enriched with DHA, flax/safflower oil blend and corn/safflower oil blend. Two SNP associated with FAE degradation and synthesis were studied. Post-treatment results showed overall that plasma FAE levels were modulated by dietary FA and were positively correlated with corresponding plasma FA levels; minor allele (A) carriers of SNP rs324420 in gene fatty acid amide hydrolase produced higher circulating oleoylethanolamide (OEA) (P=0·0209) and docosahexaenoylethanolamide (DHEA) levels (P=0·0002). In addition, elevated plasma DHEA levels in response to DHA intake tended to be associated with lower plasma OEA levels and an increased gynoid fat mass. In summary, data suggest that the metabolic and physiological responses to dietary FA may be influenced via circulating FAE. Genetic analysis of rs324420 might help identify a sub-population that appears to benefit from increased consumption of DHA and oleic acid.

  17. Interactions between dietary oil treatments and genetic variants modulate fatty acid ethanolamides in plasma and body weight composition.

    PubMed

    Pu, Shuaihua; Eck, Peter; Jenkins, David J A; Connelly, Philip W; Lamarche, Benoît; Kris-Etherton, Penny M; West, Sheila G; Liu, Xiaoran; Jones, Peter J H

    2016-03-28

    Fatty acid ethanolamides (FAE), a group of lipid mediators derived from long-chain fatty acids (FA), mediate biological activities including activation of cannabinoid receptors, stimulation of fat oxidation and regulation of satiety. However, how circulating FAE levels are influenced by FA intake in humans remains unclear. The objective of the present study was to investigate the response of six major circulating FAE to various dietary oil treatments in a five-period, cross-over, randomised, double-blind, clinical study in volunteers with abdominal obesity. The treatment oils (60 g/12 552 kJ per d (60 g/3000 kcal per d)) provided for 30 d were as follows: conventional canola oil, high oleic canola oil, high oleic canola oil enriched with DHA, flax/safflower oil blend and corn/safflower oil blend. Two SNP associated with FAE degradation and synthesis were studied. Post-treatment results showed overall that plasma FAE levels were modulated by dietary FA and were positively correlated with corresponding plasma FA levels; minor allele (A) carriers of SNP rs324420 in gene fatty acid amide hydrolase produced higher circulating oleoylethanolamide (OEA) (P=0·0209) and docosahexaenoylethanolamide (DHEA) levels (P=0·0002). In addition, elevated plasma DHEA levels in response to DHA intake tended to be associated with lower plasma OEA levels and an increased gynoid fat mass. In summary, data suggest that the metabolic and physiological responses to dietary FA may be influenced via circulating FAE. Genetic analysis of rs324420 might help identify a sub-population that appears to benefit from increased consumption of DHA and oleic acid. PMID:26806592

  18. [Characters of two gravity-related esterases in carrot callus cells].

    PubMed

    Guan, P Z; Fei, C K; Yin, J; Liu, M; Zhao, Q; Cai, W M

    1999-01-01

    On the basis of identification of two gravity-related esterases (grEST1 and grEST2) in carrot callus cells (Cai et al. 1998), we continued the study of the characteristics of these two esterases. They have the very special characteristic of SDS resistance. Their activities could be inhibited partially by deoxycholate. beta-Phenylpropionic acid, AgNO3 and CuSO4 had no inhibitory effect on their activities. The activities of grEST1 and grEST2 could be decreased by ascorbic acid and cysteine, and the influence by cysteine was particularly obvious. The molecular weights of grEST1 and grEST2 were shown to be near the ranges of 49-66 kD and 43-59 kD respectively by non-denaturing electrophoresis containing deoxycholate, Triton X-100 and SDS respectively, and the isoelectric points were approximately pH 5.4 and 4.9 respectively. Besides, grEST1 and grEST2 were found in the fraction precipitating at value between 30% and 40% saturation with (NH4)2SO4.

  19. Molecular cloning, overexpression and characterization of a novel feruloyl esterase from a soil metagenomic library.

    PubMed

    Sang, Shu Li; Li, Gang; Hu, Xiao Peng; Liu, Yu Huan

    2011-01-01

    The gene estF27, encoding a protein with feruloyl esterase activity, was cloned through functional screening from a soil metagenomic library and expressed in Escherichiacoli BL21 (DE3) with high solubility. Sequence analysis showed that estF27 encoded a protein of 291 amino acids with a predicted molecular mass of 31.16 kDa. According to the substrate specificity, EstF27 was classified as a type A feruloyl esterase. EstF27 displayed optimal activity at 40°C and pH 6.8. This enzyme was stable in a broad pH range of 5.0-10.0 over 24 h, and retained more than 50% of its activity after 96 or 120 h incubation in the presence of 3 M KCl or 5 M NaCl. The enzyme activity was slightly enhanced by the addition of Mg(2+) and Fe(3+) at a low concentration, and completely inhibited by Cu(2+). In the enzymatic hydrolysis of destarched wheat bran, EstF27 could release ferulic acid from it in the presence of xylanase from Thermomyces lanuginosus. Given its alkalitolerance, halotolerance and highly soluble expression, EstF27 is a promising candidate for industrial applications.

  20. Identification of two novel esterases from a marine metagenomic library derived from South China Sea.

    PubMed

    Chu, Xinmin; He, Haoze; Guo, Changquan; Sun, Baolin

    2008-09-01

    The demand for novel biocatalysts is increasing in modern biotechnology, which greatly stimulates the development of powerful tools to explore the genetic resources in the environment. Metagenomics, a culture independent strategy, provides an access to valuable genetic resources of the uncultured microbes. In this study, two novel esterase genes designated as estA and estB, which encoded 277- and 328-amino-acid peptides, respectively, were isolated from a marine microbial metagenomic library by functional screening, and the corresponding esterases EstA and EstB were biochemically characterized. Amino acid sequence comparison and phylogenetic analysis indicated that EstA together with other putative lipolytic enzymes was closely related to family III, and EstB with its relatives formed a subfamily of family IV. Site-directed mutagenesis showed that EstA contained classical catalytic triad made up of S146-D222-H255, whereas EstB contained an unusual catalytic triad which consisted of S-E-H, an important feature of the subfamily. EstA exhibited habitat-specific characteristics such as its high level of stability in the presence of various divalent cations and at high concentrations of NaCl. EstB displayed remarkable activity against p-nitrophenyl esters and was highly stable in 30% methanol, ethanol, dimethylformamide, and dimethyl sulfoxide, making EstB a potential candidate for industrial applications.

  1. Purification and Properties of an Esterase from the Yeast Saccharomyces cerevisiae and Identification of the Encoding Gene

    PubMed Central

    Degrassi, Giuliano; Uotila, Lasse; Klima, Raffaella; Venturi, Vittorio

    1999-01-01

    We purified an intracellular esterase that can function as an S-formylglutathione hydrolase from the yeast Saccharomyces cerevisiae. Its molecular mass was 40 kDa, as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was 5.0 by isoelectric focusing. The enzyme activity was optimal at 50°C and pH 7.0. The corresponding gene, YJLO68C, was identified by its N-terminal amino acid sequence and is not essential for cell viability. Null mutants have reduced esterase activities and grow slowly in the presence of formaldehyde. This enzyme may be involved in the detoxification of formaldehyde, which can be metabolized to S-formylglutathione by S. cerevisiae. PMID:10427036

  2. Directed evolution of an esterase: screening of enzyme libraries based on pH-indicators and a growth assay.

    PubMed

    Bornscheuer, U T; Altenbuchner, J; Meyer, H H

    1999-10-01

    In order to resolve a sterically hindered 3-hydroxy ethyl ester, which was not accepted as substrate by 20 wild-type hydrolases, a directed evolution of an esterase from Pseudomonas fluorescens (PFE) was performed. Mutations were introduced using the mutator strain Epicurian coli XL1-Red. Enzyme libraries derived from seven mutation cycles were assayed on minimal media agar plates supplemented with pH indicators (neutral red and crystal violet), thus allowing the identification of active esterase variants by the formation of a red color caused by a pH decrease due to the released acid. A further selection criteria was introduced by using the corresponding glycerol estar, because release of the carbon source glycerol facilitates growth on minimal media. By this strategy, one double mutant (A209D and L181V) of PFE was identified, which hydrolyzed the 3-hydroxy ethyl ester in a stereoselective manner (25% ee for the remaining ester, E approximate to 5).

  3. Identification of a novel carbohydrate esterase from Bjerkandera adusta: structural and function predictions through bioinformatics analysis and molecular modeling.

    PubMed

    Cuervo-Soto, Laura I; Valdés-García, Gilberto; Batista-García, Ramón; del Rayo Sánchez-Carbente, María; Balcázar-López, Edgar; Lira-Ruan, Verónica; Pastor, Nina; Folch-Mallol, Jorge Luis

    2015-03-01

    A new gene from Bjerkandera adusta strain UAMH 8258 encoding a carbohydrate esterase (designated as BacesI) was isolated and expressed in Pichia pastoris. The gene had an open reading frame of 1410 bp encoding a polypeptide of 470 amino acid residues, the first 18 serving as a secretion signal peptide. Homology and phylogenetic analyses showed that BaCesI belongs to carbohydrate esterases family 4. Three-dimensional modeling of the protein and normal mode analysis revealed a breathing mode of the active site that could be relevant for esterase activity. Furthermore, the overall negative electrostatic potential of this enzyme suggests that it degrades neutral substrates and will not act on negative substrates such as peptidoglycan or p-nitrophenol derivatives. The enzyme shows a specific activity of 1.118 U mg(-1) protein on 2-naphthyl acetate. No activity was detected on p-nitrophenol derivatives as proposed from the electrostatic potential data. The deacetylation activity of the recombinant BaCesI was confirmed by measuring the release of acetic acid from several substrates, including oat xylan, shrimp shell chitin, N-acetylglucosamine, and natural substrates such as sugar cane bagasse and grass. This makes the protein very interesting for the biofuels production industry from lignocellulosic materials and for the production of chitosan from chitin.

  4. Endophytic fungi producing of esterases: Evaluation in vitro of the enzymatic activity using pH indicator

    PubMed Central

    Lisboa, Helen Cristina Fávero; Biasetto, Carolina Rabal; de Medeiros, João Batista; Araújo, Ângela Regina; Silva, Dulce Helena Siqueira; Teles, Helder Lopes; Trevisan, Henrique Celso

    2013-01-01

    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 - carboxyl-esterases) 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 as a potential 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. PMID:24516461

  5. Identification of a novel carbohydrate esterase from Bjerkandera adusta: structural and function predictions through bioinformatics analysis and molecular modeling.

    PubMed

    Cuervo-Soto, Laura I; Valdés-García, Gilberto; Batista-García, Ramón; del Rayo Sánchez-Carbente, María; Balcázar-López, Edgar; Lira-Ruan, Verónica; Pastor, Nina; Folch-Mallol, Jorge Luis

    2015-03-01

    A new gene from Bjerkandera adusta strain UAMH 8258 encoding a carbohydrate esterase (designated as BacesI) was isolated and expressed in Pichia pastoris. The gene had an open reading frame of 1410 bp encoding a polypeptide of 470 amino acid residues, the first 18 serving as a secretion signal peptide. Homology and phylogenetic analyses showed that BaCesI belongs to carbohydrate esterases family 4. Three-dimensional modeling of the protein and normal mode analysis revealed a breathing mode of the active site that could be relevant for esterase activity. Furthermore, the overall negative electrostatic potential of this enzyme suggests that it degrades neutral substrates and will not act on negative substrates such as peptidoglycan or p-nitrophenol derivatives. The enzyme shows a specific activity of 1.118 U mg(-1) protein on 2-naphthyl acetate. No activity was detected on p-nitrophenol derivatives as proposed from the electrostatic potential data. The deacetylation activity of the recombinant BaCesI was confirmed by measuring the release of acetic acid from several substrates, including oat xylan, shrimp shell chitin, N-acetylglucosamine, and natural substrates such as sugar cane bagasse and grass. This makes the protein very interesting for the biofuels production industry from lignocellulosic materials and for the production of chitosan from chitin. PMID:25586442

  6. Feruloyl esterase activity is influenced by bile, probiotic intestinal adhesion and milk fat.

    PubMed

    Mukdsi, M C Abeijón; Argañaraz Martínez, E; Chaia, A Perez; Medina, R B

    2016-09-01

    Cinnamoyl esterases (CE) are microbial and mammalian intestinal enzymes able to release antioxidant hydroxycinnamic acids from their non-digestible ester-linked forms naturally present in vegetable foods. Previous findings showed that oral administration of Lactobacillus fermentum CRL1446 increased intestinal CE activity and improved oxidative status in mice. The aim of this work was to evaluate the in vitro CE activity of L. fermentum CRL1446 and the effect of bile on this activity, as well as strain resistance to simulated gastrointestinal tract (GIT) conditions and its ability to adhere to intestinal epithelium and influence its basal CE activity. L. fermentum CRL1446 and L. fermentum ATCC14932 (positive control for CE activity) were able to hydrolyse different synthetic hydroxycinnamates, with higher specificity toward methyl ferulate (3,853.73 and 899.19 U/g, respectively). Feruloyl esterase (FE) activity was mainly intracellular in L. fermentum CRL1446 and cell-surface associated in L. fermentum ATCC14932. Both strains tolerated simulated GIT conditions and were able to adhere ex vivo to intestinal epithelium. Pre-incubation of L. fermentum strains with bile increased FE activity in both whole cells and supernatants (~2-fold), compared to controls, suggesting that cells were permeabilised by bile, allowing more substrate to enter the cell and/or leakage of FE enzymes. Three-fold higher FE activities were detected in intestinal tissue fragments with adhered L. fermentum CRL1446 cells compared to control fragments (without bacteria), indicating that this strain provides exogenous FE activity and could stimulate esterase activity in the intestinal mucosa. Finally, we found that milk fat had a negative effect on FE activity of intestinal tissue, in absence or presence of adhered L. fermentum. These results help explaining the increase in intestinal FE activity previously observed in mice fed with L. fermentum CRL1446, and support the potential use of this strain

  7. Extracellular esterases of phylloplane yeast Pseudozyma antarctica induce defect on cuticle layer structure and water-holding ability of plant leaves.

    PubMed

    Ueda, Hirokazu; Mitsuhara, Ichiro; Tabata, Jun; Kugimiya, Soichi; Watanabe, Takashi; Suzuki, Ken; Yoshida, Shigenobu; Kitamoto, Hiroko

    2015-08-01

    Aerial plant surface (phylloplane) is a primary key habitat for many microorganisms but is generally recognized as limited in nutrient resources. Pseudozyma antarctica, a nonpathogenic yeast, is commonly isolated from plant surfaces and characterized as an esterase producer with fatty acid assimilation ability. In order to elucidate the biological functions of these esterases, culture filtrate with high esterase activity (crude enzyme) of P. antarctica was applied onto leaves of tomato and Arabidopsis. These leaves showed a wilty phenotype, which is typically associated with water deficiency. Furthermore, we confirmed that crude enzyme-treated detached leaves clearly lost their water-holding ability. In treated leaves of both plants, genes associated to abscisic acid (ABA; a plant stress hormone responding osmotic stress) were activated and accumulation of ABA was confirmed in tomato plants. Microscopic observation of treated leaf surfaces revealed that cuticle layer covering the aerial epidermis of leaves became thinner. A gas chromatography-mass spectrometry (GC-MS) analysis exhibited that fatty acids with 16 and 18 carbon chains were released in larger amounts from treated leaf surfaces, indicating that the crude enzyme has ability to degrade lipid components of cuticle layer. Among the three esterases detected in the crude enzyme, lipase A, lipase B, and P. antarctica esterase (PaE), an in vitro enzyme assay using para-nitrophenyl palmitate as substrate demonstrated that PaE was the most responsible for the degradation. These results suggest that PaE has a potential role in the extraction of fatty acids from plant surfaces, making them available for the growth of phylloplane yeasts.

  8. Identification of novel esterase-active enzymes from hot environments by use of the host bacterium Thermus thermophilus

    PubMed Central

    Leis, Benedikt; Angelov, Angel; Mientus, Markus; Li, Haijuan; Pham, Vu T. T.; Lauinger, Benjamin; Bongen, Patrick; Pietruszka, Jörg; Gonçalves, Luís G.; Santos, Helena; Liebl, Wolfgang

    2015-01-01

    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 mutant strain BL03 with multiple markerless deletions in genes for major extra- and intracellular lipolytic activities. This esterase-diminished strain 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 esterase clones in the thermophilic bacterium than in the mesophilic E. coli. From several thousand functionally screened clones only two thermostable α/β-fold hydrolase enzymes with high amino acid sequence similarity to already characterized enzymes were identifiable in E. coli. In contrast, five further fosmids were found that conferred lipolytic activities in T. thermophilus only. Four open reading frames (ORFs) were found which did not share significant similarity to known esterase enzymes but contained the conserved GXSXG motif regularly found in lipolytic 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

  9. Down-regulation of crambe fatty acid desaturase and elongase in Arabidopsis and crambe resulted in significantly increased oleic acid content in seed oil.

    PubMed

    Li, Xueyuan; Mei, Desheng; Liu, Qing; Fan, Jing; Singh, Surinder; Green, Allan; Zhou, Xue-Rong; Zhu, Li-Hua

    2016-01-01

    High oleic oil is an important industrial feedstock that has been one of the main targets for oil improvement in a number of oil crops. Crambe (Crambe abyssinica) is a dedicated oilseed crop, suitable for industrial oil production. In this study, we down-regulated the crambe fatty acid desaturase (FAD) and fatty acid elongase (FAE) genes for creating high oleic seed oil. We first cloned the crambe CaFAD2, CaFAD3 and CaFAE1 genes. Multiple copies of each of these genes were isolated, and the highly homologous sequences were used to make RNAi constructs. These constructs were first tested in Arabidopsis, which led to the elevated oleic or linoleic levels depending on the genes targeted, indicating that the RNAi constructs were effective in regulating the expression of the target genes in nonidentical but closely related species. Furthermore, down-regulation of CaFAD2 and CaFAE1 in crambe with the FAD2-FAE1 RNAi vector resulted in even more significant increase in oleic acid level in the seed oil with up to 80% compared to 13% for wild type. The high oleic trait has been stable in subsequent five generations and the GM line grew normally in greenhouse. This work has demonstrated the great potential of producing high oleic oil in crambe, thus contributing to its development into an oil crop platform for industrial oil production.

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

    SciTech Connect

    Novak, R.; Padilla, S.

    1986-04-01

    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. (/sup 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.

  11. Preliminary X-ray analysis of twinned crystals of the Q88Y25_Lacpl esterase from Lactobacillus plantarum WCFS1

    PubMed Central

    Álvarez, Yanaisis; Esteban-Torres, María; Acebrón, Iván; de las Rivas, Blanca; Muñoz, Rosario; Martínez-Ripoll, Martín; Mancheño, José M.

    2011-01-01

    Q88Y25_Lacpl is an esterase produced by the lactic acid bacterium Lactobacillus plantarum WCFS1 that shows amino-acid sequence similarity to carboxyl­esterases from the hormone-sensitive lipase family, in particular the AFEST esterase from the archaeon Archaeoglobus fulgidus and the hyperthermophilic esterase EstEI isolated from a metagenomic library. N-­terminally His6-tagged Q88Y25_Lacpl has been overexpressed in Escherichia coli BL21 (DE3) cells, purified and crystallized at 291 K using the hanging-drop vapour-diffusion method. Mass spectrometry was used to determine the purity and homogeneity of the enzyme. Crystals of His6-tagged Q88Y25_Lacpl were prepared in a solution containing 2.8 M sodium acetate trihydrate pH 7.0. X-ray diffraction data were collected to 2.24 Å resolution on beamline ID29 at the ESRF. The apparent crystal point group was 422; however, initial global analysis of the intensity statistics (data processed with high symmetry in space group I422) and subsequent tests on data processed with low symmetry (space group I4) showed that the crystals were almost perfectly merohedrally twinned. Most probably, the true space group is I4, with unit-cell parameters a = 169.05, b = 169.05, c = 183.62 Å. PMID:22102251

  12. Crystal structures of Ophiostoma piceae sterol esterase: structural insights into activation mechanism and product release.

    PubMed

    Gutiérrez-Fernández, Javier; Vaquero, María Eugenia; Prieto, Alicia; Barriuso, Jorge; Martínez, María Jesús; Hermoso, Juan A

    2014-09-01

    Sterol esterases are able to efficiently hydrolyze both sterol esters and triglycerides and to carry out synthesis reactions in the presence of organic solvents. Their high versatility makes them excellent candidates for biotechnological purposes. Sterol esterase from fungus Ophiostoma piceae (OPE) belongs to the family abH03.01 of the Candida rugosa lipase-like proteins. Crystal structures of OPE were solved in this study for the closed and open conformations. Enzyme activation involves a large displacement of the conserved lid, structural rearrangements of loop α16-α17, and formation of a dimer with a large opening. Three PEG molecules are placed in the active site, mimicking chains of the triglyceride substrate, demonstrating the position of the oxyanion hole and the three pockets that accommodate the sn-1, sn-2 and sn-3 fatty acids chains. One of them is an internal tunnel, connecting the active center with the outer surface of the enzyme 30 Å far from the catalytic Ser220. Based on our structural and biochemical results we propose a mechanism by which a great variety of different substrates can be hydrolyzed in OPE paving the way for the construction of new variants to improve the catalytic properties of these enzymes and their biotechnological applications. PMID:25108239

  13. Crystal structures of Ophiostoma piceae sterol esterase: structural insights into activation mechanism and product release.

    PubMed

    Gutiérrez-Fernández, Javier; Vaquero, María Eugenia; Prieto, Alicia; Barriuso, Jorge; Martínez, María Jesús; Hermoso, Juan A

    2014-09-01

    Sterol esterases are able to efficiently hydrolyze both sterol esters and triglycerides and to carry out synthesis reactions in the presence of organic solvents. Their high versatility makes them excellent candidates for biotechnological purposes. Sterol esterase from fungus Ophiostoma piceae (OPE) belongs to the family abH03.01 of the Candida rugosa lipase-like proteins. Crystal structures of OPE were solved in this study for the closed and open conformations. Enzyme activation involves a large displacement of the conserved lid, structural rearrangements of loop α16-α17, and formation of a dimer with a large opening. Three PEG molecules are placed in the active site, mimicking chains of the triglyceride substrate, demonstrating the position of the oxyanion hole and the three pockets that accommodate the sn-1, sn-2 and sn-3 fatty acids chains. One of them is an internal tunnel, connecting the active center with the outer surface of the enzyme 30 Å far from the catalytic Ser220. Based on our structural and biochemical results we propose a mechanism by which a great variety of different substrates can be hydrolyzed in OPE paving the way for the construction of new variants to improve the catalytic properties of these enzymes and their biotechnological applications.

  14. Purification and characterization of an esterase involved in cellulose acetate degradation by Neisseria sicca SB.

    PubMed

    Moriyoshi, K; Ohmoto, T; Ohe, T; Sakai, K

    1999-10-01

    An esterase catalyzing the hydrolysis of acetyl ester moieties in cellulose acetate was purified 1,110-fold to electrophoretic homogeneity from the culture supernatant of Neisseria sicca SB, which can assimilate cellulose acetate as the sole carbon and energy source. The purified enzyme was a monomeric protein with a molecular mass of 40 kDa and the isoelectric point was 5.3. The pH and temperature optima of the enzyme were 8.0-8.5 and 45 degrees C. The enzyme catalyzed the hydrolysis of acetyl saccharides, p-nitrophenyl esters of short-chain fatty acids, and was slightly active toward aliphatic and aromatic esters. The K(m) and Vmax for cellulose acetate (degree of substitution, 0.88) and p-nitrophenyl acetate were 0.0162% (716 microM as acetyl content in the polymer) and 36.0 microM, and 66.8 and 39.1 mumol/min/mg, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate, which indicated that the enzyme was a serine esterase.

  15. Est16, a New Esterase Isolated from a Metagenomic Library of a Microbial Consortium Specializing in Diesel Oil Degradation

    PubMed Central

    Pereira, Mariana Rangel; Mercaldi, Gustavo Fernando; Maester, Thaís Carvalho; Balan, Andrea; de Macedo Lemos, Eliana Gertrudes

    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. PMID:26214846

  16. Est16, a New Esterase Isolated from a Metagenomic Library of a Microbial Consortium Specializing in Diesel Oil Degradation.

    PubMed

    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.

  17. A New Family of Carbohydrate Esterases Is Represented by a GDSL Hydrolase/Acetylxylan Esterase from Geobacillus stearothermophilus*

    PubMed Central

    Alalouf, Onit; Balazs, Yael; Volkinshtein, Margarita; Grimpel, Yael; Shoham, Gil; Shoham, Yuval

    2011-01-01

    Acetylxylan esterases hydrolyze the ester linkages of acetyl groups at positions 2 and/or 3 of the xylose moieties in xylan and play an important role in enhancing the accessibility of xylanases to the xylan backbone. The hemicellulolytic system of the thermophilic bacterium Geobacillus stearothermophilus T-6 comprises a putative acetylxylan esterase gene, axe2. The gene product belongs to the GDSL hydrolase family and does not share sequence homology with any of the carbohydrate esterases in the CAZy Database. The axe2 gene is induced by xylose, and the purified gene product completely deacetylates xylobiose peracetate (fully acetylated) and hydrolyzes the synthetic substrates 2-naphthyl acetate, 4-nitrophenyl acetate, 4-methylumbelliferyl acetate, and phenyl acetate. The pH profiles for kcat and kcat/Km suggest the existence of two ionizable groups affecting the binding of the substrate to the enzyme. Using NMR spectroscopy, the regioselectivity of Axe2 was directly determined with the aid of one-dimensional selective total correlation spectroscopy. Methyl 2,3,4-tri-O-acetyl-β-d-xylopyranoside was rapidly deacetylated at position 2 or at positions 3 and 4 to give either diacetyl or monoacetyl intermediates, respectively; methyl 2,3,4,6-tetra-O-acetyl-β-d-glucopyranoside was initially deacetylated at position 6. In both cases, the complete hydrolysis of the intermediates occurred at a much slower rate, suggesting that the preferred substrate is the peracetate sugar form. Site-directed mutagenesis of Ser-15, His-194, and Asp-191 resulted in complete inactivation of the enzyme, consistent with their role as the catalytic triad. Overall, our results show that Axe2 is a serine acetylxylan esterase representing a new carbohydrate esterase family. PMID:21994937

  18. Characterization of a novel highly thermostable esterase from the Gram-positive soil bacterium Streptomyces lividans TK64.

    PubMed

    Wang, Baojuan; Wang, Ao; Cao, Zhengyu; Zhu, Guoping

    2016-05-01

    A novel esterase gene (estW) from soil bacterium Streptomyces lividans TK64 was successfully cloned using a pair of homologous primers. The estW gene encoded a protein (EstW) of 289 amino acid residues with a predicted molecular weight of 31.43 kDa. Sequence alignment revealed that EstW show relatively high levels of homology to other lipolytic enzymes characterized from Streptomyces and phylogenetic analysis suggested EstW belongs to the bacterial lipase/esterase family I. The estW gene was expressed at a high level in Escherichia coli and the recombinant enzyme was purified to homogeneity. The purified EstW was characterized via hydrolysis of various p-nitrophenyl esters and the best substrate was found to be p-nitrophenyl acetate (pNPA). Maximal activity of the recombinant protein was observed at pH 8.0 and 50 °C with pNPA as the substrate. The calculated activation energy (Ea ) of the esterase reaction was 9.12 kcal/mol. Half-life of EstW at 95 °C was approximately 12.5 H, making it the most thermostable esterase among all of the known lipolytic enzymes from Streptomyces, and the thermostability of EstW was similar to those of some enzymes characterized from the thermophilic bacteria. EstW exhibited relatively high tolerance to several detergents and required no cations for its maximal activity. The unique properties of EstW, namely its high thermostability and stability in the presence of organic solvents, may render it a potential candidate for industrial applications.

  19. Electrophoretic and densitometric analysis of esterase activity as an indicator of mercury toxicity

    SciTech Connect

    Benton, M.J.; Guttman, S.I.

    1995-12-31

    In an earlier experiment, esterase activity as determined by starch gel electrophoresis was absent in larval caddisflies (Nectopsyche albida) that succumbed to mercury exposure, but was present in control larvae. To test the effects of mercury exposure duration on esterase activity, additional larval N. albida were exposed under conditions identical to those in the earlier experiment, and esterase activity was determined by electrophoresis of several live individuals every 12 hours. To test the effects of mercury concentration on esterase activity, homogenates of unexposed N. albida were electrophoresed, and esterase activity was determined using esterase-specific stains spiked with various concentrations of mercury. Following both experiments, esterase activity was quantified by laser densitometry of stained electrophoresis gels, Results indicate that: (1) inorganic mercury inhibited esterase activity, (2) inhibition increased with exposure time, and (3) inhibition increased with mercury concentration. Esterase inhibition may be a causal factor in mortality related to mercury exposure. Quantification of esterase activity by densitometry of electrophoretic gels may be an alternative method of rapid toxicity assessment.

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

    PubMed

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

    2006-08-14

    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 acid, p-coumaric acid and ferulic acid from coffee pulp, apple marc and wheat straw. Their hydrolysis activity was evaluated and compared with their action on maize bran and sugar beet pulp. The specificity of both enzymes against natural and synthetic substrates was evaluated; particular attention was paid to quinic esters and lignin monomers. The efficiency of both enzymes on model substrates was studied. We show the ability of these enzymes to hydrolyze quinic esters and ester linkages between phenolic acids and lignin monomer.

  1. Identification and characterization of a novel cold-adapted esterase from a metagenomic library of mountain soil.

    PubMed

    Ko, Kyong-Cheol; Rim, Soon-Ok; Han, Yunjon; Shin, Bong Seok; Kim, Geun-Joong; Choi, Jong Hyun; Song, Jae Jun

    2012-05-01

    A novel lipolytic enzyme was isolated from a metagenomic library after demonstration of lipolytic activity on an LB agar plate containing 1% (w/v) tributyrin. A novel esterase gene (estIM1), encoding a lipolytic enzyme (EstIM1), was cloned using a shotgun method from a pFosEstIM1 clone of the metagenomic library, and the enzyme was characterized. The estIM1 gene had an open reading frame (ORF) of 936 base pairs and encoded a protein of 311 amino acids with a molecular mass 34 kDa and a pI value of 4.32. The deduced amino acid sequence was 62% identical to that of an esterase from an uncultured bacterium (ABQ11271). The amino acid sequence indicated that EstIM1 was a member of the family IV of lipolytic enzymes, all of which contain a GDSAG motif shared with similar enzymes of lactic acid microorganisms. EstIM1 was active over a temperature range of 1-50°C, at alkaline pH. The activation energy for hydrolysis of p-nitrophenyl propionate was 1.04 kcal/mol, within a temperature range of 1-40°C. The activity of EstIM1 was about 60% of maximal even at 1°C, suggesting that EstIM1 is efficiently cold-adapted. Further characterization of this cold-adapted enzyme indicated that the esterase may be very valuable in industrial applications.

  2. The Lp_3561 and Lp_3562 Enzymes Support a Functional Divergence Process in the Lipase/Esterase Toolkit from Lactobacillus plantarum

    PubMed Central

    Esteban-Torres, María; Reverón, Inés; Santamaría, Laura; Mancheño, José M.; de las Rivas, Blanca; Muñoz, Rosario

    2016-01-01

    Lactobacillus plantarum species is a good source of esterases since both lipolytic and esterase activities have been described for strains of this species. No fundamental biochemical difference exists among esterases and lipases since both share a common catalytic mechanism. L. plantarum WCFS1 possesses a protein, Lp_3561, which is 44% identical to a previously described lipase, Lp_3562. In contrast to Lp_3562, Lp_3561 was unable to degrade esters possessing a chain length higher than C4 and the triglyceride tributyrin. As in other L. plantarum esterases, the electrostatic potential surface around the active site in Lp_3561 is predicted to be basic, whereas it is essentially neutral in the Lp_3562 lipase. The fact that the genes encoding both proteins were located contiguously in the L. plantarum WCFS1 genome, suggests that they originated by tandem duplication, and therefore are paralogs as new functions have arisen during evolution. The presence of the contiguous lp_3561 and lp_3562 genes was studied among L. plantarum strains. They are located in a 8,903 bp DNA fragment that encodes proteins involved in the catabolism of sialic acid and are predicted to increase bacterial adaptability under certain growth conditions. PMID:27486450

  3. The Lp_3561 and Lp_3562 Enzymes Support a Functional Divergence Process in the Lipase/Esterase Toolkit from Lactobacillus plantarum.

    PubMed

    Esteban-Torres, María; Reverón, Inés; Santamaría, Laura; Mancheño, José M; de Las Rivas, Blanca; Muñoz, Rosario

    2016-01-01

    Lactobacillus plantarum species is a good source of esterases since both lipolytic and esterase activities have been described for strains of this species. No fundamental biochemical difference exists among esterases and lipases since both share a common catalytic mechanism. L. plantarum WCFS1 possesses a protein, Lp_3561, which is 44% identical to a previously described lipase, Lp_3562. In contrast to Lp_3562, Lp_3561 was unable to degrade esters possessing a chain length higher than C4 and the triglyceride tributyrin. As in other L. plantarum esterases, the electrostatic potential surface around the active site in Lp_3561 is predicted to be basic, whereas it is essentially neutral in the Lp_3562 lipase. The fact that the genes encoding both proteins were located contiguously in the L. plantarum WCFS1 genome, suggests that they originated by tandem duplication, and therefore are paralogs as new functions have arisen during evolution. The presence of the contiguous lp_3561 and lp_3562 genes was studied among L. plantarum strains. They are located in a 8,903 bp DNA fragment that encodes proteins involved in the catabolism of sialic acid and are predicted to increase bacterial adaptability under certain growth conditions.

  4. The Lp_3561 and Lp_3562 Enzymes Support a Functional Divergence Process in the Lipase/Esterase Toolkit from Lactobacillus plantarum.

    PubMed

    Esteban-Torres, María; Reverón, Inés; Santamaría, Laura; Mancheño, José M; de Las Rivas, Blanca; Muñoz, Rosario

    2016-01-01

    Lactobacillus plantarum species is a good source of esterases since both lipolytic and esterase activities have been described for strains of this species. No fundamental biochemical difference exists among esterases and lipases since both share a common catalytic mechanism. L. plantarum WCFS1 possesses a protein, Lp_3561, which is 44% identical to a previously described lipase, Lp_3562. In contrast to Lp_3562, Lp_3561 was unable to degrade esters possessing a chain length higher than C4 and the triglyceride tributyrin. As in other L. plantarum esterases, the electrostatic potential surface around the active site in Lp_3561 is predicted to be basic, whereas it is essentially neutral in the Lp_3562 lipase. The fact that the genes encoding both proteins were located contiguously in the L. plantarum WCFS1 genome, suggests that they originated by tandem duplication, and therefore are paralogs as new functions have arisen during evolution. The presence of the contiguous lp_3561 and lp_3562 genes was studied among L. plantarum strains. They are located in a 8,903 bp DNA fragment that encodes proteins involved in the catabolism of sialic acid and are predicted to increase bacterial adaptability under certain growth conditions. PMID:27486450

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

    SciTech Connect

    Loughna, S.; Moore, G. ); Gau, G.; Blunt, S. ); Nicolaides, K. )

    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.

  6. Impact of fumaric acid esters on cardiovascular risk factors and depression in psoriasis: a prospective pilot study.

    PubMed

    Schmieder, Astrid; Poppe, Manuel; Hametner, Christian; Meyer-Schraml, Hanna; Schaarschmidt, Marthe-Lisa; Findeisen, Peter; Benoit, Sandrine; Bauer, Boris; Schmid, Sybille; Goebeler, Matthias; Goerdt, Sergij; Ludwig-Peitsch, Wiebke K

    2015-07-01

    Patients with psoriasis have an increased risk of cardiovascular disease that is partly attributable to chronic systemic inflammation. The aim of our prospective pilot study was to investigate the impact of fumaric acid esters (FAE), a first-line systemic antipsoriatic treatment in Germany, on cardiovascular risk parameters. Participants with moderate-to-severe psoriasis from the University Medical Center Mannheim and the University Hospital Würzburg were treated with FAE for 16 weeks according to standard dosage recommendations. Disease severity, life quality and depression scores as well as biomarkers of inflammation, lipid and glucose metabolism were assessed prior to initiation of FAE and after 16 weeks. Out of 39 participants recruited, 27 completed the study. 44% of all participants and 63% of those completing the 16-week treatment achieved PASI 50 response and 27 or 37% PASI 75 response. Clinical improvement was paralleled by significant improvement in quality of life, high treatment satisfaction and significant reduction of depressive symptoms. Adverse events, most frequently mild gastrointestinal complaints, flush and lymphocytopenia occurred in 89%. FAE did not modify glucose metabolism or inflammatory parameters substantially. However, a highly significant increase in serum levels of the atheroprotective cytokine adiponectin was noted after 16 weeks (median 4.7 vs. 8.9 µg/ml; p = 0.0002). Our study demonstrates a significant beneficial impact of FAE on adiponectin, indicating a potential cardioprotective effect. It will be interesting to verify this finding in larger cohorts and to assess the long-term influence of FAE on cardiovascular risk and disease.

  7. The conversion of C'IS to C'1 esterase by plasmin and trypsin.

    PubMed

    Ratnoff, O D; Naff, G B

    1967-02-01

    The formation of C'1 esterase from C'1, the first component of complement, may be brought about by the action of plasmin or trypsin upon C'1s, a subcomponent of C'1. These enzymes also decrease the esterolytic activity of C'1 esterase. The formation of C'1 esterase was demonstrated by measuring the appearance of an agent or agents with esterolytic properties and the capacity to inactivate C'2 and C'4, attributes of C'1 esterase. The activity of the agent which evolved was blocked by serum inhibitor of C'1 esterase. The implications of these observations, that the formation of C'1 esterase during complement fixation is mediated by proteolytic processes, are under study. The possible inhibition of C'1q by soybean trypsin inhibitor is in agreement with this hypothesis.

  8. 3 Benzyl-6-chloropyrone: a suicide inhibitor of cholesterol esterase

    SciTech Connect

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

    1986-05-01

    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 /sup 14/C-cholesteryl oleate with an I/sub 50/ of approximately 150 ..mu..M. The inactivation was time-dependent and characteristic of a suicide mechanism. The ..cap alpha.. 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.

  9. Lipase and esterase-catalyzed acylation of hetero-substituted nitrogen nucleophiles in water and organic solvents.

    PubMed

    Hacking, M A; Akkus, H; van Rantwijk, F; Sheldon, R A

    2000-04-01

    The lipase- and esterase-catalyzed acylations of hydroxylamine and hydrazine derivatives with octanoic acid and ethyl octanoate are described. The influence of solvent and nucleophile on the initial reaction rate was investigated for a number of free and immobilized enzymes. Initial rates were highest in water, but the overall productivity was optimal in dioxane. Octanoic acid (250 g/L) was converted for 93% into the hydroxamic acid in 36 h with only 1% (w/w) Candida antarctica lipase B (Novozym 435) in dioxane at 40 degrees C. This translates to a catalyst productivity of 68.5 g. g(-1). day(-1) and a space time yield of 149 g. L(-1). day(-1), unprecedented figures in the direct reaction of an acid with a nitrogen nucleophile in an organic solvent.

  10. Molecular cloning and characterization of a thermostable esterase/lipase produced by a novel Anoxybacillus flavithermus strain.

    PubMed

    Chiş, Laura; Hriscu, Monica; Bica, Adriana; Toşa, Monica; Nagy, Gergely; Róna, Gergely; G Vértessy, Beata; Dan Irimie, Florin

    2013-01-01

    A thermophilic strain producing an extracellular esterase/lipase was isolated from a hot spring in Tăşnad, Romania, and was identified phenotypically and by 16S rDNA sequencing as Anoxybacillus flavithermus (GenBank ID: JQ267733). The gene encoding the putative carboxyl esterase (GenBank ID: JX494348) was cloned by direct PCR amplification from genomic DNA. The protein, consisting of 246 amino acids and having a predicted molecular weight of 28.03 kDa, is encoded by an ORF of 741 bps. Expression was achieved in Escherichia coli and a recombinant protein with esterolytic activity and estimated molecular weight of 25 kDa was recovered and purified from the periplasmic fraction by IMAC. The purified enzyme, most active at 60-65°C and in the near-neutral range (pH 6.5-8), displayed a half-life at 60°C of about 5 h. Est/Lip displayed a relative tolerance to methanol, DMSO, acetonitrile, and low detergent concentrations (SDS, Triton) increased its thermostability. Highest activity was attained with p-nitrophenyl butyrate, but the enzyme was also able to hydrolyze long chain fatty acid esters, as well as triolein. The primary sequence and predicted tridimensional structure of the enzyme are very similar to those of other Anoxybacillus and Geobacillus carboxyl esterases in a distinct, recently described lipase family. Est/Lip was highly enantioselective, with preference for the (S)-enantiomer of substrates. PMID:23759865

  11. Structural studies of a thermophilic esterase from a new Planctomycetes species, Thermogutta terrifontis.

    PubMed

    Sayer, Christopher; Isupov, Michail N; Bonch-Osmolovskaya, Elizaveta; Littlechild, Jennifer A

    2015-08-01

    Thermogutta terrifontis esterase (TtEst), a carboxyl esterase identified in the novel thermophilic bacterium T. terrifontis from the phylum Planctomycetes, has been cloned and over-expressed in Escherichia coli. The enzyme has been characterized biochemically and shown to have activity towards small p-nitrophenyl (pNP) carboxylic esters, with optimal activity for pNP-propionate. The enzyme retained 95% activity after incubation for 1 h at 80 °C. The crystal structures of the native TtEst and its complexes with the substrate analogue D-malate and the product acetate have been determined to high resolution. The bound ligands have allowed the identification of the carboxyl and alcohol binding pockets in the enzyme active site. Comparison of TtEst with structurally related enzymes provides insight into how differences in their catalytic activity can be rationalized based upon the properties of the amino acid residues in their active site pockets. The mutant enzymes L37A and L251A have been constructed to extend the substrate range of TtEst towards the larger butyrate and valerate pNP-esters. These mutant enzymes have also shown a significant increase in activity towards acetate and propionate pNP esters. A crystal structure of the L37A mutant was determined with the butyrate product bound in the carboxyl pocket of the active site. The mutant structure shows an expansion of the pocket that binds the substrate carboxyl group, which is consistent with the observed increase in activity towards pNP-butyrate. PMID:26011036

  12. Interaction between human serum esterases and environmental metal compounds.

    PubMed

    Hernández, Antonio F; Gil, Fernando; Leno, Esther; López, Olga; Rodrigo, Lourdes; Pla, Antonio

    2009-07-01

    Paraoxonase-1 (PON1) and cholinesterase (BChE) are two of the major human serum esterases. Although most of variation in PON1 activity results from genetic factors, there is growing evidence that environmental chemicals also modulate its activity. The aim of this study was to investigate whether environmental exposure to metal compounds has any influence on those esterases. A cross-sectional study was conducted in a representative sample of the general population of Andalusia, South of Spain. PON1 activity against different substrates (paraoxon, phenylacetate, diazoxon and dihydrocoumarin) and BChE were measured in serum from 536 healthy subjects. Potential associations of these esterases with metal compounds, age, sex and body mass index as well as life-style habits (smoking, alcohol drinking and food habits) were explored. Multiple linear regression analysis showed that blood lead levels were significantly associated with increased PON1 in serum regardless of the substrate used for the assay. Mercury also showed a significant and direct association with PON1 towards paraoxon and phenylacetate. In turn, cadmium and zinc levels were significantly associated with a decreased PON1 activity (zinc was associated with all PON1 activities and cadmium with PON1 towards paraoxon and diazoxon). Arsenic, nickel and manganese failed to be significantly associated with any of the PON1 activities assayed. PON1 192R alloform predicted significantly higher levels of arsenic and lead. BChE, however, was inversely associated with serum levels of manganese and zinc. These results suggest that PON1 and BChE activities are modulated by background exposure to metal compounds, which may have implications in public health given the defensive role played by both enzyme proteins against environmental toxicants. The potential underlying mechanisms merit further investigation.

  13. Esterase patterns and phylogenetic relationships of Drosophila species in the saltans subgroup (saltans group).

    PubMed

    Nascimento, A P; de Campos, Bicudo H E M

    2002-01-01

    The esterase patterns of sixteen strains from four species in the saltans subgroup were analyzed using polyacrylamide gel electrophoresis. Thirty-four esterase bands were detected. By using alpha and beta naphthyl acetates as substrates, they were classified in 18 alpha-esterases (they hydrolyse the alpha-naphtyl substrate), 15 beta-esterases (they hydrolyse the beta-naphtyl substrate) and 1 alpha/beta-esterase (it hydrolyses the alpha and beta-naphtyl substrates). Among the alpha-esterases, three were detected exclusively in males. Malathion, Eserine and pCMB were used as inhibitors in order to characterize biochemically the esterases. The results indicated the presence of cholinesterases, carboxylesterases and acetylesterases. The degree of mobility of the bands in the gels, their specificity to alpha and beta naphthyl acetates and the results of the inhibition tests allowed us to recognize tentatively nine genetic loci. Phylogenetic relationships among species inferred on the basis of the esterase patterns by PAUP 4.0b8, with neighbor-joining search and a bootstrap analysis showed that, although the four species are closely related, D. septentriosaltans, D. saltans and D. austrosaltans are closer to each other than to D. prosaltans. These results showed to be consistent with phylogenetic relationships previously inferred from inversion polymorphism.

  14. Structure of the catalytic domain of glucuronoyl esterase Cip2 from Hypocrea jecorina

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The structure of the catalytic domain of glucuronoyl esterase Cip2 from the fungus Hypocrea jecorina was determined at a resolution of 1.9 Angstroms. This is the first structure of the newly established carbohydrate esterase family 15. The structure has revealed the residues Ser278–His411–Glu301 pre...

  15. 21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... (CONTINUED) SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION Enzyme Preparations and Microorganisms § 173.140 Esterase-lipase derived from Mucor miehei. Esterase-lipase enzyme, consisting of enzyme... animals. (c) The enzyme is produced by a process which completely removes the organism Mucor miehei...

  16. 21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 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... Emerson is nonpathogenic and nontoxic in man or other animals. (c) The enzyme is produced by a...

  17. 21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 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... Emerson is nonpathogenic and nontoxic in man or other animals. (c) The enzyme is produced by a...

  18. 21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 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... Emerson is nonpathogenic and nontoxic in man or other animals. (c) The enzyme is produced by a...

  19. 21 CFR 173.140 - Esterase-lipase derived from Mucor miehei.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 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... Emerson is nonpathogenic and nontoxic in man or other animals. (c) The enzyme is produced by a...

  20. Cloning, overexpression in Escherichia coli, and characterization of a thermostable fungal acetylxylan esterase from Talaromyces emersonii.

    PubMed

    Waters, Deborah M; Murray, Patrick G; Miki, Yuta; Martínez, Angel T; Tuohy, Maria G; Faulds, Craig B

    2012-05-01

    The gene encoding an acetylxylan esterase (AXE1) from the thermophilic ascomycete Talaromyces emersonii was cloned, expressed in Escherichia coli, and characterized. This form of AXE1, rTeAXE1, exhibits increased thermostability and activity at a higher temperature than other known fungal acetyl esterases, thus having huge potential application in biomass bioconversion to high value chemicals or biofuels. PMID:22407679

  1. Production, Purification, and Properties of Extracellular Carboxyl Esterases from Bacillus subtilis NRRL 365

    PubMed Central

    Meghji, K.; Ward, O. P.; Araujo, A.

    1990-01-01

    Bacillus subtilis NRRL 365 produced high extracellular carboxyl esterase activity in submerged culture media containing wheat bran, corn steep liquor, and salts. Supplementation of this medium with glucose reduced esterase activity to 37% of that in the unsupplemented control. Esterase activity was purified by ammonium sulfate fractionation, DEAE-Sephadex A-50 ion-exchange chromatography with sodium chloride gradient elution, and preparative polyacrylamide gel electrophoresis. The resultant purified components, esterases I and II, manifested single bands following silver staining of polyacrylamide gel electrophoresis gels and had final specific activities of 80 and 520 U/mg, respectively. Molecular weights for components I and II were 36,000 and 105,000 to 110,000, respectively. Esterases I and II both had a pH optimum of 8.0, with relative activities of 10 and 85%, respectively, at pH 9.0. Kms with p-nitrophenylacetate were 0.91 mM for esterase I and 0.67 mM for esterase II. In general, patterns of enzyme inhibition were similar for both components. Differences were observed in the relative activities of esterases I and II towards p-nitrophenyl esters of acetate, propionate, and butyrate; Activity ratios for components I and II were 100:94:48 and 100:36:23, respectively. The purified components did not hydrolyze long-chain triglycerides and did not manifest proteolytic activity. Images PMID:16348375

  2. Correlation of leukocyte esterase activity and bacterial isolation from body fluids.

    PubMed Central

    Smalley, D L; Bradley, M E

    1984-01-01

    We evaluated 230 body fluid samples, of which 131 were peritoneal effluents and 99 were other body fluids. Of these, 63 dialysates were culture positive, and 54 (85.7%) of these 63 were leukocyte esterase positive. Of 99 other body fluids, 8 were both culture positive and leukocyte esterase positive. PMID:6520224

  3. A comparison of multiple esterases as biomarkers of organophosphate exposure and effect in two earthworm species.

    PubMed

    Henson-Ramsey, Heather; Schneider, Ashley; Stoskopf, Michael K

    2011-04-01

    Two different earthworm species, Eisenia fetida and Lumbricus terrestris, were exposed to 5 μg/cm(2) of malathion to evaluate their usefulness as sentinels of organophosphate exposure and to assess three different esterases, as biomarkers of malathion exposure and effect. Tissue xenobiotic burdens and esterase activity were determined for each species and each esterase in order to assess variability. E. fetida exhibited 4-fold less variability in tissue burdens than did L. terrestris and had less variable basal esterase activities. An attempt was made to correlate malathion and malaoxon tissue burdens with esterase activity post-exposure. There was no malaoxon present in the earthworm tissues. No significant correlations were determined by comparing acetylcholinesterase, butyrylcholinesterase, nor carboxylesterase activities with malathion burdens. PMID:21404045

  4. Esterase Activity and Intracellular Localization in Reconstructed Human Epidermal Cultured Skin Models

    PubMed Central

    Katayanagi, Mishina; Hashimoto, Fumie

    2015-01-01

    Background Reconstructed human epidermal culture skin models have been developed for cosmetic and pharmaceutical research. Objective This study evaluated the total and carboxyl esterase activities (i.e., Km and Vmax, respectively) and localization in two reconstructed human epidermal culture skin models (LabCyte EPI-MODEL [Japan Tissue Engineering] and EpiDerm [MatTek/Kurabo]). The usefulness of the reconstruction cultured epidermis was also verified by comparison with human and rat epidermis. Methods Homogenized epidermal samples were fractioned by centrifugation. p-nitrophenyl acetate and 4-methylumbelliferyl acetate were used as substrates of total esterase and carboxyl esterase, respectively. Results Total and carboxyl esterase activities were present in the reconstructed human epidermal culture skin models and were localized in the cytosol. Moreover, the activities and localization were the same as those in human and rat epidermis. Conclusion LabCyte EPI-MODEL and EpiDerm are potentially useful for esterase activity prediction in human epidermis. PMID:26082583

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

    PubMed

    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.

