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Sample records for acid esterase fae

  1. Identification of two feruloyl esterases in Dickeya dadantii 3937 and induction of the major feruloyl esterase and of pectate lyases by ferulic acid.

    PubMed

    Hassan, Susan; Hugouvieux-Cotte-Pattat, Nicole

    2011-02-01

    The plant-pathogenic bacterium Dickeya dadantii (formerly Erwinia chrysanthemi) produces a large array of plant cell wall-degrading enzymes. Using an in situ detection test, we showed that it produces two feruloyl esterases, FaeD and FaeT. These enzymes cleave the ester link between ferulate and the pectic or xylan chains. FaeD and FaeT belong to the carbohydrate esterase family CE10, and they are the first two feruloyl esterases to be identified in this family. Cleavage of synthetic substrates revealed strong activation of FaeD and FaeT by ferulic acid. The gene faeT appeared to be weakly expressed, and its product, FaeT, is a cytoplasmic protein. In contrast, the gene faeD is strongly induced in the presence of ferulic acid, and FaeD is an extracellular protein secreted by the Out system, responsible for pectinase secretion. The product of the adjacent gene faeR is involved in the positive control of faeD in response to ferulic acid. Moreover, ferulic acid acts in synergy with polygalacturonate to induce pectate lyases, the main virulence determinant of soft rot disease. Feruloyl esterases dissociate internal cross-links in the polysaccharide network of the plant cell wall, suppress the polysaccharide esterifications, and liberate ferulic acid, which contributes to the induction of pectate lyases. Together, these effects of feruloyl esterases could facilitate soft rot disease caused by pectinolytic bacteria.

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

    PubMed Central

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

    2002-01-01

    The faeB gene encoding a second feruloyl esterase from Aspergillus niger has been cloned and characterized. It consists of an open reading frame of 1644 bp containing one intron. The gene encodes a protein of 521 amino acids that has sequence similarity to that of an Aspergillus oryzae tannase. However, the encoded enzyme, feruloyl esterase B (FAEB), does not have tannase activity. Comparison of the physical characteristics and substrate specificity of FAEB with those of a cinnamoyl esterase from A. niger [Kroon, Faulds and Williamson (1996) Biotechnol. Appl. Biochem. 23, 255-262] suggests that they are in fact the same enzyme. The expression of faeB is specifically induced in the presence of certain aromatic compounds, but not in the presence of other constituents present in plant-cell-wall polysaccharides such as arabinoxylan or pectin. The expression profile of faeB in the presence of aromatic compounds was compared with the expression of A. niger faeA, encoding feruloyl esterase A (FAEA), and A. niger bphA, the gene encoding a benzoate-p-hydroxylase. All three genes have different subsets of aromatic compounds that induce their expression, indicating the presence of different transcription activating systems in A. niger that respond to aromatic compounds. Comparison of the activity of FAEA and FAEB on sugar-beet pectin and wheat arabinoxylan demonstrated that they are both involved in the degradation of both polysaccharides, but have opposite preferences for these substrates. FAEA is more active than FAEB towards wheat arabinoxylan, whereas FAEB is more active than FAEA towards sugar-beet pectin. PMID:11931668

  3. 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.

  4. 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.

  5. 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.

  6. Vine Trimming Shoots as Substrate for Ferulic Acid Esterases Production.

    PubMed

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

    2017-02-01

    Ferulic acid esterases (FAE) possess a large variety of biotechnological applications mainly based on their ability to release ferulic acid from lignocellulosic matrixes. The use of vine trimming shoots (VTS), an agricultural waste, as substrate for the generation of this kind of esterases represents an attractive alternative to change the consideration of VTS from residue to resource. Furthermore, xylanase, cellobiase, and cellulase activities were quantified. Six microorganisms were screened for FAE production by solid-state fermentation, and the effects of the additional supplementation and substrate size were also tested. Finally, the process was scaled-up to a horizontal bioreactor where the influence of aeration in enzymatic activities was evaluated. Thus, the optimal FAE activity (0.44 U/g dry VTS) was attained by Aspergillus terreus CECT 2808, in non-additional supplementation media, using the larger particles size of substrate (≤ 5 mm) and at a flow rate of 0.7 L/min.

  7. 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...

  8. 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

  9. 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

  10. 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...

  11. 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.

  12. 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.

  13. 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.

  14. 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 ...