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

    PubMed Central

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

    2011-01-01

    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. PMID:21887044

  7. Heterologous expression of a fungal sterol esterase/lipase in different hosts: Effect on solubility, glycosylation and production.

    PubMed

    Vaquero, María Eugenia; Barriuso, Jorge; Medrano, Francisco Javier; Prieto, Alicia; Martínez, María Jesús

    2015-12-01

    Ophiostoma piceae secretes a versatile sterol-esterase (OPE) that shows high efficiency in both hydrolysis and synthesis of triglycerides and sterol esters. This enzyme produces aggregates in aqueous solutions, but the recombinant protein, expressed in Komagataella (synonym Pichia) pastoris, showed higher catalytic efficiency because of its higher solubility. This fact owes to a modification in the N-terminal sequence of the protein expressed in Pichia pastoris, which incorporated 4-8 additional amino acids, affecting its aggregation behavior. In this study we present a newly engineered P. pastoris strain with improved protein production. We also produced the recombinant protein in the yeast Saccharomyces cerevisiae and in the prokaryotic host Escherichia coli, corroborating that the presence of these N-terminal extra amino acids affected the protein's solubility. The OPE produced in the new P. pastoris strain presented the same physicochemical properties than the old one. An inactive form of the enzyme was produced by the bacterium, but the recombinant esterase from both yeasts was active even after its enzymatic deglycosylation, suggesting that the presence of N-linked carbohydrates in the mature protein is not essential for enzyme activity. Although the yield in S. cerevisiae was lower than that obtained in P. pastoris, this work demonstrates the importance of the choice of the heterologous host for successful production of soluble and active recombinant protein. In addition, S. cerevisiae constitutes a good engineering platform for improving the properties of this biocatalyst.

  8. Three-dimensional structure of homodimeric cholesterol esterase-ligand complex at 1.4 Å resolution

    SciTech Connect

    Pletnev, V.; Addlagatta, A.; Wawrzak, Z.; Duax, W.

    2010-03-08

    The three-dimensional structure of a Candida cylindracea cholesterol esterase (ChE) homodimer (534 x 2 amino acids) in complex with a ligand of proposed formula C{sub 23}H{sub 48}O{sub 2} has been determined at 1.4 {angstrom} resolution in space group P1 using synchrotron low-temperature data. The structure refined to R = 0.136 and R{sub free} = 0.169 and has revealed new stereochemical details in addition to those detected for the apo- and holo-forms at 1.9 and 2.0 {angstrom} resolution, respectively [Ghosh et al. (1995), Structure, 3, 279-288]. The cholesterol esterase structure is a dimer with four spatially separated interfacial contact areas and two symmetry-related pairs of openings to an internal intradimer cavity. Hydrophobic active-site gorges in each subunit face each other across a central interfacial cavity. The ChE subunits have carbohydrate chains attached to their Asn314 and Asn351 residues, with two ordered N-acetyl-D-glucosoamine moieties visible at each site. The side chains of 14 residues have two alternative conformations with occupancy values of 0.5 {+-} 0.2. For each subunit the electron density in the enzyme active-site gorge is well modeled by a C{sub 23}-chain fatty acid.

  9. Heterologous expression of a fungal sterol esterase/lipase in different hosts: Effect on solubility, glycosylation and production.

    PubMed

    Vaquero, María Eugenia; Barriuso, Jorge; Medrano, Francisco Javier; Prieto, Alicia; Martínez, María Jesús

    2015-12-01

    Ophiostoma piceae secretes a versatile sterol-esterase (OPE) that shows high efficiency in both hydrolysis and synthesis of triglycerides and sterol esters. This enzyme produces aggregates in aqueous solutions, but the recombinant protein, expressed in Komagataella (synonym Pichia) pastoris, showed higher catalytic efficiency because of its higher solubility. This fact owes to a modification in the N-terminal sequence of the protein expressed in Pichia pastoris, which incorporated 4-8 additional amino acids, affecting its aggregation behavior. In this study we present a newly engineered P. pastoris strain with improved protein production. We also produced the recombinant protein in the yeast Saccharomyces cerevisiae and in the prokaryotic host Escherichia coli, corroborating that the presence of these N-terminal extra amino acids affected the protein's solubility. The OPE produced in the new P. pastoris strain presented the same physicochemical properties than the old one. An inactive form of the enzyme was produced by the bacterium, but the recombinant esterase from both yeasts was active even after its enzymatic deglycosylation, suggesting that the presence of N-linked carbohydrates in the mature protein is not essential for enzyme activity. Although the yield in S. cerevisiae was lower than that obtained in P. pastoris, this work demonstrates the importance of the choice of the heterologous host for successful production of soluble and active recombinant protein. In addition, S. cerevisiae constitutes a good engineering platform for improving the properties of this biocatalyst. PMID:25939548

  10. Esterase- and pH-responsive poly(β-amino ester)-capped mesoporous silica nanoparticles for drug delivery

    NASA Astrophysics Data System (ADS)

    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

  11. Identification of a bacterial pectin acetyl esterase in Erwinia chrysanthemi 3937.

    PubMed

    Shevchik, V E; Hugouvieux-Cotte-Pattat, N

    1997-06-01

    Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls. The structural complexity of pectin requires the combined action of several pectinases for its efficient breakdown. Three types of pectinases have so far been identified in E. chrysanthemi: two pectin methyl esterases (PemA, PemB), a polygalacturonase (PehX), and eight pectate lyases (PelA, PelB, PelC, PelD, PelE, PelL, PelZ, PelX). We report in this paper the analysis of a novel enzyme, the pectin acetyl esterase encoded by the paeY gene. No bacterial form of pectin acetyl esterases has been described previously, while plant tissues and some pectinolytic fungi were found to produce similar enzymes. The paeY gene is present in a cluster of five pectinase-encoding genes, pelA-pelE-pelD-paeY-pemA. The paeY open reading frame is 1650 bases long and encodes a 551-residue precursor protein of 60704Da, including a 25-amino-acid signal peptide. PaeY shares one region of homology with a rhamnogalacturonan acetyl esterase of Aspergillus aculeatus. To characterize the enzyme, the paeY gene was overexpressed and its protein product was purified. PaeY releases acetate from sugar-beet pectin and from various synthetic substrates. Moreover, the enzyme was shown to act in synergy with other pectinases. The de-esterification rate by PaeY increased after previous demethylation of the pectins by PemA and after depolymerization of the pectin by pectate lyases. In addition, the degradation of sugar-beet pectin by pectate lyases is favoured after the removal of methyl and acetyl groups by PemA and PaeY, respectively. The paeY gene was first identified on the basis of its regulation, which shares several characteristics with that of other pectinases. Analysis of the paeY transcription, using gene fusions, revealed that it is induced by pectic catabolic products and is affected by growth phase, oxygen limitation and catabolite repression. Regulation of pae

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

    PubMed Central

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

    2012-01-01

    Background and Aims 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. Methods 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. Key Results 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. Conclusions 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

  13. An Experimental Investigation of the Unit Charge Model of Protein Polymorphism and Its Relation to the Esterase-5 Locus of DROSOPHILA PSEUDOOBSCURA, DROSOPHILA PERSIMILIS, and DROSOPHILA MIRANDA

    PubMed Central

    Cobbs, Gary; Prakash, Satya

    1977-01-01

    The relationship between charge changes and electrophoretic mobility changes is investigated experimentally. The charge of several proteins is altered by reaction with small molecules of known structure and the change in electrophoretic mobility is measured. The method of Ferguson plots is used to separate charge and shape components of mobility differences. The average effect of an amino acid charge change on the mobility of the esterase-5 1.00 allele of Drosophila pseudoobscura is estimated to be 0.046. This estimate is then used to apply the step model of Ohta and Kimura (1973) to electrophoretic mobility data for the esterase-5 locus of D. pseudoobscura and D. miranda. The variation in electrophoretic mobility at this locus was found to be in agreement with the predictions of the step model. PMID:17248784

  14. Characterization of a Novel Alkaline Family VIII Esterase with S-Enantiomer Preference from a Compost Metagenomic Library.

    PubMed

    Lee, Hyun Woo; Jung, Won Kyeong; Kim, Yong Ho; Ryu, Bum Han; Kim, T Doohun; Kim, Jungho; Kim, Hoon

    2016-02-01

    A novel esterase gene, est7K, was isolated from a compost metagenomic library. The gene encoded a protein of 411 amino acids and the molecular mass of the Est7K was estimated to be 44,969 Da with no signal peptide. Est7K showed the highest identity of 57% to EstA3, which is an esterase from a drinking water metagenome, when compared with the enzymes with reported properties. Est7K had three motifs, SMTK, YSV, and WGG, which correspond to the typical motifs of family VIII esterases, SxxK, Yxx, and WGG, respectively. Est7K did not have the GxSxG motif in most lipolytic enzymes. Three additional motifs, LxxxPGxxW, PLGMxDTxF, and GGxG, were found to be conserved in family VIII enzymes. The results of the phylogenetic analysis and the alignment study suggest that family VIII enzymes could be classified into two subfamilies, VIII.1 and VIII.2. The purified Est7K was optimally active at 40°C and pH 10.0. It was activated to exhibit a 2.1-fold higher activity by the presence of 30% methanol. It preferred short-length p-nitrophenyl esters, particularly p-nitrophenyl butyrate, and efficiently hydrolyzed glyceryl tributyrate. It did not hydrolyze β-lactamase substrates, tertiary alcohol esters, glyceryl trioleate, fish oil, and olive oil. Est7K preferred an Senantiomer, such as (S)-methyl-3-hydroxy-2-methylpropionate, as the substrate. The tolerance to methanol and the substrate specificity may provide potential advantage in the use of the enzyme in pharmaceutical and other biotechnological processes.

  15. Branched nanotrees with immobilized acetylcholine esterase for nanobiosensor applications

    NASA Astrophysics Data System (ADS)

    Risveden, Klas; Dick, Kimberly A.; Bhand, Sunil; Rydberg, Patrik; Samuelson, Lars; Danielsson, Bengt

    2010-02-01

    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 SiNx-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.

  16. Food induced esterase phenocopies in the snail Cepaea nemoralis.

    PubMed

    Oxford, G S

    1975-12-01

    Hepatopancreatic extracts from the snail Cepaea nemoralis, assayed straight from the field, often contain three or four heavily staining esterase zones which migrate to the cathodal end of polyacrylamide disc gels during electrophoresis. Previous breeding results showed that the heavily straining zones appeared allelic but to incorporate these multibanded phenotypes, a super gene of five closely linked loci was tentatively proposed. Further breeding work again failed to demonstrate multiple zones in parents or offspring and so experiments were conducted to see whether the multi-zoned phenotypes in the wild were produced by secondary modification of single primary products. Wild snails yielding extracts containing more than two heavily staining zones were shown to possess only two such zones after three months under laboratory conditions. Also, the ingestion of nettle (Urtica dioica L.) has been demonstrated to induce extra esterase zones in laboratory-reared animals. Some of the secondarily induced zones appear identical in physical, biochemical and electrophoretic properties to the primary products of other alleles, and thus appear to be electrophoretic phenocopies. A model is suggested which could account for this phenomenon. PMID:1061709

  17. An organic-solvent-tolerant esterase from thermophilic Bacillus licheniformis S-86.

    PubMed

    Torres, Sebastián; Martínez, M Alejandra; Pandey, Ashok; Castro, Guillermo R

    2009-01-01

    A thermophile, halotolerant and organic-solvent-tolerant esterase producer Bacillus sp. S-86 strain previously isolated was found to belong to Bacillus licheniformis species through morphological, biochemical, 16S rRNA gene sequence analyses and rDNA intergenic spacers amplification (ITS-PCR). The strain can grow at 55 degrees C in presence of C2-C7 alkanols (log P=-0.86 to 2.39), and NaCl concentrations up to 15% (w/v). This bacterium showed optimal growth and esterase production at 50 degrees C. Two different molecular weight esterase activities were detected in zymographic assays. PMSF inhibited type I esterase activity, showing no inhibitory effect on type II esterase activity. B. licheniformis S-86 was able to grow in presence of hydroxylic organic-solvents like propan-2-ol, butan-1-ol and 3-methylbutan-1-ol. At a sub-lethal concentration of these solvents (392 mmoll(-1) propan-2-ol; 99 mmol l(-1) butan-1-ol, 37 mmol l(-1) 3-methylbutan-1-ol), adequate to produce 50% cell growth inhibition at 50 degrees C, an increment between 1.9 and 2.3 times was observed in type I esterase production, and between 2.2 and 3.1 times in type II esterase production. PMID:18723341

  18. Purification and properties of an esterase from organophosphate-resistant strain of the mosquito Culex quinquefasciatus.

    PubMed Central

    Merryweather, A T; Crampton, J M; Townson, H

    1990-01-01

    Organophosphate-resistant and -susceptible strains of Culex quinquefasciatus (mosquito) have been compared on the basis of their esterase activities. The homozygous resistant strain (Dar) shows two highly active esterases after starch-gel electrophoresis, of Rm 0.2 and 0.4, which are absent from susceptible strains (Apo, Mon), and which previous selection studies have shown to be inseparable from organophosphate resistance. After SDS/polyacrylamide-gel electrophoresis and silver staining of total C. quinquefasciatus proteins, a 62 kDa band is observed in strain Dar at high concentrations, and in susceptible strains in trace amounts. After Western blotting, this 62 kDa protein is recognized by antisera raised against the two esterases eluted from starch gels. After chromatofocusing of Dar proteins, the 62 kDa protein is seen to be associated with esterase activity, and of a similar pI to that observed for esterases after isoelectric focusing. Post-translational modification is not required for recognition of the 62 kDa putative esterase, since the protein is immunoprecipitated by the anti-esterase serum from products of translation of Dar mRNA in vitro. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 6. Fig. 7. PMID:2178604

  19. Distribution of esterase activity in porcine ear skin, and the effects of freezing and heat separation.

    PubMed

    Lau, Wing Man; Ng, Keng Wooi; Sakenyte, Kristina; Heard, Charles M

    2012-08-20

    Porcine ear skin is widely used to study skin permeation and absorption of ester compounds, whose permeation and absorption profiles may be directly influenced by in situ skin esterase activity. Importantly, esterase distribution and activity in porcine ear skin following common protocols of skin handling and storage have not been characterised. Thus, we have compared the distribution and hydrolytic activity of esterases in freshly excised, frozen, heated and explanted porcine ear skin. Using an esterase staining kit, esterase activity was found to be localised in the stratum corneum and viable epidermis. Under frozen storage and a common heating protocol of epidermal sheet separation, esterase staining in the skin visibly diminished. This was confirmed by a quantitative assay using HPLC to monitor the hydrolysis of aspirin, in freshly excised, frozen or heated porcine ear skin. Compared to vehicle-only control, the rate of aspirin hydrolysis was approximately three-fold higher in the presence of freshly excised skin, but no different in the presence of frozen or heated skin. Therefore, frozen and heat-separated porcine ear skin should not be used to study the permeation of ester-containing permeants, in particular co-drugs and pro-drugs, whose hydrolysis or degradation can be modulated by skin esterases.

  20. Identification of a Secreted Lipolytic Esterase in Propionibacterium freudenreichii, a Ripening Process Bacterium Involved in Emmental Cheese Lipolysis▿ †

    PubMed Central

    Dherbécourt, J.; Falentin, H.; Jardin, J.; Maillard, M.-B.; Baglinière, F.; Barloy-Hubler, F.; Thierry, A.

    2010-01-01

    Lipolysis plays an important role in the formation of cheese flavor. In Emmental cheese, the main part of lipolysis has been associated with the presence of Propionibacterium freudenreichii, a species used as a ripening culture. Our aim was to identify the most probable lipolytic esterase(s) involved in cheese lipolysis by P. freudenreichii. Since cheese lipolysis mainly occurs during P. freudenreichii growth, we hypothesized that P. freudenreichii possesses secreted lipolytic esterase(s). For 12 putative esterase genes previously identified from the genome of P. freudenreichii CIRM1, the level of expression was quantified by real-time reverse transcriptase (RT)-PCR, and the subcellular localization of esterases was predicted in silico. The esterase activity in extracellular and intracellular extracts of P. freudenreichii was characterized by zymography, and the extracellular esterases were identified by mass spectrometry. Finally, the best candidate was overexpressed in the same strain. All of the 12 genes encoding putative esterases were expressed. Esterase PF#279 was predicted to be secreted in the medium, PF#774 to be surface exposed, and the 10 remaining putative esterases to be intracellular. Zymography revealed that esterase activities in culture supernatant differed from the ones detected in intracellular extracts. PF#279 was identified as the sole esterase present in culture supernatant. Transformed P. freudenreichii CIRM1 clones overexpressing PF#279 showed 5 to 8 times more lipolytic activity on milk fat than the wild-type strain. Combining in silico, biochemical, and genetic approaches, we showed that PF#279 is the sole secreted esterase in P. freudenreichii and is active on milk fat. Therefore, it is likely a key component in cheese lipolysis by P. freudenreichii. PMID:20038704

  1. Bacteriophage 933W encodes a functional esterase downstream of the Shiga toxin 2a operon.

    PubMed

    Nübling, Simone; Eisele, Thomas; Stöber, Helen; Funk, Joschua; Polzin, Sabrina; Fischer, Lutz; Schmidt, Herbert

    2014-05-01

    In this study, the 1938bp open reading frame z1466, which is encoded directly downstream the Shiga toxin 2a (Stx2a) operon in E. coli O157:H7 phage 933W was cloned and expressed recombinantly. Purification with Ni-NTA agarose beads with subsequent SDS-PAGE revealed a 68kDa protein, designated 933Wp42-His. Analysis of 933Wp42-His demonstrated an esterase activity by activity staining of native gels using triacetin as a substrate. Purified 933Wp42-His demonstrated a Km value of about 10mM and a Vmax value of 1.667nkat/ml for 4-methylumbelliferyl-acetate (4-MUF-Ac) as a substrate. The enzyme was most active in the pH-range of 7.0-8.0, and at 50°C. Furthermore, 933Wp42-His was able to hydrolyze acetic acid from mucin, and 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac2). This is the first description of an enzymatic activity of the Stx-phage-encoded protein 933Wp42. Its role in substrate utilization during colonization and human infection is discussed.

  2. Discovery a novel organic solvent tolerant esterase from Salinispora arenicola CNP193 through genome mining.

    PubMed

    Fang, Yaowei; Wang, Shujun; Liu, Shu; Jiao, Yuliang

    2015-09-01

    An esterase gene, encoding a 325-amino-acid protein (SAestA), was mined form obligate marine actinomycete strain Salinispora arenicola CNP193 genome sequence. Phylogenetic analysis of the deduced amino acid sequence showed that the enzyme belonged to the family IV of lipolytic enzymes. The gene was cloned, expressed in Escherichia coli as a His-tagged protein, purified and characterized. The molecular weight of His-tagged SAestA is ∼38 kDa. SAestA-His6 was active in a temperature (5-40 °C) and pH range (7.0-11.0), and maximal activity was determined at pH 9.0 and 30 °C. The activity was severely inhibited by Hg(2+), Cu(2+), and Zn(2+). In particular, this enzyme showed remarkable stability in presence of organic solvents (25%, v/v) with log P>2.0 even after incubation for 7 days. All these characteristics suggested that SAestA may be a potential candidate for application in industrial processes in aqueous/organic media. PMID:26118483

  3. Combined activation of methyl paraben by light irradiation and esterase metabolism toward oxidative DNA damage.

    PubMed

    Okamoto, Yoshinori; Hayashi, Tomohiro; Matsunami, Shinpei; Ueda, Koji; Kojima, Nakao

    2008-08-01

    Methyl paraben (MP) is often used as a preservative in foods, drugs, and cosmetics because of its high reliability in safety based on the rapid excretion and nonaccumulation following administration. Light irradiation sometimes produces unexpected activity from chemicals such as MP; furthermore, there is ample opportunity for MP to be exposed to sunlight. Here, we investigated whether MP shows DNA damage after sunlight irradiation. Two major photoproducts, p-hydroxybenzoic acid (PHBA) and 3-hydroxy methyl paraben (MP-3OH), were detected after sunlight irradiation to an aqueous MP solution. Both photoproducts were inactive in the in vitro DNA damage assay that measures oxidized guanine formed in calf thymus DNA in the presence of divalent copper ion, a known mediator of oxidative DNA damage. Simulated MP metabolism using dermal tissues after light irradiation produced these two photoproducts, which reacted with a microsomal fraction (S9) of the skin. A metabolite from MP-3OH, not PHBA, caused distinct DNA damage in the in vitro assay. This active metabolite was identified as protocatechuic acid, a hydrolyzed MP-3OH product. In addition, NADH, a cellular reductant, enhanced DNA damage by approximately five times. These results suggest that reactive oxygen species generated by the redox cycle via metal ion and catechol autoxidation are participating in oxidative DNA damage. This study reveals that MP might cause skin damage involving carcinogenesis through the combined activation of sunlight irradiation and skin esterases.

  4. Identification and characterization of novel esterases from a deep-sea sediment metagenome.

    PubMed

    Jiang, Xiawei; Xu, Xuewei; Huo, Yingyi; Wu, Yuehong; Zhu, Xufen; Zhang, Xinqi; Wu, Min

    2012-03-01

    A deep-sea sediment metagenomic library was constructed and screened for lipolytic enzymes by activity-based approach. Nine novel lipolytic enzymes were identified, and the amino acid sequences shared 56% to 84% identity to other lipolytic enzymes in the database. Phylogenetic analysis showed that these enzymes belonged to family IV lipolytic enzymes. One of the lipolytic enzymes, Est6, was successfully cloned and expressed in Escherichia coli Rosetta in a soluble form. The recombinant protein was purified by Ni-nitrilotriacetic affinity chromatography column and characterized using p-nitrophenyl esters with various chain lengths. The est6 gene consisted of 909 bp that encoded 302 amino acid residues. Est6 was most similar to a lipolytic enzyme from uncultured bacterium (ACL67845, 61% identity) isolated from the South China Sea marine sediment metagenome. The characterization of Est6 revealed that it was a cold-active esterase and exhibited the highest activity toward p-nitrophenyl butyrate (C4) at 20°C and pH 7.5.

  5. Polyploid genome of Camelina sativa revealed by isolation of fatty acid synthesis genes

    PubMed Central

    2010-01-01

    Background Camelina sativa, an oilseed crop in the Brassicaceae family, has inspired renewed interest due to its potential for biofuels applications. Little is understood of the nature of the C. sativa genome, however. A study was undertaken to characterize two genes in the fatty acid biosynthesis pathway, fatty acid desaturase (FAD) 2 and fatty acid elongase (FAE) 1, which revealed unexpected complexity in the C. sativa genome. Results In C. sativa, Southern analysis indicates the presence of three copies of both FAD2 and FAE1 as well as LFY, a known single copy gene in other species. All three copies of both CsFAD2 and CsFAE1 are expressed in developing seeds, and sequence alignments show that previously described conserved sites are present, suggesting that all three copies of both genes could be functional. The regions downstream of CsFAD2 and upstream of CsFAE1 demonstrate co-linearity with the Arabidopsis genome. In addition, three expressed haplotypes were observed for six predicted single-copy genes in 454 sequencing analysis and results from flow cytometry indicate that the DNA content of C. sativa is approximately three-fold that of diploid Camelina relatives. Phylogenetic analyses further support a history of duplication and indicate that C. sativa and C. microcarpa might share a parental genome. Conclusions There is compelling evidence for triplication of the C. sativa genome, including a larger chromosome number and three-fold larger measured genome size than other Camelina relatives, three isolated copies of FAD2, FAE1, and the KCS17-FAE1 intergenic region, and three expressed haplotypes observed for six predicted single-copy genes. Based on these results, we propose that C. sativa be considered an allohexaploid. The characterization of fatty acid synthesis pathway genes will allow for the future manipulation of oil composition of this emerging biofuel crop; however, targeted manipulations of oil composition and general development of C. sativa should

  6. Purification and characterization of an esterase involved in poly(vinyl alcohol) degradation by Pseudomonas vesicularis PD.

    PubMed

    Sakai, K; Fukuba, M; Hasui, Y; Moriyoshi, K; Ohmoto, T; Fujita, T; Ohe, T

    1998-10-01

    An esterase catalyzing the hydrolysis of acetyl ester moieties in poly(vinyl alcohol) was purified 400-fold to electrophoretic homogeneity from the cytoplasmic fraction of Pseudomonas vesicularis PD, which was capable of assimilating poly(vinyl alcohol) as the sole carbon and energy source. The purified enzyme was a homodimeric protein with a molecular mass of 80 kDa and the isoelectric point was 6.8. The pH and temperature optima of the enzyme were 8.0 and 45 degrees C. The enzyme catalyzed the hydrolysis of side chains of poly(vinyl alcohol), short-chain p-nitrophenyl esters, 2-naphthyl acetate, and phenyl acetate, and was slightly active toward aliphatic esters. The enzyme was also active toward the enzymatic degradation products, acetoxy hydroxy fatty acids, of poly(vinyl alcohol). The K(m) and Vmax of poly(vinyl alcohol) (degree of polymerization, 500; saponification degree, 86.5-89.0 mol%) and p-nitrophenyl acetate were 0.381% (10.6 mM as acetyl content in the polymer) and 2.56 microM, and 6.52 and 12.6 mumol/min/mg, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate at a concentration of 5 mM, which indicated that the enzyme was a serine esterase. The pathway for the metabolism of poly(vinyl alcohol) is also discussed.

  7. Enhanced catalytic site thermal stability of cold-adapted esterase EstK by a W208Y mutation.

    PubMed

    Boyineni, Jerusha; Kim, Junyoung; Kang, Beom Sik; Lee, ChangWoo; Jang, Sei-Heon

    2014-06-01

    Hydrophobic interactions are known to play an important role for cold-adaptation of proteins; however, the role of amino acid residue, Trp, has not been systematically investigated. The extracellular esterase, EstK, which was isolated from the cold-adapted bacterium Pseudomonas mandelii, has 5 Trp residues. In this study, the effects of Trp mutation on thermal stability, catalytic activity, and conformational change of EstK were investigated. Among the 5 Trp residues, W(208) was the most crucial in maintaining structural conformation and thermal stability of the enzyme. Surprisingly, mutation of W(208) to Tyr (W(208)Y) showed an increased catalytic site thermal stability at ambient temperatures with a 13-fold increase in the activity at 40°C compared to wild-type EstK. The structure model of W(208)Y suggested that Y(208) could form a hydrogen bond with D(308), which is located next to catalytic residue H(307), stabilizing the catalytic domain. Interestingly, Tyr was conserved in the corresponding position of hyper-thermophilic esterases EstE1 and AFEST, which are active at high temperatures. Our study provides a novel insight into the engineering of the catalytic site of cold-adapted enzymes with increased thermal stability and catalytic activity at ambient temperatures.

  8. Esterase variation at three loci in meat ants.

    PubMed

    Halliday, R B

    1979-01-01

    The meat ant (Iridomyrmex purpureus) occurs in a number of color forms, with uncertain taxonomic status. Gel electrophresis of meat ant extracts, followed by nonspecific esterase staining, reveals several zones of activity. Allelic variation at three loci is proposed to account for variation in some of these zones. Two of the loci (Es-1, Es-2) appear to have recessive null alleles, whose frequencies have been estimated by the method of maximum likelihood. Geographic variation in allele frequency is attributed to behavioral and geographic subdivision of the population. Apparent disturbances in segregation ratios and deviations from Hardy-Weinberg equilibrium can be accounted for if it is argued that some nests contain more than one queen. Differences in gene frequency between sympatric populations of the red and blue forms of I. purpureus are observed, confirming their reproductive is isolation and sibling species status.

  9. Plasma B-esterase activities in European raptors.

    PubMed

    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

  10. Molecular Cloning and Characterization of a Newly Isolated Pyrethroid-Degrading Esterase Gene from a Genomic Library of Ochrobactrum anthropi YZ-1

    PubMed Central

    Song, Jinlong; Shi, Yanhua; Li, Kang; Zhao, Bin; Yan, Yanchun

    2013-01-01

    A novel pyrethroid-degrading esterase gene pytY was isolated from the genomic library of Ochrobactrum anthropi YZ-1. It possesses an open reading frame (ORF) of 897 bp. Blast search showed that its deduced amino acid sequence shares moderate identities (30% to 46%) with most homologous esterases. Phylogenetic analysis revealed that PytY is a member of the esterase VI family. pytY showed very low sequence similarity compared with reported pyrethroid-degrading genes. PytY was expressed, purified, and characterized. Enzyme assay revealed that PytY is a broad-spectrum degrading enzyme that can degrade various pyrethroids. It is a new pyrethroid-degrading gene and enriches genetic resource. Kinetic constants of Km and Vmax were 2.34 mmol·L−1 and 56.33 nmol min−1, respectively, with lambda-cyhalothrin as substrate. PytY displayed good degrading ability and stability over a broad range of temperature and pH. The optimal temperature and pH were of 35°C and 7.5. No cofactors were required for enzyme activity. The results highlighted the potential use of PytY in the elimination of pyrethroid residuals from contaminated environments. PMID:24155944

  11. Cloning, Purification and Characterization of Acetyl Xylane Esterase from Anoxybacillus flavithermus DSM 2641(T) with Activity on Low Molecular-Weight Acetates.

    PubMed

    Eminoğlu, Ayşenur; Ülker, Serdar; Sandallı, Cemal

    2015-08-01

    Family 4 carbohydrate esterases (CE-4) have deacetylate different forms of acetylated poly/oligosaccharides in nature. This family is recognized with a specific polysaccharide deacetylase domain assigned as NodB homology domain in their secondary structure. Most family 4 carbohydrate esterases have been structurally and biochemically characterized. However, this is the first study about the enzymological function of pdaB-like CE4s from thermophilic bacterium Anoxybacillus flavithermus DSM 2641(T). A. flavithermus WK1 genome harbors five putative CE4 family genes. One of them is 762 bp long and encodes a protein of 253 amino acids in length and it was used as reference sequence in this study. It was described as acetyl xylane esterase (AXE) in genome project and this AfAXE gene was amplified without signal sequence and cloned. The recombinant protein was expressed in E. coli BL21 (DE3), purified by nickel affinity chromatography and its purity was visualized on SDS-PAGE. The activity of the recombinant enzyme was shown by zymogram analysis with α-naphtyl acetate as a substrate. The enzyme was characterized spectrophotometrically using chromogenic p-nitrophenyl acetate. Optimum temperature and pH were determined as 50 °C and 7.5, respectively. Km and Vmax were determined as 0.43 mM and 3333.33 U/mg, respectively under optimum conditions. To our knowledge this is the first enzymological characterization of a pdaB-like family 4 carbohydrate esterase from the members of Anoxybacillus genus.

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

    PubMed

    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.

  13. Esterase detoxification of acetylcholinesterase inhibitors by human or rat liver in vitro

    EPA Science Inventory

    Organophosphate (OP) and N-methylcarbamate pesticides inhibit acetylcholinesterase (AChE), but differences in metabolism and detoxification can influence potency of these pesticides across and within species. Carboxylesterase (CaE) and A-esterase (paraoxonase, PON) are considered...

  14. Esterase and Malate Dehydrogenase Phenotypes in Portuguese Populations of Meloidogyne Species

    PubMed Central

    Pais, Célia S.; de O. Abrantes, Isabel M.

    1989-01-01

    Nonspecific esterases and malate dehydrogenases of 1-5 females from 40 root-knot nematode populations from Portugal were analyzed by electrophoresis in 0.4-mm-thick polyacrylamide gels. Fourteen major bands of esterase activity were detected, corresponding to 10 distinct phenotypes, Meloidogyne javanica and M. hapla had distinct species-specific phenotypes. Two phenotypes occurred in M. arenaria. The most variability was found among M. incognita populations. Of the remaining two phenotypes, one was associated with M. hispanica and the other belonged to a new species. Three malate dehydrogenase phenotypes were discerned on the basis of particular combinations of the eight main bands of activity found. As previously found, esterases were more useful than malate dehydrogenases in identification of the major Meloidogyne species. The host plant had no effect on the nematode esterase or malate dehydrogenase phenotypes. PMID:19287618

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

    SciTech Connect

    Moretto, A. . E-mail: angelo.moretto@icps.it; Nicolli, A.; Lotti, M.

    2007-03-15

    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{sub 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.

  16. A novel cold-adapted and highly salt-tolerant esterase from Alkalibacterium sp. SL3 from the sediment of a soda lake.

    PubMed

    Wang, Guozeng; Wang, Qiaohuang; Lin, Xianju; Ng, Tzi Bun; Yan, Renxiang; Lin, Juan; Ye, Xiuyun

    2016-01-01

    A novel esterase gene (estSL3) was cloned from the Alkalibacterium sp. SL3, which was isolated from the sediment of soda lake Dabusu. The 636-bp full-length gene encodes a polypeptide of 211 amino acid residues that is closely related with putative GDSL family lipases from Alkalibacterium and Enterococcus. The gene was successfully expressed in E. coli, and the recombinant protein (rEstSL3) was purified to electrophoretic homogeneity and characterized. rEstSL3 exhibited the highest activity towards pNP-acetate and had no activity towards pNP-esters with acyl chains longer than C8. The enzyme was highly cold-adapted, showing an apparent temperature optimum of 30 °C and remaining approximately 70% of the activity at 0 °C. It was active and stable over the pH range from 7 to 10, and highly salt-tolerant up to 5 M NaCl. Moreover, rEstSL3 was strongly resistant to most tested metal ions, chemical reagents, detergents and organic solvents. Amino acid composition analysis indicated that EstSL3 had fewer proline residues, hydrogen bonds and salt bridges than mesophilic and thermophilic counterparts, but more acidic amino acids and less hydrophobic amino acids when compared with other salt-tolerant esterases. The cold active, salt-tolerant and chemical-resistant properties make it a promising enzyme for basic research and industrial applications.

  17. A novel cold-adapted and highly salt-tolerant esterase from Alkalibacterium sp. SL3 from the sediment of a soda lake

    PubMed Central

    Wang, Guozeng; Wang, Qiaohuang; Lin, Xianju; Bun Ng, Tzi; Yan, Renxiang; Lin, Juan; Ye, Xiuyun

    2016-01-01

    A novel esterase gene (estSL3) was cloned from the Alkalibacterium sp. SL3, which was isolated from the sediment of soda lake Dabusu. The 636-bp full-length gene encodes a polypeptide of 211 amino acid residues that is closely related with putative GDSL family lipases from Alkalibacterium and Enterococcus. The gene was successfully expressed in E. coli, and the recombinant protein (rEstSL3) was purified to electrophoretic homogeneity and characterized. rEstSL3 exhibited the highest activity towards pNP-acetate and had no activity towards pNP-esters with acyl chains longer than C8. The enzyme was highly cold-adapted, showing an apparent temperature optimum of 30 °C and remaining approximately 70% of the activity at 0 °C. It was active and stable over the pH range from 7 to 10, and highly salt-tolerant up to 5 M NaCl. Moreover, rEstSL3 was strongly resistant to most tested metal ions, chemical reagents, detergents and organic solvents. Amino acid composition analysis indicated that EstSL3 had fewer proline residues, hydrogen bonds and salt bridges than mesophilic and thermophilic counterparts, but more acidic amino acids and less hydrophobic amino acids when compared with other salt-tolerant esterases. The cold active, salt-tolerant and chemical-resistant properties make it a promising enzyme for basic research and industrial applications. PMID:26915906

  18. Characterization and structural modeling of a new type of thermostable esterase from Thermotoga maritima.

    PubMed

    Levisson, Mark; van der Oost, John; Kengen, Servé W M

    2007-06-01

    A bioinformatic screening of the genome of the hyperthermophilic bacterium Thermotoga maritima for ester-hydrolyzing enzymes revealed a protein with typical esterase motifs, though annotated as a hypothetical protein. To confirm its putative esterase function the gene (estD) was cloned, functionally expressed in Escherichia coli and purified to homogeneity. Recombinant EstD was found to exhibit significant esterase activity with a preference for short acyl chain esters (C4-C8). The monomeric enzyme has a molecular mass of 44.5 kDa and optimal activity around 95 degrees C and at pH 7. Its thermostability is relatively high with a half-life of 1 h at 100 degrees C, but less stable compared to some other hyperthermophilic esterases. A structural model was constructed with the carboxylesterase Est30 from Geobacillus stearothermophilus as a template. The model covered most of the C-terminal part of EstD. The structure showed an alpha/beta-hydrolase fold and indicated the presence of a typical catalytic triad consisting of a serine, aspartate and histidine, which was verified by site-directed mutagenesis and inhibition studies. Phylogenetic analysis showed that EstD is only distantly related to other esterases. A comparison of the active site pentapeptide motifs revealed that EstD should be grouped into a new family of esterases (Family 10). EstD is the first characterized member of this family. PMID:17466017

  19. Production and purification of a solvent-resistant esterase from Bacillus licheniformis S-86.

    PubMed

    Torres, Sebastián; Baigorí, Mario D; Pandey, Ashok; Castro, Guillermo R

    2008-12-01

    New thermophilic and organic-solvent-tolerant Bacillus licheniformis S-86 strain is able to produce two active and solvent-stable esterases. Production of type I and II esterases was substantially enhanced when oils and surfactants were supplied as carbon sources. Grape oil (0.1% v/v) and Tween 20 to 60 (0.1% v/v) had enhanced enzyme production between 1.6- and 2.2-folds. Type II esterase was purified to homogeneity in a five-step procedure. This esterase was purified 76.7-fold with a specific activity of 135 U mg(-1). Molecular mass of the enzyme was estimated to be 38.4 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Type II esterase was active mostly on esters with short acyl chains, which allowed to classify the enzyme as a carboxylesterase with a K (m) of 80.2 mmol l(-1) and a V (max) of 256.4 micromol min(-1) mg(-1) for p-nitrophenyl acetate. Also, B. licheniformis S-86 type II esterase displayed activity in presence of water-miscible organic solvents at 50% concentration and stability after 1-h incubation. PMID:18543118

  20. Recombinant sterol esterase from Ophiostoma piceae: an improved biocatalyst expressed in Pichia pastoris

    PubMed Central

    2012-01-01

    Background The ascomycete Ophiostoma piceae produces a sterol esterase (OPE) with high affinity towards p-nitrophenol, glycerol and sterol esters. Its hydrolytic activity on natural mixtures of triglycerides and sterol esters has been proposed for pitch biocontrol in paper industry since these compounds produce important economic losses during paper pulp manufacture. Results Recently, this enzyme has been heterologously expressed in the methylotrophic yeast Pichia pastoris, and the hydrolytic activity of the recombinant protein (OPE*) studied. After the initial screening of different clones expressing the enzyme, only one was selected for showing the highest production rate. Different culture conditions were tested to improve the expression of the recombinant enzyme. Complex media were better than minimal media for production, but in any case the levels of enzymatic activity were higher (7-fold in the best case) than those obtained from O. piceae. The purified enzyme had a molecular mass of 76 kDa, higher than that reported for the native enzyme under SDS-PAGE (60 kDa). Steady-state kinetic characterization of the recombinant protein showed improved catalytic efficiency for this enzyme as compared to the native one, for all the assayed substrates (p-nitrophenol, glycerol, and cholesterol esters). Different causes for this were studied, as the increased glycosylation degree of the recombinant enzyme, their secondary structures or the oxidation of methionine residues. However, none of these could explain the improvements found in the recombinant protein. N-terminal sequencing of OPE* showed that two populations of this enzyme were expressed, having either 6 or 8 amino acid residues more than the native one. This fact affected the aggregation behaviour of the recombinant protein, as was corroborated by analytical ultracentrifugation, thus improving the catalytic efficiency of this enzyme. Conclusion P. pastoris resulted to be an optimum biofactory for the

  1. Isolation and characterization of an enzyme with esterase activity from Micropolyspora faeni.

    PubMed Central

    Bannerman, E N; Nicolet, J

    1976-01-01

    The isolation and the characterization of one of the enzymes of Micropolyspora faeni that hydrolyzes the substrate N-benzoyl-DL-phenylalanine-beta-naphthyl ester and that seems to be of medical importance are described. This enzyme (enzyme 1) was isolated with an 86-fold purification by using the following seven steps: ammonium sulfate precipitation, gel filtration through Sephadex G-150, heat treatment, chromatography on diethylaminoethyl-cellulose, rechromatography on diethylaminoethyl-Sephadex, gel filtration through Sephadex G-200, and affinity chromatography. Enzyme 1 has a molecular weight of approximately 500,000 and maximum activity at pH 7.8 to 8.0 and at 20 degrees C. The enzyme is stable between pH 7.5 and 10.5 and at temperatures up to 60 degrees C. Its activity is not inhibited by ethylenediaminetetraacetic acid. It is, however, sensitive to diisopropyl phosphofluoride and phenylmethyl sulfonyl fluoride. These properties and the ability to hydrolyze the esters of phenylalanine, tyrosine, and tryptophan without endopeptidasic activity and no marked proteolytic activity suggest that the enzyme is an esterase. Images PMID:9899

  2. Potential human cholesterol esterase inhibitor design: benefits from the molecular dynamics simulations and pharmacophore modeling studies.

    PubMed

    John, Shalini; Thangapandian, Sundarapandian; Lee, Keun Woo

    2012-01-01

    Human pancreatic cholesterol esterase (hCEase) is one of the lipases found to involve in the digestion of large and broad spectrum of substrates including triglycerides, phospholipids, cholesteryl esters, etc. The presence of bile salts is found to be very important for the activation of hCEase. Molecular dynamic simulations were performed for the apoform and bile salt complexed form of hCEase using the co-ordinates of two bile salts from bovine CEase. The stability of the systems throughout the simulation time was checked and two representative structures from the highly populated regions were selected using cluster analysis. These two representative structures were used in pharmacophore model generation. The generated pharmacophore models were validated and used in database screening. The screened hits were refined for their drug-like properties based on Lipinski's rule of five and ADMET properties. The drug-like compounds were further refined by molecular docking simulation using GOLD program based on the GOLD fitness score, mode of binding, and molecular interactions with the active site amino acids. Finally, three hits of novel scaffolds were selected as potential leads to be used in novel and potent hCEase inhibitor design. The stability of binding modes and molecular interactions of these final hits were re-assured by molecular dynamics simulations. PMID:22292952

  3. A Lactobacillus plantarum Esterase Active on a Broad Range of Phenolic Esters

    PubMed Central

    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

  4. Nature versus nurture in two highly enantioselective esterases from Bacillus cereus and Thermoanaerobacter tengcongensis.

    PubMed

    Grosse, Stephan; Bergeron, Hélène; Imura, Akihiro; Boyd, Jason; Wang, Shaozhao; Kubota, Kazuo; Miyadera, Akihiko; Sulea, Traian; Lau, Peter C K

    2010-01-01

    There is an increasing need for the use of biocatalysis to obtain enantiopure compounds as chiral building blocks for drug synthesis such as antibiotics. The principal findings of this study are: (i) the complete sequenced genomes of Bacillus cereus ATCC 14579 and Thermoanaerobacter tengcongensis MB4 contain a hitherto undescribed enantioselective and alkaliphilic esterase (BcEST and TtEST respectively) that is specific for the production of (R)-2-benzyloxy-propionic acid ethyl ester, a key intermediate in the synthesis of levofloxacin, a potent antibiotic; and (ii) directed evolution targeted for increased thermostability of BcEST produced two improved variants, but in either case the 3-5 °C increase in the apparent melting temperature (T(m)) of the mutants over the native BcEST that has a T(m) of 50 °C was outperformed by TtEST, a naturally occurring homologue with a T(m) of 65 °C. Protein modelling of BcEST mapped the S148C and K272R mutations at protein surface and the I88T and Q110L mutations at more buried locations. This work expands the repertoire of characterized members of the α/β-fold hydrolase superfamily. Further, it shows that genome mining is an economical option for new biocatalyst discovery and we provide a rare example of a naturally occurring thermostable biocatalyst that outperforms experimentally evolved homologues that carry out the same hydrolysis. PMID:21255307

  5. A Lactobacillus plantarum esterase active on a broad range of phenolic esters.

    PubMed

    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.

  6. Functional Characterization of a Novel Dactylosporangium Esterase and Its Utilization in the Asymmetric Synthesis of (R)-Methyl Mandelate.

    PubMed

    Deng, Dun; Zhang, Yun; Sun, Aijun; Hu, Yunfeng

    2016-09-01

    One novel esterase DAEst6 was identified from the genome of Dactylosporangium aurantiacum subsp. Hamdenensis NRRL 18085. DAEst6 was further characterized to be an esterase which exhibited high resistance to high pH values. Esterase DAEst6 could resolve racemic methyl mandelate and generate (R)-methyl mandelate, one key drug intermediate, with an enantiomeric excess and a conversion of 99 and 49 %, respectively, after process optimization. The optimal working condition for the preparation of (R)-methyl mandelate through DAEst6 was found to be 10-mM racemic methyl mandelate, no organic co-solvents, pH 7.5, and 40 °C, for 5 h. Our work was the first report about the functional characterization of one novel Dactylosporangium esterase and the utilization of one Dactylosporangium esterase in kinetic resolution. Dactylosporangium esterases represented by DAEst6 possess great potential in the generation of valuable chiral drug intermediates and chemicals.

  7. Structural analysis of thermostabilizing mutations of cocaine esterase

    SciTech Connect

    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.

    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.

  8. Inhibition of monocyte esterase activity by organophosphate insecticides.

    PubMed

    Lee, M J; Waters, H C

    1977-11-01

    Organophosphate insecticides, such as Vapona, Naled, and Rabon, are highly potent inhibitors of an enzyme found in human monocytes. The enzyme, a specific monocyte esterase, could be inhibited by Vapona in blood samples via airborne contamination at levels easily achieved from commercial slow-release insecticide strips. Fifty percent inhibition (I50)--as measured on the Hemalog D (Technicon Corp.)--occurred at solution concentrations of 0.22, 1.5, and 2.6 X 10(-6) g/liter for Vapona, Rabon, and Naled, respectively. Parathion (a thiophosphate) and Baygon (a carbamate) were less potent, with I50 values of 3.7 X 10(-5) and 1.5 X 10(-4) g/liter, respectively. Dursban (another thiophosphate) and Carbaryl (a carbamate) showed only marginal inhibition. Eserine, malathion, nicotine and pyrethrum had no inhibitory effect up to 0.5 g/liter. The occurrence of this effect in vivo has not yet been shown, nor is it clear what the implications of such an effect would be. The inhibition of this enzyme by airborne contaminants, however, may interfere with the proper functioning of the Hemalog D. PMID:907842

  9. Lipases and esterases from extremophiles: overview and case example of the production and purification of an esterase from Thermus thermophilus HB27.

    PubMed

    Fuciños, Pablo; González, Roberto; Atanes, Estrella; Sestelo, Ana Belén Fernández; Pérez-Guerra, Nelson; Pastrana, Lorenzo; Rúa, María Luisa

    2012-01-01

    Extremophiles are organisms that have evolved to exist in a variety of extreme environments. They fall into a number of different classes that include thermophiles, halophiles, acidophiles, alkalophiles, psychrophiles, and barophiles (piezophiles). Extremophiles have the potential to produce uniquely valuable biocatalysts that function under conditions in which usually the enzymes of their nonextremophilic counterparts could not. Among novel enzymes isolated from extremophilic microorganisms, hydrolases, and particularly lipases and esterases are experiencing a growing demand. Lipases (EC 3.1.1.3) and esterases (EC 3.1.1.1) catalyze the cleavage of ester bounds in aqueous media and the reverse reaction in organic solvents. Both lipolytic enzymes have relevant applications in food, dairy, detergent, biofuel, and pharmaceutical industries. Here, we summarize the properties of lipases and esterases from the main extremophile groups: thermophiles and hyperthermophiles, psychrophiles, halophiles, alkalophiles/acidophiles, and solvent-resistant microorganisms.We report the biomass and lipolytic activity production by Thermus thermophilus HB27 in 5-L stirred-tank bioreactor at 70°C. Suitability of thermal spring water for culture media formulation is shown. In addition, a protocol to isolate and purify a cell-bound esterase from this microorganism is described.

  10. Lipases and esterases from extremophiles: overview and case example of the production and purification of an esterase from Thermus thermophilus HB27.

    PubMed

    Fuciños, Pablo; González, Roberto; Atanes, Estrella; Sestelo, Ana Belén Fernández; Pérez-Guerra, Nelson; Pastrana, Lorenzo; Rúa, María Luisa

    2012-01-01

    Extremophiles are organisms that have evolved to exist in a variety of extreme environments. They fall into a number of different classes that include thermophiles, halophiles, acidophiles, alkalophiles, psychrophiles, and barophiles (piezophiles). Extremophiles have the potential to produce uniquely valuable biocatalysts that function under conditions in which usually the enzymes of their nonextremophilic counterparts could not. Among novel enzymes isolated from extremophilic microorganisms, hydrolases, and particularly lipases and esterases are experiencing a growing demand. Lipases (EC 3.1.1.3) and esterases (EC 3.1.1.1) catalyze the cleavage of ester bounds in aqueous media and the reverse reaction in organic solvents. Both lipolytic enzymes have relevant applications in food, dairy, detergent, biofuel, and pharmaceutical industries. Here, we summarize the properties of lipases and esterases from the main extremophile groups: thermophiles and hyperthermophiles, psychrophiles, halophiles, alkalophiles/acidophiles, and solvent-resistant microorganisms.We report the biomass and lipolytic activity production by Thermus thermophilus HB27 in 5-L stirred-tank bioreactor at 70°C. Suitability of thermal spring water for culture media formulation is shown. In addition, a protocol to isolate and purify a cell-bound esterase from this microorganism is described. PMID:22426723

  11. Oral immunization of a live attenuated Escherichia coli strain expressing a holotoxin-structured adhesin-toxoid fusion (1FaeG-FedF-LTA₂:5LTB) protected young pigs against enterotoxigenic E. coli (ETEC) infection.

    PubMed

    Ruan, Xiaosai; Zhang, Weiping

    2013-03-01

    ETEC strains expressing K88 (F4) or F18 fimbriae and enterotoxins are the predominant cause of porcine post-weaning diarrhea (PWD). PWD continues causing significant economic losses to swine producers worldwide. Vaccines effectively protecting against PWD are needed. Our recent study revealed that a tripartite adhesin-toxin monomer (FaeG-FedF-LT(A2-B)) elicited protective antibodies. In this study, we constructed a new adhesin-toxoid fusion, expressed it as a 1A:5B holotoxin-structured antigen (1FaeG-FedF-LT(192A2):5LT(B)) in an avirulent Escherichia coli strain, and evaluated its vaccine potential in pig challenge studies. Piglets orally inoculated with this live strain showed no adverse effects but developed systemic and mucosal antibodies that neutralized cholera toxin and inhibited adherence of K88 and F18 fimbriae in vitro. Moreover, the immunized piglets, when were challenged with ETEC strain 3030-2 (K88ac/LT/STb), had significant fewer bacteria colonized at small intestines and did not develop diarrhea; whereas the control piglets developed severe diarrhea and died. These results indicated the 1FaeG-FedF-LT(192A2):5LT(B) fusion antigen induced protective antiadhesin and antitoxin immunity in pigs, and suggested a live attenuated vaccine can be potentially developed against porcine ETEC diarrhea. Additionally, presenting antigens in a holotoxin structure to target host local mucosal immunity can be used in vaccine development against other enteric diseases. PMID:23375979

  12. Recombinant human C1 esterase inhibitor in the management of hereditary angioedema.

    PubMed

    Riedl, Marc

    2015-07-01

    Hereditary angioedema (HAE), a rare autosomal dominant genetic disorder, is caused by a deficiency in functional C1 esterase inhibitor (C1-INH). This potentially life-threatening condition manifests as recurrent attacks of subcutaneous and submucosal swelling of the skin, gastrointestinal tract and larynx. The management of HAE includes treatment of acute episodes, short-term prophylaxis in preparation for exposure to known triggers and long-term prophylaxis to decrease the incidence and severity of HAE attacks. Four products are approved in the USA for the treatment of acute attacks of HAE, including one human plasma-derived C1-INH therapy, a recombinant human C1-INH product (rhC1-INH), a plasma kallikrein inhibitor and a bradykinin B2 receptor antagonist. In addition, one human plasma-derived C1-INH therapy and danazol are approved for prophylaxis of HAE attacks. rhC1-INH, extracted from the milk of transgenic rabbits, is a glycoprotein of 478 amino acids with an identical amino acid sequence to the endogenous human C1-INH protein. Population pharmacokinetic analysis of rhC1-INH supports an intravenous dosing strategy of 50 U/kg (maximum 4200 U). The safety and efficacy of rhC1-INH in the treatment of acute attacks in patients with HAE were demonstrated in three randomized, double-blind, placebo-controlled studies and two open-label extension studies. In a pilot prophylaxis study, weekly administration of rhC1-INH 50 U/kg for 8 weeks reduced the incidence of HAE attacks and was well tolerated. Administration of rhC1-INH has not been associated with the development of anti-drug antibodies or antibodies to anti-host-related impurities.

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

    PubMed

    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.

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

    PubMed

    Alex, Deepthy; Shainu, Anju; Pandey, Ashok; Sukumaran, Rajeev K

    2014-01-01

    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 K m and V max of 0.01 and 1.12, respectively. The purified esterase lost its thermo and halo tolerance but interestingly, retained 97% activity in methanol. PMID:24800063

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

    PubMed Central

    Shainu, Anju; Pandey, Ashok; Sukumaran, Rajeev K.

    2014-01-01

    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. PMID:24800063

  16. The classification of esterases: an important gene family involved in insecticide resistance--a review.

    PubMed

    Montella, Isabela Reis; Schama, Renata; Valle, Denise

    2012-06-01

    The use of chemical insecticides continues to play a major role in the control of disease vector populations, which is leading to the global dissemination of insecticide resistance. A greater capacity to detoxify insecticides, due to an increase in the expression or activity of three major enzyme families, also known as metabolic resistance, is one major resistance mechanisms. The esterase family of enzymes hydrolyse ester bonds, which are present in a wide range of insecticides; therefore, these enzymes may be involved in resistance to the main chemicals employed in control programs. Historically, insecticide resistance has driven research on insect esterases and schemes for their classification. Currently, several different nomenclatures are used to describe the esterases of distinct species and a universal standard classification does not exist. The esterase gene family appears to be rapidly evolving and each insect species has a unique complement of detoxification genes with only a few orthologues across species. The examples listed in this review cover different aspects of their biochemical nature. However, they do not appear to contribute to reliably distinguish among the different resistance mechanisms. Presently, the phylogenetic criterion appears to be the best one for esterase classification. Joint genomic, biochemical and microarray studies will help unravel the classification of this complex gene family.

  17. Insecticidal properties of genetically engineered baculoviruses expressing an insect juvenile hormone esterase gene.

    PubMed Central

    Eldridge, R; O'Reilly, D R; Hammock, B D; Miller, L K

    1992-01-01

    Exploring the possibility of enhancing the properties of baculoviruses as biological control agents of insect pests, we tested the effect of expressing an insect gene (jhe) encoding juvenile hormone esterase. Juvenile hormone esterase inactivates juvenile hormone, which regulates the outcome of an insect molt. A cDNA encoding the juvenile hormone esterase of Heliothis virescens was inserted into the genome of Autographa californica nuclear polyhedrosis virus such that the gene was expressed under the control of a strong, modified viral promoter. This virus, however, naturally encodes an ecdysteroid UDP-glucosyltransferase which inactivates ecdysone, the hormone which initiates molting. Since ecdysteroid UDP-glucosyltransferase could mask the effects of jhe expression by blocking molting entirely, jhe-expressing viruses in which the ecdysteroid UDP-glucosyltransferase gene was deleted or disrupted were constructed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins from infected cells revealed several intracellular proteins and two major secreted proteins which reacted with antibodies to authentic juvenile hormone esterase. Western blot analysis coupled with tunicamycin treatment indicated that differential glycosylation was responsible for the multiple products. Hemolymph of recombinant virus-infected fourth-instar Trichoplusia ni larvae contained levels of juvenile hormone esterase activity 40-fold higher than maximal levels found in uninfected larvae. However, little or no difference in developmental characteristics, weight gain, or time of mortality was observed between insects infected with the jhe-expressing viruses and control viruses. Images PMID:1622228

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

    PubMed

    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. PMID:26369647

  19. Cloning, expression, and biochemical characterization of a cold-active GDSL-esterase of a Pseudomonas sp. S9 isolated from Spitsbergen island soil.

    PubMed

    Wicka, Monika; Wanarska, Marta; Krajewska, Ewelina; Pawlak-Szukalska, Anna; Kur, Józef; Cieśliński, Hubert

    2016-01-01

    An estS9 gene, encoding an esterase of the psychrotolerant bacterium Pseudomonas sp. S9 was cloned and sequenced. The deduced sequence revealed a protein of 636 amino acid residues with a molecular mass of 69 kDa. Further amino acid sequence analysis revealed that the EstS9 enzyme contained a G-D-S-L motif centered at a catalytic serine, an N-terminal catalytic domain and a C-terminal autotransporter domain. Two recombinant E. coli strains for production of EstS9N (a two domain enzyme) and EstS9Δ (a one domain enzyme) proteins were constructed, respectively. Both recombinant proteins were successfully produced as inclusion bodies and then purified under denaturing conditions. However, because of the low enzymatic activity of the refolded EstS9Δ protein, only the EstS9N protein was further characterized. The purified and refolded EstS9N protein was active towards short-chain p-nitrophenyl esters (C2-C8), with optimal activity for the butyrate (C4) ester. With p-nitrophenyl butyrate as the substrate, the enzyme displayed optimal activity at 35°C and pH 9.0. Additionally, the EstS9N esterase retained ~90% of its activity from 25-40°C and ~40% of its activity at 10°C. Moreover, analysis of its kinetic parameters (Km, kcat, kcat/Km) toward p-nitrophenyl butyrate determined at 15°C and 25°C confirmed that the EstS9 enzyme is cold-adapted. To the best of our knowledge, EstS9 is the third characterized cold-active GDSL-esterase and the first one confirmed to contain an autotransporter domain characteristic for enzymes secreted by the type V secretion system.

  20. VvMJE1 of the grapevine (Vitis vinifera) VvMES methylesterase family encodes for methyl jasmonate esterase and has a role in stress response.

    PubMed

    Zhao, Nan; Lin, Hong; Lan, Suque; Jia, Qidong; Chen, Xinlu; Guo, Hong; Chen, Feng

    2016-05-01

    The known members of plant methyl esterase (MES) family catalyze the hydrolysis of a C-O ester linkage of methyl esters of several phytohormones including indole-3-acetic acid, salicylic acid and jasmonic acid. The genome of grapevine (Vitis vinifera) was found to contain 15 MES genes, designated VvMES1-15. In this report, VvMES5 was selected for molecular, biochemical and structural studies. VvMES5 is most similar to tomato methyl jasmonate esterase. E. coli-expressed recombinant VvMES5 displayed methyl jasmonate (MeJA) esterase activity, it was renamed VvMJE1. Under steady-state conditions, VvMJE1 exhibited an apparent Km value of 92.9 μM with MeJA. VvMJE1 was also shown to have lower activity with methyl salicylate (MeSA), another known substrate of the MES family, and only at high concentrations of the substrate. To understand the structural basis of VvMJE1 in discriminating MeJA and MeSA, a homolog model of VvMJE1 was made using the X-ray structure of tobacco SABP2, which encodes for methyl salicylate esterase, as a template. Interestingly, two bulky residues at the binding site and near the surface of tobacco SABP2 are replaced by relatively small residues in VvMJE1. Such a change enables the accommodation of a larger substrate MeJA in VvMJE1. The expression of VvMJE1 was compared in control grape plants and grape plants treated with one of the three stresses: heat, cold and UV-B. While the expression of VvMJE1 was not affected by heat treatment, its expression was significantly up-regulated by cold treatment and UV-B treatment. This result suggests that VvMJE1 has a role in response of grape plants to these two abiotic stresses. PMID:26934101

  1. Structural Characterization and Reversal of the Natural Organophosphate Resistance of a D-Type Esterase, Saccharomyces cerevisiae S-Formylglutathione Hydrolase

    SciTech Connect

    Legler,P.; Kumaran, D.; Swaminathan, S.; Studier, F.; Millard, C.

    2008-01-01

    Saccharomyces cerevisiae expresses a 67.8 kDa homodimeric serine thioesterase, S-formylglutathione hydrolase (SFGH), that is 39.9% identical with human esterase D. Both enzymes possess significant carboxylesterase and S-formylglutathione thioesterase activity but are unusually resistant to organophosphate (OP) inhibitors. We determined the X-ray crystal structure of yeast (y) SFGH to 2.3 Angstroms resolution by multiwavelength anomalous dispersion and used the structure to guide site-specific mutagenesis experiments addressing substrate and inhibitor reactivity. Our results demonstrate a steric mechanism of OP resistance mediated by a single indole ring (W197) located in an enzyme 'acyl pocket'. The W197I substitution enhances ySFGH reactivity with paraoxon by >1000-fold (kiW197I = 16 {+-} 2 mM-1 h-1), thereby overcoming natural OP resistance. W197I increases the rate of OP inhibition under pseudo-first-order conditions but does not accelerate OP hydrolysis. The structure of the paraoxon-inhibited W197I variant was determined by molecular replacement (2.2 Angstroms); it revealed a stabilized sulfenic acid at Cys60. Wild-type (WT) ySFGH is inhibited by thiol reactive compounds and is sensitive to oxidation; thus, the cysteine sulfenic acid may play a role in the regulation of a 'D-type' esterase. The structure of the W197I variant is the first reported cysteine sulfenic acid in a serine esterase. We constructed five Cys60/W197I variants and show that introducing a positive charge near the oxyanion hole, W197I/C60R or W197I/C60K, results in a further enhancement of the rates of phosphorylation with paraoxon (ki = 42 or 80 mM-1 h-1, respectively) but does not affect the dephosphorylation of the enzyme. We also characterized three histidine substitutions near the oxyanion hole, G57H, L58H, and M162H, which significantly decrease esterase activity.

  2. ANTINOCICEPTIVE EFFECTS OF THE N-ACYLETHANOLAMINE ACID AMIDASE INHIBITOR ARN077 IN RODENT PAIN MODELS

    PubMed Central

    Sasso, Oscar; Moreno-Sanz, Guillermo; Martucci, Cataldo; Realini, Natalia; Dionisi, Mauro; Mengatto, Luisa; Duranti, Andrea; Tarozzo, Glauco; Tarzia, Giorgio; Mor, Marco; Bertorelli, Rosalia; Reggiani, Angelo; Piomelli, Daniele

    2013-01-01

    Fatty acid ethanolamides (FAEs), which include palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), are endogenous agonists of peroxisome proliferator-activated receptor-α (PPAR-α) and important regulators of the inflammatory response. They are degraded in macrophages by the lysosomal cysteine amidase, N-acylethanolamine acid amidase (NAAA). Previous studies have shown that pharmacological inhibition of NAAA activity suppresses macrophage activation in vitro and causes marked anti-inflammatory effects in vivo, which is suggestive of a role for NAAA in the control of inflammation. It is still unknown, however, whether NAAA-mediated FAE deactivation might regulate pain signaling. In the present study, we examined the effects of ARN077, a potent and selective NAAA inhibitor recently disclosed by our group, in rodent models of hyperalgesia and allodynia caused by inflammation or nerve damage. Topical administration of ARN077 attenuated, in a dose-dependent manner, heat hyperalgesia and mechanical allodynia elicited in mice by carrageenan injection or sciatic nerve ligation. The anti-nociceptive effects of ARN077 were prevented by the selective PPAR-α antagonist GW6471 and did not occur in PPAR-α-deficient mice. Furthermore, topical ARN077 reversed the allodynia caused by ultraviolet B-radiation in rats, and this effect was blocked by pretreatment with GW6471. Sciatic nerve ligation or application of the pro-inflammatory phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) decreased FAE levels in sciatic nerve and skin tissue, respectively. ARN077 reversed these biochemical effects. The results identify ARN077 as a potent inhibitor of intracellular NAAA activity, which is active in vivo by topical administration. The findings further suggest that NAAA regulates peripheral pain initiation by interrupting endogenous FAE signaling at PPAR-α. PMID:23218523

  3. Cellular function of neuropathy target esterase in lysophosphatidylcholine action

    SciTech Connect

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

    2008-11-01

    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.

  4. Improved production of Pseudomonas sp. ECU1011 acetyl esterase by medium design and fed-batch fermentation.

    PubMed

    Ju, Xin; Yu, Hui-Lei; Pan, Jiang; Xu, Jian-He

    2012-03-01

    We optimized culture medium and batch-fed fermentation conditions to enhance production of an acetyl esterase from Pseudomonas sp. ECU1011 (PSAE). This enzyme enantioselectively deacetylates α-acetoxyphenylacetic acid. The medium was redesigned by single-factor and statistical optimization. The addition of ZnSO(4) enhanced enzyme production by 37%. Yeast extract concentration was directly associated with the enzyme production. The fermentation was scaled up in a 5-l fermenter with the optimized medium, and the correlations between enzyme production and dissolved oxygen, pH, and feeding strategy were investigated. The fermentation process was highly oxygen-demanding, pH sensitive and mandelic acid-inducible. The fermentation pH was controlled at 7.5 by a pH and dissolved oxygen feedback strategy. Feeding mandelic acid as both a pH regulator and an enzyme inducer increased the enzyme production by 23%. The results of the medium redesign experiments were confirmed and explained in fed-batch culture experiments. Mathematical models describing the fermentation processes indicated that the enzyme production was strongly associated with cell growth. The optimized pH and dissolved oxygen stat fed-batch process resulted high volumetric production of PSAE (4166 U/l, 7.2-fold higher than the initial) without enantioselectivity decline. This process has potential applications for industrial production of chiral mandelic acid or its derivatives. PMID:21792565

  5. Molecular cloning and characterization of a new cold-active esterase from a deep-sea metagenomic library.

    PubMed

    Fu, Chengzhang; Hu, Yongfei; Xie, Feng; Guo, Hui; Ashforth, Elizabeth Jane; Polyak, Steven W; Zhu, Baoli; Zhang, Lixin

    2011-05-01

    A clone which conferred lipolytic activity at low temperature was identified from a fosmid library constructed from a South China Sea marine sediment sample. The gene responsible, estF, consisted of 1,080 bp that encoded 359 amino acid residues, with a typical N-terminal signal peptide of 28 amino acid residues. A phylogenetic analysis of amino acid sequence with other lipolytic enzymes revealed that EstF and seven closely related putative lipolytic enzymes comprised a unique clade in the phylogenetic tree. Moreover, these hypothetic esterases showed unique conservative sites in the amino acid sequence. The recombinant EstF was overexpressed and purified, and its biochemical properties were partially characterized. The optimal substrate for EstF to hydrolyze among a panel of p-nitrophenyl esters (C2 to C16) was p-nitrophenyl butyrate (C4), with a K(m) of 0.46 mM. Activity quickly decreased with substrates containing an acyl chain length longer than 10 carbons. We found that EstF was active in the temperature range of 0-60°C, showed the best activity at 50°C, but was unstable at 60°C. It exhibited a high level of activity in the pH range of 7.0-10.0 showing the highest activity at pH 9.0.

  6. Plant Esterase-Chitosan/Gold Nanoparticles-Graphene Nanosheet Composite-Based Biosensor for the Ultrasensitive Detection of Organophosphate Pesticides.

    PubMed

    Bao, Jing; Hou, Changjun; Chen, Mei; Li, Junjie; Huo, Danqun; Yang, Mei; Luo, Xiaogang; Lei, Yu

    2015-12-01

    As broad-spectrum pesticides, organophosphates (OPs) are widely used in agriculture all over the world. However, due to their neurotoxicity in humans and their increasing occurrence in the environment, there is growing interest in their sensitive and selective detection. This paper reports a new cost-effective plant esterase-chitosan/gold nanoparticles-graphene nanosheet (PLaE-CS/AuNPs-GNs) biosensor for the sensitive detection of methyl parathion and malathion. Highly pure plant esterase is produced from plants at low cost and shares the same inhibition mechanism with OPs as acetylcholinesterase, and then it was used to prepare PLaE-CS/AuNPs-GNs nanocomposites, which were systematically characterized using SEM, TEM, and UV-vis. The PLaE-CS/AuNPs-GNs composite-based biosensor measured as low as 50 ppt (0.19 nM) of methyl parathion and 0.5 ppb (1.51 nM) of malathion (S/N = 3) with a calibration curve up to 200 ppb (760 nM) and 500 ppb (1513.5 nM) for methyl parathion and malathion, respectively. There is also no interference observed from most of common species such as metal ions, inorganic ions, glucose, and citric acid. In addition, its applicability to OPs-contaminated real samples (carrot and apple) was also demonstrated with excellent response recovery. The developed simple, sensitive, and reliable PLaE-CS/AuNPs-GNs composite-based biosensor holds great potential in OPs detection for food and environmental safety.

  7. Plant Esterase-Chitosan/Gold Nanoparticles-Graphene Nanosheet Composite-Based Biosensor for the Ultrasensitive Detection of Organophosphate Pesticides.

    PubMed

    Bao, Jing; Hou, Changjun; Chen, Mei; Li, Junjie; Huo, Danqun; Yang, Mei; Luo, Xiaogang; Lei, Yu

    2015-12-01

    As broad-spectrum pesticides, organophosphates (OPs) are widely used in agriculture all over the world. However, due to their neurotoxicity in humans and their increasing occurrence in the environment, there is growing interest in their sensitive and selective detection. This paper reports a new cost-effective plant esterase-chitosan/gold nanoparticles-graphene nanosheet (PLaE-CS/AuNPs-GNs) biosensor for the sensitive detection of methyl parathion and malathion. Highly pure plant esterase is produced from plants at low cost and shares the same inhibition mechanism with OPs as acetylcholinesterase, and then it was used to prepare PLaE-CS/AuNPs-GNs nanocomposites, which were systematically characterized using SEM, TEM, and UV-vis. The PLaE-CS/AuNPs-GNs composite-based biosensor measured as low as 50 ppt (0.19 nM) of methyl parathion and 0.5 ppb (1.51 nM) of malathion (S/N = 3) with a calibration curve up to 200 ppb (760 nM) and 500 ppb (1513.5 nM) for methyl parathion and malathion, respectively. There is also no interference observed from most of common species such as metal ions, inorganic ions, glucose, and citric acid. In addition, its applicability to OPs-contaminated real samples (carrot and apple) was also demonstrated with excellent response recovery. The developed simple, sensitive, and reliable PLaE-CS/AuNPs-GNs composite-based biosensor holds great potential in OPs detection for food and environmental safety. PMID:26554573

  8. Immunoelectron microscopic demonstration of an esterase on the outer membrane of Xanthomonas maltophilia.

    PubMed Central

    Debette, J; Prensier, G

    1989-01-01

    Xanthomonas maltophilia (later synonym of Pseudomonas maltophilia), an ubiquitous species, is known to show proteolytic and lipolytic activities. A cell-bound esterase which hydrolyzes beta-naphthyl acetate during growth has been extracted from a strain isolated from soil. Because of its strongly hydrophobic character, the enzyme could be efficiently solubilized only by Triton X-100. This nonionic detergent must be added in polyacrylamide gels to permit migration. Polyclonal rabbit antibodies raised against the Triton-soluble esterase complex were used to localize the enzyme at the ultrastructural level. Electron microscopy of cell sections of this organism and immunogold labeling demonstrated that the enzyme was located on the outer membrane. Such an envelope-bound esterase may produce assimilable substrates for X. maltophilia which can grow in various environments. Images PMID:2495761

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

    NASA Astrophysics Data System (ADS)

    Wu, Jindan; Cai, Guoqiang; Liu, Jinqiang; Ge, Huayun; Wang, Jiping

    2014-03-01

    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.

  10. Isoenzyme status and genetic variability of serum esterases in the lesser snow goose, Anser caerulescens caerulescens.

    PubMed

    Bargiello, T A; Grossfield, J; Steele, R W; Cooke, F

    1977-08-01

    A maximum of 22 bands comprising four esterase subgroups--acetylesterase, carboxylesterase, cholinesterase, and acetylcholinesterase--were detected following electrophoresis of lesser snow goose sera on polyacrylamide gels. A minimum of seven structural genes was surmised to be involved in the biosynthesis of these enzymes following physiochemical characterizations. The genetic variability of these loci was calculated to be 1.25% average heterozygosity, while 14.3% of the loci were polymorphic. These estimates of genetic variability were substantially lower than those reported for other vertebrate species. The low degree of genetic variability found in snow goose serum esterases coupled with the extensive protein multiplicity observed may possibly reflect an adaptive strategy based on "biochemical plasticity" rather than genic heterozygosity for this species. The nature of evolutionary forces acting upon multiple enzyme systems such as esterases is discussed. The concept of "conditional neutrality" is introduced and defined within this context. PMID:921742

  11. Extraction and purification of wheat-esterase using aqueous two-phase systems of ionic liquid and salt.

    PubMed

    Jiang, Bin; Feng, Zhibiao; Liu, Chunhong; Xu, Yingcao; Li, Dongmei; Ji, Guo

    2015-05-01

    To explore a new and simple rapid extraction and purification technique for wheat-esterase, an ionic liquids (ILs)-based aqueous two-phase system (ATPS) was developed for the purification of wheat-esterase from wheat extracts. Effects of various process parameters such as the concentrations of [Bmim]BF4, the types and concentrations of phase-forming salt, the system pH and the temperature on partitioning of wheat-esterase were evaluated. The obtained data indicated that wheat-esterase was preferentially partitioned into the ILs-rich phase and the ATPS composed of 20 % [Bmim]BF4 (w/w) and 25 % (w/w) NaH2PO4(pH = 4.8) showed good selectivity on wheat-esterase. Under the optimum conditions, wheat-esterase was purified with an acceptable yield (88.93 %), but produced wheat-esterase was 4.23 times as pure. It was obvious that temperature shows little influence on the purification between 10 and 50 °C. Sephadex G-150FF revealed that the band intensity of contaminating proteins in ATPS fraction almost disappeared. Therefore, ILs-based ATPS was an effective method for partitioning and recovery of wheat-esterase from wheat crude extracts. PMID:25892786

  12. Expression of a fungal glucuronoyl esterase in Populus: effects on wood properties and saccharification efficiency.

    PubMed

    Latha Gandla, Madhavi; Derba-Maceluch, Marta; Liu, Xiaokun; Gerber, Lorenz; Master, Emma R; Mellerowicz, Ewa J; Jönsson, Leif J

    2015-04-01

    The secondary walls of angiosperms contain large amounts of glucuronoxylan that is thought to be covalently linked to lignin via ester bonds between 4-O-methyl-α-D-glucuronic acid (4-O-Me-GlcA) moieties in glucuronoxylan and alcohol groups in lignin. This linkage is proposed to be hydrolysed by glucuronoyl esterases (GCEs) secreted by wood-degrading fungi. We report effects of overexpression of a GCE from the white-rot basidiomycete Phanerochaete carnosa, PcGCE, in hybrid aspen (Populus tremula L. x tremuloides Michx.) on the wood composition and the saccharification efficiency. The recombinant enzyme, which was targeted to the plant cell wall using the signal peptide from hybrid aspen cellulase PttCel9B3, was constitutively expressed resulting in the appearance of GCE activity in protein extracts from developing wood. Diffuse reflectance FT-IR spectroscopy and pyrolysis-GC/MS analyses showed significant alternation in wood chemistry of transgenic plants including an increase in lignin content and S/G ratio, and a decrease in carbohydrate content. Sequential wood extractions confirmed a massive (+43%) increase of Klason lignin, which was accompanied by a ca. 5% decrease in cellulose, and ca. 20% decrease in wood extractives. Analysis of the monosaccharide composition using methanolysis showed a reduction of 4-O-Me-GlcA content without a change in Xyl contents in transgenic lines, suggesting that the covalent links between 4-O-Me-GlcA moieties and lignin protect these moieties from degradation. Enzymatic saccharification without pretreatment resulted in significant decreases of the yields of Gal, Glc, Xyl and Man in transgenic lines, consistent with their increased recalcitrance caused by the increased lignin content. In contrast, the enzymatic saccharification after acid pretreatment resulted in Glc yields similar to wild-type despite of their lower cellulose content. These data indicate that whereas PcGCE expression in hybrid aspen increases lignin deposition

  13. Submesoscale characteristics and transcription of a fatty acid elongase gene from a freshwater green microalgae, Myrmecia incisa Reisigl

    NASA Astrophysics Data System (ADS)

    Yu, Shuiyan; Liu, Shicheng; Li, Chunyang; Zhou, Zhigang

    2011-01-01

    Myrmecia incisa is a green coccoid freshwater microalgae, which is rich in arachidonic acid (ArA, C20: 4ω-6, δ5, 8, 11, 14), a long chain polyunsaturated fatty acid (PUFA), especially under nitrogen starvation stress. A cDNA library of M. incisa was constructed with λ phage vectors and a 545 nt expressed sequence tag (EST) was screened from this library as a putative elongase gene due to its 56% and 49% identity to Marchantia polymorpha L. and Ostreococcus tauri Courties et Chrétiennot-Dinet, respectively. Based upon this EST sequence, an elongase gene designated MiFAE was isolated from M. incisa via 5'/3' rapid amplification of cDNA ends (RACE). The cDNA sequence was 1 331 bp long and included a 33 bp 5'-untranslated region (UTR) and a 431 bp 3'-UTR with a typical poly-A tail. The 867 bp ORF encoded a predicted protein of 288 amino acids. This protein was characterized by a conserved histidine-rich box and a MYxYY motif that was present in other members of the elongase family. The genomic DNA sequence of MiFAE was found to be interrupted by three introns with splicing sites of Introns I (81 bp), II (81 bp), and III (67 bp) that conformed to the GT-AG rule. Quantitative real-time PCR showed that the transcription level of MiFAE in this microalga under nitrogen starvation was higher than that under normal condition. Prior to the ArA content accumulation, the transcription of MiFAE was enhanced, suggesting that it was possibly responsible for the ArA accumulation in this microalga cultured under nitrogen starvation conditions.

  14. Esterase-activatable β-lapachone prodrug micelles for NQO1-targeted lung cancer therapy

    PubMed Central

    Ma, Xinpeng; Huang, Xiumei; Moore, Zachary; Huang, Gang; Kilgore, Jessica A.; Wang, Yiguang; Hammer, Suntrea; Williams, Noelle S.; Boothman, David A.; Gao, Jinming

    2016-01-01

    Lung cancer is one of the most lethal forms of cancer and current chemotherapeutic strategies lack broad specificity and efficacy. Recently, β-lapachone (β-lap) was shown to be highly efficacious in killing non-small cell lung cancer (NSCLC) cells regardless of their p53, cell cycle and caspase status. Pre-clinical and clinical use of β-lap (clinical form, ARQ501 or 761) is hampered by poor pharmacokinetics and toxicity due to hemolytic anemia. Here, we report the development and preclinical evaluation of β-lap prodrug nanotherapeutics consisting of diester derivatives of β-lap encapsulated in biocompatible and biodegradable poly(ethylene glycol)-b-poly(d,l-lactic acid) (PEG-b-PLA) micelles. Compared to the parent drug, diester derivatives of β-lap showed higher drug loading densities inside PEG-b-PLA micelles. After esterase treatment, micelle-delivered β-lap-dC3 and -dC6 prodrugs were converted to β-lap. Cytotoxicity assays using A549 and H596 lung cancer cells showed that both micelle formulations maintained NAD(P)H:quinone oxidoreductase 1 (NQO1)-dependent cytotoxicity. However, antitumor efficacy study of β-lap-dC3 micelles against orthotopic A549 NSCLC xenograft-bearing mice showed significantly greater long-term survival over β-lap-dC6 micelles or β-lap-HPβCD complexes. Improved therapeutic efficacy of β-lap-dC3 micelles correlated with higher area under the concentration-time curves of β-lap in tumors, and enhanced pharmacodynamic endpoints (e.g., PARP1 hyperactivation, γH2AX, and ATP depletion). β-Lap-dC3 prodrug micelles provide a promising strategy for NQO1-targeted therapy of lung cancer with improved safety and antitumor efficacy. PMID:25542645

  15. Inhibition of polyisoprenylated methylated protein methyl esterase by synthetic musks induces cell degeneration.

    PubMed

    Ayuk-Takem, Lambert; Amissah, Felix; Aguilar, Byron J; Lamango, Nazarius S

    2014-04-01

    Synthetic fragrances are persistent environmental pollutants that tend to bioaccumulate in animal tissues. They are widely used in personal care products and cleaning agents. Worldwide production of Galaxolide and Tonalide are in excess of 4500 tons annually. Because of their widespread production and use, they have been detected in surface waters and fish in the US and Europe. Consumption of contaminated water and fish from such sources leads to bioaccumulation and eventual toxicity. Since fragrances and flavors bear structural similarities to polyisoprenes, it was of interest to determine whether toxicity by Galaxolide and Tonalide may be linked with polyisoprenylated methylated protein methyl esterase (PMPMEase) inhibition. A concentration-dependent study of PMPMEase inhibition by Galaxolide and Tonalide as well as their effects on the degeneration of cultured cells were conducted. Galaxolide and Tonalide inhibited purified porcine liver PMPMEase with Ki values of 11 and 14 μM, respectively. Galaxolide and Tonalide also induced human cancer cell degeneration with EC50 values of 26 and 98 μM (neuroblastoma SH-SY5Y cells) and 58 and 14 μM (lung cancer A549 cells), respectively. The effects on cell viability correlate well with the inhibition of PMPMEase activity in the cultured cells. Molecular docking analysis revealed that the binding interactions are most likely between the fragrance molecules and hydrophobic amino acids in the active site of the enzyme. These results appear to suggest that the reported neurotoxicity of these compounds may be associated with their inhibition of PMPMEase. Exposure to fragrances may pose a significant risk to individuals predisposed to developing degenerative disorders.

  16. SulE, a sulfonylurea herbicide de-esterification esterase from Hansschlegelia zhihuaiae S113.

    PubMed

    Hang, Bao-Jian; Hong, Qing; Xie, Xiang-Ting; Huang, Xing; Wang, Cheng-Hong; He, Jian; Li, Shun-Peng

    2012-03-01

    De-esterification is an important degradation or detoxification mechanism of sulfonylurea herbicide in microbes and plants. However, the biochemical and molecular mechanisms of sulfonylurea herbicide de-esterification are still unknown. In this study, a novel esterase gene, sulE, responsible for sulfonylurea herbicide de-esterification, was cloned from Hansschlegelia zhihuaiae S113. The gene contained an open reading frame of 1,194 bp, and a putative signal peptide at the N terminal was identified with a predicted cleavage site between Ala37 and Glu38, resulting in a 361-residue mature protein. SulE minus the signal peptide was synthesized in Escherichia coli BL21 and purified to homogeneity. SulE catalyzed the de-esterification of a variety of sulfonylurea herbicides that gave rise to the corresponding herbicidally inactive parent acid and exhibited the highest catalytic efficiency toward thifensulfuron-methyl. SulE was a dimer without the requirement of a cofactor. The activity of the enzyme was completely inhibited by Ag(+), Cd(2+), Zn(2+), methamidophos, and sodium dodecyl sulfate. A sulE-disrupted mutant strain, ΔsulE, was constructed by insertion mutation. ΔsulE lost the de-esterification ability and was more sensitive to the herbicides than the wild type of strain S113, suggesting that sulE played a vital role in the sulfonylurea herbicide resistance of the strain. The transfer of sulE into Saccharomyces cerevisiae BY4741 conferred on it the ability to de-esterify sulfonylurea herbicides and increased its resistance to the herbicides. This study has provided an excellent candidate for the mechanistic study of sulfonylurea herbicide metabolism and detoxification through de-esterification, construction of sulfonylurea herbicide-resistant transgenic crops, and bioremediation of sulfonylurea herbicide-contaminated environments.

  17. Esterases immobilized on aminosilane modified magnetic nanoparticles as a catalyst for biotransformation reactions.

    PubMed

    Alex, Deepthy; Mathew, Abraham; Sukumaran, Rajeev K

    2014-09-01

    Magnetite nanoparticles were prepared by reacting ferrous and ferric salts in presence of aqueous ammonia. The magnetic nanoparticles (MNPs) were amino functionalized by treating with 3-aminopropyl triethoxy silane (APTES) and was coupled with glutaraldehyde. A novel solvent tolerant esterase from Pseudozyma sp. NII 08165 was immobilized on the MNPs through covalent bonding to the glutaraldehyde. The magnetite nanoparticles had a size range of 10-100 nm, confirmed by DLS. Lipases immobilized on MNPs were evaluated for biotransformation reactions including synthesis of ethyl acetate and transesterification of vegetable oil for producing biodiesel. The MNP immobilized esterase had prolonged shelf life and there was no loss in enzyme activity. PMID:24968816

  18. Esterases immobilized on aminosilane modified magnetic nanoparticles as a catalyst for biotransformation reactions.

    PubMed

    Alex, Deepthy; Mathew, Abraham; Sukumaran, Rajeev K

    2014-09-01

    Magnetite nanoparticles were prepared by reacting ferrous and ferric salts in presence of aqueous ammonia. The magnetic nanoparticles (MNPs) were amino functionalized by treating with 3-aminopropyl triethoxy silane (APTES) and was coupled with glutaraldehyde. A novel solvent tolerant esterase from Pseudozyma sp. NII 08165 was immobilized on the MNPs through covalent bonding to the glutaraldehyde. The magnetite nanoparticles had a size range of 10-100 nm, confirmed by DLS. Lipases immobilized on MNPs were evaluated for biotransformation reactions including synthesis of ethyl acetate and transesterification of vegetable oil for producing biodiesel. The MNP immobilized esterase had prolonged shelf life and there was no loss in enzyme activity.

  19. [Erythropoietin-forming and esterase activity of rat kidney subcellular fractions during stimulation of erythropoiesis].

    PubMed

    Novikov, N M; Voronkov, S F; Voloshchenko, L G; Mikhaĭlova, S N

    1977-01-01

    Stimulation of erythropoiesis in rats (hemolytic-phenylhydrazine and acute posthemorrhagic anemia, effect of hypoxic hypoxia) was accompanied by an increased erythropoietine-formating activity in kidney microsomes and light mitochondria. The phenomenon correlated with an increased esterase activity in hypotonic supernatant of kidney homogenate mainly due to the enzymatic fraction, corresponding to alpha2-globulin by its mobility. Histochemical examination of kidney showed that the most distinct alterations in the esterase activity were observed in epithelial cells of nephron proximal part and in capillary endothelium.

  20. The Chlamydia trachomatis CT149 protein exhibits esterase activity in vitro and catalyzes cholesteryl ester hydrolysis when expressed in HeLa cells

    PubMed Central

    Peters, Jan; Onguri, Vijaya; Nishimoto, Satoru K.; Marion, Tony N.; Byrne, Gerald I.

    2012-01-01

    Chlamydia, like other intracellular bacteria, are auxotrophic for a variety of essential metabolites and obtain cholesterol and fatty acids from their eukaryotic host cell, however not many Chlamydia-specific enzymes have been identified that are involved in lipid metabolism. In silico analysis of one candidate C. trachomatis enzyme, annotated as a conserved putative hydrolase (CT149), identified two lipase/esterase GXSXG motifs, and a potential cholesterol recognition/interaction amino acid consensus (CRAC) sequence. His-tag purified recombinant CT149 exhibited ester hydrolysis activity in a nitrophenyl acetate-based cell-free assay system. When cholesteryl linoleate was used as substrate, ester hydrolysis occurred and production of cholesterol was detected by high performance liquid chromatography. Exogenous expression of transfected CT149 in HeLa cells resulted in a significant decrease of cytoplasmic cholesteryl esters within 48 hrs. These results demonstrate that CT149 has cholesterol esterase activity and is likely to contribute to the hydrolysis of eukaryotic cholesteryl esters during intracellular chlamydial growth. PMID:22940277

  1. A cluster of at least three esterase genes in Lucilia cuprina includes malathion carboxylesterase and two other esterases implicated in resistance to organophosphates

    SciTech Connect

    Smyth, K.A. |; Russell, R.J.; Oakeshott, J.G.

    1994-12-01

    Three distinct malathion carboxylesterase (MCE) phenotypes have been identified among strains of Lucilia cuprina. The high-activity phenotype shows 1.6- and 3.3-fold more MCE specific activity than the intermediate- and low-activity phenotypes, respectively. Flies with high MCE activity are 1000-fold more resistant to malathion than flies with either low or intermediate MCE phenotypes, which are equally susceptible. High and low MCE specific activity are allelic and encoded by the Rmal gene on chromosome 4. Rmal is clustered within one map unit of two other esterase genes, Rop1 and E9, which are implicated in resistance to other organophosphate insecticides. Intermediate MCE specific activity is also inherited within the cluster, although its allelism to Rmal, Rop1, or E9 is unclear. The cluster does not contain the gene for the hemolymph esterase E4, which maps 6.1 map units from Rop1, on the other side of the bubbled wing marker. The cluster appears to be homologous to part of a tandem array of 11 esterase genes on chromosome 3R of Drosophila melanogaster. 41 refs., 4 figs., 2 tabs.

  2. Effects of extract of soapnut Sapindus emarginatus on esterases and phosphatases of the vector mosquito, Aedes aegypti (Diptera: Culicidae).

    PubMed

    Koodalingam, Arunagirinathan; Mullainadhan, Periasamy; Arumugam, Munusamy

    2011-04-01

    Our earlier investigations with kernels from the soapnut Sapindus emarginatus revealed it as a new source of botanical biocide with potent antimosquito activity, as evident from the proven unique ability of the aqueous kernel extract to kill all the developmental stages of three important vector mosquito species, Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. This extract was also found to be safe for two non-target aquatic insects. As a sequel to these findings, we have further examined quantitative and qualitative changes in total proteins, esterases, and phosphatases in whole body homogenates of fourth instar larvae and pupae of A. aegypti exposed to this extract at an appropriate threshold time for its lethal effect to gain insights into the impact of the botanical biocide on biochemical characteristics of the target vector mosquito at two distinct developmental stages. The profiles of proteins, esterases (acetylcholinesterse, α- and β-carboxylesterases), and phosphatases (acid and alkaline) exhibited distinct patterns of variation during normal development of fourth instar larvae and pupae, indicating intrinsic difference in biochemical features between these two developmental stages of A. aegypti. Upon exposure of the larvae to the extract, significant reduction in the activities of acetylcholinesterse, β-carboxylesterase, and acid phosphatases were recorded, whereas the total proteins, α-carboxylesterase and alkaline phosphatase activities were unaffected. By contrast, only alkaline phosphatase activity was significantly affected in pupae exposed to the extract. Analysis of these enzymes in native PAGE revealed that they exist in isoforms in both the larvae and pupae. The alterations in the levels of enzymatic activities observed from the quantitative assays of various enzymes were reflected by the respective zymograms with perceptible differences in the intensity and the number of bands detected especially with β-carboxylesterase, acid

  3. Esterase detoxification of acetylcholinesterase inhibitors using human liver samples in vitro

    EPA Science Inventory

    Organophosphate (OP) and N-methylcarbamate pesticides inhibit acetylcholinesterase (AChE), but differences in metabolism and detoxification can influence potency of these pesticides across and within species. Carboxylesterase (CaE) and A-esterase (paraoxonase, PON1) are consider...

  4. Purification, crystallization and preliminary X-ray analysis of an acetylxylan esterase from Bacillus pumilus.

    PubMed

    Benini, S; Degrassi, G; Krastanova, I; Lamba, D; Venturi, V

    2001-12-01

    The gene encoding for acetylxylan esterase from Bacillus pumilus has been cloned and expressed in Escherichia coli. The recombinant protein has been purified to homogeneity and crystallized. The crystals obtained are of regular shape of dimensions 0.05 x 0.05 x 0.05 mm with R32 symmetry and diffract to 2.0 A using synchrotron radiation.

  5. Tissue-specific inhibition and recovery of esterase activities in Lumbricus terrestris experimentally exposed to chlorpyrifos.

    PubMed

    Vejares, Sandra González; Sabat, Pablo; Sanchez-Hernandez, Juan C

    2010-04-01

    Exposure and effect assessment of organophosphate (OP) pesticides generally involves the use of cholinesterase (ChE) inhibition. In earthworm, this enzyme activity is often measured in homogenates from the whole organism. Here we examine the tissue-specific response of ChE and carboxylesterase (CE) activities in Lumbricus terrestris experimentally exposed to chlorpyrifos-spiked field soils. Esterases were measured in different gut segments and in the seminal vesicles of earthworms following acute exposure (2 d) to the OP and during 35d of a recovery period. We found that inhibition of both esterase activities was dependent on the tissue. Cholinesterase activity decreased in the pharynx, crop, foregut and seminal vesicles in a concentration-dependent way, whereas CE activity (4-nitrophenyl valerate) was strongly inhibited in these tissues. Gizzard CE activity was not inhibited by the OP, even an increase of enzyme activity was evident during the recovery period. These results suggest that both esterases should be determined jointly in selected tissues of earthworms. Moreover, the high levels of gut CE activity and its inhibition and recovery dynamic following OP exposure suggest that this esterase could play an important role as an enzymatic barrier against OP uptake from the ingested contaminated soil. PMID:20045489

  6. Phylogenetic classification of Aureobasidium pullulans strains for production of feruloyl esterase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective was to phylogenetically classify diverse strains of A. pullulans and determine their production of feruloyl esterase. Seventeen strains from the A. pullulans literature were phylogenetically classified. Phenotypic traits of color variation and endo-ß-1,4-xylanase overproduction were as...

  7. Evolution and homologous recombination of the hemagglutinin-esterase gene sequences from porcine torovirus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of the present study was to gain new insights into the evolution, homologous recombination and selection pressures imposed on the porcine torovirus (PToV), by examining changes in the hemagglutinin-esterase (HE) gene. The most recent common ancestor of PToV was estimated to have emerge...

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

    PubMed Central

    2015-01-01

    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. PMID:27335975

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

    PubMed Central

    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

  10. Electrochemical biosensor for carbofuran pesticide based on esterases from Eupenicillium shearii FREI-39 endophytic fungus.

    PubMed

    Grawe, Gregory Ferreira; de Oliveira, Tássia Regina; de Andrade Narciso, Esther; Moccelini, Sally Katiuce; Terezo, Ailton José; Soares, Marcos Antonio; Castilho, Marilza

    2015-01-15

    In this work, a biosensor was constructed by physical adsorption of the isolated endophytic fungus Eupenicillium shearii FREI-39 esterase on halloysite, using graphite powder, multi-walled carbon nanotubes and mineral oil for the determination of carbofuran pesticide by inhibition of the esterase using square-wave voltammetry (SWV). Specific esterase activities were determined each 2 days over a period of 15 days of growth in four different inoculation media. The highest specific activity was found on 6th day, with 33.08 U on PDA broth. The best performance of the proposed biosensor was obtained using 0.5 U esterase activity. The carbofuran concentration response was linear in the range from 5.0 to 100.0 µg L(-1) (r=0.9986) with detection and quantification limits of 1.69 µg L(-1) and 5.13 µg L(-1), respectively. A recovery study of carbofuran in spiked water samples showed values ranging from 103.8±6.7% to 106.7±9.7%. The biosensor showed good repeatability and reproducibility and remained stable for a period of 20 weeks. The determination of carbofuran in spiked water samples using the proposed biosensor was satisfactory when compared to the chromatographic reference method. The results showed no significant difference at the 95% confidence level with t-test statistics. The application of enzymes from endophytic fungi in constructing biosensors broadens the biotechnological importance of these microorganisms.

  11. Chaperone-like activities of {alpha}-synuclein: {alpha}-Synuclein assists enzyme activities of esterases

    SciTech Connect

    Ahn, Misun; Kim, SeungBum; Kang, Mira; Ryu, Yeonwoo . E-mail: ywryu@ajou.ac.kr; Doohun Kim, T. . E-mail: doohunkim@ajou.ac.kr

    2006-08-11

    {alpha}-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 {alpha}-synuclein has not yet been known. Here we have shown that {alpha}-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 {alpha}-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 {alpha}-synuclein. Our results indicate that {alpha}-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. Absence of "A"-esterase activity in the serum of a patient with Tangier disease.

    PubMed

    Mackness, M I; Peuchant, E; Dumon, M F; Walker, C H; Clerc, M

    1989-12-01

    The levels of apolipoprotein A-I, A-II and B in subjects who are homozygous or heterozygous for Tangier disease are reported and compared with the amount of "A"-esterase in the serum. The "A"-esterases hydrolyse toxic organophosphate pesticides and are currently classified by the nomenclature committee of the International Union of Biochemistry as arylesterases (EC 3.1.1.2) although recent evidence has cast doubt on this classification. The apolipoprotein data are consistent with previous data reported for a number of Tangier patients. The homozygote has a marked reduction in apo A-I and A-II levels and a 30% reduction in apo B. The heterozygotes have about a 50% reduction of apo A-I, a slight reduction in apo A-II and no change in apo B. These apolipoprotein values correspond to a marked reduction in HDL cholesterol for the homozygote and substantial reductions in the heterozygotes. The "A"-esterase activity is zero in one homozygote while heterozygotes have about 5% of the levels in control subjects. Arylesterase activity appears to be essentially normal. The data thus support previous observations that the HDL "A"-esterase activity is greatly reduced in those conditions where HDL apo A-I is markedly reduced, e.g., in "Fish-eye" Disease.

  13. Gel-electrophoretic identification of hen brain neurotoxic esterase, labelled with tritiated di-isopropyl phosphorofluoridate.

    PubMed Central

    Williams, D G; Johnson, M K

    1981-01-01

    The particulate fraction from hen brain was labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated by polyacrylamide-gel electrophoresis. Four radioactive protein bands (1--4) of molecular weights 155000, 92000, 60000, and 30000 were resolved. Most of the labelling of bands 2, 3 and 4 was inhibited by preincubation with Paraoxon. The residue in band 4 was sensitive to pH 5.2. Successive treatments with Paraoxon and pH 5.2 resulted in the abolition of bands 3 and 4. Bands 1 and 2 contained one and two polypeptides respectively, whose labelling was sensitive to Mipafox, but one, in band 2, was sensitive to higher concentrations of Paraoxon. The concentrations of the other two polypeptides were 6.7 and 1.95 pmol of DiPF bound/g of brain in bands 1 and 2 respectively. Both were as sensitive to Mipafox as neurotoxic esterase and were also sensitive to phenyl benzylcarbamate. 4-Nitrophenyl di-n-pentylphosphinate given in vivo inhibited neurotoxic esterase and the labelling of the band-1 polypeptide by 82% and 84% respectively, but inhibited the labelling of the band 2 polypeptide by 51%. The phosphinate in vitro produced 98% inhibition of the labelling of the band-1 polypeptide, with only 26% inhibition of the band-2 polypeptide, under conditions sufficient to inhibit neurotoxic esterase totally. Both neurotoxic esterase and the band-1 polypeptide were found in the forebrain at 1.74-fold their concentration in the rest of the brain, whereas the band-2 polypeptide was uniformly distributed. The evidence indicates that the Mipafox-sensitive polypeptide in band 1 is the [3H]DiPF-labelled active-site subunit of neurotoxic esterase. The catalytic-centre activity of the enzyme for phenyl valerate hydrolysis was found to be 2.6 x 10(5) min-1. PMID:7340807

  14. Association of esterases with insecticide resistance in Culex quinquefasciatus (Diptera: Culicidae).

    PubMed

    Gordon, Jennifer R; Ottea, James

    2012-06-01

    The southern house mosquito, Culex quinquefasciatus Say, is a competent vector of human disease and an important target of mosquito abatement programs. However, these management programs have been compromised by development of insecticide resistance. In the current study, susceptibilities to naled and resmethrin, two adulticides used in mosquito abatement, were monitored using a topical and contact bioassay, respectively, in five field- collected populations of C. quinquefasciatus (MARC, HOOD1, HOOD2, MINLOVE, and THIB). Frequencies of resistance, measured as survival after treatment with discriminating concentrations (i.e., sufficient to kill > 90% of a reference susceptible strain) were high (88.0-96.8%) in all field collections treated with naled, but were variable (3.3-94.2%) with resmethrin. In addition, esterase activities in mosquitoes from these collections were quantified using alpha-naphthyl acetate and ranged from 1.08 to 3.39 micromol alpha-naphthol produced min(-1) mg prot(-1). Heightened activities were associated with decreased insecticide susceptibility in HOOD1, THIB, and MINLOVE but not HOOD2. Esterases were visualized using native polyacrylamide gel electrophoresis, and intra- and interstrain differences in banding patterns were detected. In addition, esterases from MINLOVE mosquitoes were more numerous and intensely staining when compared with those from a laboratory-susceptible strain. Finally, naled synergized the toxicity of resmethrin in populations with decreased insecticide susceptibility and increased esterase activity by 2.5-(MINLOVE) to three-fold (THIB). Results from this study will allow management strategies for populations of C. quinquefasciatus to be optimized, and provide a foundation for further studies exploring use of esterase inhibitors as synergists of pyrethroid toxicity. PMID:22812138

  15. Molecular cloning and characterization of two thermostable carboxyl esterases from Geobacillus stearothermophilus.

    PubMed

    Ewis, Hosam E; Abdelal, Ahmed T; Lu, Chung-Dar

    2004-03-31

    Screening of the genomic libraries of Geobacillus stearothermophilus ATCC12980 and ATCC7954 for esterase/lipase activity led to the isolation of two positive clones. The results of subclonings and sequence analyses identified two genes, est30 and est55, encoding two different carboxylesterases, and genetic rearrangement in the est55 locus was revealed from genomic comparison. The est30 gene encodes a polypeptide of 248 amino acids with a calculated molecular mass of 28338 Da, and the est55 gene encodes a polypeptide of 499 amino acids with a calculated molecular mass of 54867 Da. Both enzymes were purified to near homogeneity from recombinant strains of Escherichia coli. The results of enzyme characterization showed that while both enzymes possess optimal activities with short chain acyl derivatives, Est55 has a broader pH tolerance (pH 8-9) and optimal temperature range (30-60 degrees C) than Est30. The activation energy of Est55 (35.7 kJ/mol) was found to be significantly lower than that of Est30 (101.9 kJ/mol). Both enzymes were stable at 60 degrees C for more than 2 h; at 70 degrees C, the half-life for thermal inactivation was 40 and 180 min for Est55 and Est30, respectively. With p-nitrophenyl caproate as the substrate and assayed at 60 degrees C, Est55 had K(m) and k(cat) values of 0.5 microM and 39758 s(-1) while Est30 exhibited values of 2.16 microM and 38 s(-1). Inhibition studies indicated that both Est30 and Est55 were strongly inhibited by phenylmethanesulfonyl fluoride, p-hydroxymercuribenzoate, and tosyl-l-phenylalanine, consistent with the proposed presence of Ser-His-Glu catalytic triad of the alpha/beta hydrolase family. The enzymatic properties of Est30 and Est55 reported here warrant the potential applications of these enzymes in biotechnological industries. PMID:15033540

  16. Esterase SeE of Streptococcus equi ssp. equi is a Novel Non-specific Carboxylic Ester Hydrolase

    PubMed Central

    Xie, Gang; Liu, Mengyao; Zhu, Hui; Lei, Benfang

    2009-01-01

    Extracellular carboxylic ester hydrolases are produced by many bacterial pathogens and have been shown recently to be important for virulence of some pathogens. However, these hydrolases are poorly characterized in enzymatic activity. This study prepared and characterized the secreted ester hydrolase of Streptococcus equi ssp. equi (designated SeE for S. equi esterase). SeE hydrolyzes ethyl acetate, acetylsalicylic acid, and tributyrin but not ethyl butyrate. This substrate specificity pattern does not match those of the three conventional types of non-specific carboxylic ester hydrolases (carboxylesterases, arylesterases, and acetylesterases). To determine whether SeE has lipase activity, a number of triglycerides and vinyl esters were tested in SeE-catalyzed hydrolysis. SeE does not hydrolyze triglycerides and vinyl esters of long chain carboxylic acids nor display interfacial activation, indicating that SeE is not a lipase. Like the conventional carboxylesterases, SeE is inhibited by diisopropylfluorophosphate. These findings indicate that SeE is a novel non-specific carboxylic ester hydrolase that has broader substrate specificity than the conventional carboxylesterases. PMID:19054107

  17. Characterization of Heterologously Expressed Acetyl Xylan Esterase1 Isolated from the Anaerobic Rumen Fungus Neocallimastix frontalis PMA02

    PubMed Central

    Kwon, Mi; Song, Jaeyong; Park, Hong-Seog; Park, Hyunjin; Chang, Jongsoo

    2016-01-01

    Acetyl xylan esterase (AXE), which hydrolyzes the ester linkages of the naturally acetylated xylan and thus known to have an important role for hemicellulose degradation, was isolated from the anaerobic rumen fungus Neocallimastix frontatlis PMA02, heterologously expressed in Escherichi coli (E.coli) and characterized. The full-length cDNA encoding NfAXE1 was 1,494 bp, of which 978 bp constituted an open reading frame. The estimated molecular weight of NfAXE1 was 36.5 kDa with 326 amino acid residues, and the calculated isoelectric point was 4.54. The secondary protein structure was predicted to consist of nine α-helixes and 12 β-strands. The enzyme expressed in E.coli had the highest activity at 40°C and pH 8. The purified recombinant NfAXE1 had a specific activity of 100.1 U/mg when p-nitrophenyl acetate (p-NA) was used as a substrate at 40°C, optimum temperature. The amount of liberated acetic acids were the highest and the lowest when p-NA and acetylated birchwood xylan were used as substrates, respectively. The amount of xylose released from acetylated birchwod xylan was increased by 1.4 fold when NfAXE1 was mixed with xylanase in a reaction cocktail, implying a synergistic effect of NfAXE1 with xylanase on hemicellulose degradation. PMID:27383808

  18. Chemotactic activity from rabbit peritoneal neutrophils. Lack of identity with N-acetyl-DL-phenylalanine beta-napthyl esterase.

    PubMed

    Tsung, P K; Showell, H J; Kegeles, S W; Becker, E L

    1976-08-12

    The chemotactic and N-acetyl-DL-phenylalanine beta-naphthyl esterase activities of rabbit peritoneal neutrophils are separable from each other by both DEAE cellulose and Sephadex G-100 column chromatography. Partially purified esterase obtained from DEAE-cellulose chromatography had molecular weight of 70 000. However, the partially purified fraction contained chemotactic activities with major activity in molecular weight of 28000 and minor activities in the molecular weights of 45000, 21900, 14500 and 10500. Esterase activity is inhibited by 10(-7) M p-nitrophenylethyl-5-chloropentylphosphonate but chemotactic activity is not.

  19. Use of 'small but smart' libraries to enhance the enantioselectivity of an esterase from Bacillus stearothermophilus towards tetrahydrofuran-3-yl acetate.

    PubMed

    Nobili, Alberto; Gall, Markus G; Pavlidis, Ioannis V; Thompson, Mark L; Schmidt, Marlen; Bornscheuer, Uwe T

    2013-07-01

    Two libraries of simultaneous double mutations in the active site region of an esterase from Bacillus stearothermophilus were constructed to improve the enantioselectivity in the hydrolysis of tetrahydrofuran-3-yl acetate. As screening of large mutant libraries is hampered by the necessity for GC/MS analysis, mutant libraries were designed according to a 'small but smart' concept. The design of focused libraries was based on data derived from a structural alignment of 3317 amino acid sequences of α/β-hydrolase fold enzymes with the bioinformatic tool 3DM. In this way, the number of mutants to be screened was substantially reduced as compared with a standard site-saturation mutagenesis approach. Whereas the wild-type esterase showed only poor enantioselectivity (E = 4.3) in the hydrolysis of (S)-tetrahydrofuran-3-yl acetate, the best variants obtained with this approach showed increased E-values of up to 10.4. Furthermore, some variants with inverted enantiopreference were found. PMID:23331978

  20. Improving enantioselectivity towards tertiary alcohols using mutants of Bacillus sp. BP-7 esterase EstBP7 holding a rare GGG(X)-oxyanion hole.

    PubMed

    Fillat, Amanda; Romea, Pedro; Urpí, Fèlix; Pastor, F I Javier; Diaz, Pilar

    2014-05-01

    Lipases and esterases are important biocatalysts for synthetic organic fine chemistry. An esterase from Bacillus sp. BP-7 (EstBP7) bears in its amino acid sequence a rare GGG(A)X oxyanion hole motif, where an uncommon threonine (T) is found at the third position. Detection of this pattern motivated evaluation of the ability of EstBP7 for conversion of tertiary alcohols. The enzyme was engineered in order to optimize its performance to provide important chiral building blocks: five variants with mutations in the oxyanion hole motif were created to investigate the influence on activity and enantioselectivity in the kinetic resolution of eight acetates of tertiary alcohols. Wild-type enzyme converted all esters of tertiary alcohols assayed with low enantioselectivity, whereas some of the mutants displayed significantly increased E-values. One of the mutants (EstBP7-AGA; Mut 5) showed an E >100 towards a complex tertiary alcohol acetate (2-(4-pyridyl)but-3-yn-2-yl acetate) at low reaction temperature (4 °C). Therefore, the catalytic toolbox was expanded for biocatalysis of optically pure tertiary alcohols valuable for the pharmaceutical industry. PMID:24407449

  1. Förster resonance energy transfer studies of luminescent gold nanoparticles functionalized with ruthenium(II) and rhenium(I) complexes: modulation via esterase hydrolysis.

    PubMed

    Leung, Frankie Chi-Ming; Tam, Anthony Yiu-Yan; Au, Vonika Ka-Man; Li, Mei-Jin; Yam, Vivian Wing-Wah

    2014-05-14

    A number of ruthenium(II) and rhenium(I) bipyridine complexes functionalized with lipoic acid moieties have been synthesized and characterized. Functionalization of gold nanoparticles with these chromophoric ruthenium(II) and rhenium(I) complexes has resulted in interesting supramolecular assemblies with Förster resonance energy transfer (FRET) properties that could be modulated via esterase hydrolysis. The luminescence of the metal complex chromophores was turned on upon cleavage of the ester bond linkage by esterase to reduce the efficiency of FRET quenching. The prepared nanoassembly conjugates have been characterized by transmission electron microscopy (TEM), energy-dispersive X-ray analysis (EDX), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), UV-visible spectroscopy, and emission spectroscopy. The quenching mechanism has also been studied by transient absorption and time-resolved emission decay measurements. The FRET efficiencies were found to vary with the nature of the chromophores and the length of the spacer between the donor (transition metal complexes) and the acceptor (gold nanoparticles).

  2. Polypeptide having acetyl xylan esterase activity and uses thereof

    DOEpatents

    Schoonneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Los, Alrik Pieter

    2015-10-20

    The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 82% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 82% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

  3. Fumaric acid esters prevent the NLRP3 inflammasome-mediated and ATP-triggered pyroptosis of differentiated THP-1 cells.

    PubMed

    Miglio, Gianluca; Veglia, Eleonora; Fantozzi, Roberto

    2015-09-01

    Fumaric acid esters (FAEs) exert therapeutic effects in patients with psoriasis and multiple sclerosis, however their mode of action remains elusive. Pyroptosis is a caspase-1-dependent pro-inflammatory form of programmed cell death, mediated by the activation of inflammasomes. To understand the pharmacological basis of the therapeutic effects of FAEs, the anti-pyroptotic activity of dimethyl fumarate (DMF) and its hydrolysis metabolite monomethyl fumarate (MMF) was studied in a model of NLRP3 inflammasome-mediated pyroptosis of human macrophages. Phorbol myristate acetate-differentiated THP-1 cells were exposed to lipopolysaccharide (5 μg/ml; 4h), then pulsed with ATP (5mM; 1h). MMF, DMF, or parthenolide (positive control) were added 1h before the ATP pulse. The pyroptotic cell death was evaluated by morphological examination and quantified by measuring the lactate dehydrogenase leakage. The ATP-triggered death of THP-1 cells (60.4 ± 4.0%) was significantly (P<0.01) prevented by DMF, in a time- and concentration-dependent manner (pIC50 and maximal effect were 6.6 and 67.6 ± 1.2%, respectively). MMF was less efficacious than DMF. These effects were accompanied by a decreased intracellular activation of caspase-1 and interleukin-1β release from ATP-treated cells, thus suggesting that FAEs antagonise the effects of ATP by preventing the activation of the pyroptotic molecular cascade leading to cell death. These results indicate that FAEs are endowed with anti-pyroptotic activity, which may contribute to their therapeutic effects. PMID:26096886

  4. An esterase on the outer membrane of Pseudomonas aeruginosa for the hydrolysis of long chain acyl esters.

    PubMed

    Ohkawa, I; Shiga, S; Kageyama, M

    1979-09-01

    A new esterase activity which hydrolyzes palmitoyl-CoA was found in the membrane fraction of Pseudomonas aeruginosa. All the 11 strains of P. aeruginosa tested possessed this esterase activity. The esterase was constitutive and was fully active on the intact cell bodies toward substrates in the medium. It was located on the outer membrane of the cell envelope, and was not released into the culture medium. This activity was designated as OM (outer membrane) esterase. OM esterase was solubilized from the cell envelope with EDTA-Triton X-100 and purified 690-fold. It was a minor component of the outer membrane. Its molecular weight was approximately 55,000. The activity was rather stable to heat, a wide range of pH, and treatment with detergents and organic solvents. No cofactors were required. The pH optimum of the reaction was 8.5. Among various acyl-CoAs, only long chain (C12--C18) thioesters were hydrolyzed. OM esterase also hydrolyzed some kinds of oxy-esters such as p-nitrophenyl acyl esters, monoacyl esters of sucrose and Tween 80 (polyoxyethylene sorbitan monooleate). On the other hand, triglycerides, phospholipids, or hydrophobic monoesters were not hydrolyzed at all. Thus, this enzyme seems to have specificity for long chain acyl esters with hydrophilic groups, whether thio- or oxy-ester. Mutants deficient in this esterase activity were isolated. These mutants were unable to grow on Tween 80 as a sole carbon source. This suggests a possible role of OM esterase in the utilization of acyl esters as carbon sources.

  5. Cocaine metabolism: cocaine and norcocaine hydrolysis by liver and serum esterases.

    PubMed

    Stewart, D J; Inaba, T; Lucassen, M; Kalow, W

    1979-04-01

    The hydrolysis of cocaine and its N-demethylated product, norcocaine, by esterases was examined in liver and serum. Both liver and serum enzymatically formed ecgonine methyl ester from cocaine. The liver enzyme had a much lower affinity for cocaine than that of serum, indicating that a different form of esterase was present in liver. The liver enzyme had a similar affinity for both norcocaine and cocaine. Likewise, the serum enzyme showed similar affinities for both substrates. The Vmax estimates, however, were consistently higher for norcocaine than cocaine in both liver and serum. Benzoyl ecgonine, a major metabolite of cocaine formed by hydrolysis, was not produced enzymatically in either serum or liver; the rate of spontaneous formation at physiological pH suggests that this metabolite may arise nonenzymatically in the body.

  6. A New Strategy for Fluorogenic Esterase Probes Displaying Low Levels of Non-specific Hydrolysis.

    PubMed

    Kim, Sungwoo; Kim, Hyunjin; Choi, Yongdoo; Kim, Youngmi

    2015-06-26

    A new design for fluorescence probes of esterase activity that features a carboxylate-side pro-fluorophore is demonstrated with boron dipyrromethene (BODIPY)-based probes 1 a and 1 b. Because the design relies on the enzyme-catalyzed hydrolysis of an ester group that is not electronically activated, these probes exhibit a stability to background hydrolysis that is far superior to classical alcohol-side profluorophore-based probes, large signal-to-noise ratios, reduced sensitivity to pH variations, and high enzymatic reactivity. The utility of probe 1 a was established with a real-time fluorescence imaging experiment of endogenous esterase activity that does not require washing of the extracellular medium. PMID:26033618

  7. Pseudomonas aeruginosa biofilm growth inhibition on medical plastic materials by immobilized esterases and acylase.

    PubMed

    Kisch, Johannes Martin; Utpatel, Christian; Hilterhaus, Lutz; Streit, Wolfgang R; Liese, Andreas

    2014-09-01

    Biofilms are matrix-encapsulated cell aggregates that cause problems in technical and health-related areas; for example, 65 % of all human infections are biofilm associated. This is mainly due to their ameliorated resistance against antimicrobials and immune systems. Pseudomonas aeruginosa, a biofilm-forming organism, is commonly responsible for nosocomial infections. Biofilm development is partly mediated by signal molecules, such as acyl-homoserine lactones (AHLs) in Gram-negative bacteria. We applied horse liver esterase, porcine kidney acylase, and porcine liver esterase; these can hydrolyze AHLs, thereby inhibiting biofilm formation. As biofilm infections are often related to foreign material introduced into the human body, we immobilized the enzymes on medical plastic materials. Biofilm formation was quantified by Crystal Violet staining and confocal laser scanning microscopy, revealing up to 97 % (on silicone), 54 % (on polyvinyl chloride), and 77 % (on polyurethane) reduced biomass after 68 h growth.

  8. Application of glutaraldehyde for the staining of esterase-active cells with carboxyfluorescein diacetate.

    PubMed

    Morono, Yuki; Takano, Suguru; Miyanaga, Kazuhiko; Tanji, Yasunori; Unno, Hajime; Hori, Katsutoshi

    2004-03-01

    Staining of esterase-active bacteria with carboxyfluorescein diacetate (CFDA) has been used to evaluate the viability of various types of cell. However, the outer membrane of Gram-negative bacteria prevents CFDA from permeating into the cell. Although EDTA can increase the permeability of the outer membrane allowing CFDA to enter the cells, it was experimentally confirmed that there is still considerable difficulty in visualizing viable cells due to passive diffusion of carboxyfluorescein (CF), a hydrolyzed product of CFDA, out of the cells. We found that glutaraldehyde enhances the discriminative recognition of esterase-active Gram-negative bacteria under microscopic observation by improving the efficacy of staining. We believe the successful staining in the presence of glutaraldehyde is due to two separate effects: an increase in the permeability of CFDA into the cell and prevention of leakage of CF out of the cell.

  9. Esterase D enhances type I interferon signal transduction to suppress foot-and-mouth disease virus replication.

    PubMed

    Li, Weiwei; Zhu, Zixiang; Cao, Weijun; Yang, Fan; Zhang, Xiangle; Li, Dan; Zhang, Keshan; Li, Pengfei; Mao, Ruoqing; Liu, Xiangtao; Zheng, Haixue

    2016-07-01

    The enzymatic activities of esterase D (ESD) are involved in many human diseases. However, no antiviral property of ESD has been described to date. Foot-and-mouth disease virus (FMDV) is the etiological agent of foot-and-mouth disease. In this study, we showed that FMDV infection triggered ESD expression. Overexpression of ESD significantly suppressed FMDV replication and knockdown of ESD expression enhanced virus replication, showing an essential antiviral role of ESD. Furthermore, we found that Sendai-virus-induced interferon (IFN) signaling was enhanced by upregulation of ESD, and ESD promoted activation of the IFN-β promoter simulated by IFN regulatory factor (IRF)3 or its upstream molecules (retinoic acid-inducible gene-I, melanoma differentiation-associated protein 5, virus-induced signaling adaptor and TANK binding kinase 1). Detailed analysis revealed that ESD protein enhanced IRF3 phosphorylation during FMDV infection. Overexpression of ESD also promoted the expression of various antiviral interferon-stimulated genes (ISGs) and knockdown of ESD impaired the expression of these antiviral genes during FMDV infection. Our findings demonstrate a new mechanism evolved by ESD to enhance type I IFN signal transduction and suppress viral replication during FMDV infection. PMID:27267271

  10. Cloning and Characterization of a Novel Esterase from Rhodococcus sp. for Highly Enantioselective Synthesis of a Chiral Cilastatin Precursor

    PubMed Central

    Zhang, Yan; Pan, Jiang; Luan, Zheng-Jiao; Park, Sunghoon

    2014-01-01

    A novel nonheme chloroperoxidase (RhEst1), with promiscuous esterase activity for enantioselective hydrolysis of ethyl (S)-2,2-dimethylcyclopropanecarboxylate, was identified from a shotgun library of Rhodococcus sp. strain ECU1013. RhEst1 was overexpressed in Escherichia coli BL21(DE3), purified to homogeneity, and functionally characterized. Fingerprinting analysis revealed that RhEst1 prefers para-nitrophenyl (pNP) esters of short-chain acyl groups. pNP esters with a cyclic acyl moiety, especially that with a cyclobutanyl group, were also substrates for RhEst1. The Km values for methyl 2,2-dimethylcyclopropanecarboxylate (DmCpCm) and ethyl 2,2-dimethylcyclopropane carboxylate (DmCpCe) were 0.25 and 0.43 mM, respectively. RhEst1 could serve as an efficient hydrolase for the bioproduction of optically pure (S)-2,2-dimethyl cyclopropane carboxylic acid (DmCpCa), which is an important chiral building block for cilastatin. As much as 0.5 M DmCpCe was enantioselectively hydrolyzed into (S)-DmCpCa, with a molar yield of 47.8% and an enantiomeric excess (ee) of 97.5%, indicating an extremely high enantioselectivity (E = 240) of this novel and unique biocatalyst for green manufacturing of highly valuable chiral chemicals. PMID:25239898

  11. Relationship between the esterase paraoxonase-1 (PON1) and metal concentrations in the whole blood of Inuit in Canada.

    PubMed

    Laird, Brian D; Goncharov, Alexey B; Ayotte, Pierre; Chan, Hing Man

    2015-02-01

    The esterase paraoxonase (PON1), a major component of high-density lipoprotein (HDL), protects against the development of atherosclerosis in humans. Although variation in PON1 activity is primarily governed by PON1 genotype, there is growing evidence that environmental chemicals may also modulate its activity. This cross-sectional study aimed to determine whether environmental exposure to various metals is associated with PON1 activity in Inuit people routinely exposed to mercury (Hg), cadmium (Cd), lead (Pb), and (Se) selenium. PON1 activity and metal concentrations were measured in blood collected from 2172 healthy participants. Sociodemographic, anthropometric and lifestyle variables were also assessed. The associations between PON1 activity and blood metal concentrations, HDL, omega-3 fatty acid blood levels, age, sex, body mass index (BMI), and lifestyle habits (e.g. smoking and alcohol consumption) were explored via multiple linear regression. PON1 activity was positively associated with Se blood concentration (β=0.056, P=0.001) but was negatively associated with Cd blood concentration (β=-0.025, P<0.001). No association was observed between PON1 activity and Hg or Pb blood concentrations. Our results suggest that: PON1 activity is modulated by metal exposure, and Inuit traditional foods may confer health benefit by increasing PON1 activity via higher Se intakes. These findings underline that current environmental metal exposures among Inuit living in the Canadian Arctic are associated with paraoxonase activity, a toxicologically-relevant biochemical parameter. PMID:25260045

  12. Esterase D enhances type I interferon signal transduction to suppress foot-and-mouth disease virus replication.

    PubMed

    Li, Weiwei; Zhu, Zixiang; Cao, Weijun; Yang, Fan; Zhang, Xiangle; Li, Dan; Zhang, Keshan; Li, Pengfei; Mao, Ruoqing; Liu, Xiangtao; Zheng, Haixue

    2016-07-01

    The enzymatic activities of esterase D (ESD) are involved in many human diseases. However, no antiviral property of ESD has been described to date. Foot-and-mouth disease virus (FMDV) is the etiological agent of foot-and-mouth disease. In this study, we showed that FMDV infection triggered ESD expression. Overexpression of ESD significantly suppressed FMDV replication and knockdown of ESD expression enhanced virus replication, showing an essential antiviral role of ESD. Furthermore, we found that Sendai-virus-induced interferon (IFN) signaling was enhanced by upregulation of ESD, and ESD promoted activation of the IFN-β promoter simulated by IFN regulatory factor (IRF)3 or its upstream molecules (retinoic acid-inducible gene-I, melanoma differentiation-associated protein 5, virus-induced signaling adaptor and TANK binding kinase 1). Detailed analysis revealed that ESD protein enhanced IRF3 phosphorylation during FMDV infection. Overexpression of ESD also promoted the expression of various antiviral interferon-stimulated genes (ISGs) and knockdown of ESD impaired the expression of these antiviral genes during FMDV infection. Our findings demonstrate a new mechanism evolved by ESD to enhance type I IFN signal transduction and suppress viral replication during FMDV infection.

  13. Relationship between the esterase paraoxonase-1 (PON1) and metal concentrations in the whole blood of Inuit in Canada.

    PubMed

    Laird, Brian D; Goncharov, Alexey B; Ayotte, Pierre; Chan, Hing Man

    2015-02-01

    The esterase paraoxonase (PON1), a major component of high-density lipoprotein (HDL), protects against the development of atherosclerosis in humans. Although variation in PON1 activity is primarily governed by PON1 genotype, there is growing evidence that environmental chemicals may also modulate its activity. This cross-sectional study aimed to determine whether environmental exposure to various metals is associated with PON1 activity in Inuit people routinely exposed to mercury (Hg), cadmium (Cd), lead (Pb), and (Se) selenium. PON1 activity and metal concentrations were measured in blood collected from 2172 healthy participants. Sociodemographic, anthropometric and lifestyle variables were also assessed. The associations between PON1 activity and blood metal concentrations, HDL, omega-3 fatty acid blood levels, age, sex, body mass index (BMI), and lifestyle habits (e.g. smoking and alcohol consumption) were explored via multiple linear regression. PON1 activity was positively associated with Se blood concentration (β=0.056, P=0.001) but was negatively associated with Cd blood concentration (β=-0.025, P<0.001). No association was observed between PON1 activity and Hg or Pb blood concentrations. Our results suggest that: PON1 activity is modulated by metal exposure, and Inuit traditional foods may confer health benefit by increasing PON1 activity via higher Se intakes. These findings underline that current environmental metal exposures among Inuit living in the Canadian Arctic are associated with paraoxonase activity, a toxicologically-relevant biochemical parameter.

  14. Genetic diversity analysis of Capsicum spp germplasm bank accessions based on α/β-esterase polymorphism.

    PubMed

    Monteiro, E R; Bronzato, A R; Orasmo, G R; Lopes, A C A; Gomes, R L F; Mangolin, C A; Machado, M F P S

    2013-04-12

    Genetic diversity and structure were analyzed in 10 accessions belonging to Banco Ativo de Germoplasma de Capsicum located at Federal University of Piauí in northwestern Brazil that receives pepper samples grown in community gardens in various regions and Brazilian states. Selections were made from seeds of C. chinense (4 accessions), C. annuum (5 accessions), and C. baccatum (1 accession). Samples consisting of leaves were collected from 4-10 plants of each accession (a total of 85 plants). Native polyacrylamide gel electrophoresis was used to identify α- and β-esterase polymorphisms. Polymorphism was clearly detected in 5 loci. Sixteen alleles were found at 5 α/β-esterase loci of the three Capsicum species. In the C. chinense samples, the highest HO and HE values were 0.3625 and 0.4395, respectively, whereas in C. annuum samples, HO and HE values were 0.2980 and 0.3310, respectively; the estimated HO and HE values in C. chinense samples were higher than those detected in C. annuum samples. A deficit of homozygous individuals was found in C. chinense (FIS = -0.6978) and C. annuum (FIS = 0.7750). Genetic differentiation between C. chinense and C. annuum at these loci was high (FST = 0.1867) indicating that C. chinense and C. annuum are genetically structured species for α/β- esterase isozymes. The esterase analysis showed high genetic diversity among the C. chinense and C. annuum samples and very high genetic differentiation (FST = 0.6321) among the C. chinense and C. annuum samples and the C. baccatum accession.

  15. Heterologous Expression and Biochemical Characterisation of Fourteen Esterases from Helicoverpa armigera

    PubMed Central

    Li, Yongqiang; Coppin, Chris W.; Devonshire, Alan L.; Scott, Colin; East, Peter; Russell, Robyn J.; Oakeshott, John G.

    2013-01-01

    Esterases have recurrently been implicated in insecticide resistance in Helicoverpa armigera but little is known about the underlying molecular mechanisms. We used a baculovirus system to express 14 of 30 full-length esterase genes so far identified from midgut cDNA libraries of this species. All 14 produced esterase isozymes after native PAGE and the isozymes for seven of them migrated to two regions of the gel previously associated with both organophosphate and pyrethroid resistance in various strains. Thirteen of the enzymes obtained in sufficient yield for further analysis all showed tight binding to organophosphates and low but measurable organophosphate hydrolase activity. However there was no clear difference in activity between the isozymes from regions associated with resistance and those from elsewhere in the zymogram, or between eight of the isozymes from a phylogenetic clade previously associated with resistance in proteomic and quantitative rtPCR experiments and five others not so associated. By contrast, the enzymes differed markedly in their activities against nine pyrethroid isomers and the enzymes with highest activity for the most insecticidal isomers were from regions of the gel and, in some cases, the phylogeny that had previously been associated with pyrethroid resistance. Phospholipase treatment confirmed predictions from sequence analysis that three of the isozymes were GPI anchored. This unusual feature among carboxylesterases has previously been suggested to underpin an association that some authors have noted between esterases and resistance to the Cry1Ac toxin from Bacillus thuringiensis. However these three isozymes did not migrate to the zymogram region previously associated with Cry1Ac resistance. PMID:23799064

  16. Genetic diversity analysis of Capsicum spp germplasm bank accessions based on α/β-esterase polymorphism.

    PubMed

    Monteiro, E R; Bronzato, A R; Orasmo, G R; Lopes, A C A; Gomes, R L F; Mangolin, C A; Machado, M F P S

    2013-01-01

    Genetic diversity and structure were analyzed in 10 accessions belonging to Banco Ativo de Germoplasma de Capsicum located at Federal University of Piauí in northwestern Brazil that receives pepper samples grown in community gardens in various regions and Brazilian states. Selections were made from seeds of C. chinense (4 accessions), C. annuum (5 accessions), and C. baccatum (1 accession). Samples consisting of leaves were collected from 4-10 plants of each accession (a total of 85 plants). Native polyacrylamide gel electrophoresis was used to identify α- and β-esterase polymorphisms. Polymorphism was clearly detected in 5 loci. Sixteen alleles were found at 5 α/β-esterase loci of the three Capsicum species. In the C. chinense samples, the highest HO and HE values were 0.3625 and 0.4395, respectively, whereas in C. annuum samples, HO and HE values were 0.2980 and 0.3310, respectively; the estimated HO and HE values in C. chinense samples were higher than those detected in C. annuum samples. A deficit of homozygous individuals was found in C. chinense (FIS = -0.6978) and C. annuum (FIS = 0.7750). Genetic differentiation between C. chinense and C. annuum at these loci was high (FST = 0.1867) indicating that C. chinense and C. annuum are genetically structured species for α/β- esterase isozymes. The esterase analysis showed high genetic diversity among the C. chinense and C. annuum samples and very high genetic differentiation (FST = 0.6321) among the C. chinense and C. annuum samples and the C. baccatum accession. PMID:23661440

  17. Isolation and Characterization of a Novel Cold-Adapted Esterase, MtEst45, from Microbulbifer thermotolerans DAU221

    PubMed Central

    Lee, Yong-Suk

    2016-01-01

    A novel esterase, MtEst45, was isolated from a fosmid genomic library of Microbulbifer thermotolerans DAU221. The encoding gene is predicted to have a mass of 45,564 Da and encodes 495 amino acids, excluding a 21 amino acid signal peptide. MtEst45 showed a low amino acid identity (approximately 23–24%) compared with other lipolytic enzymes belonging to Family III, a closely related bacterial lipolytic enzyme family. MtEst45 also showed a conserved GXSXG motif, G131IS133YG135, which was reported as active site of known lipolytic enzymes, and the putative catalytic triad composed of D237 and H265. Because these mutants of MtEst45, which was S133A, D237N, and H265L, had no activity, these catalytic triad is deemed essential for the enzyme catalysis. MtEst45 was overexpressed in Escherichia coli BL21 (DE3) and purified via His-tag affinity chromatography. The optimal pH and temperature of MtEst45 were estimated to be 8.17 and 46.27°C by response surface methodology, respectively. Additionally, MtEst45 was also active between 1 and 15°C. The optimal hydrolysis substrate for MtEst45 among p-nitrophenyl esters (C2–C18) was p-nitrophenyl butyrate, and the Km and Vmax values were 0.0998 mM and 550 μmol/min/mg of protein, respectively. MtEst45 was strongly inhibited by Hg2+, Zn2+, and Cu2+ ions; by phenylmethanesulfonyl fluoride; and by β-mercaptoethanol. Ca2+ did not affect the enzyme's activity. These biochemical properties, sequence identity, and phylogenetic analysis suggest that MtEst45 represents a novel and valuable bacterial lipolytic enzyme family and is useful for biotechnological applications. PMID:26973604

  18. Hormone-sensitive lipase is a cholesterol esterase of the intestinal mucosa.

    PubMed

    Grober, Jacques; Lucas, Stéphanie; Sörhede-Winzell, Maria; Zaghini, Isabelle; Mairal, Aline; Contreras, Juan-Antonio; Besnard, Philippe; Holm, Cecilia; Langin, Dominique

    2003-02-21

    The identity of the enzymes responsible for lipase and cholesterol esterase activities in the small intestinal mucosa is not known. Because hormone-sensitive lipase (HSL) catalyzes the hydrolysis of acylglycerols and cholesteryl esters, we sought to determine whether HSL could be involved. HSL mRNA and protein were detected in all segments of the small intestine by Northern and Western blot analyses, respectively. Immunocytochemistry experiments revealed that HSL was expressed in the differentiated enterocytes of the villi and was absent in the undifferentiated cells of the crypt. Diacylglycerol lipase and cholesterol esterase activities were found in the different segments. Analysis of gut from HSL-null mice showed that diacylglycerol lipase activity was unchanged in the duodenum and reduced in jejunum. Neutral cholesterol esterase activity was totally abolished in duodenum, jejunum, and ileum of HSL-null mice. Analysis of HSL mRNA structure showed two types of transcripts expressed in equal amounts with alternative 5'-ends transcribed from two exons. This work demonstrates that HSL is expressed in the mucosa of the small intestine. The results also reveal that the enzyme participates in acylglycerol hydrolysis in jejunal enterocytes and cholesteryl ester hydrolysis throughout the small intestine. PMID:12482847

  19. Newly Identified Thermostable Esterase from Sulfobacillus acidophilus: Properties and Performance in Phthalate Ester Degradation

    PubMed Central

    Zhang, Xiao-Yan; Fan, Xiang; Qiu, Yong-Jun; Li, Cheng-Yuan; Xing, Shuai; Zheng, Yi-Tao

    2014-01-01

    EstS1, a newly identified thermostable esterase from Sulfobacillus acidophilus DSM10332, was heterologously expressed in Escherichia coli and shown to enzymatically degrade phthalate esters (PAEs) to their corresponding monoalkyl PAEs. The optimal pH and temperature of the esterase were found to be 8.0 and 70°C, respectively. The half-life of EstS1 at 60°C was 15 h, indicating that the enzyme had good thermostability. The specificity constant (kcat/Km) of the enzyme for p-nitrophenyl butyrate was as high as 6,770 mM−1 s−1. The potential value of EstS1 was demonstrated by its ability to effectively hydrolyze 35 to 82% of PAEs (10 mM) within 2 min at 37°C, with all substrates being completely degraded within 24 h. At 60°C, the time required for complete hydrolysis of most PAEs was reduced by half. To our knowledge, this enzyme is a new esterase identified from thermophiles that is able to degrade various PAEs at high temperatures. PMID:25149523

  20. An Open Receptor-Binding Cavity of Hemagglutinin-Esterase-Fusion Glycoprotein from Newly-Identified Influenza D Virus: Basis for Its Broad Cell Tropism

    PubMed Central

    Song, Hao; Qi, Jianxun; Khedri, Zahra; Diaz, Sandra; Yu, Hai; Chen, Xi; Varki, Ajit; Shi, Yi; Gao, George F.

    2016-01-01

    Influenza viruses cause seasonal flu each year and pandemics or epidemic sporadically, posing a major threat to public health. Recently, a new influenza D virus (IDV) was isolated from pigs and cattle. Here, we reveal that the IDV utilizes 9-O-acetylated sialic acids as its receptor for virus entry. Then, we determined the crystal structures of hemagglutinin-esterase-fusion glycoprotein (HEF) of IDV both in its free form and in complex with the receptor and enzymatic substrate analogs. The IDV HEF shows an extremely similar structural fold as the human-infecting influenza C virus (ICV) HEF. However, IDV HEF has an open receptor-binding cavity to accommodate diverse extended glycan moieties. This structural difference provides an explanation for the phenomenon that the IDV has a broad cell tropism. As IDV HEF is structurally and functionally similar to ICV HEF, our findings highlight the potential threat of the virus to public health. PMID:26816272

  1. An Open Receptor-Binding Cavity of Hemagglutinin-Esterase-Fusion Glycoprotein from Newly-Identified Influenza D Virus: Basis for Its Broad Cell Tropism.

    PubMed

    Song, Hao; Qi, Jianxun; Khedri, Zahra; Diaz, Sandra; Yu, Hai; Chen, Xi; Varki, Ajit; Shi, Yi; Gao, George F

    2016-01-01

    Influenza viruses cause seasonal flu each year and pandemics or epidemic sporadically, posing a major threat to public health. Recently, a new influenza D virus (IDV) was isolated from pigs and cattle. Here, we reveal that the IDV utilizes 9-O-acetylated sialic acids as its receptor for virus entry. Then, we determined the crystal structures of hemagglutinin-esterase-fusion glycoprotein (HEF) of IDV both in its free form and in complex with the receptor and enzymatic substrate analogs. The IDV HEF shows an extremely similar structural fold as the human-infecting influenza C virus (ICV) HEF. However, IDV HEF has an open receptor-binding cavity to accommodate diverse extended glycan moieties. This structural difference provides an explanation for the phenomenon that the IDV has a broad cell tropism. As IDV HEF is structurally and functionally similar to ICV HEF, our findings highlight the potential threat of the virus to public health. PMID:26816272

  2. Morphological and physiological changes in esterase and lipid peroxidation of two bean cultivars pre-soaked with potassium nitrate under salt stress.

    PubMed

    Shaddad, Mohamed A K; Abd El-Baki, Gaber K; Doaa, Mostafa; Al-Shimaa, Rafat

    2015-12-01

    Two broad bean cultivars (Vicia faba CV Nobaria3 and Vicia faba CV Sakha3) were obtained from Mallwi Agriculture Research Center, El Minia Governorate, Egypt. The seeds were divided into two groups, the first group soaked with distilled water, while the second group were soaked with 3 mM KNO₃, respectively, for 4 hours. Seeds were sown and left to grow for 3 weeks then treated with different concentrations of NaCl (0.0, 40, 80, 120 and 160 mM) by top irrigation, then they left to grow further for 65 days from sowing. Plant samples were collected for some measurements: leaf area, plant height, root length, fresh and dry weight, photosynthetic pigments, carotenoids, soluble sugars, soluble proteins, total free amino acids, esterase enzyme, as well as MDA (malondialdehyde) content. Salinity reduced both fresh and dry weight in two broad bean cultivars, this reduction were more pronounced in Sakha3 than Nobaria3. Seed pre-soaking with KNO₃resulted in enhancement of fresh and dry weight production in both cultivars especially at 40 mM NaCl. Photosynthetic pigments were substantially affected by salt treatment while the carotenoids were increased, seed pre-soaking with KNO₃improved these components. The soluble sugars, amino acids as well as soluble proteins showed various responses with increasing salinity in the cultivars, seed pre-soaking with KNO₃has improved these parameters to some extent. The shoots of two cultivars exhibited significant accumulation of MDA, compared to roots exposed to the highest salinity levels. Pre-soaking seeds with KNO₃did not improve MDA in shoots but enhanced it in roots, however, in most cases still lower than the absolute control. The assessment of the esterase isozyme profiles on 7.5% native polyacrylamide gel revealed the presence of 13 isoforms in two faba bean plants in response to KNO₃pre-soaking and treatments with different concentrations of NaCl. PMID:26616374

  3. Organophosphorus compound esterase profiles as predictors of therapeutic and toxic effects.

    PubMed

    Makhaeva, Galina F; Radchenko, Eugene V; Palyulin, Vladimir A; Rudakova, Elena V; Aksinenko, Alexey Yu; Sokolov, Vladimir B; Zefirov, Nikolay S; Richardson, Rudy J

    2013-03-25

    Certain organophosphorus compounds (OPCs) inhibit various serine esterases (EOHs) via phosphorylation of their active site serines. We focused on 4 EOHs of particular toxicological interest: acetylcholinesterase (AChE: acute neurotoxicity; cognition enhancement), butyrylcholinesterase (BChE: inhibition of drug metabolism and/or stoichiometric scavenging of EOH inhibitors; cognition enhancement), carboxylesterase (CaE: inhibition of drug metabolism and/or stoichiometric scavenging of EOH inhibitors), and neuropathy target esterase (NTE: delayed neurotoxicity, OPIDN). The relative degree of inhibition of these EOHs constitutes the "esterase profile" of an OPC and serves as a major determinant of its net physiological effects. Thus, understanding and controlling the esterase profile of OPC activity and selectivity toward these 4 target enzymes is a significant undertaking. In the present study, we analyzed the inhibitor properties of 52 OPCs against the 4 EOHs, along with pairwise and multitarget selectivities between them, using 2 QSAR approaches: Hansch modeling and Molecular Field Topology Analysis (MFTA). The general formula of the OPCs was (RO)(2)P(O)X, where R = alkyl, X = - SCH(Hal)COOEt (Hal = Cl, Br), -SCHCl(2), -SCH(2)Br, -OCH(CF(3))R(1) (R(1) = C(6)H(5), CF(3), COOEt, COOMe). The Hansch model showed that increasing neuropathic potential correlated with rising R hydrophobicity; moreover, OPC binding to scavenger EOHs (BChE and CaE) had different effects on potential acute and delayed neurotoxicity. Predicted protective roles of BChE and CaE against acute toxicity were enhanced with increasing hydrophobicity, but projected protection against OPIDN was decreased. Next, Molecular Field Topology Analysis (MFTA) models were built, considering atomic descriptors, e.g., effective charge, van der Waals radius of environment, and group lipophilicity. Activity/selectivity maps confirmed predictions from Hansch models and revealed other structural factors affecting

  4. Fumaric acid esters promote neuronal survival upon ischemic stress through activation of the Nrf2 but not HIF-1 signaling pathway.

    PubMed

    Lin-Holderer, Jiemeng; Li, Lexiao; Gruneberg, Daniel; Marti, Hugo H; Kunze, Reiner

    2016-06-01

    Oxidative stress is a hallmark of ischemic stroke pathogenesis causing neuronal malfunction and cell death. Up-regulation of anti-oxidative genes through activation of the NF-E2-related transcription factor 2 (Nrf2) is one of the key mechanisms in cellular defense against oxidative stress. Fumaric acid esters (FAEs) represent a class of anti-oxidative and anti-inflammatory molecules that are already in clinical use for multiple sclerosis therapy. Purpose of this study was to investigate whether FAEs promote neuronal survival upon ischemia, and analyze putative underlying molecular mechanisms in neurons. Murine organotypic hippocampal slice cultures, and two neuronal cell lines were treated with dimethyl fumarate (DMF) and monomethyl fumarate (MMF). Ischemic conditions were generated by exposing cells and slice cultures to oxygen-glucose deprivation (OGD), and cell death was determined through propidium iodide staining. Treatment with both DMF and MMF immediately after OGD during reoxygenation strongly reduced cell death in hippocampal cultures ex vivo. Both DMF and MMF promoted neuronal survival in HT-22 and SH-SY5Y cell lines exposed to ischemic stress. DMF but not MMF activated the anti-oxidative Nrf2 pathway in neurons. Accordingly, Nrf2 knockdown in murine neurons abrogated the protective effect of DMF but not MMF. Moreover, FAEs did not activate the hypoxia-inducible factor (HIF) pathway suggesting that this pathway may not significantly contribute to FAE mediated neuroprotection. Our results may provide the basis for a new therapeutic approach to treat ischemic pathologies such as stroke with a drug that already has a broad safety record in humans. PMID:26801077

  5. Novel Redox-Dependent Esterase Activity (EC 3.1.1.2) for DJ-1: Implications for Parkinson's Disease.

    PubMed

    Vázquez-Mayorga, Emmanuel; Díaz-Sánchez, Ángel G; Dagda, Ruben K; Domínguez-Solís, Carlos A; Dagda, Raul Y; Coronado-Ramírez, Cynthia K; Martínez-Martínez, Alejandro

    2016-01-01

    Mutations the in human DJ-1 (hDJ-1) gene are associated with early-onset autosomal recessive forms of Parkinson's disease (PD). hDJ-1/parkinsonism associated deglycase (PARK7) is a cytoprotective multi-functional protein that contains a conserved cysteine-protease domain. Given that cysteine-proteases can act on both amide and ester substrates, we surmised that hDJ-1 possessed cysteine-mediated esterase activity. To test this hypothesis, hDJ-1 was overexpressed, purified and tested for activity towards 4-nitrophenyl acetate (pNPA) as µmol of pNPA hydrolyzed/min/mg·protein (U/mg protein). hDJ-1 showed maximum reaction velocity esterase activity (Vmax = 235.10 ± 12.00 U/mg protein), with a sigmoidal fit (S0.5 = 0.55 ± 0.040 mM) and apparent positive cooperativity (Hill coefficient of 2.05 ± 0.28). A PD-associated mutant of DJ-1 (M26I) lacked activity. Unlike its protease activity which is inactivated by reactive oxygen species (ROS), esterase activity of hDJ-1 is enhanced upon exposure to low concentrations of hydrogen peroxide (<10 µM) and plateaus at elevated concentrations (>100 µM) suggesting that its activity is resistant to oxidative stress. Esterase activity of DJ-1 requires oxidation of catalytic cysteines, as chemically protecting cysteines blocked its activity whereas an oxido-mimetic mutant of DJ-1 (C106D) exhibited robust esterase activity. Molecular docking studies suggest that C106 and L126 within its catalytic site interact with esterase substrates. Overall, our data show that hDJ-1 contains intrinsic redox-sensitive esterase activity that is abolished in a PD-associated mutant form of the hDJ-1 protein. PMID:27556455

  6. Cloning, Expression and Characterization of a Thermostable Esterase HydS14 from Actinomadura sp. Strain S14 in Pichia pastoris

    PubMed Central

    Sriyapai, Pichapak; Kawai, Fusako; Siripoke, Somjai; Chansiri, Kosum; Sriyapai, Thayat

    2015-01-01

    A thermostable esterase gene (hydS14) was cloned from an Actinomadura sp. S14 gene library. The gene is 777 bp in length and encodes a polypeptide of 258 amino acid residues with no signal peptide, no N-glycosylation site and a predicted molecular mass of 26,604 Da. The encoded protein contains the pentapeptide motif (GYSLG) and catalytic triad (Ser88-Asp208-His235) of the esterase/lipase superfamily. The HydS14 sequence shows 46%–64% identity to 23 sequences from actinomycetes (23 α/β-hydrolases), has three conserved regions, and contains the novel motif (GY(F)SLG), which distinguishes it from other clusters in the α/β-hydrolase structural superfamily. A plasmid containing the coding region (pPICZαA-hydS14) was used to express HydS14 in Pichia pastoris under the control of the AOXI promoter. The recombinant HydS14 collected from the supernatant had a molecular mass of ~30 kDa, which agrees with its predicted molecular mass without N-glycosylation. HydS14 had an optimum temperature of approximately 70 °C and an optimum pH of 8.0. HydS14 was stable at 50 and 60 °C for 120 min, with residual activities of above 80% and above 90%, respectively, as well as 50% activity at pH 6.0–8.0 and pH 9.0, respectively. The enzyme showed higher activity with p-nitrophenyl-C2 and C4. The Km and Vmax values for p-nitrophenyl-C4 were 0.21 ± 0.02 mM and 37.07 ± 1.04 μmol/min/mg, respectively. The enzyme was active toward short-chain p-nitrophenyl ester (C2–C6), displaying optimal activity with p-nitrophenyl-C4 (Kcat/Km = 11.74 mM−1·S−1). In summary, HydS14 is a thermostable esterase from Actinomadura sp. S14 that has been cloned and expressed for the first time in Pichia pastoris. PMID:26075873

  7. Cloning, Expression and Characterization of a Thermostable Esterase HydS14 from Actinomadura sp. Strain S14 in Pichia pastoris.

    PubMed

    Sriyapai, Pichapak; Kawai, Fusako; Siripoke, Somjai; Chansiri, Kosum; Sriyapai, Thayat

    2015-01-01

    A thermostable esterase gene (hydS14) was cloned from an Actinomadura sp. S14 gene library. The gene is 777 bp in length and encodes a polypeptide of 258 amino acid residues with no signal peptide, no N-glycosylation site and a predicted molecular mass of 26,604 Da. The encoded protein contains the pentapeptide motif (GYSLG) and catalytic triad (Ser88-Asp208-His235) of the esterase/lipase superfamily. The HydS14 sequence shows 46%-64% identity to 23 sequences from actinomycetes (23 α/β-hydrolases), has three conserved regions, and contains the novel motif (GY(F)SLG), which distinguishes it from other clusters in the α/β-hydrolase structural superfamily. A plasmid containing the coding region (pPICZαA-hydS14) was used to express HydS14 in Pichia pastoris under the control of the AOXI promoter. The recombinant HydS14 collected from the supernatant had a molecular mass of ~30 kDa, which agrees with its predicted molecular mass without N-glycosylation. HydS14 had an optimum temperature of approximately 70 °C and an optimum pH of 8.0. HydS14 was stable at 50 and 60 °C for 120 min, with residual activities of above 80% and above 90%, respectively, as well as 50% activity at pH 6.0-8.0 and pH 9.0, respectively. The enzyme showed higher activity with p-nitrophenyl-C2 and C4. The Km and Vmax values for p-nitrophenyl-C4 were 0.21 ± 0.02 mM and 37.07 ± 1.04 μmol/min/mg, respectively. The enzyme was active toward short-chain p-nitrophenyl ester (C2-C6), displaying optimal activity with p-nitrophenyl-C4 (Kcat/Km = 11.74 mM(-1) · S(-1)). In summary, HydS14 is a thermostable esterase from Actinomadura sp. S14 that has been cloned and expressed for the first time in Pichia pastoris. PMID:26075873

  8. [Studies on sialidase and esterase in influenza viruses].

    PubMed

    Cabezas, J A

    1991-01-01

    The main contributions of the author and collaborators about sialidase (EC 3.2.1.18) of influenza virus types A and B and O-acetylesterase (EC 3.1.1.53) of type C are summarized. After a short introduction on the topic, the negative results obtained by the author on inhibitors are commented. Then, the peculiarities of the three procedures assayed, based on the NADH determination as a measurement for the sialidase activity, are discussed. The spectrofluorimetric measurement of NADH concentration is a more sensitive and convenient procedure than that by spectrophotometry, although it is less sensitive than that based on bioluminiscence. Sialidase activity is generally higher in influenza virus type A than in type B; however, some differences have been found between the three sub-types A analysed. Furthermore, thermal stability and stability against changes in the pH values are higher for influenza virus from ducks, followed by those from humans and, finally, by those from pigs. O-acetylesterase of influenza virus type C shows a broad specificity; it acts on O-acetyl-containing compounds which may not be sialic acids. It seems that this enzyme might contribute to facilitate the action of sialidase of influenza virus types A and B. The peculiarities of influenza virus type C suggest to include this type as a new genus in the future classification of viruses.

  9. 4-Hydroxy-N-propyl-1,8-naphthalimide esters: New fluorescence-based assay for analysing lipase and esterase activity.

    PubMed

    Nalder, Tim D; Ashton, Trent D; Pfeffer, Frederick M; Marshall, Susan N; Barrow, Colin J

    2016-01-01

    Research using 1,8-naphthalimide derivatives has expanded rapidly in recent years owing to their cell-permeable nature, ability to target certain cellular locations and fluorescent properties. Here we describe the synthesis of three new esters of 4-hydroxy-N-propyl-1,8-naphthalimide (NAP) and the development of a simple and sensitive assay protocol to measure the activity of carboxylester hydrolases. The NAP fluorophore was esterified with short (butyrate), medium (octanoate) and long (palmitate) chain fatty acids. The esters were spectroscopically characterised and their properties investigated for their suitability as assay substrates. The esters were found to be relatively stable under the conditions of the assay and levels of spontaneous hydrolysis were negligible. Non-specific hydrolysis by proteins such as bovine serum albumin was also minimal. A simple and rapid assay methodology was developed and used to analyse a range of commercially available enzymes that included enzymes defined as lipases, esterases and phospholipases. Clear differences were observed between the enzyme classes with respect to the hydrolysis of the various chain length esters, with lipases preferentially hydrolysing the medium chain ester, whereas esterases reacted more favourably with the short ester. The assay was found to be highly sensitive with the fluorophore detectable to the low nM range. These esters provide alternate substrates from established coumarin-based fluorophores, possessing distinctly different excitation (447 nm) and emission (555 nm) optima. Absorbing at 440-450 nm also offers the flexibility of analysis by UV-visible spectrophotometry. This represents the first instance of a naphthalimide-derived compound being used to analyse these enzymes.

  10. Biochemical studies on a versatile esterase that is most catalytically active with polyaromatic esters

    PubMed Central

    Martínez-Martínez, Mónica; Lores, Iván; Peña-García, Carlina; Bargiela, Rafael; Reyes-Duarte, Dolores; Guazzaroni, María-Eugenia; Peláez, Ana Isabel; Sánchez, Jesús; Ferrer, Manuel

    2014-01-01

    Herein, we applied a community genomic approach using a naphthalene-enriched community (CN1) to isolate a versatile esterase (CN1E1) from the α/β-hydrolase family. The protein shares low-to-medium identity (≤ 57%) with known esterase/lipase-like proteins. The enzyme is most active at 25–30°C and pH 8.5; it retains approximately 55% of its activity at 4°C and less than 8% at ≥ 55°C, which indicates that it is a cold-adapted enzyme. CN1E1 has a distinct substrate preference compared with other α/β-hydrolases because it is catalytically most active for hydrolysing polyaromatic hydrocarbon (phenanthrene, anthracene, naphthalene, benzoyl, protocatechuate and phthalate) esters (7200–21 000 units g−1 protein at 40°C and pH 8.0). The enzyme also accepts 44 structurally different common esters with different levels of enantio-selectivity (1.0–55 000 units g−1 protein), including (±)-menthyl-acetate, (±)-neomenthyl acetate, (±)-pantolactone, (±)-methyl-mandelate, (±)-methyl-lactate and (±)-glycidyl 4-nitrobenzoate (in that order). The results provide the first biochemical evidence suggesting that such broad-spectrum esterases may be an ecological advantage for bacteria that mineralize recalcitrant pollutants (including oil refinery products, plasticizers and pesticides) as carbon sources under pollution pressure. They also offer a new tool for the stereo-assembly (i.e. through ester bonds) of multi-aromatic molecules with benzene rings that are useful for biology, chemistry and materials sciences for cases in which enzyme methods are not yet available. PMID:24418210

  11. Biochemical studies on a versatile esterase that is most catalytically active with polyaromatic esters.

    PubMed

    Martínez-Martínez, Mónica; Lores, Iván; Peña-García, Carlina; Bargiela, Rafael; Reyes-Duarte, Dolores; Guazzaroni, María-Eugenia; Peláez, Ana Isabel; Sánchez, Jesús; Ferrer, Manuel

    2014-03-01

    Herein, we applied a community genomic approach using a naphthalene-enriched community (CN1) to isolate a versatile esterase (CN1E1) from the α/β-hydrolase family. The protein shares low-to-medium identity (≤ 57%) with known esterase/lipase-like proteins. The enzyme is most active at 25-30°C and pH 8.5; it retains approximately 55% of its activity at 4°C and less than 8% at ≥ 55°C, which indicates that it is a cold-adapted enzyme. CN1E1 has a distinct substrate preference compared with other α/β-hydrolases because it is catalytically most active for hydrolysing polyaromatic hydrocarbon (phenanthrene, anthracene, naphthalene, benzoyl, protocatechuate and phthalate) esters (7200-21 000 units g(-1) protein at 40°C and pH 8.0). The enzyme also accepts 44 structurally different common esters with different levels of enantio-selectivity (1.0-55 000 units g(-1) protein), including (±)-menthyl-acetate, (±)-neomenthyl acetate, (±)-pantolactone, (±)-methyl-mandelate, (±)-methyl-lactate and (±)-glycidyl 4-nitrobenzoate (in that order). The results provide the first biochemical evidence suggesting that such broad-spectrum esterases may be an ecological advantage for bacteria that mineralize recalcitrant pollutants (including oil refinery products, plasticizers and pesticides) as carbon sources under pollution pressure. They also offer a new tool for the stereo-assembly (i.e. through ester bonds) of multi-aromatic molecules with benzene rings that are useful for biology, chemistry and materials sciences for cases in which enzyme methods are not yet available.

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

    SciTech Connect

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

    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.

  13. p-Nitrophenylacetate hydrolysis by honey bee esterases: kinetics and inhibition.

    PubMed

    Spoonamore, J E; Frohlich, D R; Wells, M A

    1993-03-01

    1. The kinetics and inhibition of p-nitrophenylacetate hydrolysis by cytosolic esterases of 1-day old female honey bees, Apis mellifera L., were studied. 2. The calculated values obtained were Km = 2.27 x 10(-5)M and Vmax = 2.48 x 10(-8) mol/s per mg protein. 3. The inhibition mechanisms examined for four organophosphorus insecticides were highly competitive in nature and based on competitive inhibition coefficients the order of toxicity was naled > dichlorvos > cis-mevinphos = trans-mevinphos. 4. Comparisons are made with the alfalfa leafcutting bee, Megachile rotundata (Fab). PMID:8498090

  14. Functional characterization of a novel microbial esterase identified from the Indian Ocean and its use in the stereoselective preparation of ( R)-methyl mandelate

    NASA Astrophysics Data System (ADS)

    Liang, Jiayuan; Sun, Aijun; Zhang, Yun; Deng, Dun; Wang, Yongfei; Ma, Sanmei; Hu, Yunfeng

    2016-11-01

    Genomic mining has identified a novel microbial alkaline esterase from the Indian Ocean. This esterase was overexpressed in E. coli BL21 (DE3) and further functionally characterized. Under optimal conditions (10 mmol/L substrate, pH 6.0, 2 h at 40 °C), this esterase can hydrolyze racemic methyl mandelate to ( R)-methyl mandelate with very high optical purity ( e.e. >99%) and yield (nearly 90%). Interestingly, the stereoselectivity of this esterase is opposite to that of two previously reported lipases that can generate ( S)-methyl mandelate through the hydrolysis of racemic methyl mandelate. No organic solvents or other additives were required to optimize the optical purity and production of the final chiral product ( R)-methyl mandelate, which can potentially simplify the production procedure of ( R)-methyl mandelate catalyzed by esterase.

  15. Insertion of an esterase gene into a specific locust pathogen (Metarhizium acridum) enables it to infect caterpillars.

    PubMed

    Wang, Sibao; Fang, Weiguo; Wang, Chengshu; St Leger, Raymond J

    2011-06-01

    An enduring theme in pathogenic microbiology is poor understanding of the mechanisms of host specificity. Metarhizium is a cosmopolitan genus of invertebrate pathogens that contains generalist species with broad host ranges such as M. robertsii (formerly known as M. anisopliae var. anisopliae) as well as specialists such as the acridid-specific grasshopper pathogen M. acridum. During growth on caterpillar (Manduca sexta) cuticle, M. robertsii up-regulates a gene (Mest1) that is absent in M. acridum and most other fungi. Disrupting M. robertsii Mest1 reduced virulence and overexpression increased virulence to caterpillars (Galleria mellonella and M. sexta), while virulence to grasshoppers (Melanoplus femurrubrum) was unaffected. When Mest1 was transferred to M. acridum under control of its native M. robertsii promoter, the transformants killed and colonized caterpillars in a similar fashion to M. robertsii. MEST1 localized exclusively to lipid droplets in M. robertsii conidia and infection structures was up-regulated during nutrient deprivation and had esterase activity against lipids with short chain fatty acids. The mobilization of stored lipids was delayed in the Mest1 disruptant mutant. Overall, our results suggest that expression of Mest1 allows rapid hydrolysis of stored lipids, and promotes germination and infection structure formation by M. robertsii during nutrient deprivation and invasion, while Mest1 expression in M. acridum broadens its host range by bypassing the regulatory signals found on natural hosts that trigger the mobilization of endogenous nutrient reserves. This study suggests that speciation in an insect pathogen could potentially be driven by host shifts resulting from changes in a single gene.

  16. Insertion of an Esterase Gene into a Specific Locust Pathogen (Metarhizium acridum) Enables It to Infect Caterpillars

    PubMed Central

    Wang, Sibao; Fang, Weiguo; Wang, Chengshu; St. Leger, Raymond J.

    2011-01-01

    An enduring theme in pathogenic microbiology is poor understanding of the mechanisms of host specificity. Metarhizium is a cosmopolitan genus of invertebrate pathogens that contains generalist species with broad host ranges such as M. robertsii (formerly known as M. anisopliae var. anisopliae) as well as specialists such as the acridid-specific grasshopper pathogen M. acridum. During growth on caterpillar (Manduca sexta) cuticle, M. robertsii up-regulates a gene (Mest1) that is absent in M. acridum and most other fungi. Disrupting M. robertsii Mest1 reduced virulence and overexpression increased virulence to caterpillars (Galleria mellonella and M. sexta), while virulence to grasshoppers (Melanoplus femurrubrum) was unaffected. When Mest1 was transferred to M. acridum under control of its native M. robertsii promoter, the transformants killed and colonized caterpillars in a similar fashion to M. robertsii. MEST1 localized exclusively to lipid droplets in M. robertsii conidia and infection structures was up-regulated during nutrient deprivation and had esterase activity against lipids with short chain fatty acids. The mobilization of stored lipids was delayed in the Mest1 disruptant mutant. Overall, our results suggest that expression of Mest1 allows rapid hydrolysis of stored lipids, and promotes germination and infection structure formation by M. robertsii during nutrient deprivation and invasion, while Mest1 expression in M. acridum broadens its host range by bypassing the regulatory signals found on natural hosts that trigger the mobilization of endogenous nutrient reserves. This study suggests that speciation in an insect pathogen could potentially be driven by host shifts resulting from changes in a single gene. PMID:21731492

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

    SciTech Connect

    Wood, S. J.; Li, X. -L.; Cotta, M. A.; Biely, P.; Duke, N. E. C.; Schiffer, M.; Pokkuluri, P. R.; Biosciences Division; National Center for Agricultural Utilization Research; Slovak Academy of Sciences

    2008-01-01

    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 {angstrom} resolution. This is the first enzyme with glucoronoyl esterase activity to be crystallized; its structure will be valuable in lignocellulose-degradation research.

  18. Design and production of peptides mimicking the active site of serine esterases with covalent binding to the organophosphorous poison soman. Annual report, 1 July 1984-30 June 1985

    SciTech Connect

    Seltzman, H.H.

    1985-12-09

    The objective of this research program is to design, synthesize, and test peptides and peptide mimics that will scavange soman in vivo and thereby provide protection against this CW agent. The test compounds were designed to mimic the active site of serine esterases (AChE), which are the natural targets of soman, enabling them to react with soman and thus protect endogenous AChE. Cyclodextrins derivatized with peptide functional groups and their equivalents such as imidazole, histamine, ethylene diamine, diethylene triamine, catechol, and ethane dithiol were synthesized for testing. The synthesis of precursors to cyclohexapeptides containing histidine, serine, and aspartic acid, which are amino acids that have been implicated in the active site of numerous esterases, were pursued. Testing of the ability of alpha-, beta, and gamma-cyclodextrins to protect AChE frominactivation by soman was carried out in vitro. From this group of compounds, beta-cyclodextrin was observed to preserve the activity of AChE in a dose response manner achieving a 72.1% preservation of activity when present in 200,000 fold excess versus soman after only ten minutes incubation time (beta-cyclodextrin + soman). Neither alpha, nor gamma-cyclodextrin showed any protective effect at the same doses. The test results suggest that beta cyclodextrin is uniquely suited to scavange soman. Improved scavanging might be achieved with the modified cyclodextrins prepared above for testing.

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

    SciTech Connect

    Hiraki, Toshiki; Shibayama, Naoya; Yoon, Young-Ho; Yun, Kyung-Mook; Hamamoto, Toshiro; Tame, Jeremy R. H.; Park, Sam-Yong

    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%.

  20. Entamoeba histolytica: soluble and membrane-associated neutral sphingomyelinase-C and other unidentified esterase activity.

    PubMed

    Vargas-Villarreal, Javier; Palacios-Corona, Rebeca; Hernández-Luna, Carlos; Mata-Cárdenas, Benito David; Torres de la Cruz, Victor M; Cortés-Gutiérrez, Elva I; González-Salazar, Francisco; Garza-González, Jesús Norberto; Escobedo-Guajardo, Brenda Leticia; Said-Fernández, Salvador

    2010-08-01

    Sphingomyelinase (SMase) activity was measured in Entamoeba histolytica particulate and soluble subcellular fractions. The effects on SMase of incubation time, total protein concentration, pH, and several divalent cations were determined. SMase-C and other unidentified esterase activity were detected in soluble and particulate fractions. SMase-C was 94.5-96.0% higher than the unidentified esterase activity. Soluble and insoluble SMase-C specific activities increased with protein dose and incubation time. Soluble and insoluble SMase-C activities were maximum at pH 7.5 and were dependent on Mg(2+), Mn(2+), or Co(2+), and inhibited by Zn(2+), Hg(2+), Ca(2+), and EDTA. SMase-C was active in the pH range of 3-10 and its maximum activity was at pH 7.5. The soluble and insoluble SMases have remarkably similar physicochemical properties, strongly suggesting that E. histolytica has just one isoform of neutral SMase-C that had not been described before and might be essential for E. histolytica metabolism or virulence. PMID:20350542

  1. Esterase activity of carbonic anhydrases serves as surrogate for selecting antibodies blocking hydratase activity.

    PubMed

    Uda, Narasimha Rao; Seibert, Volker; Stenner-Liewen, Frank; Müller, Philipp; Herzig, Petra; Gondi, Gabor; Zeidler, Reinhard; van Dijk, Marc; Zippelius, Alfred; Renner, Christoph

    2015-12-01

    Carbonic anhydrase 9 (CA9) and carbonic anhydrase 12 (CA12) were proposed as potential targets for cancer therapy more than 20 years ago. However, to date, there are only very few antibodies that have been described to specifically target CA9 and CA12 and also block the enzymatic activity of their targets. One of the early stage bottlenecks in identifying CA9- and CA12-inhibiting antibodies has been the lack of a high-throughput screening system that would allow for rapid assessment of inhibition of the targeted carbon dioxide hydratase activity of carbonic anhydrases. In this study, we show that measuring the esterase activity of carbonic anhydrase offers a robust and inexpensive screening method for identifying antibody candidates that block both hydratase and esterase activities of carbonic anhydrase's. To our knowledge, this is the first implementation of a facile surrogate-screening assay to identify potential therapeutic antibodies that block the clinically relevant hydratase activity of carbonic anhydrases. PMID:25775095

  2. Leukocyte esterase urine strips for the screening of men with urethritis--use in developing countries.

    PubMed Central

    Tyndall, M W; Nasio, J; Maitha, G; Ndinya-Achola, J O; Plummer, F A; Sellors, J W; Luinstra, K E; Jang, D; Mahony, J B; Chernesky, M A

    1994-01-01

    BACKGROUND AND OBJECTIVES--The leukocyte esterase (LE) strip is a useful tool for the screening of men with urethritis. In developing countries, where laboratory facilities are limited, and sexually transmitted diseases endemic, simple and inexpensive diagnostic tests which perform well, would be of great value. METHODS--Men presenting with urethritis to a referral clinic for sexually transmitted diseases in Nairobi, Kenya participated in this cohort analytical study. First-void urine was collected for LE dipstick testing as part of the diagnostic work-up. The results of the dipstick measurement were compared with the laboratory detection of Chlamydia trachomatis and Neisseria gonorrhoeae. RESULTS--Of 200 men with symptoms of urethritis, 33 (17%) had a pathogen detected from the urethra or the urine. Chlamydia was detected in urine by PCR in 22 (11%), and gonorrhoea was cultured from the urethra in 11 (6%). Esterase activity (trace or greater) had a sensitivity of 76%, a specificity of 80%, a positive predictive value of 42% and a negative predictive value of 94% for the presence of chlamydia or gonorrhoea. CONCLUSIONS--The use of the LE dipstick for the screening of men with symptomatic urethritis can improve diagnostic accuracy and reduce the amount of empiric antimicrobial therapy. The low detection rate of chlamydia in these men with a clinical diagnosis of nongonococcal urethritis needs further study. PMID:8300096

  3. Crystal structure of human esterase D: a potential genetic marker of retinoblastoma

    SciTech Connect

    Wu, Dong; Li, Yang; Song, Gaojie; Zhang, David; Shaw, Neil; Liu, Zhi-Jie

    2009-07-10

    Retinoblastoma (RB), a carcinoma of the retina, is caused by mutations in the long arm of chromosome 13, band 13q14. The esterase D (ESD) gene maps at a similar location as the RB gene locus and therefore serves as a potential marker for the prognosis of retinoblastoma. Because very little is known about the structure and function of ESD, we determined the 3-dimensional structure of the enzyme at 1.5 {angstrom} resolution using X-ray crystallography. ESD shows a single domain with an {alpha}/{beta}-hydrolase fold. A number of insertions are observed in the canonical {alpha}/{beta}-hydrolase fold. The active site is located in a positively charged, shallow cleft on the surface lined by a number of aromatic residues. Superimposition studies helped identify the typical catalytic triad residues -- Ser-153, His264, and Asp230 -- involved in catalysis. Mutagenesis of any of the catalytic triad residues to alanine abolished the enzyme activity. Backbone amides of Leu54 and Met150 are involved in the formation of the oxyanion hole. Interestingly, a M150A mutation increased the enzyme activity by 62%. The structure of human ESD determined in this study will aid the elucidation of the physiological role of the enzyme in the human body and will assist in the early diagnosis of retinoblastoma. Wu, D., Li, Y., Song, G., Zhang, D., Shaw, N., Liu, Z. J. Crystal structure of human esterase D: a potential genetic marker of retinoblastoma.

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

    PubMed Central

    Juhl, P Benjamin; Trodler, Peter; Tyagi, Sadhna; Pleiss, Jürgen

    2009-01-01

    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. PMID:19493341

  5. Functional Analysis of Esterase TCE2 Gene from Tetranychus cinnabarinus (Boisduval) involved in Acaricide Resistance

    PubMed Central

    Shi, Li; Wei, Peng; Wang, Xiangzun; Shen, Guangmao; Zhang, Jiao; Xiao, Wei; Xu, Zhifeng; Xu, Qiang; He, Lin

    2016-01-01

    The carmine spider mite, Tetranychus cinnabarinus is an important pest of crops and vegetables worldwide, and it has the ability to develop resistance against acaricides rapidly. Our previous study identified an esterase gene (designated TCE2) over-expressed in resistant mites. To investigate this gene’s function in resistance, the expression levels of TCE2 in susceptible, abamectin-, fenpropathrin-, and cyflumetofen-resistant strains were knocked down (65.02%, 63.14%, 57.82%, and 63.99%, respectively) via RNA interference. The bioassay data showed that the resistant levels to three acaricides were significantly decreased after the down-regulation of TCE2, indicating a correlation between the expression of TCE2 and the acaricide-resistance in T. cinnabarinus. TCE2 gene was then re-engineered for heterologous expression in Escherichia coli. The recombinant TCE2 exhibited α-naphthyl acetate activity (483.3 ± 71.8 nmol/mg pro. min−1), and the activity of this enzyme could be inhibited by abamectin, fenpropathrin, and cyflumetofen, respectively. HPLC and GC results showed that 10 μg of the recombinant TCE2 could effectively decompose 21.23% fenpropathrin and 49.70% cyflumetofen within 2 hours. This is the first report of a successful heterologous expression of an esterase gene from mites. This study provides direct evidence that TCE2 is a functional gene involved in acaricide resistance in T. cinnabarinus. PMID:26725309

  6. Analysis of QTLs for erucic acid and oil content in seeds on A8 chromosome and the linkage drag between the alleles for the two traits in Brassica napus.

    PubMed

    Cao, Zhengying; Tian, Fang; Wang, Nian; Jiang, Congcong; Lin, Bing; Xia, Wei; Shi, Jiaqin; Long, Yan; Zhang, Chunyu; Meng, Jinling

    2010-04-01

    The history of canola breeding began with the discovery of germplasm with low erucic acid content in seeds of spring forage cultivar in the 1950's. FAE1 mutations led to a dramatic decrease of the seed erucic acid content in Arabidopsis thaliana. The products of the two FAE1 loci, BnA8.FAE1 and BnC3.FAE1, showed additive effects to the level of erucic acid content in oilseed rape. Previous research believed that the pleiotropy of FAE1 was responsible for the decrease in seed oil content along with the reduction of seed erucic acid content in the modern cultivars. TN DH population was developed from a canola cultivar Tapidor and a Chinese traditional cultivar Ningyou7. The population had been tested in 10 and 11 environments to map QTLs for the erucic acid content and oil content in seeds. As the map resolution increased, a novel QTL for seed erucic acid content was revealed, after Meta-analysis, 7 cM away from the most significant seed erucic acid content QTL where BnA8.FAE1 is located. Seven independent QTLs for seed oil content (qOC) were detected around the two seed erucic acid content QTLs (qEA) across 39.20 cM on linkage group A8. Two of the qOCs co-localized with the two qEAs, respectively, and were detected in a single environment. The other five qOCs were detected in 10 of 11 environments independent of qEAs. Alleles from Tapidor in all the QTLs at the 0-39.20 cM region contributed negative effects to either erucic acid content or oil content in seeds. Parallel, genotyping showed that on 5 of the 7 QTLs regions, Tapidor alleles had the same genotypes with that in 'Liho', the original low seed erucic acid content source. Through rounds of crossbreeding with oil-cropped cultivars and intensive selection for multi generations, Tapidor still had the inferior alleles for low seed oil content from 'Liho', the forage rape. This showed a strong linkage drag of low seed oil content, which was controlled by the five qEA-independent qOCs, with low seed erucic acid

  7. Role of bifidobacteria in the hydrolysis of chlorogenic acid

    PubMed Central

    Raimondi, Stefano; Anighoro, Andrew; Quartieri, Andrea; Amaretti, Alberto; Tomás-Barberán, Francisco A; Rastelli, Giulio; Rossi, Maddalena

    2015-01-01

    This study aimed to explore the capability of potentially probiotic bifidobacteria to hydrolyze chlorogenic acid into caffeic acid (CA), and to recognize the enzymes involved in this reaction. Bifidobacterium strains belonging to eight species occurring in the human gut were screened. The hydrolysis seemed peculiar of Bifidobacterium animalis, whereas the other species failed to release CA. Intracellular feruloyl esterase activity capable of hydrolyzing chlorogenic acid was detected only in B. animalis. In silico research among bifidobacteria esterases identified Balat_0669 as the cytosolic enzyme likely responsible of CA release in B. animalis. Comparative modeling of Balat_0669 and molecular docking studies support its role in chlorogenic acid hydrolysis. Expression, purification, and functional characterization of Balat_0669 in Escherichia coli were obtained as further validation. A possible role of B. animalis in the activation of hydroxycinnamic acids was demonstrated and new perspectives were opened in the development of new probiotics, specifically selected for the enhanced bioconversion of phytochemicals into bioactive compounds. PMID:25515139

  8. Crystallization and Preliminary X-ray Diffraction Analysis of the Glucuronoyl Esterase Catalytic Domain from Hypocrea jecorina

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The catalytic domain of the glucuronoyl esterase from Hypocrea jecorina (anamorph Trichoderma reesei) was over-expressed, purified, and crystallized by sitting-drop vapor-diffusion method using 1.4 M sodium/potassium phosphate pH 6.9. Crystals had space group P212121 and X-ray diffraction data were...

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

    EPA Science Inventory

    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...

  10. Statistical optimization of medium components and physicochemical parameters to simultaneously enhance bacterial growth and esterase production by Bacillus thuringiensis.

    PubMed

    Mazzucotelli, Cintia Anabela; Moreira, María del Rosario; Ansorena, María Roberta

    2016-01-01

    Bacillus thuringiensis is a genus extensively studied because of its high potential for biotechnological application, principally in biocontrol techniques. However, the optimization of esterase production by this strain has been scarcely studied. The aim of this work was to select and optimize the physicochemical and nutritional parameters that significantly influence the growth and esterase production of B. thuringiensis. To this purpose, 6 nutritional factors and 2 physicochemical parameters were evaluated using a Plackett-Burman design. Significant variables were optimized using a Box-Behnken design and through the desirability function to select the levels of the variables that simultaneously maximize microbial growth and esterase production. The optimum conditions resulting from simultaneous optimization of the responses under study were found to be 1 g/L glucose, 15 g/L peptone, and 3.25 g/L NaCl. Under these optimal conditions, it was possible to achieve a 2.5 log CFU/mL increase in bacterial growth and a 113-fold increase in esterase productivity, compared with minimal medium without agitation.

  11. Novel feruloyl esterase from Lactobacillus fermentum NRRL B-1932 and analysis of the recombinant enzyme produced in Escherichia coli.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using agar plates containing ethyl ferulate as the sole carbon source, 33 Lactobacillus strains were screened for feruloyl esterase (FE) activity. Among a dozen species showing a clearing zone on the opaque plate containing ethyl ferulate, Lactobacillus fermentum NRRL B-1932 demonstrated the stronge...

  12. Monitoring Lipase/Esterase Activity by Stopped Flow in a Sequential Injection Analysis System Using p-Nitrophenyl Butyrate

    PubMed Central

    Pliego, Jorge; Mateos, Juan Carlos; Rodriguez, Jorge; Valero, Francisco; Baeza, Mireia; Femat, Ricardo; Camacho, Rosa; Sandoval, Georgina; Herrera-López, Enrique J.

    2015-01-01

    Lipases and esterases are biocatalysts used at the laboratory and industrial level. To obtain the maximum yield in a bioprocess, it is important to measure key variables, such as enzymatic activity. The conventional method for monitoring hydrolytic activity is to take out a sample from the bioreactor to be analyzed off-line at the laboratory. The disadvantage of this approach is the long time required to recover the information from the process, hindering the possibility to develop control systems. New strategies to monitor lipase/esterase activity are necessary. In this context and in the first approach, we proposed a lab-made sequential injection analysis system to analyze off-line samples from shake flasks. Lipase/esterase activity was determined using p-nitrophenyl butyrate as the substrate. The sequential injection analysis allowed us to measure the hydrolytic activity from a sample without dilution in a linear range from 0.05–1.60 U/mL, with the capability to reach sample dilutions up to 1000 times, a sampling frequency of five samples/h, with a kinetic reaction of 5 min and a relative standard deviation of 8.75%. The results are promising to monitor lipase/esterase activity in real time, in which optimization and control strategies can be designed. PMID:25633600

  13. Monitoring lipase/esterase activity by stopped flow in a sequential injection analysis system using p-nitrophenyl butyrate.

    PubMed

    Pliego, Jorge; Mateos, Juan Carlos; Rodriguez, Jorge; Valero, Francisco; Baeza, Mireia; Femat, Ricardo; Camacho, Rosa; Sandoval, Georgina; Herrera-López, Enrique J

    2015-01-01

    Lipases and esterases are biocatalysts used at the laboratory and industrial level. To obtain the maximum yield in a bioprocess, it is important to measure key variables, such as enzymatic activity. The conventional method for monitoring hydrolytic activity is to take out a sample from the bioreactor to be analyzed off-line at the laboratory. The disadvantage of this approach is the long time required to recover the information from the process, hindering the possibility to develop control systems. New strategies to monitor lipase/esterase activity are necessary. In this context and in the first approach, we proposed a lab-made sequential injection analysis system to analyze off-line samples from shake flasks. Lipase/esterase activity was determined using p-nitrophenyl butyrate as the substrate. The sequential injection analysis allowed us to measure the hydrolytic activity from a sample without dilution in a linear range from 0.05-1.60 U/mL, with the capability to reach sample dilutions up to 1000 times, a sampling frequency of five samples/h, with a kinetic reaction of 5 min and a relative standard deviation of 8.75%. The results are promising to monitor lipase/esterase activity in real time, in which optimization and control strategies can be designed. PMID:25633600

  14. Combined effects of carbonation with heating and fatty acid esters on inactivation and growth inhibition of various bacillus spores.

    PubMed

    Klangpetch, Wannaporn; Nakai, Tomoe; Noma, Seiji; Igura, Noriyuki; Shimoda, Mitsuya

    2013-09-01

    The effects of carbonation treatment (1 to 5 MPa, 30 min) plus heat treatment (30 to 80°C, 30 min) in the presence of various fatty acid esters (FAEs; 0.05 and 0.1%, wt/vol) on counts of viable Bacillus subtilis spores were investigated. FAEs or carbonation alone had no inactivation or growth inhibition effects on B. subtilis spores. However, carbonation plus heat (CH; 80°C, 5 MPa, 30 min) in the presence of mono- and diglycerol fatty acid esters markedly decreased counts of viable spores, and the spore counts did not change during storage for 30 days. The greatest decrease in viable spore counts occurred in the presence of monoglycerol fatty acid esters. Under CH conditions, inactivation and/or growth inhibition occurred at only 80°C and increased with increasing pressure. The greatest decrease in spore counts (more than 4 log units) occurred with CH (80°C, 5 MPa, 30 min) in the presence of monoglycerol fatty acid esters. However, this treatment was less effective against Bacillus coagulans and Geobacillus stearothermophilus spores. PMID:23992501

  15. Esterase detoxication of acetylcholinesterase inhibitors using human liver samples in vitro.

    PubMed

    Moser, Virginia C; Padilla, Stephanie

    2016-04-15

    Organophosphorus (OP) and N-methylcarbamate pesticides inhibit acetylcholinesterase (AChE), but differences in metabolism and detoxication can influence potency of these pesticides across and within species. Carboxylesterase (CaE) and A-esterase (paraoxonase, PON1) are considered factors underlying age-related sensitivity differences. We used an in vitro system to measure detoxication of AChE-inhibiting pesticides mediated via these esterases. Recombinant human AChE was used as a bioassay of inhibitor concentration following incubation with detoxifying tissue: liver plus Ca(+2) (to stimulate PON1s, measuring activity of both esterases) or EGTA (to inhibit PON1s, thereby measuring CaE activity). AChE inhibitory concentrations of aldicarb, chlorpyrifos oxon, malaoxon, methamidophos, oxamyl, paraoxon, and methylparaoxon were incubated with liver homogenates from adult male rat or one of 20 commercially provided human (11-83 years of age) liver samples. Detoxication was defined as the difference in inhibition produced by the pesticide alone and inhibition measured in combination with liver plus Ca(+2) or liver plus EGTA. Generally, rat liver produced more detoxication than did the human samples. There were large detoxication differences across human samples for some pesticides (especially malaoxon, chlorpyrifos oxon) but not for others (e.g., aldicarb, methamidophos); for the most part these differences did not correlate with age or sex. Chlorpyrifos oxon was fully detoxified only in the presence of Ca(+2) in both rat and human livers. Detoxication of paraoxon and methylparaoxon in rat liver was greater with Ca(+2), but humans showed less differentiation than rats between Ca(+2) and EGTA conditions. This suggests the importance of PON1 detoxication for these three OPs in the rat, but mostly only for chlorpyrifos oxon in human samples. Malaoxon was detoxified similarly with Ca(+2) or EGTA, and the differences across humans correlated with metabolism of p

  16. Esterases activity in the axolotl Ambystoma mexicanum exposed to chlorpyrifos and its implication to motor activity.

    PubMed

    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

  17. Esterase mediated resistance against synthetic pyrethroids in field populations of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in Punjab districts of India.

    PubMed

    Singh, Nirbhay Kumar; Rath, Shitanshu S

    2014-08-29

    Detection of resistance levels against cypermethrin and deltamethrin, the most commonly used synthetic pyrethroids (SP), in Rhipicephalus (Boophilus) microplus collected from thirteen districts of Punjab (India) was carried out using adult immersion test. The regression graphs of probit mortality of ticks plotted against log values of concentrations of drugs were utilized for the determination of slope of mortality, lethal concentration for 50% (LC50), 95% (LC95) and resistance factor (RF). On the basis of the data generated on variables (mortality, egg mass weight, reproductive index and percentage inhibition of oviposition) the resistance levels were categorized. Against cypermethrin RFs of 1.48-11.22 were recorded in 12 isolates whereas, one isolate was susceptible. Resistance factors against deltamethrin were 2.4-38.54 and all 13 isolates were found to be resistant. Quantitative analysis of general esterase activity (measured by the production of the metabolite naphthol) revealed a range of 3.34 ± 0.30-13.75 ± 1.33 and 1.31 ± 0.15-8.09 ± 0.68 μmol/min/mg protein for α and β-esterase activity, respectively in different field isolates. Further, multiple pairwise comparisons of the mean values with susceptible strain (Tukey, P = 0.05) revealed significant elevated levels of both α-esterase and β-esterase in nine tick isolates resistant to both deltamethrin and cypermethrin. The data generated on acaricide resistant status and esterase mediated mechanism in ticks will help in formulating tick control strategy for the region.

  18. Leucocyte esterase, glucose and C-reactive protein in the diagnosis of prosthetic joint infections: a prospective study.

    PubMed

    De Vecchi, E; Villa, F; Bortolin, M; Toscano, M; Tacchini, L; Romanò, C L; Drago, L

    2016-06-01

    Analysis of joint fluid is of paramount importance for the diagnosis of prosthetic joint infections. Different markers of inflammation and/or infection in joint fluid have been proposed for diagnosis of these infections. In this study we evaluated the performance of leucocyte esterase, C-reactive protein (CRP) and glucose assays in synovial fluids from 129 patients with septic (n = 27) or aseptic (n = 102) prosthetic joint failure. Samples were collected in serum tubes and centrifuged to limit the presence of corpuscle interfering with the assays. Determinations of leucocyte esterase and glucose were carried out by means of enzymatic colorimetric reactions performed on strips for urine analysis. Tests were considered positive when graded + or ++ whereas traces or absence of colour were considered negative. CRP was measured using an automated turbidimetric method and considered suggestive for infections when >10 mg/L. Leucocyte esterase was positive in 25/27 infected patients and negative in 99/102 not infected patients (sensitivity 92.6%, specificity 97.0%). CRP was higher than the threshold in 22/27 infected patients and in 6/102 not infected patients (sensitivity: 81.5%; specificity: 94.1%) whereas glucose showed the lowest sensitivity (77.8%) and specificity (81.4%), being negative in 21/27 and 19/102 infected and not infected patients, respectively. CRP led to a correct diagnosis in 19 of 22 patients with discordant esterase and glucose results. In conclusion, evaluation of leucocyte esterase, glucose and CRP may represent a useful tool for rapid diagnosis of prosthetic joint infections. PMID:27040804

  19. Analysing deltamethrin susceptibility and pyrethroid esterase activity variations in sylvatic and domestic Triatoma infestans at the embryonic stage

    PubMed Central

    Santo-Orihuela, Pablo Luis; Carvajal, Guillermo; Picollo, María Inés; Vassena, Claudia Viviana

    2013-01-01

    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). PMID:24402155

  20. Structure of EstA esterase from psychrotrophic Pseudoalteromonas sp. 643A covalently inhibited by monoethylphosphonate

    SciTech Connect

    Brzuszkiewicz, Anna; Nowak, Elzbieta; Dauter, Zbigniew; Dauter, Miroslawa; Cieslinski, Hubert; Dlugolecka, Anna; Kur, Józef

    2010-10-28

    The crystal structure of the esterase EstA from the cold-adapted bacterium Pseudoalteromonas sp. 643A was determined in a covalently inhibited form at a resolution of 1.35 {angstrom}. The enzyme has a typical SGNH hydrolase structure consisting of a single domain containing a five-stranded {beta}-sheet, with three helices at the convex side and two helices at the concave side of the sheet, and is ornamented with a couple of very short helices at the domain edges. The active site is located in a groove and contains the classic catalytic triad of Ser, His and Asp. In the structure of the crystal soaked in diethyl p-nitrophenyl phosphate (DNP), the catalytic serine is covalently connected to a phosphonate moiety that clearly has only one ethyl group. This is the only example in the Protein Data Bank of a DNP-inhibited enzyme with covalently bound monoethylphosphate.

  1. Potential of Ophiostoma piceae sterol esterase for biotechnologically relevant hydrolysis reactions

    PubMed Central

    Barba Cedillo, Víctor; Prieto, Alicia; Martínez, María Jesús

    2013-01-01

    The ascomycete Ophiostoma piceae produces a sterol esterase (OPE) with high affinity toward p-nitrophenol, glycerol, and sterol esters. Recently, this enzyme has been heterologously expressed in the methylotrophic yeast Pichia pastoris under the AOX1 methanol-inducible promoter (PAOX1) using sorbitol as co-susbtrate, and the hydrolytic activity of the recombinant protein (OPE*) turned out to be improved from a kinetic point of view. In this study, we analyze the effects of sorbitol during the expression of OPE*, at first added as an additional carbon source, and methanol as inducer. The O. piceae enzyme was successfully used for PVAc hydrolysis, suggesting its potential applicability in recycled paper production to decrease stickies problems. PMID:23138020

  2. Bioassay technique using nonspecific esterase activities of Tetrahymena pyriformis for screening and assessing cytotoxicity of xenobiotics

    SciTech Connect

    Bogaerts, P.; Senaud, J.; Bohatier, J. |

    1998-08-01

    A simple and rapid test for screening and assessing the cytotoxicity of xenobiotics was developed with Tetrahymena pyriformis. The method estimates the activities of nonspecific esterases of a cell by concentrating within it a specific amount of fluorescence associated with fluorescein dye. The 2-h median effective concentration (EC50) values of 10 inorganic and eight organic substances are presented and compared to those of three other bioassays: the conventional T. pyriformis proliferation rate 9-h median inhibitory concentrations, the Microtox 30-min EC50s, and the Daphnia magna 4-methylumbelliferyl {beta}-D galactoside 1-h EC50s. A highly significant correlation was found between the results obtained with the fluorescein diacetate test and those obtained with the growth inhibition and Microtox tests. This in vivo enzymatic test showed high sensitivity to all compounds tested except Cr{sup 6+} and sodium dodecyl sulfate.

  3. Adsorption of microbial esterases on Bacillus subtilis-templated cobalt oxide nanoparticles.

    PubMed

    Jang, Eunjin; Ryu, Bum Han; Shim, Hyun-Woo; Ju, Hansol; Kim, Dong-Wan; Kim, T Doohun

    2014-04-01

    Due to low diffusion rates and large surface areas, nanomaterials have received great interest as supporting materials for enzyme immobilization. Here, the preparation of a cobalt oxide nanoparticle using Bacillus subtilis as a biological template and use of the nanostructure for microbial esterase immobilization is described. Morphological features and size distributions were investigated using electron microscopy (EM) and dynamic light scattering (DLS). Catalytic properties of enzyme-coated nanostructures were investigated using 4-methylumbelliferyl acetate and p-nitrophenyl (PNP) acetate as model substrates. Enzyme-coated nanostructures were observed to retain ∼85% of the initial activity after 15 successive reaction cycles, and enzyme immobilization processes could be repeated four times without a loss of immobilization potential. The present work demonstrates that B. subtilis-templated cobalt oxide nanoparticles have the potential to be used as biocompatible immobilization materials, and are promising candidates for the preparation of effective nanobiocatalysts.

  4. A tripartite fusion, FaeG-FedF-LT(192)A2:B, of enterotoxigenic Escherichia coli (ETEC) elicits antibodies that neutralize cholera toxin, inhibit adherence of K88 (F4) and F18 fimbriae, and protect pigs against K88ac/heat-labile toxin infection.

    PubMed

    Ruan, Xiaosai; Liu, Mei; Casey, Thomas A; Zhang, Weiping

    2011-10-01

    Enterotoxigenic Escherichia coli (ETEC) strains expressing K88 (F4) or F18 fimbriae and heat-labile (LT) and/or heat-stable (ST) toxins are the major cause of diarrhea in young pigs. Effective vaccines inducing antiadhesin (anti-K88 and anti-F18) and antitoxin (anti-LT and anti-ST) immunity would provide broad protection to young pigs against ETEC. In this study, we genetically fused nucleotides coding for peptides from K88ac major subunit FaeG, F18 minor subunit FedF, and LT toxoid (LT(192)) A2 and B subunits for a tripartite adhesin-adhesin-toxoid fusion (FaeG-FedF-LT(192)A2:B). This fusion was used for immunizations in mice and pigs to assess the induction of antiadhesin and antitoxin antibodies. In addition, protection by the elicited antiadhesin and antitoxin antibodies against a porcine ETEC strain was evaluated in a gnotobiotic piglet challenge model. The data showed that this FaeG-FedF-LT(192)A2:B fusion elicited anti-K88, anti-F18, and anti-LT antibodies in immunized mice and pigs. In addition, the anti-porcine antibodies elicited neutralized cholera toxin and inhibited adherence against both K88 and F18 fimbriae. Moreover, immunized piglets were protected when challenged with ETEC strain 30302 (K88ac/LT/STb) and did not develop clinical disease. In contrast, all control nonvaccinated piglets developed severe diarrhea and dehydration after being challenged with the same ETEC strain. This study clearly demonstrated that this FaeG-FedF-LT(192)A2:B fusion antigen elicited antibodies that neutralized LT toxin and inhibited the adherence of K88 and F18 fimbrial E. coli strains and that this fusion could serve as an antigen for vaccines against porcine ETEC diarrhea. In addition, the adhesin-toxoid fusion approach used in this study may provide important information for developing effective vaccines against human ETEC diarrhea. PMID:21813665

  5. Enzyme induced biodegradation of polycarbonate-polyurethanes: dose dependence effect of cholesterol esterase.

    PubMed

    Tang, Y W; Labow, R S; Santerre, J P

    2003-05-01

    The current study has investigated the influence of esterase activity (80-400units/ml) on the biodegradation of polycarbonate-urethanes (PCNUs) by cholesterol esterase (CE), with a particular interest in studying the influence of different hard segment structures and their contribution to sensitizing the polymer towards enzyme catalyzed hydrolysis. Polycarbonate based polyurethanes were synthesized with varying hard segment content as well as hard segment chemistry based on three different diisocyanates, 1,6-hexane diisocyanate (HDI), 4,4'-methylene bisphenyl diisocyanate (MDI) and 4,4-methylene biscyclohexyl diisocyanate (HMDI). The effect of different chemistry on surface contact angle was measured in order to define the relative chemical nature of the surfaces. The enzyme dose response was found to be lower when hard segment content in the polymer was high. There was a very strong dependence on enzyme concentration for polyurethanes with different hard segment chemistry, despite the fact that the nature of the hydrolysable polycarbonate segment remained the same. The PCNU which showed the most dramatic dependence on enzyme concentration was synthesized with HMDI. At low enzyme concentration (80units/ml) this material was the most stable of the polymers while at elevated CE concentration (400units/ml) the polymer underwent a catastrophic breakdown. The findings suggested that protein binding on the surfaces was saturated even though enzyme degradation did not achieve saturation on any of the surfaces. The role of protein binding in modulating the hydrolytic action of the enzymes at different activity levels highlights a need for further study in this area.

  6. Positive and negative selection in the beta-esterase gene cluster of the Drosophila melanogaster subgroup.

    PubMed

    Balakirev, Evgeniy S; Anisimova, Maria; Ayala, Francisco J

    2006-04-01

    We examine the pattern of molecular evolution of the beta-esterase gene cluster, including the Est-6 and psiEst-6 genes, in eight species of the Drosophila melanogaster subgroup. Using maximum likelihood estimates of nonsynonymous/synonymous rate ratios, we show that the majority of Est-6 sites evolves under strong (48% of sites) or moderate (50% of sites) negative selection and a minority of sites (1.5%) is under significant positive selection. Est-6 sites likely to be under positive selection are associated with increased intraspecific variability. One positively selected site is responsible for the EST-6 F/S allozyme polymorphism; the same site is responsible for the EST-6 functional divergence between species of the melanogaster subgroup. For psiEst-6 83.7% sites evolve under negative selection, 16% sites evolve neutrally, and 0.3% sites are under positive selection. The positively selected sites of psiEst-6 are located at the beginning and at the end of the gene, where there is reduced divergence between D. melanogaster and D. simulans; these regions of psiEst-6 could be involved in regulation or some other function. Branch-site-specific analysis shows that the evolution of the melanogaster subgroup underwent episodic positive selection. Collating the present data with previous results for the beta-esterase genes, we propose that positive and negative selection are involved in a complex relationship that may be typical of the divergence of duplicate genes as one or both duplicates evolve a new function.

  7. C1-esterase inhibitor treatment: preclinical safety aspects on the potential prothrombotic risk.

    PubMed

    Schürmann, Daniel; Herzog, Eva; Raquet, Elmar; Nolte, Marc W; May, Frauke; Müller-Cohrs, Jochen; Björkqvist, Jenny; Dickneite, Gerhard; Pragst, Ingo

    2014-11-01

    Human plasma-derived C1-esterase inhibitor (C1-INH) is an efficacious and safe treatment for hereditary angioedema. However, thrombotic events in subjects treated with C1-INH at recommended or off-label, high doses have been reported. In this study, we addressed the potential prothrombotic risk of C1-INH treatment in high doses using a non-clinical rabbit model. Following intravenous infusion of C1-INH to rabbits at doses up to 800 IU/kg, the exposure and the pharmacodynamic efficacy of C1-INH in rabbits were confirmed by activity measurements of C1-esterase, and coagulation factors XIa and XIIa, respectively. Potential prothrombotic effects were assessed following induction of venous and arterial thrombosis using in vivo models of venous and arterial stasis, complemented by various in vitro assays of coagulation markers. Administration of C1-INH at doses up to 800 IU/kg did not potentiate thrombus formation during venous stasis. In contrast, inhibition of arterial occlusion was observed upon C1-INH administration when compared with isotonic saline treatment, indicating antithrombotic rather than prothrombotic activity of high dose C1-INH treatment in vivo. This was further confirmed in vitro by decreased thrombin generation, increased activated partial thromboplastin time, clotting time and clot formation time, and inhibition of platelet aggregation. No relevant changes in fibrinolysis or in the levels of thrombin-antithrombin complexes, and prothrombin fragment 1+2 were observed upon high dose C1-INH treatment. The data suggest that treatment of healthy rabbits with high doses of C1-INH could potentially inhibit coagulation and thrombus formation rather than induce a prothrombotic risk.

  8. Functional characterization of an α-esterase gene involving malathion detoxification in Bactrocera dorsalis (Hendel).

    PubMed

    Wang, Luo-Luo; Lu, Xue-Ping; Meng, Li-Wei; Huang, Yong; Wei, Dong; Jiang, Hong-Bo; Smagghe, Guy; Wang, Jin-Jun

    2016-06-01

    Extensive use of insecticides in many orchards has prompted resistance development in the oriental fruit fly, Bactrocera dorsalis (Hendel). In this study, a laboratory selected strain of B. dorsalis (MR) with a 21-fold higher resistance to malathion was used to examine the resistance mechanisms to this organophosphate insecticide. Carboxylesterase (CarE) was found to be involved in malathion resistance in B. dorsalis from the synergism bioassay by CarE-specific inhibitor triphenylphosphate (TPP). Molecular studies further identified a previously uncharacterized α-esterase gene, BdCarE2, that may function in the development of malathion resistance in B. dorsalis via gene upregulation. This gene is predominantly expressed in the Malpighian tubules, a key insect tissue for detoxification. The transcript levels of BdCarE2 were also compared between the MR and a malathion-susceptible (MS) strain of B. dorsalis, and it was significantly more abundant in the MR strain. No sequence mutation or gene copy changes were detected between the two strains. Functional studies using RNA interference (RNAi)-mediated knockdown of BdCarE2 significantly increased the malathion susceptibility in the adult files. Furthermore, heterologous expression of BdCarE2 combined with cytotoxicity assay in Sf9 cells demonstrated that BdCarE2 could probably detoxify malathion. Taken together, the current study bring new molecular evidence supporting the involvement of CarE-mediated metabolism in resistance development against malathion in B. dorsalis and also provide bases on functional analysis of insect α-esterase associated with insecticide resistance. PMID:27155483

  9. Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

    PubMed Central

    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. Propoxur-induced acetylcholine esterase inhibition and impairment of cognitive function: attenuation by Withania somnifera.

    PubMed

    Yadav, C S; Kumar, V; Suke, S G; Ahmed, R S; Mediratta, P K; Banerjee, B D

    2010-04-01

    Propoxur (2-isopropoxyphenyl N-methylcarbamate) is widely used as an acaricide in agriculture and public health programs. Studies have shown that sub-chronic exposure to propoxur can cause oxidative stress and immuno-suppression in rats. Carbamates are also known to exhibit inhibitory effect on cholinesterase activity, which is directly related to their cholinergic effects. In the present study, the effect of Withania somnifera (Ashwagandha), a widely used herbal drug possessing anti-stress and immunomodulatory properties was studied on propoxur-induced acetylcholine esterase inhibition and impairment of cognitive function in rats. Male Wistar rats were divided into four groups. Group I was treated with olive oil and served as control. Group II was administered orally with propoxur (10 mg/kg b.wt.) in olive oil, group III received a combination of propoxur (10 mg/kg b.wt.) and W. somnifera (100 mg/kg b.wt.) suspension and group IV W. somnifera (100 mg/kg b.wt.) only. All animals were treated for 30 days. Cognitive behaviour was assessed by transfer latency using elevated plus maze. Blood and brain acetylcholine esterase (AChE) activity was also assessed. Oral administration of propoxur (10 mg/kg b.wt.) resulted in a significant reduction of brain and blood AChE activity. A significant prolongation of the acquisition as well as retention transfer latency was observed in propoxur-treated rats. Oral treatment of W. somnifera exerts protective effect and attenuates AChE inhibition and cognitive impairment caused by sub-chronic exposure to propoxur.

  11. GDSL esterase/lipase genes in Brassica rapa L.: genome-wide identification and expression analysis.

    PubMed

    Dong, Xiangshu; Yi, Hankuil; Han, Ching-Tack; Nou, Ill-Sup; Hur, Yoonkang

    2016-04-01

    GDSL esterase/lipase proteins (GELPs), a very large subfamily of lipolytic enzymes, have been identified in microbes and many plants, but only a few have been characterized with respect to their roles in growth, development, and stress responses. In Brassica crops, as in many other species, genome-wide systematic analysis and functional studies of these genes are still lacking. As a first step to study their function in B. rapa ssp. pekinensis (Chinese cabbage), we comprehensively identified all GELP genes in the genome. We found a total of 121 Brassica rapa GDSL esterase/lipase protein genes (BrGELPs), forming three clades in the phylogenetic analysis (two major and one minor), with an asymmetrical chromosomal distribution. Most BrGELPs possess four strictly conserved residues (Ser-Gly-Asn-His) in four separate conserved regions, along with short conserved and clade-specific blocks, suggesting functional diversification of these proteins. Detailed expression profiling revealed that BrGELPs were expressed in various tissues, including floral organs, implying that BrGELPs play diverse roles in various tissues and during development. Ten percent of BrGELPs were specifically expressed in fertile buds, rather than male-sterile buds, implying their involvement in pollen development. Analyses of EXL6 (extracellular lipase 6) expression and its co-expressed genes in both B. rapa and Arabidopsis, as well as knockdown of this gene in Arabidopsis, revealed that this gene plays an important role in pollen development in both species. The data described in this study will facilitate future investigations of other BrGELP functions.

  12. Characterization of Esterases Produced by a Ruminal Bacterium Identified as Butyrivibrio fibrisolvens1

    PubMed Central

    Lanz, Wayne W.; Williams, Phletus P.

    1973-01-01

    An obligately anaerobic ruminal bacterial isolate was selected from 18 tributyrin-degrading isolates and identified as Butyrivibrio fibrisolvens strain 53. The culture in late exponential phase contained enzymes which could be released by sonic disruption. These enzymes degraded substrates at a rate in the order 1-naphthyl acetate (NA) > 1-naphthyl butyrate > 1-naphthyl propionate but did not degrade 1-naphthyl palmitate or 1-naphthyl phosphate. The enzymes on NA were neither stimulated nor inhibited by CoCl2, MgCl2, and MnCl (each varied from 10−6 to 10−4 M). CaCl at 10−3 M stimulated esterase activity by 16%. Aliphatic substrates were hydrolyzed at a rate in the order triacetin > tributyrin > tripropionin, and ethyl acetate > ethyl formate. Similarly, aromatic fluorescein diesters were degraded at a rate in the order acetyl > propionyl > caproyl > butyryl > capryl > lauryl. Polyacrylamide gel electrophoretic zymograms indicated that the enzyme composite contained cathodally migrating bands. By column chromatography, these enzymes were separated into six NA-degrading fractions. Fraction V contained an esterase which had an optimal temperature of 39 C, a Km of 7.6 × 10−4 on NA, and a molecular weight of about 66,000. This enzyme was inhibited by paraoxon (41%, 10−4 M), eserine (17%, 10−2 M), NaF (17%, 10−2 M), and diisopropyl fluorophosphate (62%, 10−4 M) but not by 1-naphthyl N-methyl carbamate at 8.4 × 10−4 M. PMID:4734862

  13. GDSL esterase/lipase genes in Brassica rapa L.: genome-wide identification and expression analysis.

    PubMed

    Dong, Xiangshu; Yi, Hankuil; Han, Ching-Tack; Nou, Ill-Sup; Hur, Yoonkang

    2016-04-01

    GDSL esterase/lipase proteins (GELPs), a very large subfamily of lipolytic enzymes, have been identified in microbes and many plants, but only a few have been characterized with respect to their roles in growth, development, and stress responses. In Brassica crops, as in many other species, genome-wide systematic analysis and functional studies of these genes are still lacking. As a first step to study their function in B. rapa ssp. pekinensis (Chinese cabbage), we comprehensively identified all GELP genes in the genome. We found a total of 121 Brassica rapa GDSL esterase/lipase protein genes (BrGELPs), forming three clades in the phylogenetic analysis (two major and one minor), with an asymmetrical chromosomal distribution. Most BrGELPs possess four strictly conserved residues (Ser-Gly-Asn-His) in four separate conserved regions, along with short conserved and clade-specific blocks, suggesting functional diversification of these proteins. Detailed expression profiling revealed that BrGELPs were expressed in various tissues, including floral organs, implying that BrGELPs play diverse roles in various tissues and during development. Ten percent of BrGELPs were specifically expressed in fertile buds, rather than male-sterile buds, implying their involvement in pollen development. Analyses of EXL6 (extracellular lipase 6) expression and its co-expressed genes in both B. rapa and Arabidopsis, as well as knockdown of this gene in Arabidopsis, revealed that this gene plays an important role in pollen development in both species. The data described in this study will facilitate future investigations of other BrGELP functions. PMID:26423069

  14. Tissue distribution, characterization and in vitro inhibition of B-esterases in the earwig Forficula auricularia.

    PubMed

    Malagnoux, Laure; Capowiez, Yvan; Rault, Magali

    2014-10-01

    Earwigs are important natural enemies of numerous pests in pome fruit orchards worldwide. Studying the effects of agricultural practices on these biological control agents is important for understanding its vulnerability in the field. The aim of this study was to characterize the B-esterase activities in the European earwig Forficula auricularia and to evaluate in vitro its sensitivity to organophosphate and carbamate pesticides. Acetylcholinesterase (AChE) activity was mainly measured with 1.5 mM acetylthiocholine as the substrate in the microsomal fraction of earwig heads (70% of total AChE activity). Carboxylesterase (CbE) activities were measured with three substrates [5 mM 4-nitrophenyl acetate (4-NPA), 1mM 4-nitrophenyl valerate (4-NPV), and 2 mM α-naphtyl acetate (α-NA)] to examine different isoenzymes, which were present mainly in the cytosolic fraction (about 70-88% of total activities) of all earwig tissues. CbE activity was higher than AChE activity, especially with α-NA, then 4-NPA and lastly 4-NPV. Chlorpyrifos-oxon an organophosphate, and carbaryl a carbamate pesticide, inhibited AChE and CbE activities in a concentration-dependent manner. Earwig CbE activities showed a stronger sensitivity to organophosphate than AChE, with the strongest effect for chlorpyrifos-oxon on male carboxylesterase activities. CbE and AChE showed about the same sensitivity to carbamate pesticides regardless of sex. These results suggest that B-type esterases in the European earwig F.auricularia are suitable biomarkers of pesticide exposure.

  15. Histochemical studies on genetical control of hormonal enzyme inducibility in the mouse. I. Non-specific esterase activity and regional histology of the epididymis.

    PubMed Central

    Blecher, S R; Kirkeby, S

    1978-01-01

    As a base line for future cell genetical studies the authors record the distribution of non-specific esterase reaction in the various histologically distinguishable cell types of the mouse epididymis. The findings are correlated with previous descriptions of the lobar structure of the organ. Assuming the sequence of lobes of the head to be as implied in these classical descriptions, the esterase activity of the epithelial cells gradates between strong to weak several times along the length of the epididymal duct. The relationship of the lobes to each other, as seen in transverse sections, is described. Methodological studies using different fixatives indicate that apparent similarity of esterase reaction at different sites may camouflage an underlying difference in the nature of the esterases at these sites. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 PMID:564339

  16. Glutaraldehyde cross-linking of immobilized thermophilic esterase on hydrophobic macroporous resin for application in poly(ε-caprolactone) synthesis.

    PubMed

    Wang, Min; Shi, Hui; Wu, Di; Han, Haobo; Zhang, Jianxu; Xing, Zhen; Wang, Shuang; Li, Quanshun

    2014-01-01

    The immobilized thermophilic esterase from Archaeoglobus fulgidus was successfully constructed through the glutaraldehyde-mediated covalent coupling after its physical adsorption on a hydrophobic macroporous resin, Sepabeads EC-OD. Through 0.05% glutaraldehyde treatment, the prevention of enzyme leaching and the maintenance of catalytic activity could be simultaneously realized. Using the enzymatic ring-opening polymerization of ε-caprolactone as a model, effects of organic solvents and reaction temperature on the monomer conversion and product molecular weight were systematically investigated. After the optimization of reaction conditions, products were obtained with 100% monomer conversion and Mn values lower than 1010 g/mol. Furthermore, the cross‑linked immobilized thermophilic esterase exhibited an excellent operational stability, with monomer conversion values exceeding 90% over the course of 12 batch reactions, still more than 80% after 16 batch reactions. PMID:25006789

  17. Enantioselective Resolution of (±)-1-Phenylethanol and (±)-1-Phenylethyl Acetate by a Novel Esterase from Bacillus sp. SCSIO 15121.

    PubMed

    Liang, Jiayuan; Zhang, Yun; Sun, Aijun; Deng, Dun; Hu, Yunfeng

    2016-02-01

    A novel microbial esterase BSE01281 identified from the Indian Ocean was cloned, expressed, and functionally characterized. Esterase BSE01281 could enanoselectively resolve (±)-1-phenylethanol and (±)-1-phenylethyl acetate through two types of enzymatic reactions. After the optimization of enzymatic reactions, BSE01281 could efficiently generate (R)-1-phenylethyl acetate with high enantiomeric excess (>99%) and high conversion (42%) after 96 h trans-esterification reactions. Additionally, BSE01281 could also produce (R)-1-phenylethanol (e.e. > 99%) and (S)-1-phenylethyl acetate (e.e. > 95%) at a conversion of 49% through direct hydrolysis of inexpensive racemic 1-phenylethyl acetate for 8 h. Optically pure (R)-1-phenylethanol generated from direct enzymatic hydrolysis of racemic 1-phenylethyl acetate by BSE01281 is not easily prepared by dehydrogenases, which generally follow the "Prelog's rule" and give (S)-1-phenylethanol instead.

  18. Lactobacillus fermentum CRL1446 Ameliorates Oxidative and Metabolic Parameters by Increasing Intestinal Feruloyl Esterase Activity and Modulating Microbiota in Caloric-Restricted Mice

    PubMed Central

    Russo, Matias; Fabersani, Emanuel; Abeijón-Mukdsi, María C.; Ross, Romina; Fontana, Cecilia; Benítez-Páez, Alfonso; Gauffin-Cano, Paola; Medina, Roxana B.

    2016-01-01

    The purpose of this study was to determine whether the administration of the feruloyl esterase (FE)-producing strain Lactobacillus fermentum CRL1446 enhances metabolic and oxidative parameters in caloric-restricted (CR) mice. Balb/c male mice were divided into ad libitum fed Group (ALF Group), CR diet Group (CR Group) and CR diet plus L. fermentum Group (CR-Lf Group). CR diet was administered during 45 days and CRL1446 strain was given in the dose of 108 cells/mL/day/mouse. FE activity was determined in intestinal mucosa and content at Day 1, 20 and 45. Triglyceride, total cholesterol, glucose, thiobarbituric acid reactive substances (TBARS) levels and glutathione reductase activity were determined in plasma. Gut microbiota was evaluated by high-throughput sequencing of 16S rRNA gene amplicons. At Day 45, total intestinal FE activity in CR-Lf Group was higher (p = 0.020) than in CR and ALF groups and an improvement in both metabolic (reductions in triglyceride (p = 0.0025), total cholesterol (p = 0.005) and glucose (p < 0.0001) levels) and oxidative (decrease of TBARS levels and increase of plasmatic glutathione reductase activity (p = 0.006)) parameters was observed, compared to ALF Group. CR diet increased abundance of Bacteroidetes and CRL1446 administration increased abundance of Bifidobacterium and Lactobacillus genus. L. fermentun CRL1446 exerted a bifidogenic effect under CR conditions. PMID:27399766

  19. Lactobacillus fermentum CRL1446 Ameliorates Oxidative and Metabolic Parameters by Increasing Intestinal Feruloyl Esterase Activity and Modulating Microbiota in Caloric-Restricted Mice.

    PubMed

    Russo, Matias; Fabersani, Emanuel; Abeijón-Mukdsi, María C; Ross, Romina; Fontana, Cecilia; Benítez-Páez, Alfonso; Gauffin-Cano, Paola; Medina, Roxana B

    2016-01-01

    The purpose of this study was to determine whether the administration of the feruloyl esterase (FE)-producing strain Lactobacillus fermentum CRL1446 enhances metabolic and oxidative parameters in caloric-restricted (CR) mice. Balb/c male mice were divided into ad libitum fed Group (ALF Group), CR diet Group (CR Group) and CR diet plus L. fermentum Group (CR-Lf Group). CR diet was administered during 45 days and CRL1446 strain was given in the dose of 10⁸ cells/mL/day/mouse. FE activity was determined in intestinal mucosa and content at Day 1, 20 and 45. Triglyceride, total cholesterol, glucose, thiobarbituric acid reactive substances (TBARS) levels and glutathione reductase activity were determined in plasma. Gut microbiota was evaluated by high-throughput sequencing of 16S rRNA gene amplicons. At Day 45, total intestinal FE activity in CR-Lf Group was higher (p = 0.020) than in CR and ALF groups and an improvement in both metabolic (reductions in triglyceride (p = 0.0025), total cholesterol (p = 0.005) and glucose (p < 0.0001) levels) and oxidative (decrease of TBARS levels and increase of plasmatic glutathione reductase activity (p = 0.006)) parameters was observed, compared to ALF Group. CR diet increased abundance of Bacteroidetes and CRL1446 administration increased abundance of Bifidobacterium and Lactobacillus genus. L. fermentun CRL1446 exerted a bifidogenic effect under CR conditions. PMID:27399766

  20. Esterase LpEst1 from Lactobacillus plantarum: A Novel and Atypical Member of the αβ Hydrolase Superfamily of Enzymes

    PubMed Central

    Cortés-Cabrera, Álvaro; Gago, Federico; Acebrón, Iván; Benavente, Rocío; Mardo, Karin; de las Rivas, Blanca; Muñoz, Rosario; Mancheño, José M.

    2014-01-01

    The genome of the lactic acid bacterium Lactobacillus plantarum WCFS1 reveals the presence of a rich repertoire of esterases and lipases highlighting their important role in cellular metabolism. Among them is the carboxylesterase LpEst1 a bacterial enzyme related to the mammalian hormone-sensitive lipase, which is known to play a central role in energy homeostasis. In this study, the crystal structure of LpEst1 has been determined at 2.05 Å resolution; it exhibits an αβ-hydrolase fold, consisting of a central β-sheet surrounded by α-helices, endowed with novel topological features. The structure reveals a dimeric assembly not comparable with any other enzyme from the bacterial hormone-sensitive lipase family, probably echoing the specific structural features of the participating subunits. Biophysical studies including analytical gel filtration and ultracentrifugation support the dimeric nature of LpEst1. Structural and mutational analyses of the substrate-binding pocket and active site together with biochemical studies provided insights for understanding the substrate profile of LpEst1 and suggested for the first time the conserved Asp173, which is adjacent to the nucleophile, as a key element in the stabilization of the loop where the oxyanion hole resides. PMID:24663330

  1. The Structure of a Novel Thermophilic Esterase from the Planctomycetes Species, Thermogutta terrifontis Reveals an Open Active Site Due to a Minimal ‘Cap’ Domain

    PubMed Central

    Sayer, Christopher; Szabo, Zalan; Isupov, Michail N.; Ingham, Colin; Littlechild, Jennifer A.

    2015-01-01

    A carboxyl esterase (TtEst2) has been identified in a novel thermophilic bacterium, Thermogutta terrifontis from the phylum Planctomycetes and has been cloned and over-expressed in Escherichia coli. The enzyme has been characterized biochemically and shown to have activity toward small p-nitrophenyl (pNP) carboxylic esters with optimal activity for pNP-acetate. The enzyme shows moderate thermostability retaining 75% activity after incubation for 30 min at 70°C. The crystal structures have been determined for the native TtEst2 and its complexes with the carboxylic acid products propionate, butyrate, and valerate. TtEst2 differs from most enzymes of the α/β-hydrolase family 3 as it lacks the majority of the ‘cap’ domain and its active site cavity is exposed to the solvent. The bound ligands have allowed the identification of the carboxyl pocket in the enzyme active site. Comparison of TtEst2 with structurally related enzymes has given insight into how differences in their substrate preference can be rationalized based upon the properties of their active site pockets. PMID:26635762

  2. Identification and characterization of an esterase involved in malathion resistance in the head louse Pediculus humanus capitis.

    PubMed

    Kwon, Deok Ho; Kim, Ju Hyeon; Kim, Young Ho; Yoon, Kyong Sup; Clark, J Marshall; Lee, Si Hyeock

    2014-06-01

    Enhanced malathion carboxylesterase (MCE) activity was previously reported to be involved in malathion resistance in the head louse Pediculus humanus capitis (Gao et al., 2006 [8]). To identify MCE, the transcriptional profiles of all five esterases that had been annotated to be catalytically active were determined and compared between the malathion-resistant (BR-HL) and malathion-susceptible (KR-HL) strains of head lice. An esterase gene, designated HLCbE3, exhibited approximately 5.4-fold higher transcription levels, whereas remaining four esterases did not exhibit a significant increase in their transcription in BR-HL, indicating that HLCbE3 may be the putative MCE. Comparison of the entire cDNA sequences of HLCbE3 revealed no sequence differences between the BR-HL and KR-HL strains and suggested that no single nucleotide polymorphism is associated with enhanced MCE activity. Two copies of the HLCbE3 gene were observed in BR-HL, implying that the over-transcription of HLCbE3 is due to the combination of a gene duplication and up-regulated transcription. Knockdown of HLCbE3 expression by RNA interference in the BR-HL strain led to increases in malathion susceptibility, confirming the identity of HLCbE3 as a MCE responsible for malathion resistance in the head louse. Phylogenetic analysis suggested that HLCbE3 is a typical dietary esterase and belongs to a clade containing various MCEs involved in malathion resistance. PMID:24974112

  3. Exploitation of heterosis for carotenoid content in African marigold (Tagetes erecta L.) and its correlation with esterase polymorphism.

    PubMed

    Sreekala, C; Raghava, S P S

    2003-02-01

    African marigold (Tagetes erecta L.), a major source of carotenoids, is also grown as a cut flower and a garden flower in addition to being grown for its medicinal values. We studied gene action, combining ability and heterosis, aiming at genetic improvement of T. erecta for enhanced carotenoid content in petals, and report for the first time that heterosis can be exploited for total carotenoids and its commercially important fractions. Total content of carotenoids and lutein appears to be governed by dominance (or non-additive) gene action, while content of xanthophyll esters is governed by both additive and dominance (or non-additive) gene actions. Specific combining ability variance was predominant for all the three traits. General and specific combining abilities and heterosis were highly significant. Heterobeltiosis was also positive. General combining ability (GCA) variances were not significantly correlated to performance per se. There was also no correlation between performance per se of normal petalled pollen parents and the performance of crosses made between male-sterile (female) and male-fertile (pollen) parents. These findings suggest that carotenoid content should not be the only criterion considered in the selection of parental lines. Studies on esterase in seeds and peroxidase in seedlings revealed a relatively high level of polymorphism in esterase with a total of 14 isoforms, whereas peroxidase showed low polymorphism. Similarity indices between different parental combinations, calculated based on seed esterase polymorphism, showed a significant negative correlation (r = -0.479, P = 0.05) with heterosis for carotenoid content. This indicates that the selection of parents with wider variation in their esterase profiles may possibly be exploited for genetic enhancement of carotenoids in T. erecta.

  4. Identification and characterization of a periplasmic trilactone esterase, Cee, revealed unique features of ferric enterobactin acquisition in Campylobacter

    PubMed Central

    Zeng, Ximin; Mo, Yiming; Xu, Fuzhou; Lin, Jun

    2012-01-01

    Summary Ferric enterobactin (FeEnt) acquisition is a highly efficient and conserved iron scavenging system in Gram-negative bacteria. Recently, we have characterized two FeEnt receptors (CfrA and CfrB) in Campylobacter jejuni and C. coli, the enteric human pathogens that do not produce any siderophores. In this study, whole genome sequencing and comparative genomic analysis identified a unique Ent trilactone esterase Cee (Cj1376) in C. jejuni. Genomic analysis and biochemical assay strongly suggested that Cee is the sole trilactone esterase in C. jejuni. Thin layer chromatography and HPLC analyses showed high efficiency of the purified Cee to hydrolyze Ent. Three Cee homologs previously characterized from other bacteria (IroE, IroD, and Fes) were also purified and analyzed together with Cee, indicating that Cee, Fes, and IroD displayed similar hydrolysis dynamics for both apo and ferric forms of Ent while IroE catalyzed Ent inefficiently. Unlike cytoplasmic Fes and IroD, Cee is localized in the periplasm as demonstrated by immunoblotting using Cee-specific antibodies. Genetic manipulation of diverse Campylobacter strains demonstrated that Cee is not only essential for CfrB-dependent FeEnt acquisition but also involved in CfrA-dependent pathway. Together, this study identified and characterized a novel periplasmic trilactone esterase and suggested a new model of FeEnt acquisition in Campylobacter. PMID:23278903

  5. Immobilization of thermoalkalophilic recombinant esterase enzyme by entrapment in silicate coated Ca-alginate beads and its hydrolytic properties.

    PubMed

    Gülay, Seçkin; Şanlı-Mohamed, Gülşah

    2012-04-01

    Thermoalkalophilic esterase enzyme from Balçova (Agamemnon) geothermal site were aimed to be immobilized effectively via a simple and cost-effective protocol in silicate coated Calcium alginate (Ca-alginate) beads by entrapment. The optimal immobilization conditions of enzyme in Ca-alginate beads were investigated and obtained with 2% alginate using 0.5mg/ml enzyme and 0.7 M CaCl(2) solution. In order to prevent enzyme from leaking out of the gel beads, Ca-alginate beads were then coated with silicate. Enzyme loading efficiency and immobilization yield for silicate coated beads was determined as 98.1% and 71.27%, respectively and compared with non-coated ones which were 68.5% and 45.80%, respectively. Surface morphologies, structure and elemental analysis of both silicate coated and non-coated alginate beads were also compared using Fourier Transform Infrared Spectroscopy (FT-IR) and Scanning Electron Microscope (SEM) equipped with Energy-dispersive X-ray spectroscopy (EDX). Moreover, silicate coated alginate beads enhanced reusability of esterase in continuous processes compared to non-coated beads. The hydrolytic properties of free and immobilized enzyme in terms of storage and thermal stability as well as the effects of the temperature and pH were determined. It was observed that operational, thermal and storage stabilities of the esterase were increased with immobilization. PMID:22309712

  6. Immobilization of thermoalkalophilic recombinant esterase enzyme by entrapment in silicate coated Ca-alginate beads and its hydrolytic properties.

    PubMed

    Gülay, Seçkin; Şanlı-Mohamed, Gülşah

    2012-04-01

    Thermoalkalophilic esterase enzyme from Balçova (Agamemnon) geothermal site were aimed to be immobilized effectively via a simple and cost-effective protocol in silicate coated Calcium alginate (Ca-alginate) beads by entrapment. The optimal immobilization conditions of enzyme in Ca-alginate beads were investigated and obtained with 2% alginate using 0.5mg/ml enzyme and 0.7 M CaCl(2) solution. In order to prevent enzyme from leaking out of the gel beads, Ca-alginate beads were then coated with silicate. Enzyme loading efficiency and immobilization yield for silicate coated beads was determined as 98.1% and 71.27%, respectively and compared with non-coated ones which were 68.5% and 45.80%, respectively. Surface morphologies, structure and elemental analysis of both silicate coated and non-coated alginate beads were also compared using Fourier Transform Infrared Spectroscopy (FT-IR) and Scanning Electron Microscope (SEM) equipped with Energy-dispersive X-ray spectroscopy (EDX). Moreover, silicate coated alginate beads enhanced reusability of esterase in continuous processes compared to non-coated beads. The hydrolytic properties of free and immobilized enzyme in terms of storage and thermal stability as well as the effects of the temperature and pH were determined. It was observed that operational, thermal and storage stabilities of the esterase were increased with immobilization.

  7. Separating esterase targets of organophosphorus compounds in the brain by preparative chromatography.

    PubMed

    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.

  8. Evolutionary endocrinology of juvenile hormone esterase in Gryllus assimilis: direct and correlated responses to selection.

    PubMed

    Zera, A J; Zhang, C

    1995-11-01

    Hemolymph juvenile hormone esterase (JHE) activity on the third day of the last stadium in the cricket, Gryllus assimilis, exhibited a significant response to selection in each of six replicate lines. Mean realized heritability was 0.26 +/- 0.04. The response was due to changes in whole-organism enzyme activity as well as to changes in the proportion of enzyme allocated to the hemolymph compartment. In vivo juvenile hormone metabolism differed between some lines selected for high vs. low enzyme activity. Only minimal differences were observed between lines with respect to hemolymph protein concentration or whole-cricket activity of juvenile hormone epoxide hydrolase, the other major JH-degrading enzyme. Dramatic correlated responses to selection, equal in magnitude to the direct response, were observed for JHE activity on each of three other days of the last juvenile stadium. In contrast, no correlated responses in JHE activity were observed in adults. This indicates that JHE activities throughout the last stadium will evolve as a highly correlated unit independent of adult activities and the evolution of endocrine mechanisms regulating juvenile development can be decoupled from those controlling adult reproduction. This study represents the first quantitative-genetic analysis of naturally occurring endocrine variation in an insect species.

  9. Subunit Stabilization and Polyethylene Glycolation of Cocaine Esterase Improves In Vivo Residence TimeS⃞

    PubMed Central

    Narasimhan, Diwahar; Collins, Gregory T.; Nance, Mark R.; Nichols, Joseph; Edwald, Elin; Chan, Jimmy; Ko, Mei-Chuan; Woods, James H.; Tesmer, John J. G.

    2011-01-01

    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. PMID:21890748

  10. Bacterial cocaine esterase: a protein-based therapy for cocaine overdose and addiction

    PubMed Central

    Narasimhan, Diwahar; Woods, James H; Sunahara, Roger K

    2012-01-01

    Cocaine is highly addictive and there are no pharmacotherapeutic drugs available to treat acute cocaine toxicity or chronic abuse. Antagonizing an inhibitor such as cocaine using a small molecule has proven difficult. The alternative approach is to modify cocaine’s pharmacokinetic properties by sequestering or hydrolyzing it in serum and limiting access to its sites of action. We took advantage of a bacterial esterase (CocE) that has evolved to hydrolyze cocaine and have developed it as a therapeutic that rapidly and specifically clears cocaine from the subject. Native enzyme was unstable at 37°C, thus limiting CocE’s potential. Innovative computational methods based on the protein’s structure helped elucidate its mechanism of destabilization. Novel protein engineering methodologies were applied to substantially improve its stability in vitro and in vivo. These improvements rendered CocE as a powerful and efficacious therapeutic to treat cocaine intoxication and lead the way towards developing a therapy for addiction. PMID:22300094

  11. Discovery of potent carbonic anhydrase and acetylcholine esterase inhibitors: novel sulfamoylcarbamates and sulfamides derived from acetophenones.

    PubMed

    Akıncıoğlu, Akın; Akıncıoğlu, Hülya; Gülçin, İlhami; Durdagi, Serdar; Supuran, Claudiu T; Göksu, Süleyman

    2015-07-01

    In this study, several novel sulfamides were synthesized and evaluated for their acetylcholine esterase (AChE) and human carbonic anhydrase I, and II isoenzymes (hCA I and II) inhibition profiles. Reductive amination of methoxyacetophenones was used for the synthesis of amines. Amines were converted to sulfamoylcarbamates with chlorosulfonyl isocyanate (CSI) in the presence of BnOH. Pd-C catalyzed hydrogenolysis of sulfamoylcarbamates afforded sulfamides. These novel compounds were good inhibitors of the cytosolic hCA I, and hCA II with Ki values in the range of 45.9±8.9-687.5±84.3 pM for hCA I, and 48.80±8.2-672.2±71.9pM for hCA II. The inhibitory effects of the synthesized novel compounds on AChE were also investigated. The Ki values of these compounds were in the range of 4.52±0.61-38.28±6.84pM for AChE. These results show that hCA I, II, and AChE were effectively inhibited by the novel sulfamoylcarbamates 17-21 and sulfamide derivatives 22-26. All investigated compounds were docked within the active sites of the corresponding enzymes revealing the reasons of the effective inhibitory activity. PMID:25921269

  12. Are PECTIN ESTERASE INHIBITOR Genes Involved in Mediating Resistance to Rhynchosporium commune in Barley?

    PubMed Central

    Marzin, Stephan; Hanemann, Anja; Sharma, Shailendra; Hensel, Götz; Kumlehn, Jochen; Schweizer, Günther; Röder, Marion S.

    2016-01-01

    A family of putative PECTIN ESTERASE INHIBITOR (PEI) genes, which were detected in the genomic region co-segregating with the resistance gene Rrs2 against scald caused by Rhynchosporium commune in barley, were characterized and tested for their possible involvement in mediating resistance to the pathogen by complementation and overexpression analysis. The sequences of the respective genes were derived from two BAC contigs originating from the susceptible cultivar ‘Morex’. For the genes HvPEI2, HvPEI3, HvPEI4 and HvPEI6, specific haplotypes for 18 resistant and 23 susceptible cultivars were detected after PCR-amplification and haplotype-specific CAPS-markers were developed. None of the tested candidate genes HvPEI2, HvPEI3 and HvPEI4 alone conferred a high resistance level in transgenic over-expression plants, though an improvement of the resistance level was observed especially with OE-lines for gene HvPEI4. These results do not confirm but also do not exclude an involvement of the PEI gene family in the response to the pathogen. A candidate for the resistance gene Rrs2 could not be identified yet. It is possible that Rrs2 is a PEI gene or another type of gene which has not been detected in the susceptible cultivar ‘Morex’ or the full resistance reaction requires the presence of several PEI genes. PMID:26937960

  13. A cold-adapted, solvent and salt tolerant esterase from marine bacterium Psychrobacter pacificensis.

    PubMed

    Wu, Gaobing; Zhang, Xiangnan; Wei, Lu; Wu, Guojie; Kumar, Ashok; Mao, Tao; Liu, Ziduo

    2015-11-01

    Lipolytic enzymes with unique physico-chemical characteristics are gaining more attention for their immense industrial importance. In this study, a novel lipolytic enzyme (Est11) was cloned from the genomic library of a marine bacterium Psychrobacter pacificensis. The enzyme was expressed in Escherichia coli and purified to homogeneity with molecular mass of 32.9kDa. The recombinant Est11 was able to hydrolyze short chain esters (C2-C8) and displayed an optimum activity against butyrate ester (C4). The optimal temperature and pH were 25°C and 7.5, respectively. Est11 retained more than 70% of its original activity at 10°C, suggesting that it was a cold-active esterase. The enzyme was highly active and stable at high concentration of NaCl (5M). Further, incubation with ethanol, isopropanol, propanediol, DMSO, acetonitrile, and glycerol rendered remarkable positive effects on Est11 activity. Typically, even at the concentration of 30% (v/v), ethanol, DMSO, and propanediol increased Est11 activity by 1.3, 2.0, and 2.4-folds, respectively. This new robust enzyme with remarkable properties like cold-adaptability, exceptional tolerance to salt and organic solvents provides us a promising candidate to meet the needs of some harsh industrial processes. PMID:26231332

  14. Acquired C1 esterase inhibitor deficiency in lymphomas: prevalence, symptoms, and response to treatment.

    PubMed

    Bekos, Christine; Perkmann, Thomas; Krauth, Maria; Raderer, Markus; Lechner, Klaus; Jaeger, Ulrich

    2016-09-01

    We retrospectively studied the prevalence of C1 esterase inhibitor (C1 INH) deficiency in 131 patients with various lymphomas. We determined C1 INH activity, C1 INH antigen, and C4 concentration at diagnosis and after chemotherapy. In follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL) consecutive patients were studied. In these entities, the prevalence of C1 INH deficiency was 10.2% in DLBCL, 4.1% in CLL, and 0% in FL and Hodgkin lymphoma. In indolent lymphomas, we identified only single cases of C1 INH deficiency, predominantly in splenic marginal zone lymphomas (SMZL) (four cases). Only three patients were symptomatic while the majority (11 cases) was asymptomatic. In DLBCL patients who were successfully treated with chemotherapy, complete normalization of C1 INH activity and C4 was observed. In contrast, C1 INH deficiency remained in SMZL patients after splenectomy. We conclude that C1 INH deficiency in lymphomas is frequently asymptomatic and responsive to immunochemotherapy.

  15. Subunit Stabilization and Polyethylene Glycolation of Cocaine Esterase Improves In Vivo Residence Time

    SciTech Connect

    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.

    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.

  16. A thermostable bacterial cocaine esterase rapidly eliminates cocaine from brain in nonhuman primates

    PubMed Central

    Howell, L L; Nye, J A; Stehouwer, J S; Voll, R J; Mun, J; Narasimhan, D; Nichols, J; Sunahara, R; Goodman, M M; Carroll, F I; Woods, J H

    2014-01-01

    A long-acting, thermostable bacterial cocaine esterase (CocE) has been identified that rapidly degrades cocaine with a KM of 1.33+0.085 μM. In vivo evaluation of CocE has shown protection against convulsant and lethal effects of cocaine in rodents, confirming the therapeutic potential of CocE against cocaine overdose. However, the current study is the first to evaluate the effects of CocE on cocaine brain levels. Positron emission tomogrpahy neuroimaging of [11C]cocaine was used to evaluate the time course of cocaine elimination from brain in the presence and absence of CocE in nonhuman primates. Systemic administration of CocE eliminated cocaine from the rhesus-monkey brain approximately three times faster than control conditions via peripheral actions through attenuating the input function from blood plasma. The efficiency of this process is sufficient to alleviate or prevent adverse central nervous system effects induced by cocaine. Although the present study used tracer doses of cocaine to access brain clearance, these findings further support the development of CocE for the treatment of acute cocaine toxicity. PMID:24984194

  17. HIGHLY METHYL ESTERIFIED SEEDS Is a Pectin Methyl Esterase Involved in Embryo Development1[OPEN

    PubMed Central

    Levesque-Tremblay, Gabriel; Müller, Kerstin; Mansfield, Shawn D.; Haughn, George W.

    2015-01-01

    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. PMID:25572606

  18. Identification and characterization of barley mutants lacking glycine decarboxylase and carboxyl esterase activities

    SciTech Connect

    Blackwell, R.; Lewis, K.; Lea, P. )

    1990-05-01

    A barley mutant has been isolated, from a selection of fifty air-sensitive seed-lines, using a standard gel stain technique which lacks carboxyl esterase activity, but has normal levels of carbonic anhydrase. In addition, two barley mutants lacking the ability to convert glycine to serine in the mitochondria, have been characterized. Both plants accumulate glycine in air and are unable to metabolize ({sup 14}C)glycine in the short-term. When ({sup 14}C)glycine was supplied over 2h LaPr 85/55 metabolized 90%, whereas the second mutant (LaPr 87/30) metabolized 10%. Results indicate that the mutation in LaPr 85/55 is almost certainly in the glycine transporter into the mitochondrion. The mutation in LaPr 87/30 has been shown, using western blotting, to be in both the P and H proteins, two of four proteins which comprise glycine decarboxylase (P, H, T and L).

  19. Activity and dynamics of an enzyme, pig liver esterase, in near-anhydrous conditions

    SciTech Connect

    Lopez, Murielle; Kurkal-Siebert, V; Dunn, Rachel V.; Tehei, M; Finney, J.L.; Smith, Jeremy C; Daniel, R. M.

    2010-10-01

    Water is widely assumed to be essential for life, although the exact molecular basis of this requirement is unclear. Water facilitates protein motions, and although enzyme activity has been demonstrated at low hydrations in organic solvents, such nonaqueous solvents may allow the necessary motions for catalysis. To examine enzyme function in the absence of solvation and bypass diffusional constraints we have tested the ability of an enzyme, pig liver esterase, to catalyze alcoholysis as an anhydrous powder, in a reaction system of defined water content and where the substrates and products are gaseous. At hydrations of 3 ( 2) molecules of water per molecule of enzyme, activity is several orders-of-magnitude greater than nonenzymatic catalysis. Neutron spectroscopy indicates that the fast ( nanosecond) global anharmonic dynamics of the anhydrous functional enzyme are suppressed. This indicates that neither hydration water nor fast anharmonic dynamics are required for catalysis by this enzyme, implying that one of the biological requirements of water may lie with its role as a diffusion medium rather than any of its more specific properties.

  20. Modeling the tertiary structure of the patatin domain of neuropathy target esterase.

    PubMed

    Wijeyesakere, Sanjeeva J; Richardson, Rudy J; Stuckey, Jeanne A

    2007-04-01

    Neuropathy target esterase (NTE) is a transmembrane protein of unknown function whose specific chemical modification by certain organophosphorus (OP) compounds leads to distal axonopathy. Therefore, solving the 3D structure of NTE would advance the understanding of its pathogenic and physiologic roles. In this study, the tertiary structures of the patatin (catalytic) domain and the N-terminal transmembrane domain of NTE were modeled using the crystal structures of patatin (PDB ID 1oxw) and moricin (PDB ID 1kv4) as templates. Sequence alignments and secondary structure predictions were obtained from the INUB server (Buffalo, NY). O and PyMol were used to build the PNTE and NTE TMD chains from these sequence alignments. The PNTE model was refined in the presence of water using the crystallography and NMR system, while the NTE TMD model was refined in vacuo using the GROMOS implementation in the Swiss PDB viewer. The modeled active site of NTE was found to consist of a Ser966-Asp1086 catalytic dyad, which is characteristic of phospholipase A2 enzymes. The Ser966 Ogamma was located 2.93 A from the Odelta2 of Asp1086. In addition, our NTE model was found to contain a single N-terminal transmembrane domain. This modeling effort provided structural and mechanistic predictions about the catalytic domain of NTE that are being verified via experimental techniques.

  1. Subunit stabilization and polyethylene glycolation of cocaine esterase improves in vivo residence time.

    PubMed

    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

    2011-12-01

    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. PMID:21890748

  2. A thermostable bacterial cocaine esterase rapidly eliminates cocaine from brain in nonhuman primates.

    PubMed

    Howell, L L; Nye, J A; Stehouwer, J S; Voll, R J; Mun, J; Narasimhan, D; Nichols, J; Sunahara, R; Goodman, M M; Carroll, F I; Woods, J H

    2014-01-01

    A long-acting, thermostable bacterial cocaine esterase (CocE) has been identified that rapidly degrades cocaine with a K(M) of 1.33+0.085 μM. In vivo evaluation of CocE has shown protection against convulsant and lethal effects of cocaine in rodents, confirming the therapeutic potential of CocE against cocaine overdose. However, the current study is the first to evaluate the effects of CocE on cocaine brain levels. Positron emission tomogrpahy neuroimaging of [(11)C]cocaine was used to evaluate the time course of cocaine elimination from brain in the presence and absence of CocE in nonhuman primates. Systemic administration of CocE eliminated cocaine from the rhesus-monkey brain approximately three times faster than control conditions via peripheral actions through attenuating the input function from blood plasma. The efficiency of this process is sufficient to alleviate or prevent adverse central nervous system effects induced by cocaine. Although the present study used tracer doses of cocaine to access brain clearance, these findings further support the development of CocE for the treatment of acute cocaine toxicity. PMID:24984194

  3. Immobilization and Biochemical Properties of the Enantioselective Recombinant NStcI Esterase of Aspergillus nidulans

    PubMed Central

    Peña-Montes, Carolina; Mondragón-Tintor, María Elena; Castro-Rodríguez, José Augusto; Bustos-Jaimes, Ismael; Navarro-Ocaña, Arturo; Farrés, Amelia

    2013-01-01

    The recombinant NStcI A. nidulans esterase was adsorbed on Accurel MP1000, where protein yield and immobilization efficiency were 42.48% and 81.94%, respectively. Storage stability test at 4°C and RT showed 100% of residual activity after 40 days at both temperatures. The biocatalyst retains more than 70% of its initial activity after 3 cycles of repeated use. Biochemical properties of this new biocatalyst were obtained. Maximum activity was achieved at pH 11 and 30°C, while the best stability was observed with the pH between 9 and 11 at 40°C. NStcI thermostability was increased after immobilization, as it retained 47.5% of its initial activity after 1 h at 60°C, while the free enzyme under the same conditions displayed no activity. NStcI preserved 70% of its initial activity in 100% hexane after 72 h. Enzymatic kinetic resolution of (R,S)-1-phenylethanol was chosen as model reaction, using vinyl acetate as acyl donor. After optimization of reaction parameters, the highest possible conversion (42%) was reached at 37°C, aw of 0.07, and 120 h of bioconversion in hexane with an enantiomeric excess of 71.7%. NStcI has selectivity for (R)-enantiomer. The obtained E value (31.3) is in the range considered useful to resolve enantiomeric mixtures. PMID:23781330

  4. Synthesis and Structure–Activity Relationships of N-(2-Oxo-3-oxetanyl)amides as N-Acylethanolamine-hydrolyzing Acid Amidase Inhibitors

    PubMed Central

    Solorzano, Carlos; Antonietti, Francesca; Duranti, Andrea; Tontini, Andrea; Rivara, Silvia; Lodola, Alessio; Vacondio, Federica; Tarzia, Giorgio; Piomelli, Daniele; Mor, Marco

    2010-01-01

    The fatty acid ethanolamides (FAEs) are a family of bioactive lipid mediators that include the endogenous agonist of peroxisome proliferator-activated receptor-α, palmitoylethanolamide (PEA). FAEs are hydrolyzed intracellularly by either fatty acid amide hydrolase or N-acylethanolamine-hydrolyzing acid amidase (NAAA). Selective inhibition of NAAA by (S)-N-(2-oxo-3-oxetanyl)-3-phenylpropionamide [(S)-OOPP, 7a] prevents PEA degradation in mouse leukocytes and attenuates responses to proinflammatory stimuli. Starting from the structure of 7a a series of β-lactones was prepared and tested on recombinant rat NAAA to explore structure-activity relationships (SARs) for this class of inhibitors and improve their in vitro potency. Following the hypothesis that these compounds inhibit NAAA by acylation of the catalytic cysteine, we identified several requirements for recognition at the active site and obtained new potent inhibitors. In particular, (S)-N-(2-oxo-3-oxetanyl)biphenyl-4-carboxamide (7h) was more potent than 7a at inhibiting recombinant rat NAAA activity (7a, IC50 = 420 nM; 7h, IC50 = 115 nM) in vitro and at reducing carrageenan-induced leukocyte infiltration in vivo. PMID:20604568

  5. Novel Redox-Dependent Esterase Activity (EC 3.1.1.2) for DJ-1: Implications for Parkinson’s Disease

    PubMed Central

    Vázquez-Mayorga, Emmanuel; Díaz-Sánchez, Ángel G.; Dagda, Ruben K.; Domínguez-Solís, Carlos A.; Dagda, Raul Y.; Coronado-Ramírez, Cynthia K.; Martínez-Martínez, Alejandro

    2016-01-01

    Mutations the in human DJ-1 (hDJ-1) gene are associated with early-onset autosomal recessive forms of Parkinson’s disease (PD). hDJ-1/parkinsonism associated deglycase (PARK7) is a cytoprotective multi-functional protein that contains a conserved cysteine-protease domain. Given that cysteine-proteases can act on both amide and ester substrates, we surmised that hDJ-1 possessed cysteine-mediated esterase activity. To test this hypothesis, hDJ-1 was overexpressed, purified and tested for activity towards 4-nitrophenyl acetate (pNPA) as µmol of pNPA hydrolyzed/min/mg·protein (U/mg protein). hDJ-1 showed maximum reaction velocity esterase activity (Vmax = 235.10 ± 12.00 U/mg protein), with a sigmoidal fit (S0.5 = 0.55 ± 0.040 mM) and apparent positive cooperativity (Hill coefficient of 2.05 ± 0.28). A PD-associated mutant of DJ-1 (M26I) lacked activity. Unlike its protease activity which is inactivated by reactive oxygen species (ROS), esterase activity of hDJ-1 is enhanced upon exposure to low concentrations of hydrogen peroxide (<10 µM) and plateaus at elevated concentrations (>100 µM) suggesting that its activity is resistant to oxidative stress. Esterase activity of DJ-1 requires oxidation of catalytic cysteines, as chemically protecting cysteines blocked its activity whereas an oxido-mimetic mutant of DJ-1 (C106D) exhibited robust esterase activity. Molecular docking studies suggest that C106 and L126 within its catalytic site interact with esterase substrates. Overall, our data show that hDJ-1 contains intrinsic redox-sensitive esterase activity that is abolished in a PD-associated mutant form of the hDJ-1 protein. PMID:27556455

  6. Genetically engineered Oenococcus oeni strains to highlight the impact of estA2 and estA7 esterase genes on wine ester profile.

    PubMed

    Darsonval, M; Alexandre, H; Grandvalet, C

    2016-12-01

    Besides deacidifying wine, Oenococcus oeni bring significant changes in the chemical composition of wine by releasing esters by the action of their own esterases. The impact of O. oeni esterases remains relatively unexplored. Four esterase genes were identified from O. oeni genome (estA2, estA7, estC, and estB). The dual objective of this study was, first to use a genetic tool enabling the expression of esterase genes in enological conditions and, second, to investigate the impact of O. oeni esterase gene expression during winemaking on wine aromatic profile. Both estA2 and estA7 genes were successfully cloned and expressed in O. oeni and recombinant strains were inoculated in Aligoté wine to initiate malolactic fermentation (MLF). Ester profile of experimental wine was established by SPME-GC-MS. EstA2 caused significant decreases in the concentrations of isoamyl acetate, ethyl hexanoate, isobutyl acetate, and hexyl acetate, by 42.7%, 23.4%, 51.5%, and 28.9%, respectively. EstA2 has preferential hydrolytic activity toward acetate esters from higher alcohols. EstA7 has synthetic activity toward hexyl acetate with a significant 22.7% increase. This study reports the first efficient expression system enabling the production of a functional protein in O. oeni in enological conditions. PMID:27554142

  7. Insecticide resistance status, esterase activity, and electromorphs from mosquito populations of Culex quinquefasciatus Say (Diptera: Culicidae), in Houston (Harris County), Texas.

    PubMed

    Pietrantonio, P V; Gibson, G; Nawrocki, S; Carrier, F; Knight, W P

    2000-06-01

    Culex quinquefasciatus Say is a vector of St. Louis encephalitis (SLE) in Texas. This disease is endemic and prevalent in the Houston area. Disease prevention through mosquito control is mainly targeted against adults by application of a resmethrin-piperonyl butoxide formulation (Scourge). Immature mosquitoes were collected from eight areas in Harris County during 1998. The susceptibility status of these populations to Scourge, malathion, and resmethrin, the latter alone or with an esterase inhibitor as a synergist, was determined using a bottle assay with females. The population structure was investigated by electrophoretic analysis of esterases and their activity. Individual females were also analyzed for esterase activity by plate assay and for isoenzyme pattern by native PAGE. Bioassays indicated high levels of resistance to malathion in all areas. In addition, the effectiveness of Scourge in mosquitoes from area 51 deteriorated throughout the season. A localized, distinctive esterase pattern and activity level was observed in mosquitoes from different areas. Overall, the frequency of esterases Est alpha 2 (A2)/Est beta 2 (B2) was higher than that of Est beta 1 (B1). Altogether, these results indicate the onset of a fragile situation for mosquito control that should be further analyzed to effectively maintain the SLE prevention program for Harris County.

  8. Interactions in vitro of some organophosphoramidates with neuropathy target esterase and acetylcholinesterase of hen brain.

    PubMed

    Jokanovic, M; Johnson, M K

    1993-03-01

    For organophosphates or phosphonates to initiate delayed neuropathy two steps are necessary: (1) progressive covalent reaction with neuropathy target esterase (NTE) to produce a form of inhibited NTE which can be reactivated by incubation with aqueous potassium fluoride (KF) and (2) progressive "aging" of inhibited NTE to a form which can no longer be reactivated by KF. However, it has been shown recently that certain N-unsubstituted organophosphoro-monoamidates (analogues of methamidophos) cause delayed neuropathy even though the inhibited NTE appeared not to have aged (Johnson et al. (1991). Arch. Toxicol., 65, 618-624). In order to study the generality of this phenomenon, we have examined some N-substituted compounds. We report in vitro studies of inhibition and reactivation and aging of both NTE and acetylcholinesterase (AChE) prior to toxicological tests. All the compounds studied were less inhibitory to both NTE and AChE in concentrated rather than in dilute suspensions of EDTA-washed brain particles without added cofactors. There was an apparent disposal of up to 100 mumoles of test compound by particles from 95 mg hen brain, which is far greater than can be explained by covalent binding. The activity is distinct from calcium-dependent "A" esterase. Several N-alkyl phosphoromonoamidates were found to be potent and selective inhibitors of NTE: second-order rate constant for O-n-pentyl N-benzylphosphoramido-fluoridate (Cmpd 6) = 5.6 x 10(7) M-1 min-1 at 37 degrees, which is about 100x higher than for acetylcholinesterase (AChE). Inhibited NTE and AChE from several chiral phosphoromono-amidates did not reactivate spontaneously (21 hours at 37 degrees). Virtually 100% reactivation by KF of AChE inhibited by phosphoromonoamidates was achieved at all times tested. Acetylcholinesterase inhibited by 2,5-dichlorophenyl N,N'-di-n-butylphosphorodiamidate was 42-56% reactivated by incubation with KF (192 mM in pH 5.2 buffer for 30 minutes at 37 degrees). We believe this

  9. The thrombogenicity of C1 esterase inhibitor (human): review of the evidence.

    PubMed

    Crowther, Mark; Bauer, Kenneth A; Kaplan, Allen P

    2014-01-01

    Thromboembolic events associated with human plasma-derived C1 esterase inhibitor (C1-INH) use in patients with hereditary angioedema (HAE) have been reported in the U.S. Food and Drug Administration (FDA) Adverse Event Reporting System database. The purpose of this article is to review and assess the strength of available evidence regarding the thrombogenicity of human plasma-derived C1-INH. A PubMed search was conducted of English language articles from January 1990 to December 2013 reporting the thrombogenicity of C1-INH. Original research articles were selected if the following criteria were met: (1) C1-INH was the focus of the study and (2) the authors addressed the pro- or antithrombotic potential of C1-INH. Additional articles on the clinical use of C1-INH in disease states other than HAE were obtained using reference lists of selected articles. Pivotal studies and prescribing information for C1-INH products were also reviewed. Limited animal and clinical data suggest that C1-INH, particularly at high doses of up to 500 U/kg (compared with the U.S. FDA-approved 20-U/kg dose), may be prothrombotic. In contrast, C1-INH has been used in some patients with myocardial infarction, ischemic stroke, sepsis, and capillary leak syndrome at off-label supratherapeutic doses (up to 100 U/kg) without evidence of a thrombogenic effect. Based on our review, thromboembolic events reported with C1-INH use are rare and patients with HAE who experienced such events often have underlying thromboembolic risk factors.

  10. Biochemical comparison of four commercially available C1 esterase inhibitor concentrates for treatment of hereditary angioedema

    PubMed Central

    Feussner, Annette; Kalina, Uwe; Hofmann, Peter; Machnig, Thomas; Henkel, Georg

    2014-01-01

    Background For safe and efficacious treatment of hereditary angioedema, C1 esterase inhibitor (C1-INH) concentrates should have high purity and high amounts of functional protein. As no pharmacopoeia requirements exist for C1-INH concentrate lot release, biochemical characteristics as declared by the manufacturers may not be compared directly. This study compared the characteristics and purity profiles of four commercially available C1-INH concentrates. Study Design and Methods The analysis included one transgenic (Ruconest) and three plasma-derived (Berinert, Cetor, Cinryze) C1-INH concentrates. C1-INH antigen concentration was determined by nephelometry, total protein (specific activity) with a Bradford assay, purity by size-exclusion chromatography and gel electrophoresis, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was performed. Results Functionality (inversely proportional to antigen-to-activity ratio) was lowest for Ruconest (1.67), followed by Cetor (1.42), Berinert (1.24), and Cinryze (1.22). Specific activity (U/mg) and purity (%) were highest in Ruconest (12.13; 98.6) and Berinert (11.57; 97.0), followed by Cinryze (10.41; 89.5) and Cetor (9.01; 88.6). Main protein bands were found for all plasma-derived products at approximately 105 kDa, and for Ruconest, at approximately 98 kDa. Additional bands in the plasma-derived products were α1-antichymotrypsin, ceruloplasmin, Factor C3 (Cinryze/Cetor), and immunoglobulin heavy constant mu (Berinert). Conclusion Ruconest has a very high purity profile but is not identical to the human C1-INH protein. Of the plasma-derived products, Berinert has the highest purity profile. The impact of the nontherapeutic proteins identified has not yet been evaluated. For harmonization of the analysis for drug release, we recommend the establishment of regulatory requirements for purity determination and the implementation of threshold levels in C1-INH concentrates. PMID:24805006

  11. Efficacy of C1 esterase inhibitor concentrate in treatment of cutaneous attacks of hereditary angioedema

    PubMed Central

    Bork, Konrad; Craig, Timothy J.; Bernstein, Jonathan A.; Feuersenger, Henrike; Machnig, Thomas

    2015-01-01

    Background: Although treatment with C1 esterase inhibitor (C1-INH) concentrate is well established for hereditary angioedema (HAE) attacks in general, data that assess its efficacy for cutaneous attack treatment are sparse. Objective: To assess efficacy of plasma-derived, nanofiltered C1-INH concentrate for cutaneous attack treatment by comparing treated attacks from the uncontrolled I.M.P.A.C.T.2 study with historical data for untreated attacks. Methods: Cutaneous attack data from patients with HAE who were treated for cutaneous edema with 20 IU/kg body weight C1-INH concentrate in the uncontrolled I.M.P.A.C.T.2 study (38 patients) were compared with data from untreated patients from an historical data base (46 patients) and included subset analyses for facial edema (treated group, 21 patients; untreated group, 33 patients) and peripheral edema (30 patients in each group). Average attack duration (AAD) per patient was the efficacy end point used to compare treated and untreated patients. Differences were assessed with a Wilcoxon test (primary analysis) and a log-rank test; AAD per patient was analyzed descriptively and graphically with Kaplan-Meier curves. Results: The AAD per patient of all cutaneous attacks or facial and peripheral cutaneous attack subsets was significantly faster with C1-INH treatment than without treatment (Wilcoxon and log-rank tests, both p < 0.0001 for all comparisons). Mean AADs per patient for all, facial, and peripheral attacks were 2.04, 1.45, and 2.16 days, respectively, in the C1-INH–treated group, and were 3.74, 4.45, and 2.98 days, respectively, in the untreated group. Kaplan-Meier curves corroborated the observed group differences. Conclusion: Treatment of cutaneous HAE attacks (all attacks or facial and peripheral attack subsets) with 20 IU/kg C1-INH concentrate provided faster attack resolution compared with no treatment. PMID:25803207

  12. Organophosphates induce distal axonal damage, but not brain oedema, by inactivating neuropathy target esterase

    SciTech Connect

    Read, David J.; Li Yong; Chao, Moses V.; Cavanagh, John B.; Glynn, Paul

    2010-05-15

    Single doses of organophosphorus compounds (OP) which covalently inhibit neuropathy target esterase (NTE) can induce lower-limb paralysis and distal damage in long nerve axons. Clinical signs of neuropathy are evident 3 weeks post-OP dose in humans, cats and chickens. By contrast, clinical neuropathy in mice following acute dosing with OPs or any other toxic compound has never been reported. Moreover, dosing mice with ethyloctylphosphonofluoridate (EOPF) - an extremely potent NTE inhibitor - causes a different (subacute) neurotoxicity with brain oedema. These observations have raised the possibility that mice are intrinsically resistant to neuropathies induced by acute toxic insult, but may incur brain oedema, rather than distal axonal damage, when NTE is inactivated. Here we provide the first report that hind-limb dysfunction and extensive axonal damage can occur in mice 3 weeks after acute dosing with a toxic compound, bromophenylacetylurea. Three weeks after acutely dosing mice with neuropathic OPs no clinical signs were observed, but distal lesions were present in the longest spinal sensory axons. Similar lesions were evident in undosed nestin-cre:NTEfl/fl mice in which NTE had been genetically-deleted from neural tissue. The extent of OP-induced axonal damage in mice was related to the duration of NTE inactivation and, as reported in chickens, was promoted by post-dosing with phenylmethanesulfonylfluoride. However, phenyldipentylphosphinate, another promoting compound in chickens, itself induced in mice lesions different from the neuropathic OP type. Finally, EOPF induced subacute neurotoxicity with brain oedema in both wild-type and nestin-cre:NTEfl/fl mice indicating that the molecular target for this effect is not neural NTE.

  13. Strategies for increasing heterologous expression of a thermostable esterase from Archaeoglobus fulgidus in Escherichia coli.

    PubMed

    Kim, Jinyeong; Kim, Seul I; Hong, Eunsoo; Ryu, Yeonwoo

    2016-11-01

    Heterologous proteins expressed in bacteria are used for numerous biotechnological applications. Escherichia coli is the most commonly used host for heterologous protein expression because of its many advantages. Researchers have been studying proteins from extremophiles heterologously expressed in E. coli because the proteins of extremophiles are strongly resistant to extreme conditions. In a previous study, a thermostable esterase Est-AF was isolated from Archaeoglobus fulgidus and expressed in E. coli. However, further studies of Est-AF were difficult owing to its low expression levels in E. coli. In this study, we used various strategies, such as changing the expression vector and host strain, codon optimization, and optimization of induction conditions, to increase the expression of Est-AF. Through codon optimization and by changing the vector and host strain, Est-AF expression was increased from 31.50 ± 0.35 mg/L to 61.75 ± 0.28 mg/L. The optimized expression system consisted of a codon-optimized Est-AF gene in a pET28a(+)-based expression plasmid in E. coli Rosetta cells. The expression level was further increased by optimizing the induction conditions. The optimized conditions were induction with 0.4 mM isopropyl-b-d-1-thiogalactoside (IPTG) at 37 °C for 5 h. Under these conditions, the expression level of Est-AF was increased from 31.5 ± 0.35 mg/L to 119.52 ± 0.34 mg/L. PMID:27449918

  14. Nanofiltered C1 esterase inhibitor (human) for hereditary angioedema attacks in pregnant women.

    PubMed

    Baker, James W; Craig, Timothy J; Riedl, Marc A; Banerji, Aleena; Fitts, David; Kalfus, Ira N; Uknis, Marc E

    2013-01-01

    Data are limited on hereditary angioedema (HAE) in pregnant women and the safety and efficacy of therapies for treatment and prevention of HAE attacks during pregnancy. Prospective studies are unlikely given the rarity of HAE and ethical considerations regarding enrollment of pregnant female subjects in clinical trials. A retrospective analysis of clinical trial and compassionate-use data was conducted to identify subjects who received nanofiltered C1 esterase inhibitor (C1 INH-nf; human) during pregnancy. This study evaluates the efficacy and safety of human C1 INH-nf for treatment and prevention of HAE attacks in pregnant women. Data from pregnant subjects enrolled in either open-label extensions of two randomized, double-blind, placebo-controlled trials of C1 INH-nf or in a compassionate-use program were retrospectively analyzed for efficacy (e.g., total attacks, attack frequency during prophylaxis, and monthly attack rates) and safety (e.g., pregnancy outcomes and adverse events). C1 INH-nf was administered as acute treatment, preprocedural prophylaxis, or routine prophylaxis. C1 INH-nf prophylaxis substantially reduced monthly attack rates. Of 16 subjects, 13 delivered 14 healthy neonates (1 set of twins). Two adverse fetal outcomes were reported; neither was considered by the principal investigator to be related to C1 INH-nf. One subject's pregnancy outcome was unknown. This analysis shows a favorable risk-benefit profile for C1 INH-nf for managing HAE during pregnancy. NCT Identifier: NCT00438815; NCT00462709. PMID:23484892

  15. Avoidance behaviour response and esterase inhibition in the earthworm, Lumbricus terrestris, after exposure to chlorpyrifos.

    PubMed

    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. PMID:23435687

  16. Polyisoprenylated methylated protein methyl esterase overexpression and hyperactivity promotes lung cancer progression

    PubMed Central

    Amissah, Felix; Duverna, Randolph; Aguilar, Byron J; Poku, Rosemary A; Kiros, Gebre-Egziabher; Lamango, Nazarius S

    2014-01-01

    The involvement of hyperactive polyisoprenylated proteins in cancers has stimulated the search for drugs to target and suppress their excessive activities. Polyisoprenylated methylated protein methyl esterase (PMPMEase) inhibition has been shown to modulate polyisoprenylated protein function. For PMPMEase inhibition to be effective against cancers, polyisoprenylated proteins, the signaling pathways they mediate and/or PMPMEase must be overexpressed, hyperactive and be involved in at least some cases of cancer. PMPMEase activity in lung cancer cells and its expression in lung cancer cells and cancer tissues were investigated. PMPMEase was found to be overexpressed and significantly more active in lung cancer A549 and H460 cells than in normal lung fibroblasts. In a tissue microarray study, PMPMEase immunoreactivity was found to be significantly higher in lung cancer tissues compared to the normal controls (p < 0.0001). The mean scores ± SEM were 118.8 ± 7.7 (normal), 232.1 ± 25.1 (small-cell lung carcinomas), 352.1 ± 9.4 (squamous cell carcinomas), 311.7 ± 9.8 (adenocarcinomas), 350.0 ± 24.2 (papillary adenocarcinomas), 334.7 ± 30.1 (adenosquamous carcinomas), 321.9 ± 39.7 (bronchioloalveolar carcinomas), and 331.3 ± 85.0 (large-cell carcinomas). Treatment of lung cancer cells with L-28, a specific PMPMEase inhibitor, resulted in concentration-dependent cell death (EC50 of 8.5 μM for A549 and 2.8 μM for H460 cells). PMPMEase inhibition disrupted actin filament assembly, significantly inhibited cell migration and altered the transcription of cancer-related genes. These results indicate that elevated PMPMEase activity spur cell growth and migration, implying the possible use of PMPMEase as a protein biomarker and drug target for lung cancer. PMID:24660102

  17. Enlarging the substrate portfolio of the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius.

    PubMed

    Pennacchio, Angela; Mandrich, Luigi; Manco, Giuseppe; Trincone, Antonio

    2015-09-01

    The enzymatic regioselective hydrolysis of (a) acetylated mono- to tetrasaccharides of different nature, (b) of acetylated aryl glycosides and (c) of different acetylated nucleosides was studied enlarging the portfolio of substrates that can be employed by the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius. The reactions were optimised to the extent that the amount of enzyme needed was lowered of two orders of magnitude with respect to the previously reported reactions, namely from 4000 to 40 U of enzyme per reaction. New additional solvents were screened and dramatic changes in regioselectivity were observed depending on the amount and type of solvent used. For example, in the presence of 10 % DMF, only two α-D-glucose products 6-OH and 4,6-OH (in a 76:24 ratio) were detected, whereas with 25 % DMF, at least four products of similar amount were observed. This versatility adds specific value to the biocatalyst making possible the design of biocatalytic reactions with different hydrophobic ester substrates. As an additional remarkable example, EST2 catalysed with a good yield and high regioselectivity the hydrolysis of p-nitrophenyl β-D-xylopyranoside triacetate producing only the monoacetylated derivative with acetyl group in 3-O-position, in 2 min. The results with nucleosides as substrates are particularly interesting. The peracetates of 3',5'-di-O-acetylthymidine are converted almost quantitatively (95 %) to the monoacetylated derivative possessing free secondary OH; this regioselectivity is complementary to hydrolysis/alcoholysis reactions catalysed by CAL-B lipase or to other microbial hydrolytic biocatalysts, generally giving products with free primary OH groups. A docking analysis was undertaken with all analysed substrates suggesting a structural interpretation of the results. In most of cases, the best pose of the selected substrate was in line with the observed regioselectivity. PMID:26216109

  18. Neuropathy target esterase in mouse whole blood as a biomarker of exposure to neuropathic organophosphorus compounds.

    PubMed

    Makhaeva, Galina F; Rudakova, Elena V; Sigolaeva, Larisa V; Kurochkin, Ilya N; Richardson, Rudy J

    2016-11-01

    The adult hen is the standard animal model for testing organophosphorus (OP) compounds for organophosphorus compound-induced delayed neurotoxicity (OPIDN). Recently, we developed a mouse model for biochemical assessment of the neuropathic potential of OP compounds based on brain neuropathy target esterase (NTE) and acetylcholinesterase (AChE) inhibition. We carried out the present work to further develop the mouse model by testing the hypothesis that whole blood NTE inhibition could be used as a biochemical marker for exposure to neuropathic OP compounds. Because brain NTE and AChE inhibition are biomarkers of OPIDN and acute cholinergic toxicity, respectively, we compared NTE and AChE 20-min IC50 values as well as ED50 values 1 h after single intraperitoneal (i.p.) injections of increasing doses of two neuropathic OP compounds that differed in acute toxicity potency. We found good agreement between the brain and blood for in vitro sensitivity of each enzyme as well for the ratios IC50 (AChE)/IC50 (NTE). Both OP compounds inhibited AChE and NTE in the mouse brain and blood dose-dependently, and brain and blood inhibitions in vivo were well correlated for each enzyme. For both OP compounds, the ratio ED50 (AChE)/ED50 (NTE) in blood corresponded to that in the brain despite the somewhat higher sensitivity of blood enzymes. Thus, our results indicate that mouse blood NTE could serve as a biomarker of exposure to neuropathic OP compounds. Moreover, the data suggest that relative inhibition of blood NTE and AChE provide a way to assess the likelihood that OP compound exposure in a susceptible species would produce cholinergic and/or delayed neuropathic effects. Copyright © 2016 John Wiley & Sons, Ltd.

  19. Interdomain Hydrophobic Interactions Modulate the Thermostability of Microbial Esterases from the Hormone-Sensitive Lipase Family*

    PubMed Central

    Li, Ping-Yi; Chen, Xiu-Lan; Ji, Peng; Li, Chun-Yang; Wang, Peng; Zhang, Yi; Xie, Bin-Bin; Qin, Qi-Long; Su, Hai-Nan; Zhou, Bai-Cheng; Zhang, Yu-Zhong; Zhang, Xi-Ying

    2015-01-01

    Microbial hormone-sensitive lipases (HSLs) contain a CAP domain and a catalytic domain. However, it remains unclear how the CAP domain interacts with the catalytic domain to maintain the stability of microbial HSLs. Here, we isolated an HSL esterase, E40, from a marine sedimental metagenomic library. E40 exhibited the maximal activity at 45 °C and was quite thermolabile, with a half-life of only 2 min at 40 °C, which may be an adaptation of E40 to the permanently cold sediment environment. The structure of E40 was solved to study its thermolability. Structural analysis showed that E40 lacks the interdomain hydrophobic interactions between loop 1 of the CAP domain and α7 of the catalytic domain compared with its thermostable homologs. Mutational analysis showed that the introduction of hydrophobic residues Trp202 and Phe203 in α7 significantly improved E40 stability and that a further introduction of hydrophobic residues in loop 1 made E40 more thermostable because of the formation of interdomain hydrophobic interactions. Altogether, the results indicate that the absence of interdomain hydrophobic interactions between loop 1 and α7 leads to the thermolability of E40. In addition, a comparative analysis of the structures of E40 and other thermolabile and thermostable HSLs suggests that the interdomain hydrophobic interactions between loop 1 and α7 are a key element for the thermostability of microbial HSLs. Therefore, this study not only illustrates the structural element leading to the thermolability of E40 but also reveals a structural determinant for HSL thermostability. PMID:25771540

  20. Design of Fexofenadine Prodrugs Based on Tissue-Specific Esterase Activity and Their Dissimilar Recognition by P-Glycoprotein.

    PubMed

    Ohura, Kayoko; Nakada, Yuichiro; Kotani, Shunsuke; Imai, Teruko

    2015-09-01

    The aim of this study was to develop a suitable prodrug for fexofenadine (FXD), a model parent drug, that is resistant to intestinal esterase but converted to FXD by hepatic esterase. Carboxylesterases (CESs), human carboxylesterase 1 (hCE1) and human carboxylesterase 2 (hCE2), are the major esterases in human liver and intestine, respectively. These two CESs show quite different substrate specificities, and especially, hCE2 poorly hydrolyzes prodrugs with large acyl groups. FXD contains a carboxyl group and is poorly absorbed because of low membrane permeability and efflux by P-glycoprotein (P-gp). Therefore, two potential FXD prodrugs, ethyl-FXD and 2-hydroxyethyl-FXD, were synthesized by substitution of the carboxyl group in FXD. Both derivatives were resistant to intestinal hydrolysis, indicating their absorption as intact prodrugs. Ethyl-FXD was hydrolyzed by hepatic hCE1, but 2-hydroxyethyl-FXD was not. Both derivatives showed high membrane permeability in human P-gp-negative LLC-PK1 cells. In LLC-GA5-COL300 cells overexpressing human P-gp, ethyl-FXD was transported by P-gp, but its efflux was easily saturated. Whereas 2-hydroxyethyl-FXD showed more efficient P-gp-mediated transport than FXD. Although the structure of 2-hydroxyethyl-FXD only differs from ethyl-FXD by substitution of a hydroxyl group, 2-hydroxyethyl-FXD is unsuitable as a prodrug. However, ethyl-FXD is a good candidate prodrug because of good intestinal absorption and hepatic conversion by hCE1.

  1. Pegylation improves the pharmacokinetics and bioavailability of small-molecule drugs hydrolyzable by esterases: a study of phospho-Ibuprofen.

    PubMed

    Mattheolabakis, George; Wong, Chi C; Sun, Yu; Amella, Carol A; Richards, Robert; Constantinides, Panayiotis P; Rigas, Basil

    2014-10-01

    Esterase hydrolysis of drugs can accelerate their elimination, thereby limiting their efficacy. Polyethylene glycol (PEG) covalently attached to drugs (pegylation) is known to improve the efficiency of many drugs. Using as a test agent the novel phospho-ibuprofen (PI), we examined whether pegylation of PI could abrogate its hydrolytic degradation by esterases; PI, known to inhibit colon cancer growth, has a carboxylic ester hydrolyzable by carboxylesterases (CES). We covalently attached mPEG-2000 to PI (PI-PEG) and studied its stability by exposing it to cells overexpressing CES and by administering it to mice. We also evaluated PI-PEG's anticancer efficacy in human colon cancer xenografts and in Apc(min/+) mice. PI-PEG was stable in the presence of cells overexpressing CES1 or CES2, whereas PI was extensively hydrolyzed (90.2 ± 0.7%, 14.3 ± 1.1%, mean ± S.E.M.). In mice, PI was nearly completely hydrolyzed. Intravenous administration of PI-PEG resulted in significant levels in blood and in colon cancer xenografts (xenograft values in parentheses): area under the curve for 0-24 hours = 2351 (2621) (nmol/g) × h; Cmax = 1965 (886) nmol/g; Tmax = 0.08 (2) hour. The blood levels of ibuprofen, its main hydrolytic product, were minimal. Compared with controls, PI-PEG inhibited the growth of the xenografts by 74.8% (P < 0.01) and reduced intestinal tumor multiplicity in Apc(min/+) mice by 73.1% (P < 0.01), prolonging their survival (100% versus 55.1% of controls; P = 0.013). Pegylation protects PI from esterase hydrolysis and improves its pharmacokinetics. In preclinical models of colon cancer, PI-PEG is a safe and efficacious agent that merits further evaluation.

  2. A membrane-bound esterase PA2949 from Pseudomonas aeruginosa is expressed and purified from Escherichia coli.

    PubMed

    Kovacic, Filip; Bleffert, Florian; Caliskan, Muttalip; Wilhelm, Susanne; Granzin, Joachim; Batra-Safferling, Renu; Jaeger, Karl-Erich

    2016-05-01

    Pseudomonas aeruginosa strain 1001 produces an esterase (EstA) that can hydrolyse the racemic methyl ester of β-acetylthioisobutyrate to produce the (D)-enantiomer, which serves as a precursor of captopril, a drug used for treatment of hypertension. We show here that PA2949 from P. aeruginosa PA01, a homologue of EstA, can efficiently be expressed in an enzymatically active form in E. coli. The enzyme is membrane-associated as demonstrated by cell fractionation studies. PA2949 was purified to homogeneity after solubilisation with the nonionic detergent, Triton X-100, and was shown to possess a conserved esterase catalytic triad consisting of Ser137-His258-Asp286. Our results should allow the development of an expression and purification strategy to produce this biotechnologically relevant esterase in a pure form with a high yield.

  3. A membrane-bound esterase PA2949 from Pseudomonas aeruginosa is expressed and purified from Escherichia coli.

    PubMed

    Kovacic, Filip; Bleffert, Florian; Caliskan, Muttalip; Wilhelm, Susanne; Granzin, Joachim; Batra-Safferling, Renu; Jaeger, Karl-Erich

    2016-05-01

    Pseudomonas aeruginosa strain 1001 produces an esterase (EstA) that can hydrolyse the racemic methyl ester of β-acetylthioisobutyrate to produce the (D)-enantiomer, which serves as a precursor of captopril, a drug used for treatment of hypertension. We show here that PA2949 from P. aeruginosa PA01, a homologue of EstA, can efficiently be expressed in an enzymatically active form in E. coli. The enzyme is membrane-associated as demonstrated by cell fractionation studies. PA2949 was purified to homogeneity after solubilisation with the nonionic detergent, Triton X-100, and was shown to possess a conserved esterase catalytic triad consisting of Ser137-His258-Asp286. Our results should allow the development of an expression and purification strategy to produce this biotechnologically relevant esterase in a pure form with a high yield. PMID:27419054

  4. The kinetics and inhibition of p-nitrophenylacetate-hydrolysing esterases in a solitary bee, Megachile rotundata (Fab.).

    PubMed

    Frohlich, D R; Boeker, E A; Brindley, W A

    1990-05-01

    1. The kinetics and inhibition of p-nitrophenylacetate hydrolysis by cytosolic esterases of female alfalfa leafcutting bees, Megachile rotundata (Fab.) was examined. 2. For p-nitrophenylacetate, the Km = 1.24 x 10(-4) M and Vmax = 2.29 x 10(-9) mol/s per mg protein. 3. Regarding four organophosphate insecticides, the mechanism of inhibition in all cases was mixed (competitive and uncompetitive) and, based on inhibition constants, the order of toxicity was naled greater than paraoxon greater than trichlorfon greater than oxydemeton methyl. 4. Comparisons are made to the honey bee, Apis mellifera. PMID:2349807

  5. Hereditary and acquired angioedema: problems and progress: proceedings of the third C1 esterase inhibitor deficiency workshop and beyond.

    PubMed

    Agostoni, Angelo; Aygören-Pürsün, Emel; Binkley, Karen E; Blanch, Alvaro; Bork, Konrad; Bouillet, Laurence; Bucher, Christoph; Castaldo, Anthony J; Cicardi, Marco; Davis, Alvin E; De Carolis, Caterina; Drouet, Christian; Duponchel, Christiane; Farkas, Henriette; Fáy, Kálmán; Fekete, Béla; Fischer, Bettina; Fontana, Luigi; Füst, George; Giacomelli, Roberto; Gröner, Albrecht; Hack, C Erik; Harmat, George; Jakenfelds, John; Juers, Mathias; Kalmár, Lajos; Kaposi, Pál N; Karádi, István; Kitzinger, Arianna; Kollár, Tímea; Kreuz, Wolfhart; Lakatos, Peter; Longhurst, Hilary J; Lopez-Trascasa, Margarita; Martinez-Saguer, Inmaculada; Monnier, Nicole; Nagy, István; Németh, Eva; Nielsen, Erik Waage; Nuijens, Jan H; O'grady, Caroline; Pappalardo, Emanuela; Penna, Vincenzo; Perricone, Carlo; Perricone, Roberto; Rauch, Ursula; Roche, Olga; Rusicke, Eva; Späth, Peter J; Szendei, George; Takács, Edit; Tordai, Attila; Truedsson, Lennart; Varga, Lilian; Visy, Beáta; Williams, Kayla; Zanichelli, Andrea; Zingale, Lorenza

    2004-09-01

    Hereditary angioedema (HAE), a rare but life-threatening condition, manifests as acute attacks of facial, laryngeal, genital, or peripheral swelling or abdominal pain secondary to intra-abdominal edema. Resulting from mutations affecting C1 esterase inhibitor (C1-INH), inhibitor of the first complement system component, attacks are not histamine-mediated and do not respond to antihistamines or corticosteroids. Low awareness and resemblance to other disorders often delay diagnosis; despite availability of C1-INH replacement in some countries, no approved, safe acute attack therapy exists in the United States. The biennial C1 Esterase Inhibitor Deficiency Workshops resulted from a European initiative for better knowledge and treatment of HAE and related diseases. This supplement contains work presented at the third workshop and expanded content toward a definitive picture of angioedema in the absence of allergy. Most notably, it includes cumulative genetic investigations; multinational laboratory diagnosis recommendations; current pathogenesis hypotheses; suggested prophylaxis and acute attack treatment, including home treatment; future treatment options; and analysis of patient subpopulations, including pediatric patients and patients whose angioedema worsened during pregnancy or hormone administration. Causes and management of acquired angioedema and a new type of angioedema with normal C1-INH are also discussed. Collaborative patient and physician efforts, crucial in rare diseases, are emphasized. This supplement seeks to raise awareness and aid diagnosis of HAE, optimize treatment for all patients, and provide a platform for further research in this rare, partially understood disorder.

  6. Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding.

    PubMed

    Chen, Qi; Luan, Zheng-Jiao; Yu, Hui-Lei; Cheng, Xiaolin; Xu, Jian-He

    2015-11-01

    A new carboxylic esterase RhEst1 which catalyzes the hydrolysis of (S)-(+)-2,2-dimethylcyclopropanecarboxylate (S-DmCpCe), the key chiral building block of cilastatin, was identified and subsequently crystallized in our previous work. Mutant RhEst1A147I/V148F/G254A was found to show a 5-fold increase in the catalytic activity. In this work, molecular dynamic simulations were performed to elucidate the molecular determinant of the enzyme activity. Our simulations show that the substrate binds much more strongly in the A147I/V148F/G254A mutant than in wild type, with more hydrogen bonds formed between the substrate and the catalytic triad and the oxyanion hole. The OH group of the catalytic residue Ser101 in the mutant is better positioned to initiate the nucleophilic attack on S-DmCpCe. Interestingly, the "170-179" loop which is involved in shaping the catalytic sites and facilitating the product release shows remarkable dynamic differences in the two systems. Based on the simulation results, six residues were identified as potential "hot-spots" for further experimental testing. Consequently, the G126S and R133L mutants show higher catalytic efficiency as compared with the wild type. This work provides molecular-level insights into the substrate binding mechanism of carboxylic esterase RhEst1, facilitating future experimental efforts toward developing more efficient RhEst1 variants for industrial applications.

  7. Enhancement of acetyl xylan esterase activity on cellulose acetate through fusion to a family 3 cellulose binding module.

    PubMed

    Mai-Gisondi, Galina; Turunen, Ossi; Pastinen, Ossi; Pahimanolis, Nikolaos; Master, Emma R

    2015-11-01

    The current study investigates the potential to increase the activity of a family 1 carbohydrate esterase on cellulose acetate through fusion to a family 3 carbohydrate binding module (CBM). Specifically, CtCBM3 from Clostridium thermocellum was fused to the carboxyl terminus of the acetyl xylan esterase (AnAXE) from Aspergillus nidulans, and active forms of both AnAXE and AnAXE-CtCBM3 were produced in Pichia pastoris. CtCBM3 fusion had negligible impact on the thermostability or regioselectivity of AnAXE; activities towards acetylated corncob xylan, 4-methylumbelliferyl acetate, p-nitrophenyl acetate, and cellobiose octaacetate were also unchanged. By contrast, the activity of AnAXE-CtCBM3 on cellulose acetate increased by two to four times over 24 h, with greater differences observed at earlier time points. Binding studies using microcrystalline cellulose (Avicel) and a commercial source of cellulose acetate confirmed functional production of the CtCBM3 domain; affinity gel electrophoresis using acetylated xylan also verified the selectivity of CtCBM3 binding to cellulose. Notably, gains in enzyme activity on cellulose acetate appeared to exceed gains in substrate binding, suggesting that fusion to CtCBM3 increases functional associations between the enzyme and insoluble, high molecular weight cellulosic substrates.

  8. Genetic variability in the natural populations of Lasioderma serricorne (F.) (Coleoptera: Anobiidae), detected by RAPD markers and by esterase isozymes.

    PubMed

    Coelho-Bortolo, T; Mangolin, C A; Lapenta, A S

    2016-02-01

    Lasioderma serricorne (F.) is a small cosmopolitan beetle regarded as a destructive pest of several stored products such as grains, flour, spices, dried fruit and tobacco. Chemical insecticides are one of the measures used against the pest. However, intensive insecticide use has resulted in the appearance of resistant insect populations. Therefore, for the elaboration of more effective control programs, it is necessary to know the biological aspects of L. serricorne. Among these aspects, the genetic variability knowledge is very important and may help in the development of new control methods. The objective of this study was to evaluate the genetic variability of 11 natural populations of L. serricorne collected respectively in three and four towns in the states of Paraná and São Paulo, Brazil, using 20 primers random amplified polymorphic DNA (RAPD) and polymorphisms of esterases. These primers produced 352 polymorphic bands. Electrophoretic analysis of esterases allowed the identification of four polymorphic loci (Est-2, Est-4, Est-5 and Est-6) and 18 alleles. Results show that populations are genetically differentiated and there is a high level of genetic variability within populations. The high degree of genetic differentiation is not directly correlated to geographical distance. Thus, our data indicate that movement of infested commodities may contribute to the dissemination of L. serricorne, facilitating gene flow.

  9. Temephos resistance and esterase activity in the mosquito Aedes aegypti in Havana, Cuba increased dramatically between 2006 and 2008.

    PubMed

    Bisset, J A; Rodríguez, M M; Ricardo, Y; Ranson, H; Pérez, O; Moya, M; Vázquez, A

    2011-09-01

    Aedes aegypti (L.) (Diptera: Culicidae) control programmes in Cuba rely on the application of the organophosphate temephos for larval control. Hence, the monitoring of resistance to this insecticide is an essential component of such programmes. Here, 15 field populations from different municipalities of Havana City were assayed for resistance to temephos. High levels of resistance were detected in all strains and resistance ratios were highly correlated with esterase activity (P = 0.00001). Populations from three municipalities were tested in both 2006 and 2008; resistance and esterase activities both significantly increased during this 2-year period. Synergist studies demonstrated that neither glutathione transferases nor monooxygenases were associated with the increase in resistance to temephos in this period. The duration of the efficacy of commercial formulations of temephos in controlling Ae. aegypti populations in Havana City was reduced by the high level of temephos resistance observed; hence these data are of clear operational significance for the dengue control programme in Cuba. New integrated strategies to avoid further increases in temephos resistance in Cuba are necessary.

  10. A carboxylesterase, Esterase-6, modulates sensory physiological and behavioral response dynamics to pheromone in Drosophila

    PubMed Central

    2012-01-01

    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 antennae. Our results

  11. Thermodynamically based solvent design for enzymatic saccharide acylation with hydroxycinnamic acids in non-conventional media.

    PubMed

    Zeuner, Birgitte; Kontogeorgis, Georgios M; Riisager, Anders; Meyer, Anne S

    2012-02-15

    Enzyme-catalyzed synthesis has been widely studied with lipases (EC 3.1.1.3), but feruloyl esterases (FAEs; EC 3.1.1.73) may provide advantages such as higher substrate affinity and regioselectivity in the synthesis of hydroxycinnamate saccharide esters. These compounds are interesting because of their amphiphilicity and antioxidative potential. Synthetic reactions using mono- or disaccharides as one of the substrates may moreover direct new routes for biomass upgrading in the biorefinery. The paper reviews the available data for enzymatic hydroxycinnamate saccharide ester synthesis in organic solvent systems as well as other enzymatic hydroxycinnamate acylations in ionic liquid systems. The choice of solvent system is highly decisive for enzyme stability, selectivity, and reaction yields in these synthesis reactions. To increase the understanding of the reaction environment and to facilitate solvent screening as a crucial part of the reaction design, the review explores the use of activity coefficient models for describing these systems and - more importantly - the use of group contribution model UNIFAC and quantum chemistry based COSMO-RS for thermodynamic predictions and preliminary solvent screening. Surfactant-free microemulsions of a hydrocarbon, a polar alcohol, and water are interesting solvent systems because they accommodate different substrate and product solubilities and maintain enzyme stability. Ionic liquids may provide advantages as solvents in terms of increased substrate and product solubility, higher reactivity and selectivity, as well as tunable physicochemical properties, but their design should be carefully considered in relation to enzyme stability. The treatise shows that thermodynamic modeling tools for solvent design provide a new toolbox to design enzyme-catalyzed synthetic reactions from biomass sources. PMID:22154740

  12. Isomer-specific comparisons of the hydrolysis of synthetic pyrethroids and their fluorogenic analogues by esterases from the cotton bollworm Helicoverpa armigera.

    PubMed

    Yuan, G; Li, Y; Farnsworth, C A; Coppin, C W; Devonshire, A L; Scott, C; Russell, R J; Wu, Y; Oakeshott, J G

    2015-06-01

    The low aqueous solubility and chiral complexity of synthetic pyrethroids, together with large differences between isomers in their insecticidal potency, have hindered the development of meaningful assays of their metabolism and metabolic resistance to them. To overcome these problems, Shan and Hammock (2001) [7] therefore developed fluorogenic and more water-soluble analogues of all the individual isomers of the commonly used Type 2 pyrethroids, cypermethrin and fenvalerate. The analogues have now been used in several studies of esterase-based metabolism and metabolic resistance. Here we test the validity of these analogues by quantitatively comparing their hydrolysis by a battery of 22 heterologously expressed insect esterases with the hydrolysis of the corresponding pyrethroid isomers by these esterases in an HPLC assay recently developed by Teese et al. (2013) [14]. We find a strong, albeit not complete, correlation (r = 0.7) between rates for the two sets of substrates. The three most potent isomers tested were all relatively slowly degraded in both sets of data but three esterases previously associated with pyrethroid resistance in Helicoverpa armigera did not show higher activities for these isomers than did allelic enzymes derived from susceptible H. armigera. Given their amenability to continuous assays at low substrate concentrations in microplate format, and ready detection of product, we endorse the ongoing utility of the analogues in many metabolic studies of pyrethroids. PMID:26047117

  13. Induction of cutinolytic esterase activity during saprophytic growth of cucurbit pathogens, Fusarium solani f. sp. cucurbitae races one and two (Nectria haematococca MPI and MPV, respectively).

    PubMed

    Hawthorne, B T; Rees-George, J; Crowhurst, R N

    2001-01-15

    Cutins from fruit of Cucurbita maxima and Cucurbita moschata cultivars, apple and a C(16) alcohol (hexadecanol) were used to induce cutinolytic esterase activity during saprophytic growth of strains of the two cucurbit pathogens, Fusarium solani f. sp. cucurbitae, race 1 (Nectria haematococca mating population (MPI) and F. solani f. sp. cucurbitae, race 2 (MPV). Four strains of MPV and 11 strains of MPI were were included in the study. Although we were primarily interested in the two cucurbit pathogens (MPI and MPV), six strains of the pea pathogen F. solani f. sp. pisi (MPVI) were included to provide a comparison since most of the knowledge on cutinase activity in N. haematococca has come from a study of that group. Cutinolytic esterase was induced in all strains from both MPV and MPVI but was not detected in any of the 11 strains from MPI regardless of the induction conditions. The amount of cutinolytic esterase activity induced in the MPV strains differed according to the strain and both the source and the amount of cutin used in the induction medium. Information on the influence of cutin source and pH on the induction of cutinolytic esterase activity during saprophytic growth of strains from MPV demonstrates that the gene is regulated differently from that in MPVI.

  14. Isomer-specific comparisons of the hydrolysis of synthetic pyrethroids and their fluorogenic analogues by esterases from the cotton bollworm Helicoverpa armigera.

    PubMed

    Yuan, G; Li, Y; Farnsworth, C A; Coppin, C W; Devonshire, A L; Scott, C; Russell, R J; Wu, Y; Oakeshott, J G

    2015-06-01

    The low aqueous solubility and chiral complexity of synthetic pyrethroids, together with large differences between isomers in their insecticidal potency, have hindered the development of meaningful assays of their metabolism and metabolic resistance to them. To overcome these problems, Shan and Hammock (2001) [7] therefore developed fluorogenic and more water-soluble analogues of all the individual isomers of the commonly used Type 2 pyrethroids, cypermethrin and fenvalerate. The analogues have now been used in several studies of esterase-based metabolism and metabolic resistance. Here we test the validity of these analogues by quantitatively comparing their hydrolysis by a battery of 22 heterologously expressed insect esterases with the hydrolysis of the corresponding pyrethroid isomers by these esterases in an HPLC assay recently developed by Teese et al. (2013) [14]. We find a strong, albeit not complete, correlation (r = 0.7) between rates for the two sets of substrates. The three most potent isomers tested were all relatively slowly degraded in both sets of data but three esterases previously associated with pyrethroid resistance in Helicoverpa armigera did not show higher activities for these isomers than did allelic enzymes derived from susceptible H. armigera. Given their amenability to continuous assays at low substrate concentrations in microplate format, and ready detection of product, we endorse the ongoing utility of the analogues in many metabolic studies of pyrethroids.

  15. Interactive toxicity of chlorpyrifos and parathion in neonatal rats: Role of esterases in exposure sequence-dependent toxicity

    SciTech Connect

    Kacham, R.; Karanth, S.; Baireddy, P.; Liu, J.; Pope, C. . E-mail: carey.pope@okstate.edu

    2006-01-15

    We previously reported that sequence of exposure to chlorpyrifos and parathion in adult rats can markedly influence toxic outcome. In the present study, we evaluated the interactive toxicity of chlorpyrifos (8 mg/kg, po) and parathion (0.5 mg/kg, po) in neonatal (7 days old) rats. Rats were exposed to the insecticides either concurrently or sequentially (separated by 4 h) and sacrificed at 4, 8, and 24 h after the first exposure for biochemical measurements (cholinesterase activity in brain, plasma, and diaphragm and carboxylesterase activity in plasma and liver). The concurrently-exposed group showed more cumulative lethality (15/24) than either of the sequential dosing groups. With sequential dosing, rats treated initially with chlorpyrifos prior to parathion (C/P) exhibited higher lethality (7/23) compared to those treated with parathion before chlorpyrifos (P/C; 1/24). At 8 h after initial dosing, brain cholinesterase inhibition was significantly greater in the C/P group (59%) compared to the P/C group (28%). Diaphragm and plasma cholinesterase activity also followed a relatively similar pattern of inhibition. Carboxylesterase inhibition in plasma and liver was relatively similar among the treatment groups across time-points. Similar sequence-dependent differences in brain cholinesterase inhibition were also noted with lower binary exposures to chlorpyrifos (2 mg/kg) and parathion (0.35 mg/kg). In vitro and ex vivo studies compared relative oxon detoxification of carboxylesterases (calcium-insensitive) and A-esterases (calcium-sensitive) in liver homogenates from untreated and insecticide pretreated rats. Using tissues from untreated rats, carboxylesterases detoxified both chlorpyrifos oxon and paraoxon, while A-esterases only detoxified chlorpyrifos oxon. With parathion pretreatment, A-esterases still detoxified chlorpyrifos oxon while liver from chlorpyrifos pretreated rats had little apparent effect on paraoxon. We conclude that while neonatal rats are less

  16. Role of the N terminus in enzyme activity, stability and specificity in thermophilic esterases belonging to the HSL family.

    PubMed

    Mandrich, Luigi; Merone, Luigia; Pezzullo, Margherita; Cipolla, Laura; Nicotra, Francesco; Rossi, Mosè; Manco, Giuseppe

    2005-01-21

    A superposition between the structures of Alicyclobacillus acidocaldarius esterase 2 (EST2) and Burkholderia cepacia lipase, the latter complexed with a phosphonate inhibitor, allowed us to hypothesize for the EST2 N terminus a role in restricting the access to the active site and therefore in modulating substrate specificity. In order to test this hypothesis we generated by site-directed mutagenesis some truncated versions of EST2 and its double mutant M211S/R215L (S/L) at the N terminus. In parallel, an analysis of the Sulfolobus solfataricus P2 genome allowed us to identify a gene coding for a putative esterase of the HSL family having a natural deletion of the corresponding region. The product of this gene and the above-mentioned EST2 mutants were expressed in Escherichia coli, purified and characterised. These studies support the notion that the N terminus affects substrate specificity other than several other enzyme parameters. Although the deletions afforded a tenfold and 550-fold decrease in catalytic efficiency towards the best substrate pNP-hexanoate at 50 degrees C for EST2 and S/L, respectively, the analysis of the specific activities with different triacylglycerols with respect to pNP-hexanoate showed that their ratios were higher for deleted versus non-deleted enzymes, on all tested substrates. In particular, the above ratios for glyceryl tridecanoate were 30-fold and 14-fold higher in S/L and EST2 deleted forms, respectively, compared with their full-length versions. This behaviour was confirmed by the analysis of the S.solfataricus esterase, which showed similar specific activities on pNP-hexanoate and triacylglycerols; in addition, higher activities on the latter substrates were observed in comparison with EST2, S/L and their deleted forms. Finally, a dramatic effect on thermophilicity and thermostability in the EST2 deleted forms was observed. This is the first report highlighting the importance of the "cap" domain in the HSL family, since the N

  17. Determination of organophosphorus pesticide residues in vegetables by an enzyme inhibition method using α-naphthyl acetate esterase extracted from wheat flour*

    PubMed Central

    Wang, Jun-liang; Xia, Qing; Zhang, An-ping; Hu, Xiao-yan; Lin, Chun-mian

    2012-01-01

    The widespread use of organophosphorus pesticides (OPs) poses a great threat to human health and has made the detection of OP residues in food an important task, especially in view of the fact that easy and rapid detection methods are needed. Because OPs have inhibitory effects on the activity of α-naphthyl acetate esterase (ANAE) in plants, in this work we evaluated the possibility of detecting OPs in vegetables with ANAE extracted from commercial flour. The limits of detection (LODs) obtained for methamidophos, dichlorvos, phoxim, dimethoate, and malathion in lettuce samples with crude ANAE were 0.17, 0.11, 0.11, 0.96, and 1.70 mg/kg, respectively. Based on the maximum residue limits (MRLs) for OPs in food stipulated by Chinese laws which are 0.05, 0.20, 0.05, 1.00, and 8.00 mg/kg for methamidophos, dichlorvos, phoxim, dimethoate, and malathion, respectively, the esterase inhibition method with crude ANAE had sufficient sensitivity to detect the residues of dichlorvos, dimethoate, and malathion in lettuce, but it could not be used to guarantee the safety of the same samples if methamidophos or phoxim residue was present. The sensitivity of the method was improved by the use of esterase purified by ammonium sulfate salting-out. The LODs obtained for methamidophos and phoxim with purified esterase were lower than the MRLs for these OPs in food. This is a very promising method for the detection of OP residues in vegetables using crude or purified esterase because of its cheapness, sensitivity, and convenience. PMID:22467368

  18. Esterase and glutathione S-transferase levels associated with synthetic pyrethroid resistance in Hyalomma anatolicum and Rhipicephalus microplus ticks from Punjab, India.

    PubMed

    Nandi, Abhijit; Jyoti; Singh, Harkirat; Singh, Nirbhay Kumar

    2015-05-01

    Larval packet test was used for assessment of resistance status against cypermethrin and deltamethrin in Hyalomma anatolicum and Rhipicephalus microplus from various districts of Punjab (India). Among the various field isolates of H. anatolicum susceptible status was recorded against cypermethrin in all isolates, whereas against deltamethrin resistance status (level I-III) was recorded. In R. microplus lower resistance levels (I-II) were recorded against cypermethrin in comparison to deltamethrin (level I-IV). Quantitative analysis of general esterase activity revealed a range of 4.21 ± 0.46 to 6.05 ± 0.55 and 2.23 ± 0.23 to 2.66 ± 0.24 µmol/min/mg protein for α- and β-esterase activity, respectively, in different field isolates of H. anatolicum and the increase in comparison to susceptible was not significant (P > 0.05). In contrast to H. anatolicum, the α- and β-esterase activity in all field isolates (except Jalandhar) of R. microplus was higher (range of 3.89 ± 0.26 to 10.85 ± 0.47 and 1.75 ± 0.08 to 5.87 ± 0.29 µmol/min/mg protein, respectively) (P < 0.001). The glutathione-S-transferase (GST) activity in field isolates of H. anatolicum and R. microplus was in the range of 0.01 ± 0.001 to 0.03 ± 0.001 and 0.02 ± 0.0003 to 0.03 ± 0.001 mM/mg/min. The enzyme ratios (α-and β-esterase and GST) and RR95 against deltamethrin of H. anatolicum isolates were correlated (P < 0.05), whereas in R. microplus only α-and β-esterase and RR50 against deltamethrin were correlated (P < 0.05).

  19. Detection of carboxylesterase and esterase activity in culturable gut bacterial flora isolated from diamondback moth, Plutella xylostella (Linnaeus), from India and its possible role in indoxacarb degradation.

    PubMed

    Ramya, Shanivarsanthe Leelesh; Venkatesan, Thiruvengadam; Srinivasa Murthy, Kottilingam; Jalali, Sushil Kumar; Verghese, Abraham

    2016-01-01

    Diamondback moth (DBM), Plutella xylostella (Linnaeus), is a notorious pest of brassica crops worldwide and is resistant to all groups of insecticides. The insect system harbors diverse groups of microbiota, which in turn helps in enzymatic degradation of xenobiotic-like insecticides. The present study aimed to determine the diversity of gut microflora in DBM, quantify esterase activity and elucidate their possible role in degradation of indoxacarb. We screened 11 geographic populations of DBM in India and analyzed them for bacterial diversity. The culturable gut bacterial flora underwent molecular characterization with 16S rRNA. We obtained 25 bacterial isolates from larvae (n=13) and adults (n=12) of DBM. In larval gut isolates, gammaproteobacteria was the most abundant (76%), followed by bacilli (15.4%). Molecular characterization placed adult gut bacterial strains into three major classes based on abundance: gammaproteobacteria (66%), bacilli (16.7%) and flavobacteria (16.7%). Esterase activity from 19 gut bacterial isolates ranged from 0.072 to 2.32μmol/min/mg protein. Esterase bands were observed in 15 bacterial strains and the banding pattern differed in Bacillus cereus - KC985225 and Pantoea agglomerans - KC985229. The bands were characterized as carboxylesterase with profenofos used as an inhibitor. Minimal media study showed that B. cereus degraded indoxacarb up to 20%, so it could use indoxacarb for metabolism and growth. Furthermore, esterase activity was greater with minimal media than control media: 1.87 versus 0.26μmol/min/mg protein. Apart from the insect esterases, bacterial carboxylesterase may aid in the degradation of insecticides in DBM. PMID:26991291

  20. Increase of gluthatione S-transferase, carboxyl esterase and carbonyl reductase in Fasciola hepatica recovered from triclabendazole treated sheep.

    PubMed

    Scarcella, S; Solana, M V; Fernandez, V; Lamenza, P; Ceballos, L; Solana, H

    2013-10-01

    Fasciolasis is a zoonotic parasitic disease caused by Fasciola hepatica and its control is mainly based on the use of triclabendazole (TCBZ). Parasite resistance to different anthelmintics is growing worldwide, including the resistance of F. hepatica to TCBZ. In the present work we evaluate "in vivo" the activity of xenobiotic metabolizing enzymes of phase I (carboxyl esterases) and phase II (glutathione S-transferases and carbonyl reductases) recovered of flukes from sheep treated with TCBZ. All three enzymes showed increased activity in TCBZ flukes returning 60h post-treatment at similar to baseline unexposed flukes. TCBZ action may induce secondary oxidative stress, which may explain the observed increment in activities of the analyzed enzymes as a defensive mechanism. The enzymes analyzed are candidates to participate actively in the development of resistance at TCBZ in F. hepatica.

  1. Human neural stem cells expressing carboxyl esterase target and inhibit tumor growth of lung cancer brain metastases.

    PubMed

    Hong, S H; Lee, H J; An, J; Lim, I; Borlongan, C; Aboody, K S; Kim, S U

    2013-12-01

    Neural stem cells (NSCs) led to the development of a novel strategy for delivering therapeutic genes to brain tumors. Human NSCs expressing rabbit carboxyl esterase (F3.CE), which activates CPT-11, significantly inhibit the growth of A549 human non-small cell lung adenocarcinoma cells in the presence of CPT-11 in vitro and in vivo. F3.CE cells migrated selectively into the brain metastases located in the opposite hemisphere. The treatment also significantly decreased tumor volume in immune-deficient mice bearing lung cancer when F3.CE cells were transplanted into the contralateral hemisphere. The survival of tumor-bearing animals was significantly prolonged by the treatment with F3.CE and CPT-11. This strategy could be considered as an effective treatment regimen for lung cancer brain metastases.

  2. Simultaneous determination of curcumin diethyl disuccinate and its active metabolite curcumin in rat plasma by LC-MS/MS: Application of esterase inhibitors in the stabilization of an ester-containing prodrug.

    PubMed

    Ratnatilaka Na Bhuket, Pahweenvaj; Niwattisaiwong, Nuansri; Limpikirati, Patanachai; Khemawoot, Phisit; Towiwat, Pasarapa; Ongpipattanakul, Boonsri; Rojsitthisak, Pornchai

    2016-10-15

    Four esterase inhibitors, ethylenediamine tetraacetic acid disodium (Na2EDTA), sodium fluoride (NaF), bis(4-nitrophenyl) phosphate (BNPP) and phenylmethanesulfonyl fluoride (PMSF), were evaluated for their inhibitory effects on enzymatic hydrolysis of labile phenolate esters in curcumin diethyl disuccinate (CDD), a prodrug of curcumin (CUR), in rat plasma. BNPP and PMSF at 10mM exhibited stabilization by preventing degradation of CDD. BNPP at a final concentration of 10mM was subsequently selected to prevent ex vivo metabolism of CDD throughout LC-MS/MS analysis of CDD and CUR in rat plasma. A simple protein precipitation technique using acetonitrile as a precipitating agent was used to extract CDD, CUR and dimethylcurcumin (DMC), an internal standard, from rat plasma. Chromatographic separation was performed on a Halo C8 column (4.6×50mm, 2.7μm) using an isocratic mobile phase containing acetonitrile-0.2% formic acid in water (73:27v/v) with a flow rate of 0.4mLmin(-1). An AB SCIEX QTRAP(®) 6500 mass spectrometer was operated using a positive ion electrospray mode for ionization and detection of analytes and internal standard. Calibration curves for CDD and CUR were established using 50μL of rat plasma over the concentration range of 1-500ngmL(-1). The developed method was fully validated according to US Food and Drug Administration (FDA) guidelines for selectivity, sensitivity, linearity, accuracy, precision, dilution integrity, recovery, matrix effect, and stability. The validated method was applied to evaluate the pharmacokinetics of CDD and CUR in rats after a single intravenous dose of 40mgkg(-1). The method using BNPP as an esterase inhibitor was successful in determining the remaining CDD in rat plasma. The pharmacokinetic results indicate that CDD in rats is converted instantaneously to CUR after intravenous administration and a higher CUR plasma concentration at 5min is achieved in comparison with direct intravenous injection of CUR. PMID:27595650

  3. Neurotoxic esterase (NTE) assay: optimized conditions based on detergent-induced shifts in the phenol/4-aminoantipyrine chromophore spectrum.

    PubMed

    Kayyali, U S; Moore, T B; Randall, J C; Richardson, R J

    1991-01-01

    An assay for neurotoxic esterase (neuropathy target esterase, NTE) was developed by Johnson (1,2) to assess the delayed neurotoxic potential of organophosphorus compounds. NTE activity is calculated from the rate of phenyl valerate hydrolysis resistant to paraoxon and sensitive to mipafox inhibition under specified conditions of inhibitor concentrations, pH, temperature, and incubation times with inhibitors and substrate. The amount of phenol produced is measured colorimetrically after its oxidative coupling with 4-aminoantipyrine to yield 4-N-(1,4-benzoquinoneimine)-antipyrine, a chromophore with a wavelength of maximum absorbance (lambda m) 510 nm and corresponding molar absorptivity (molar extinction coefficient, epsilon) equal to 13,900 M-1cm-1. The assay was improved and simplified later by Johnson (3) without any change in the lambda m or epsilon, even though the chromophore solvent was altered by adding the detergent, sodium dodecyl sulfate (SDS). The present work demonstrates that when the NTE assay is performed according to the improved procedure, with a final [SDS] of 3.0 mg/mL, the lambda m of the chromophore in the assay mixture is shifted from 510 to 490 nm. The same shift in the chromophore lambda m is observed when phenol standards are coupled with 4-aminoantipyrine in solutions containing 3.0 mg/mL SDS. A systematic investigation of the dependence of the lambda m of the chromophore on [SDS] in the assay mixture revealed that the spectral shift increases rapidly at an [SDS] greater than the apparent critical micelle concentration (CMC; estimated to be 0.53 mg/mL under these conditions) and begins to plateau at [SDS] greater than 10 mg/mL.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Isolation and characterization of EstC, a new cold-active esterase from Streptomyces coelicolor A3(2).

    PubMed

    Brault, Guillaume; Shareck, François; Hurtubise, Yves; Lépine, François; Doucet, Nicolas

    2012-01-01

    The genome sequence of Streptomyces coelicolor A3(2) contains more than 50 genes coding for putative lipolytic enzymes. Many studies have shown the capacity of this actinomycete to store important reserves of intracellular triacylglycerols in nutrient depletion situations. In the present study, we used genome mining of S. coelicolor to identify genes coding for putative, non-secreted esterases/lipases. Two genes were cloned and successfully overexpressed in E. coli as His-tagged fusion proteins. One of the recombinant enzymes, EstC, showed interesting cold-active esterase activity with a strong potential for the production of valuable esters. The purified enzyme displayed optimal activity at 35°C and was cold-active with retention of 25% relative activity at 10°C. Its optimal pH was 8.5-9 but the enzyme kept more than 75% of its maximal activity between pH 7.5 and 10. EstC also showed remarkable tolerance over a wide range of pH values, retaining almost full residual activity between pH 6-11. The enzyme was active toward short-chain p-nitrophenyl esters (C2-C12), displaying optimal activity with the valerate (C5) ester (k(cat)/K(m) = 737±77 s(-1) mM(-1)). The enzyme was also very active toward short chain triglycerides such as triacetin (C2:0) and tributyrin (C4:0), in addition to showing good primary alcohol and organic solvent tolerance, suggesting it could function as an interesting candidate for organic synthesis of short-chain esters such as flavors.

  5. A Thermally Stable Form of Bacterial Cocaine Esterase: A Potential Therapeutic Agent for Treatment of Cocaine Abuse

    SciTech Connect

    Brim, Remy L.; Nance, Mark R.; Youngstrom, Daniel W.; Narasimhan, Diwahar; Zhan, Chang-Guo; Tesmer, John J.G.; Sunahara, Roger K.; Woods, James H.

    2010-09-03

    Rhodococcal cocaine esterase (CocE) is an attractive potential treatment for both cocaine overdose and cocaine addiction. CocE directly degrades cocaine into inactive products, whereas traditional small-molecule approaches require blockade of the inhibitory action of cocaine on a diverse array of monoamine transporters and ion channels. The usefulness of wild-type (wt) cocaine esterase is hampered by its inactivation at 37 C. Herein, we characterize the most thermostable form of this enzyme to date, CocE-L169K/G173Q. In vitro kinetic analyses reveal that CocE-L169K/G173Q displays a half-life of 2.9 days at 37 C, which represents a 340-fold improvement over wt and is 15-fold greater than previously reported mutants. Crystallographic analyses of CocE-L169K/G173Q, determined at 1.6-{angstrom} resolution, suggest that stabilization involves enhanced domain-domain interactions involving van der Waals interactions and hydrogen bonding. In vivo rodent studies reveal that intravenous pretreatment with CocE-L169K/G173Q in mice provides protection from cocaine-induced lethality for longer time periods before cocaine administration than wt CocE. Furthermore, intravenous administration (pretreatment) of CocE-L169K/G173Q prevents self-administration of cocaine in a time-dependent manner. Termination of the in vivo effects of CoCE seems to be dependent on, but not proportional to, its clearance from plasma as its half-life is approximately 2.3 h and similar to that of wt CocE (2.2 h). Taken together these data suggest that CocE-L169K/G173Q possesses many of the properties of a biological therapeutic for treating cocaine abuse but requires additional development to improve its serum half-life.

  6. Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding

    DOE PAGES

    Chen, Qi; Luan, Zheng -Jiao; Yu, Hui -Lei; Cheng, Xiaolin; Xu, Jian -He

    2015-10-31

    A new carboxylic esterase RhEst1 which catalyzes the hydrolysis of (S)-(+)-2,2-dimethylcyclopropanecarboxylate (S-DmCpCe), the key chiral building block of cilastatin, was identified and subsequently crystallized in our previous work. Mutant RhEst1A147I/V148F/G254A was found to show a 5-fold increase in the catalytic activity. In this work, molecular dynamic simulations were performed to elucidate the molecular determinant of the enzyme activity. Our simulations show that the substrate binds much more strongly in the A147I/V148F/G254A mutant than in wild type, with more hydrogen bonds formed between the substrate and the catalytic triad and the oxyanion hole. The OH group of the catalytic residue Ser101more » in the mutant is better positioned to initiate the nucleophilic attack on S-DmCpCe. Interestingly, the "170-179" loop which is involved in shaping the catalytic sites and facilitating the product release shows remarkable dynamic differences in the two systems. Based on the simulation results, six residues were identified as potential "hot-spots" for further experimental testing. Consequently, the G126S and R133L mutants show higher catalytic efficiency as compared with the wild type. In conclusion, this work provides molecular-level insights into the substrate binding mechanism of carboxylic esterase RhEst1, facilitating future experimental efforts toward developing more efficient RhEst1 variants for industrial applications.« less

  7. Extracellular production of Streptomyces lividans acetyl xylan esterase A in Escherichia coli for rapid detection of activity.

    PubMed

    Nisole, Audrey; Lussier, François-Xavier; Morley, Krista L; Shareck, François; Kazlauskas, Romas J; Dupont, Claude; Pelletier, Joelle N

    2006-04-01

    Acetyl xylan esterase A (AxeA) from Streptomyces lividans belongs to a large family of industrially relevant polysaccharide esterases. AxeA and its truncated form containing only the catalytically competent domain, AxeA(tr), catalyze both the deacetylation of xylan and the N-deacetylation of chitosan. This broad substrate specificity lends additional interest to their characterization and production. Here, we report three systems for extracellular production of AxeA(tr): secretion from the native host S. lividans with the native signal peptide, extracellular production in Escherichia coli with the native signal peptide, and in E. coli with the OmpA signal peptide. Over five to seven days of a shake flask culture, the native host S. lividans with the native signal peptide secreted AxeA(tr) into the extracellular medium in high yield (388 mg/L) with specific activity of 19 U/mg corresponding to a total of 7000 U/L. Over one day of shake flask culture, E. coli with the native secretion signal peptide produced 84-fold less in the extracellular medium (4.6 mg/L), but the specific activity was higher (100 U/mg) corresponding to a total of 460 U/L. A similar E. coli culture using the OmpA signal peptide, produced 10mg/L with a specific activity of 68 U/mg, corresponding to a total of 680 U/L. In 96-well microtiter plates, extracellular production with E. coli gave approximately 30 and approximately 86 microg/mL in S. lividans. Expression in S. lividans with the native signal peptide is best for high level production, while expression in E. coli using the OmpA secretion signal peptide is best for high-throughput expression and screening of variants in microtiter plate format.

  8. Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding

    SciTech Connect

    Chen, Qi; Luan, Zheng -Jiao; Yu, Hui -Lei; Cheng, Xiaolin; Xu, Jian -He

    2015-10-31

    A new carboxylic esterase RhEst1 which catalyzes the hydrolysis of (S)-(+)-2,2-dimethylcyclopropanecarboxylate (S-DmCpCe), the key chiral building block of cilastatin, was identified and subsequently crystallized in our previous work. Mutant RhEst1A147I/V148F/G254A was found to show a 5-fold increase in the catalytic activity. In this work, molecular dynamic simulations were performed to elucidate the molecular determinant of the enzyme activity. Our simulations show that the substrate binds much more strongly in the A147I/V148F/G254A mutant than in wild type, with more hydrogen bonds formed between the substrate and the catalytic triad and the oxyanion hole. The OH group of the catalytic residue Ser101 in the mutant is better positioned to initiate the nucleophilic attack on S-DmCpCe. Interestingly, the "170-179" loop which is involved in shaping the catalytic sites and facilitating the product release shows remarkable dynamic differences in the two systems. Based on the simulation results, six residues were identified as potential "hot-spots" for further experimental testing. Consequently, the G126S and R133L mutants show higher catalytic efficiency as compared with the wild type. In conclusion, this work provides molecular-level insights into the substrate binding mechanism of carboxylic esterase RhEst1, facilitating future experimental efforts toward developing more efficient RhEst1 variants for industrial applications.

  9. Amelioration of the cardiovascular effects of cocaine in rhesus monkeys by a long-acting mutant form of cocaine esterase.

    PubMed

    Collins, Gregory T; Carey, Kathy A; Narasimhan, Diwahar; Nichols, Joseph; Berlin, Aaron A; Lukacs, Nicholas W; Sunahara, Roger K; Woods, James H; Ko, Mei-Chuan

    2011-04-01

    A long-acting mutant form of a naturally occurring bacterial cocaine esterase (T172R/G173Q CocE; double mutant CocE (DM CocE)) has previously been shown to antagonize the reinforcing, convulsant, and lethal effects of cocaine in rodents. However, the effectiveness and therapeutic characteristics of DM CocE in nonhuman primates, in a more clinically relevant context, are unknown. The current studies were aimed at (1) characterizing the cardiovascular effects of cocaine in freely moving rhesus monkeys, (2) evaluating the capacity of DM CocE to ameliorate these cocaine-induced cardiovascular effects when administered 10 min after cocaine, and (3) assessing the immunological responses of monkeys to DM CocE following repeated administration. Intravenous administration of cocaine produced dose-dependent increases in mean arterial pressure (MAP) and heart rate (HR) that persisted throughout the 2-h observation period following a dose of 3.2 mg/kg cocaine. Cocaine failed to produce reliable changes in electrocardiograph (ECG) parameters, body temperature, and locomotor activity. DM CocE produced a rapid and dose-dependent amelioration of the cardiovascular effects, with saline-like MAP measures restored within 5-10 min, and saline-like HR measures restored within 20-40 min of DM CocE administration. Although administration of DM CocE produced increases in anti-CocE antibodies, they did not appear to have a neutralizing effect on the capacity of DM CocE to reverse the cardiovascular effects of cocaine. In conclusion, these findings in monkeys provide strong evidence to suggest that highly efficient cocaine esterases, such as DM CocE, can provide a potential therapeutic option for treatment of acute cocaine intoxication in humans. PMID:21289605

  10. The Use of Plasma-Derived Complement C1-Esterase Inhibitor Concentrate (Berinert®) in the Treatment of Angiotensin Converting Enzyme-Inhibitor Related Angioedema

    PubMed Central

    Hermanrud, Thorbjørn; Duus, Nicolaj; Bygum, Anette; Rasmussen, Eva Rye

    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 (ACEi-AE) of the hypopharynx that completely resolved rapidly after the infusion of plasma-derived C1-inhibitor concentrate adding to the sparse reports in the existing literature. PMID:27123347

  11. Identification and characterization of carboxyl esterases of gill chamber-associated microbiota in the deep-sea shrimp Rimicaris exoculata by using functional metagenomics.

    PubMed

    Alcaide, María; Tchigvintsev, Anatoli; Martínez-Martínez, Mónica; Popovic, Ana; Reva, Oleg N; Lafraya, Álvaro; Bargiela, Rafael; Nechitaylo, Taras Y; Matesanz, Ruth; Cambon-Bonavita, Marie-Anne; Jebbar, Mohamed; Yakimov, Michail M; Savchenko, Alexei; Golyshina, Olga V; Yakunin, Alexander F; Golyshin, Peter N; Ferrer, Manuel

    2015-03-01

    The shrimp Rimicaris exoculata dominates the fauna in deep-sea hydrothermal vent sites along the Mid-Atlantic Ridge (depth, 2,320 m). Here, we identified and biochemically characterized three carboxyl esterases from microbial communities inhabiting the R. exoculata gill that were isolated by naive screens of a gill chamber metagenomic library. These proteins exhibit low to moderate identity to known esterase sequences (≤52%) and to each other (11.9 to 63.7%) and appear to have originated from unknown species or from genera of Proteobacteria related to Thiothrix/Leucothrix (MGS-RG1/RG2) and to the Rhodobacteraceae group (MGS-RG3). A library of 131 esters and 31 additional esterase/lipase preparations was used to evaluate the activity profiles of these enzymes. All 3 of these enzymes had greater esterase than lipase activity and exhibited specific activities with ester substrates (≤356 U mg(-1)) in the range of similar enzymes. MGS-RG3 was inhibited by salts and pressure and had a low optimal temperature (30°C), and its substrate profile clustered within a group of low-activity and substrate-restricted marine enzymes. In contrast, MGS-RG1 and MGS-RG2 were most active at 45 to 50°C and were salt activated and barotolerant. They also exhibited wider substrate profiles that were close to those of highly active promiscuous enzymes from a marine hydrothermal vent (MGS-RG2) and from a cold brackish lake (MGS-RG1). The data presented are discussed in the context of promoting the examination of enzyme activities of taxa found in habitats that have been neglected for enzyme prospecting; the enzymes found in these taxa may reflect distinct habitat-specific adaptations and may constitute new sources of rare reaction specificities. PMID:25595762

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

    PubMed

    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.

  13. Multifunctionality and diversity of GDSL esterase/lipase gene family in rice (Oryza sativa L. japonica) genome: new insights from bioinformatics analysis

    PubMed Central

    2012-01-01

    Background GDSL esterases/lipases are a newly discovered subclass of lipolytic enzymes that are very important and attractive research subjects because of their multifunctional properties, such as broad substrate specificity and regiospecificity. Compared with the current knowledge regarding these enzymes in bacteria, our understanding of the plant GDSL enzymes is very limited, although the GDSL gene family in plant species include numerous members in many fully sequenced plant genomes. Only two genes from a large rice GDSL esterase/lipase gene family were previously characterised, and the majority of the members remain unknown. In the present study, we describe the rice OsGELP (Oryza sativa GDSL esterase/lipase protein) gene family at the genomic and proteomic levels, and use this knowledge to provide insights into the multifunctionality of the rice OsGELP enzymes. Results In this study, an extensive bioinformatics analysis identified 114 genes in the rice OsGELP gene family. A complete overview of this family in rice is presented, including the chromosome locations, gene structures, phylogeny, and protein motifs. Among the OsGELPs and the plant GDSL esterase/lipase proteins of known functions, 41 motifs were found that represent the core secondary structure elements or appear specifically in different phylogenetic subclades. The specification and distribution of identified putative conserved clade-common and -specific peptide motifs, and their location on the predicted protein three dimensional structure may possibly signify their functional roles. Potentially important regions for substrate specificity are highlighted, in accordance with protein three-dimensional model and location of the phylogenetic specific conserved motifs. The differential expression of some representative genes were confirmed by quantitative real-time PCR. The phylogenetic analysis, together with protein motif architectures, and the expression profiling were analysed to predict the

  14. Textural properties of gelling system of low-methoxy pectins produced by demethoxylating reaction of pectin methyl esterase.

    PubMed

    Kim, Y; Yoo, Y-H; Kim, K-O; Park, J-B; Yoo, S-H

    2008-06-01

    After deesterification of commercial pectins with a pectin methyl esterase (PME), their gelling properties were characterized using instrumental texture analysis. The final degree of esterification (DE) of the high- and low-methoxy pectins reached approximately 6% after the PME treatment, while deesterification of low-methoxy amidated pectin stopped at 18% DE. Furthermore, DE of high-methoxy pectin was tailored to be 40%, which is equivalent to the DE of commercial low-methoxy pectin. As a result, significant changes in molecular weight (Mw) distribution were observed in the PME-treated pectins. The texture profile analysis showed that PME modification drastically increased hardness, gumminess, and chewiness, while decreasing cohesiveness and adhesiveness of the pectin gels (P < 0.05). The pectin gel with relatively high peak molecular weight (Mp, 3.5 x 10(5)) and low DE (6), which was produced from high-methoxy pectin, exhibited the greatest hardness, gumminess, chewiness, and resilience. The hardness of low-methoxy amidated pectin increased over 300% after PME deesterification, suggesting that the effects of amide substitution could be reinforced when DE is even lower. The partial least square regression analysis indicated that the Mw and DE of the pectin molecule are the most crucial factors for hardness, chewiness, gumminess, and resilience of gel matrix.

  15. Self-administered C1 esterase inhibitor concentrates for the management of hereditary angioedema: usability and patient acceptance.

    PubMed

    Li, Huamin Henry

    2016-01-01

    Hereditary angioedema (HAE) is a rare genetic disease characterized by episodic subcutaneous or submucosal swelling. The primary cause for the most common form of HAE is a deficiency in functional C1 esterase inhibitor (C1-INH). The swelling caused by HAE can be painful, disfiguring, and life-threatening. It reduces daily function and compromises the quality of life of affected individuals and their caregivers. Among different treatment strategies, replacement with C1-INH concentrates is employed for on-demand treatment of acute attacks and long-term prophylaxis. Three human plasma-derived C1-INH preparations are approved for HAE treatment in the US, the European Union, or both regions: Cinryze(®), Berinert(®), and Cetor(®); however, only Cinryze is approved for long-term prophylaxis. Postmarketing studies have shown that home therapy (self-administered or administered by a caregiver) is a convenient and safe option preferred by many HAE patients. In this review, we summarize the role of self-administered plasma-derived C1-INH concentrate therapy with Cinryze at home in the prophylaxis of HAE. PMID:27660422

  16. Potato tuber pectin structure is influenced by pectin methyl esterase activity and impacts on cooked potato texture

    PubMed Central

    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.

    2011-01-01

    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 method and a sloughing assay, revealing major differences between the potato types. Differences in pectin structure between potato types with different textural properties were revealed using monoclonal antibodies specific for different pectic epitopes. Chemical analysis of tuber pectin clearly demonstrated that, in tubers containing a higher level of total PME activity, there was a reduced degree of methylation of cell wall pectin and consistently higher peak force and work done values during the fracture of cooked tuber samples, demonstrating the link between PME activity, the degree of methylation of cell wall pectin, and cooked tuber textural properties. PMID:20855456

  17. Flexibility and Stability Trade-Off in Active Site of Cold-Adapted Pseudomonas mandelii Esterase EstK.

    PubMed

    Truongvan, Ngoc; Jang, Sei-Heon; Lee, ChangWoo

    2016-06-28

    Cold-adapted enzymes exhibit enhanced conformational flexibility, especially in their active sites, as compared with their warmer-temperature counterparts. However, the mechanism by which cold-adapted enzymes maintain their active site stability is largely unknown. In this study, we investigated the role of conserved D308-Y309 residues located in the same loop as the catalytic H307 residue in the cold-adapted esterase EstK from Pseudomonas mandelii. Mutation of D308 and/or Y309 to Ala or deletion resulted in increased conformational flexibility. Particularly, the D308A or Y309A mutant showed enhanced substrate affinity and catalytic rate, as compared with wild-type EstK, via enlargement of the active site. However, all mutant EstK enzymes exhibited reduced thermal stability. The effect of mutation was greater for D308 than Y309. These results indicate that D308 is not preferable for substrate selection and catalytic activity, whereas hydrogen bond formation involving D308 is critical for active site stabilization. Taken together, conformation of the EstK active site is constrained via flexibility-stability trade-off for enzyme catalysis and thermal stability. Our study provides further insights into active site stabilization of cold-adapted enzymes. PMID:27259687

  18. Inhibition, recovery and oxime-induced reactivation of muscle esterases following chlorpyrifos exposure in the earthworm Lumbricus terrestris.

    PubMed

    Collange, B; Wheelock, C E; Rault, M; Mazzia, C; Capowiez, Y; Sanchez-Hernandez, J C

    2010-06-01

    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 (< or = 1 week); resulting a valuable approach for assessing multiple OP exposure episodes in the field. PMID:20334963

  19. Self-administered C1 esterase inhibitor concentrates for the management of hereditary angioedema: usability and patient acceptance

    PubMed Central

    Li, Huamin Henry

    2016-01-01

    Hereditary angioedema (HAE) is a rare genetic disease characterized by episodic subcutaneous or submucosal swelling. The primary cause for the most common form of HAE is a deficiency in functional C1 esterase inhibitor (C1-INH). The swelling caused by HAE can be painful, disfiguring, and life-threatening. It reduces daily function and compromises the quality of life of affected individuals and their caregivers. Among different treatment strategies, replacement with C1-INH concentrates is employed for on-demand treatment of acute attacks and long-term prophylaxis. Three human plasma-derived C1-INH preparations are approved for HAE treatment in the US, the European Union, or both regions: Cinryze®, Berinert®, and Cetor®; however, only Cinryze is approved for long-term prophylaxis. Postmarketing studies have shown that home therapy (self-administered or administered by a caregiver) is a convenient and safe option preferred by many HAE patients. In this review, we summarize the role of self-administered plasma-derived C1-INH concentrate therapy with Cinryze at home in the prophylaxis of HAE. PMID:27660422

  20. Food-induced esterase electromorphs in Carinarion spp. and their effects on taxonomic data analysis (Gastropoda, Pulmonata, Arionidae).

    PubMed

    Jordaens, K; Van Riel, P; Verhagen, R; Backeljau, T

    1999-03-01

    Nonspecific esterases (EST) are often used to measure genetic variation, yet they may be influenced by environmental factors such as food, climate and age. This may produce misleading similarity indices and genetic diversity estimates (i.e., clone or strain diversities in uniparental organisms). Therefore, polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF) were used to investigate environmental effects on the EST variation in natural Carinarion populations, as well as in 45 individuals that were raised individually on carrots to produce offspring by selfing. Food effects on EST profiles in these progenies were examined by raising them on different food items (lettuce, nettle, or paper). Our results indicated that: (i) Arion (Carinarion) fasciatus and A. (C.) silvaticus show species-specific EST profiles, (ii) A. fasciatus-like outcrossers most probably are conspecific with A. fasciatus s.s., (iii) not all EST variation has a Mendelian basis since lettuce and nettle altered EST profiles, and (iv) food effects on EST profiles differed strongly between individuals. Although food-induced EST profiles did not affect taxonomic interpretations, they did inflate genetic diversity estimates and thus provided misleading population-genetic data.

  1. Effects of juvenile hormone (JH) analog insecticides on larval development and JH esterase activity in two spodopterans.

    PubMed

    El-Sheikh, El-Sayed A; Kamita, Shizuo G; Hammock, Bruce D

    2016-03-01

    Juvenile hormone analog (JHA) insecticides are biological and structural mimics of JH, a key insect developmental hormone. Toxic and anti-developmental effects of the JHA insecticides methoprene, fenoxycarb, and pyriproxyfen were investigated on the larval and pupal stages of Spodoptera littoralis and Spodoptera frugiperda. Bioassays showed that fenoxycarb has the highest toxicity and fastest speed of kill in 2nd instar S. littoralis. All three JHAs affected the development of 6th instar (i.e., final instar) and pupal S. frugiperda. JH esterase (JHE) is a critical enzyme that helps to regulate JH levels during insect development. JHE activity in the last instar S. littoralis and S. frugiperda was 11 and 23 nmol min(-1) ml(-1) hemolymph, respectively. Methoprene and pyriproxyfen showed poor inhibition of JHE activity from these insects, whereas fenoxycarb showed stronger inhibition. The inhibitory activity of fenoxycarb, however, was more than 1000-fold lower than that of OTFP, a highly potent inhibitor of JHEs. Surprisingly, topical application of methoprene, fenoxycarb or pyriproxyfen on 6th instars of S. littoralis and S. frugiperda prevented the dramatic reduction in JHE activity that was found in control insects. Our findings suggest that JHAs may function as JH agonists that play a disruptive role or a hormonal replacement role in S. littoralis and S. frugiperda. PMID:26969437

  2. Correlation between the In Vitro Functionality of Stored Platelets and the Cytosolic Esterase-Induced Fluorescence Intensity with CMFDA.

    PubMed

    Wang, Jiexi; Yi, Xiaoyang; Liu, Minxia; Zhou, Qian; Ren, Suping; Wang, Yan; Yang, Chao; Zhou, Jianwei; Han, Ying

    2015-01-01

    It has been hypothesized that the cytosolic esterase-induced fluorescence intensity (CEIFI) from carboxy dimethyl fluorescein diacetate (CMFDA) in platelets may related to platelet functions. In the present study, we measured the change of CEIFI in platelets during storage, and examined the correlations of CEIFI with the in vitro functionality of stored platelets, including the ADP-induced aggregation activity, hypotonic shock response, expression of CD62P as well as platelet apoptosis. The CEIFI of fresh platelets, when tested at 10 μM CMFDA, the mean fluorescence intensity index (MFI) was 305.9 ± 49.9 (N = 80). After 1-day storage, it was 203.8 ± 34.4, the CEIFI of the stored platelets started to decline significantly, and reduced to 112.7 ±27.7 after 7-day storage. The change in CEIFI is highly correlated to all four functional parameters measured, with the correlation coefficients being 0.9813, 0.9848, -0.9945 and -0.9847 for the ADP-induced aggregation activity, hypotonic shock response (HSR), expression of CD62P and platelet apoptosis respectively. The above results show that the CEIFI measurement of platelets represents well the viability and functional state of in vitro stored platelets. This may be used as a convenient new method for quality evaluation for stored platelets if this result can be further validated by the following clinical trials.

  3. The ectopic expression of a pectin methyl esterase inhibitor increases pectin methyl esterification and limits fungal diseases in wheat.

    PubMed

    Volpi, Chiara; Janni, Michela; Lionetti, Vincenzo; Bellincampi, Daniela; Favaron, Francesco; D'Ovidio, Renato

    2011-09-01

    Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.

  4. Luminescence and antibacterial studies of silver nanoparticles using the esterases-containing latex of E. Tirucalli plant via green route

    NASA Astrophysics Data System (ADS)

    Sudheerkumar, K. H.; Dhananjaya, N.; Reddy Yadav, L. S.

    2016-04-01

    Silver nanoparticles (Ag NPs) synthesized from silver nitrate solutions using the esterase-containing latex of the E. Tirucalli plant widely found in a large region in Karnataka, India. Plant-mediated synthesis of nanoparticles is a green chemistry approach that intercom-nects nanotechnology and plant biotechnology. The effect of extract concentration, contact time, and temperature on the reaction rate and the shape of the Ag nanoparticles was investigated. The nanoparticles have been characterized by powder X-ray diffraction, UV-visible spectroscopy, photoluminescence spectroscopy and morphology by scanning electron microscope, transmission electron microscopy, as a function of the ratio of silver ions to reducing agent molecules. Powder X-ray diffraction patterns show that the crystal structure obtained is face-centered cubic (fcc). The morphology of the silver nanoparticle was uniform with well-distributed elliptical particles with a range from 15 to 25nm. Ag NPs exhibit significant antibacterial activity against Bacillus cereus using the agar well diffusion method.

  5. Self-administered C1 esterase inhibitor concentrates for the management of hereditary angioedema: usability and patient acceptance

    PubMed Central

    Li, Huamin Henry

    2016-01-01

    Hereditary angioedema (HAE) is a rare genetic disease characterized by episodic subcutaneous or submucosal swelling. The primary cause for the most common form of HAE is a deficiency in functional C1 esterase inhibitor (C1-INH). The swelling caused by HAE can be painful, disfiguring, and life-threatening. It reduces daily function and compromises the quality of life of affected individuals and their caregivers. Among different treatment strategies, replacement with C1-INH concentrates is employed for on-demand treatment of acute attacks and long-term prophylaxis. Three human plasma-derived C1-INH preparations are approved for HAE treatment in the US, the European Union, or both regions: Cinryze®, Berinert®, and Cetor®; however, only Cinryze is approved for long-term prophylaxis. Postmarketing studies have shown that home therapy (self-administered or administered by a caregiver) is a convenient and safe option preferred by many HAE patients. In this review, we summarize the role of self-administered plasma-derived C1-INH concentrate therapy with Cinryze at home in the prophylaxis of HAE.

  6. Determination of fungal activity in modified wood by means of micro-calorimetry and determination of total esterase activity

    PubMed Central

    Verma, Pradeep; Dyckmans, Jens; Militz, Holger

    2008-01-01

    Beech and pine wood blocks were treated with 1,3-dimethylol-4,5-dihydroxyethylen urea (DMDHEU) to increasing weight percent gains (WPG). The resistance of the treated specimens against Trametes versicolor and Coniophora puteana, determined as mass loss, increased with increasing WPG of DMDHEU. Metabolic activity of the fungi in the wood blocks was assessed as total esterase activity (TEA) based on the hydrolysis of fluorescein diacetate and as heat or energy production determined by isothermal micro-calorimetry. Both methods revealed that the fungal activity was related with the WPG and the mass loss caused by the fungi. Still, fungal activity was detected even in wood blocks of the highest WPG and showed that the treatment was not toxic to the fungi. Energy production showed a higher consistency with the mass loss after decay than TEA; higher mass loss was more stringently reflected by higher heat production rate. Heat production did not proceed linearly, possibly due to the inhibition of fungal activity by an excess of carbon dioxide. PMID:18542949

  7. A hemagglutinin-esterase-expressing salmonid alphavirus replicon protects Atlantic salmon (Salmo salar) against infectious salmon anemia (ISA).

    PubMed

    Wolf, Astrid; Hodneland, Kjartan; Frost, Petter; Braaen, Stine; Rimstad, Espen

    2013-01-11

    A replicon expression system based on the salmonid alphavirus (SAV) that encodes the infectious salmon anemia virus (ISAV) hemagglutinin-esterase (HE) was constructed and found to be an efficacious vaccine against infectious salmon anemia (ISA). Following a single intramuscular immunization, Atlantic salmon (Salmo salar) were effectively protected against subsequent ISAV challenge. Additional replicons coding for the ISAV fusion glycoprotein (F) or the ISAV matrix protein (M) were created and tested in combination with the replicon that encodes the HE. The ISAV HE was confirmed as a potent antigen, but neither the F nor the M proteins were found to be essential for immunization-induced protection. Innate immune response induced at the site of vaccination illustrated the immunogenicity of the SAV-based replicon and its ability to activate antiviral responses in Atlantic salmon. The successful testing of the SAV-based replicon as a vaccine model against ISA showed that the replicon approach may represent a novel immunization technology for the aquaculture industry. It offers potential benefits in terms of safety, efficacy, flexibility, and vaccine production complexity.

  8. A novel cold active esterase derived from Colombian high Andean forest soil metagenome.

    PubMed

    Jiménez, Diego Javier; Montaña, José Salvador; Alvarez, Diana; Baena, Sandra

    2012-01-01

    In order to search new lipolytic enzymes and conduct bioprospecting of microbial communities from high Andean forest soil, a metagenomic library of approximately 20,000 clones was constructed in Escherichia coli using plasmid p-Bluescript II SK+. The library covered 80 Mb of the metagenomic DNA mainly from Proteobacteria, Actinobacteria and Acidobacteria. Two clones with lipolytic activity in tributyrin as a substrate were recovered. Clone BAA3G2 (pSK-estGX1) was selected and the entire 4.6 Kb insert sequence was determined. The sequence had a GC content of 70.6% and could be derived from an undescribed Actinobacteria genome. One open reading frame encoded a polypeptide of 210 amino acids (gene estGX1) with a molecular mass of 22.4 kDa that contained the pentapeptide G-P-S-G-G near the N-terminus essential for lipase activity and the putative catalytic triad was identified, also a putative ribosomal binding site located 18 bp upstream the estGX1 ATG start codon was identified. The phylogenetic analysis suggested that the protein belonged to a new lipase family. The secreted enzyme showed a preference for short length fatty acids, with specific activity against p-nitrophenyl-butyrate (0.142 U/mg of total protein), it was cold active with relative activity of 30% at 10°C and moderately thermo active with relative activity of 80% at 50°C and had a pH optimum of 8.0 at 40°C. PMID:22806812

  9. Coexpressing Escherichia coli cyclopropane synthase with Sterculia foetida Lysophosphatidic acid acyltransferase enhances cyclopropane fatty acid accumulation.

    PubMed

    Yu, Xiao-Hong; Prakash, Richa Rawat; Sweet, Marie; Shanklin, John

    2014-01-01

    Cyclopropane fatty acids (CPAs) are desirable as renewable chemical feedstocks for the production of paints, plastics, and lubricants. Toward our goal of creating a CPA-accumulating crop, we expressed nine higher plant cyclopropane synthase (CPS) enzymes in the seeds of fad2fae1 Arabidopsis (Arabidopsis thaliana) and observed accumulation of less than 1% CPA. Surprisingly, expression of the Escherichia coli CPS gene resulted in the accumulation of up to 9.1% CPA in the seed. Coexpression of a Sterculia foetida lysophosphatidic acid acyltransferase (SfLPAT) increases CPA accumulation up to 35% in individual T1 seeds. However, seeds with more than 9% CPA exhibit wrinkled seed morphology and reduced size and oil accumulation. Seeds with more than 11% CPA exhibit strongly decreased seed germination and establishment, and no seeds with CPA more than 15% germinated. That previous reports suggest that plant CPS prefers the stereospecific numbering (sn)-1 position whereas E. coli CPS acts on sn-2 of phospholipids prompted us to investigate the preferred positions of CPS on phosphatidylcholine (PC) and triacylglycerol. Unexpectedly, in planta, E. coli CPS acts primarily on the sn-1 position of PC; coexpression of SfLPAT results in the incorporation of CPA at the sn-2 position of lysophosphatidic acid. This enables a cycle that enriches CPA at both sn-1 and sn-2 positions of PC and results in increased accumulation of CPA. These data provide proof of principle that CPA can accumulate to high levels in transgenic seeds and sets the stage for the identification of factors that will facilitate the movement of CPA from PC into triacylglycerol to produce viable seeds with additional CPA accumulation. PMID:24204024

  10. Neuropathy target esterase inhibitors: enantiomeric separation and stereospecificity of 2-substituted-4H-1,3,2-benzodioxaphosphorin 2-oxides.

    PubMed

    Wu, S Y; Casida, J E

    1994-01-01

    2-Substituted-4H-1,3,2-benzodioxaphosphorin 2-oxides (2-substituted-BDPOs) are known to be potent neuropathy target esterase (NTE) inhibitors (I50s for the racemates of 0.2-3 nM) when the 2-substituents are n-alkyl (C5-C12), N-alkoxy (C7-C10), or p-n-alkylbenzyl (C3 and C4). The list of potent inhibitors (I50s < 3 nM) is expanded by the new n-alkylamino (C9) and n-alkylthio (C5, C7, and C9) analogs reported here. The optimal chain length of the 2-substituent is about 10 atoms in the alkylamino and alkylthio series as in our previous study on alkyl and alkoxy moieties. In contrast, an I50 of 60 nM is reported for o-methylphenoxy-BDPO, the neuropathic metabolite of tri-o-cresyl phosphate (TOCP). In addition to substituent effects, each of these compounds contains two enantiomers of unknown stereospecificity as NTE inhibitors. Separation by chiral HPLC with the CHIRALCEL OC column and hexane-2-propanol eluent gives individual enantiomers of > 98% e.e. and a stereospecificity for NTE inhibition depending on the type and chain length of the 2-substituent; e.g., the ratio for inhibitory potency of the individual enantiomers is 1.7-fold for nonylthio, 1255-fold for nonylamino, and 9-fold for the TOCP metabolite. In comparing enantiomeric pairs of BDPOs with alkyl, alkoxy, alkylamino, alkylthio, benzyl, p-butylbenzyl, o-methylphenoxy, or phenyl as the 2-substituent, the more retained enantiomer in HPLC is always the better NTE inhibitor (in a series of twenty-two pairs) and housefly toxicant (based on two pairs) than the less retained one.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8155829

  11. Potential therapeutic benefit of C1-esterase inhibitor in neuromyelitis optica evaluated in vitro and in an experimental rat model.

    PubMed

    Tradtrantip, Lukmanee; Asavapanumas, Nithi; Phuan, Puay-Wah; Verkman, A S

    2014-01-01

    Neuromyelitis optica (NMO) is an autoimmune demyelinating disease of the central nervous system in which binding of anti-aquaporin-4 (AQP4) autoantibodies (NMO-IgG) to astrocytes causes complement-dependent cytotoxicity (CDC) and inflammation resulting in oligodendrocyte and neuronal injury. There is compelling evidence for a central role of complement in NMO pathogenesis. Here, we evaluated the potential of C1-esterase inhibitor (C1-inh) for complement-targeted therapy of NMO. C1-inh is an anti-inflammatory plasma protein with serine protease inhibition activity that has a broad range of biological activities on the contact (kallikrein), coagulation, fibrinolytic and complement systems. C1-inh is approved for therapy of hereditary angioedema (HAE) and has been studied in a small safety trial in acute NMO relapses (NCT 01759602). In vitro assays of NMO-IgG-dependent CDC showed C1-inh inhibition of human and rat complement, but with predicted minimal complement inhibition activity at a dose of 2000 units in humans. Inhibition of complement by C1-inh was potentiated by ∼10-fold by polysulfated macromolecules including heparin and dextran sulfate. In rats, intravenous C1-inh at a dose 30-fold greater than that approved to treat HAE inhibited serum complement activity by <5%, even when supplemented with heparin. Also, high-dose C1-inh did not reduce pathology in a rat model of NMO produced by intracerebral injection of NMO-IgG. Therefore, although C1r and C1s are targets of C1-inh, our in vitro data with human serum and in vivo data in rats suggest that the complement inhibition activity of C1-inh in serum is too low to confer clinical benefit in NMO.

  12. Overexpression of sinapine esterase BnSCE3 in oilseed rape seeds triggers global changes in seed metabolism.

    PubMed

    Clauss, Kathleen; von Roepenack-Lahaye, Edda; Böttcher, Christoph; Roth, Mary R; Welti, Ruth; Erban, Alexander; Kopka, Joachim; Scheel, Dierk; Milkowski, Carsten; Strack, Dieter

    2011-03-01

    Sinapine (O-sinapoylcholine) is the predominant phenolic compound in a complex group of sinapate esters in seeds of oilseed rape (Brassica napus). Sinapine has antinutritive activity and prevents the use of seed protein for food and feed. A strategy was developed to lower its content in seeds by expressing an enzyme that hydrolyzes sinapine in developing rape seeds. During early stages of seedling development, a sinapine esterase (BnSCE3) hydrolyzes sinapine, releasing choline and sinapate. A portion of choline enters the phospholipid metabolism, and sinapate is routed via 1-O-sinapoyl-β-glucose into sinapoylmalate. Transgenic oilseed rape lines were generated expressing BnSCE3 under the control of a seed-specific promoter. Two distinct single-copy transgene insertion lines were isolated and propagated to generate homozygous lines, which were subjected to comprehensive phenotyping. Sinapine levels of transgenic seeds were less than 5% of wild-type levels, whereas choline levels were increased. Weight, size, and water content of transgenic seeds were significantly higher than those of wild-type seeds. Seed quality parameters, such as fiber and glucosinolate levels, and agronomically important traits, such as oil and protein contents, differed only slightly, except that amounts of hemicellulose and cellulose were about 30% higher in transgenic compared with wild-type seeds. Electron microscopic examination revealed that a fraction of the transgenic seeds had morphological alterations, characterized by large cavities near the embryonic tissue. Transgenic seedlings were larger than wild-type seedlings, and young seedlings exhibited longer hypocotyls. Examination of metabolic profiles of transgenic seeds indicated that besides suppression of sinapine accumulation, there were other dramatic differences in primary and secondary metabolism. Mapping of these changes onto metabolic pathways revealed global effects of the transgenic BnSCE3 expression on seed metabolism. PMID

  13. Treatment with C1-esterase inhibitor concentrate in type I or II hereditary angioedema: a systematic literature review.

    PubMed

    Bork, Konrad; Steffensen, Isabella; Machnig, Thomas

    2013-01-01

    Hereditary angioedema (HAE) due to C1 esterase inhibitor (HAE-C1-INH) deficiency is a rare genetic disorder presenting with recurrent episodes of skin swellings, abdominal pain attacks, and potentially fatal laryngeal edema. This study was designed to review the efficacy and safety of pasteurized, human, plasma-derived C1-INH concentrate for the treatment of patients with HAE-C1-INH. A systematic search of electronic databases up to December 2011 was performed without language or date restrictions. Two reviewers completed the study selection using predefined inclusion criteria, tabulated, and analyzed the data. The data were inappropriate for meta-analysis; thus, a qualitative synthesis was performed. We identified 89 studies (≍2000 patients) that investigated C1-INH. Replacement therapy with C1-INH significantly shortened time to onset of symptom relief in HAE attacks compared with placebo in a randomized controlled trial, and similar improvements were consistently reported in observational and descriptive studies, accompanied by improvements in patients' quality of life. C1-INH has been shown to be effective for patients receiving home therapy and short- and long-term prophylaxis. Treatment with C1-INH was generally well tolerated. Administration of C1-INH was not associated with transmission of viruses or development of autoantibodies irrespective of treatment duration. This research provides additional confirmation of the efficacy of C1-INH in the treatment and prevention of HAE attacks. C1-INH is generally safe and well tolerated and has an excellent safety record for over 25 years of clinical use.

  14. An antennal carboxylesterase from Drosophila melanogaster, esterase 6, is a candidate odorant-degrading enzyme toward food odorants.

    PubMed

    Chertemps, Thomas; Younus, Faisal; Steiner, Claudia; Durand, Nicolas; Coppin, Chris W; Pandey, Gunjan; Oakeshott, John G; Maïbèche, Martine

    2015-01-01

    Reception of odorant molecules within insect olfactory organs involves several sequential steps, including their transport through the sensillar lymph, interaction with the respective sensory receptors, and subsequent inactivation. Odorant-degrading enzymes (ODEs) putatively play a role in signal dynamics by rapid degradation of odorants in the vicinity of the receptors, but this hypothesis is mainly supported by in vitro results. We have recently shown that an extracellular carboxylesterase, esterase-6 (EST-6), is involved in the physiological and behavioral dynamics of the response of Drosophila melanogaster to its volatile pheromone ester, cis-vaccenyl acetate. However, as the expression pattern of the Est-6 gene in the antennae is not restricted to the pheromone responding sensilla, we tested here if EST-6 could play a broader function in the antennae. We found that recombinant EST-6 is able to efficiently hydrolyse several volatile esters that would be emitted by its natural food in vitro. Electrophysiological comparisons of mutant Est-6 null flies and a control strain (on the same genetic background) showed that the dynamics of the antennal response to these compounds is influenced by EST-6, with the antennae of the null mutants showing prolonged activity in response to them. Antennal responses to the strongest odorant, pentyl acetate, were then studied in more detail, showing that the repolarization dynamics were modified even at low doses but without modification of the detection threshold. Behavioral choice experiments with pentyl acetate also showed differences between genotypes; attraction to this compound was observed at a lower dose among the null than control flies. As EST-6 is able to degrade various bioactive odorants emitted by food and plays a role in the response to these compounds, we hypothesize a role as an ODE for this enzyme toward food volatiles. PMID:26594178

  15. An antennal carboxylesterase from Drosophila melanogaster, esterase 6, is a candidate odorant-degrading enzyme toward food odorants

    PubMed Central

    Chertemps, Thomas; Younus, Faisal; Steiner, Claudia; Durand, Nicolas; Coppin, Chris W.; Pandey, Gunjan; Oakeshott, John G.; Maïbèche, Martine

    2015-01-01

    Reception of odorant molecules within insect olfactory organs involves several sequential steps, including their transport through the sensillar lymph, interaction with the respective sensory receptors, and subsequent inactivation. Odorant-degrading enzymes (ODEs) putatively play a role in signal dynamics by rapid degradation of odorants in the vicinity of the receptors, but this hypothesis is mainly supported by in vitro results. We have recently shown that an extracellular carboxylesterase, esterase-6 (EST-6), is involved in the physiological and behavioral dynamics of the response of Drosophila melanogaster to its volatile pheromone ester, cis-vaccenyl acetate. However, as the expression pattern of the Est-6 gene in the antennae is not restricted to the pheromone responding sensilla, we tested here if EST-6 could play a broader function in the antennae. We found that recombinant EST-6 is able to efficiently hydrolyse several volatile esters that would be emitted by its natural food in vitro. Electrophysiological comparisons of mutant Est-6 null flies and a control strain (on the same genetic background) showed that the dynamics of the antennal response to these compounds is influenced by EST-6, with the antennae of the null mutants showing prolonged activity in response to them. Antennal responses to the strongest odorant, pentyl acetate, were then studied in more detail, showing that the repolarization dynamics were modified even at low doses but without modification of the detection threshold. Behavioral choice experiments with pentyl acetate also showed differences between genotypes; attraction to this compound was observed at a lower dose among the null than control flies. As EST-6 is able to degrade various bioactive odorants emitted by food and plays a role in the response to these compounds, we hypothesize a role as an ODE for this enzyme toward food volatiles. PMID:26594178

  16. Extending the story of very-long-chain fatty acid elongation.

    PubMed

    Haslam, Tegan M; Kunst, Ljerka

    2013-09-01

    Very-long-chain fatty acids (VLCFAs) are essential molecules produced by all plant cells, and are components or precursors of numerous specialized metabolites synthesized in specific cell types. VLCFAs are elongated by an endoplasmic reticulum-localized fatty acid elongation complex of four core enzymes, which sequentially add two carbon units to a growing acyl chain. Identification and characterization of these enzymes in Arabidopsis thaliana has revealed that three of the four enzymes act as generalists, contributing to all metabolic pathways that require VLCFAs. A fourth component, the condensing enzyme, provides substrate specificity and determines the amount of product synthesized by the entire complex. Land plants have two families of condensing enzymes, FATTY ACID ELONGATION 1 (FAE1)-type ketoacyl-CoA synthases (KCSs) and ELONGATION DEFECTIVE-LIKEs (ELO-LIKEs). Our current knowledge of the specific roles of different condensing enzymes is incomplete, as is our understanding of the biological function of a recently characterized family of proteins, CER2-LIKEs, which contribute to condensing enzyme function. More broadly, the stoichiometry and quaternary structure of the fatty acid elongase complex remains poorly understood, and specific phylogenetic and biochemical questions persist for each component of the complex. Investigation of VLCFA elongation in different organisms, structural biochemistry, and cell biology approaches stand to greatly benefit this field of plant biology.

  17. Extending the story of very-long-chain fatty acid elongation.

    PubMed

    Haslam, Tegan M; Kunst, Ljerka

    2013-09-01

    Very-long-chain fatty acids (VLCFAs) are essential molecules produced by all plant cells, and are components or precursors of numerous specialized metabolites synthesized in specific cell types. VLCFAs are elongated by an endoplasmic reticulum-localized fatty acid elongation complex of four core enzymes, which sequentially add two carbon units to a growing acyl chain. Identification and characterization of these enzymes in Arabidopsis thaliana has revealed that three of the four enzymes act as generalists, contributing to all metabolic pathways that require VLCFAs. A fourth component, the condensing enzyme, provides substrate specificity and determines the amount of product synthesized by the entire complex. Land plants have two families of condensing enzymes, FATTY ACID ELONGATION 1 (FAE1)-type ketoacyl-CoA synthases (KCSs) and ELONGATION DEFECTIVE-LIKEs (ELO-LIKEs). Our current knowledge of the specific roles of different condensing enzymes is incomplete, as is our understanding of the biological function of a recently characterized family of proteins, CER2-LIKEs, which contribute to condensing enzyme function. More broadly, the stoichiometry and quaternary structure of the fatty acid elongase complex remains poorly understood, and specific phylogenetic and biochemical questions persist for each component of the complex. Investigation of VLCFA elongation in different organisms, structural biochemistry, and cell biology approaches stand to greatly benefit this field of plant biology. PMID:23849117

  18. Geranyl acetate esterase controls and regulates the level of geraniol in lemongrass (Cymbopogon flexuosus Nees ex Steud.) mutant cv. GRL-1 leaves.

    PubMed

    Ganjewala, Deepak; Luthra, Rajesh

    2009-01-01

    Essential oil isolated from lemongrass (Cymbopogon flexuosus) mutant cv. GRL-1 leaves is mainly composed of geraniol (G) and geranyl acetate (GA). The proportion of G and GA markedly fluctuates during leaf development. The proportions of GA and G in the essential oil recorded at day 10 after leaf emergence were approximately 59% and approximately 33% respectively. However, the level of GA went down from approximately 59 to approximately 3% whereas the level of G rose from approximately 33 to approximately 91% during the leaf growth period from day 10 to day 50. However, the decline in the level of GA was most pronounced in the early (day 10 to day 30) stage of leaf growth. The trend of changes in the proportion of GA and G has clearly indicated the role of an esterase that must be involved in the conversion of GA to G during leaf development. We isolated an esterase from leaves of different ages that converts GA into G and has been given the name geranyl acetate esterase (GAE). The GAE activity markedly varied during the leaf development cycle; it was closely correlated with the monoterpene (GA and G) composition throughout leaf development. GAE appeared as several isoenzymes but only three (GAE-I, GAE-II, and GAE-III) of them had significant GA cleaving activity. The GAE isoenzymes pattern was greatly influenced by the leaf developmental stages and so their GA cleaving activities. Like the GAE activity, GAE isoenzyme patterns were also found to be consistent with the monoterpene (GA and G) composition. GAE had an optimum pH at 8.5 and temperature at 30 degrees C. Besides GAE, a compound with phosphatase activity capable of hydrolyzing geranyl diphosphate (GPP) to produce geraniol has also been isolated.

  19. A jojoba beta-Ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants.

    PubMed

    Lassner, M W; Lardizabal, K; Metz, J G

    1996-02-01

    beta-Ketoacyl-coenzyme A (CoA) synthase (KCS) catalyzes the condensation of malonyl-CoA with long-chain acyl-CoA. This reaction is the initial step of the microsomal fatty acyl-CoA elongation pathway responsible for formation of very long chain fatty acids (VLCFAs, or fatty acids with chain lengths > 18 carbons). Manipulation of this pathway is significant for agriculture, because it is the basis of conversion of high erucic acid rapeseed into canola. High erucic acid rapeseed oil, used as an industrial feedstock, is rich in VLCFAs, whereas the edible oil extracted from canola is essentially devoid of VLCFAs. Here, we report the cloning of a cDNA from developing jojoba embryos involved in microsomal fatty acid elongation. The jojoba cDNA is homologous to the recently cloned Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene that has been suggested to encode KCS. We characterize the jojoba enzyme and present biochemical data indicating that the jojoba cDNA does indeed encode KCS. Transformation of low erucic acid rapeseed with the jojoba cDNA restored KCS activity to developing embryos and altered the transgenic seed oil composition to contain high levels of VLCFAs. The data reveal the key role KCS plays in determining the chain lengths of fatty acids found in seed oils.

  20. Kinetics of the inhibitory interaction of organophosphorus neuropathy inducers and non-inducers in soluble esterases in the avian nervous system

    SciTech Connect

    Mangas, Iris; Vilanova, Eugenio; Estevez, Jorge

    2011-11-15

    Some published studies suggest that low level exposure to organophosphorus esters (OPs) may cause neurological and neurobehavioral effects at long term exposure. These effects cannot be explained by action on known targets. In this work, the interactions (inhibition, spontaneous reactivation and 'ongoing inhibition') of two model OPs (paraoxon, non neuropathy-inducer, and mipafox, neuropathy-inducer) with the chicken brain soluble esterases were evaluated. The best-fitting kinetic model with both inhibitors was compatible with three enzymatic components. The amplitudes (proportions) of the components detected with mipafox were similar to those obtained with paraoxon. These observations confirm the consistency of the results and the model applied and may be considered an external validation. The most sensitive component (E{alpha}) for paraoxon (11-23% of activity, I{sub 50} (30 min) = 9-11 nM) is also the most sensitive for mipafox (I{sub 50} (30 min) = 4 nM). This component is spontaneously reactivated after inhibition with paraoxon. The second sensitive component to paraoxon (E{beta}, 71-84% of activity; I{sub 50} (30 min) = 1216 nM) is practically resistant to mipafox. The third component (E{gamma}, 5-8% of activity) is paraoxon resistant and has I{sub 50} (30 min) of 3.4 {mu}M with mipafox, similar to NTE (neuropathy target esterase). The role of these esterases remains unknown. Their high sensitivity suggests that they may either play a role in toxicity in low-level long-term exposure of organophosphate compounds or have a protective effect related with the spontaneous reactivation. They will have to be considered in further metabolic and toxicological studies. -- Research Highlights: Black-Right-Pointing-Pointer Paraoxon and mipafox interactions have been evaluated with chicken soluble brain esterases. Black-Right-Pointing-Pointer The paraoxon inhibition was analyzed considering the simultaneous spontaneous reactivation. Black-Right-Pointing-Pointer The best