  15. Expression of a fungal ferulic acid esterase in alfalfa modifies cell wall digestibility

    PubMed Central

    2014-01-01

    Background Alfalfa (Medicago sativa) is an important forage crop in North America owing to its high biomass production, perennial nature and ability to fix nitrogen. Feruloyl esterase (EC 3.1.1.73) hydrolyzes ester linkages in plant cell walls and has the potential to further improve alfalfa as biomass for biofuel production. Results In this study, faeB [GenBank:AJ309807] was synthesized at GenScript and sub-cloned into a novel pEACH vector containing different signaling peptides to target type B ferulic acid esterase (FAEB) proteins to the apoplast, chloroplast, endoplasmic reticulum and vacuole. Four constructs harboring faeB were transiently expressed in Nicotiana leaves, with FAEB accumulating at high levels in all target sites, except chloroplast. Stable transformed lines of alfalfa were subsequently obtained using Agrobacterium tumefaciens (LBA4404). Out of 136 transgenic plants regenerated, 18 independent lines exhibited FAEB activity. Subsequent in vitro digestibility and Fourier transformed infrared spectroscopy (FTIR) analysis of FAEB-expressing lines showed that they possessed modified cell wall morphology and composition with a reduction in ester linkages and elevated lignin content. Consequently, they were more recalcitrant to digestion by mixed ruminal microorganisms. Interestingly, delignification by alkaline peroxide treatment followed by exposure to a commercial cellulase mixture resulted in higher glucose release from transgenic lines as compared to the control line. Conclusion Modifying cell wall crosslinking has the potential to lower recalcitrance of holocellulose, but also exhibited unintended consequences on alfalfa cell wall digestibility due to elevated lignin content. The combination of efficient delignification treatment (alkaline peroxide) and transgenic esterase activity complement each other towards efficient and effective digestion of transgenic lines. PMID:24650274

  16. An Aspergillus niger esterase (ferulic acid esterase III) and a recombinant Pseudomonas fluorescens subsp. cellulosa esterase (Xy1D) release a 5-5' ferulic dehydrodimer (diferulic acid) from barley and wheat cell walls.

    PubMed Central

    Bartolomé, B; Faulds, C B; Kroon, P A; Waldron, K; Gilbert, H J; Hazlewood, G; Williamson, G

    1997-01-01

    Diferulate esters strengthen and cross-link primary plant cell walls and help to defend the plant from invading microbes. Phenolics also limit the degradation of plant cell walls by saprophytic microbes and by anaerobic microorganisms in the rumen. We show that incubation of wheat and barley cell walls with ferulic acid esterase from Aspergillus niger (FAE-III) or Pseudomonas fluorescens (Xy1D), together with either xylanase I from Aspergillus niger, Trichoderma viride xylanase, or xylanase from Pseudomonas fluorescens (XylA), leads to release of the ferulate dimer 5-5' diFA [(E,E)-4,4'-dihydroxy-5,5'-dimethoxy-3,3'-bicinnamic acid]. Direct saponification of the cell walls without enzyme treatment released the following five identifiable ferulate dimers (in order of abundance): (Z)-beta-(4-[(E)-2-carboxyvinyl]-2-methoxyphenoxy)-4-hydroxy-3-methoxycinnamic acid, trans-5-[(E)-2-carboxyvinyl]-2-(4-hydroxy-3-methoxy-phenyl) -7-methoxy-2, 3-dihydrobenzofuran-3-carboxylic acid, 5-5' diFA, (E,E)-4, 4'-dihydroxy-3, 5'-dimethoxy-beta, 3'-bicinnamic acid, and trans-7-hydroxy-1-(4-hydroxy-3-methoxyphenyl) -6-methoxy-1, 2-dihydronaphthalene-2, 3-dicarboxylic acid. Incubation of the wheat or barley cell walls with xylanase, followed by saponification of the solubilized fraction, yielded 5-5'diFA and, in some cases, certain of the above dimers, depending on the xylanase used. These experiments demonstrate that FAE-III and XYLD specifically release only esters of 5-5'diFA from either xylanase-treated or insoluble fractions of cell walls, even though other esterified dimers were solubilized by preincubation with xylanase. It is also concluded that the esterified dimer content of the xylanase-solubilized fraction depends on the source of the xylanase. PMID:8979352

  17. 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.

  18. 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.

  19. From Classical to High Throughput Screening Methods for Feruloyl Esterases: A Review.

    PubMed

    Ramírez-Velasco, Lorena; Armendáriz-Ruiz, Mariana; Rodríguez-González, Jorge Alberto; Müller-Santos, Marcelo; Asaff-Torres, Ali; Mateos-Díaz, Juan Carlos

    2016-01-01

    Feruloyl esterases (FAEs) are a diverse group of hydrolases widely distributed in plants and microorganisms which catalyzes the cleavage and formation of ester bonds between plant cell wall polysaccharides and phenolic acids. FAEs have gained importance in biofuel, medicine and food industries due to their capability of acting on a large range of substrates for cleaving ester bonds and synthesizing highadded value molecules through esterification and transesterification reactions. During the past two decades extensive studies have been carried out on the production, characterization and classification of FAEs, however only a few reports of suitable High Throughput Screening assays for this kind of enzymes have been reported. This review is focused on a concise but complete revision of classical to High Throughput Screening methods for FAEs, highlighting its advantages and disadvantages, and finally suggesting future perspectives for this important research field.

  20. 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

  1. Contribution of Disulfide Bridges to the Thermostability of a Type A Feruloyl Esterase from Aspergillus usamii.

    PubMed

    Yin, Xin; Hu, Die; Li, Jian-Fang; He, Yao; Zhu, Tian-Di; Wu, Min-Chen

    2015-01-01

    The contribution of disulfide bridges to the thermostability of a type A feruloyl esterase (AuFaeA) from Aspergillus usamii E001 was studied by introducing an extra disulfide bridge or eliminating a native one from the enzyme. MODIP and DbD, two computational tools that can predict the possible disulfide bridges in proteins for thermostability improvement, and molecular dynamics (MD) simulations were used to design the extra disulfide bridge. One residue pair A126-N152 was chosen, and the respective amino acid residues were mutated to cysteine. The wild-type AuFaeA and its variants were expressed in Pichia pastoris GS115. The temperature optimum of the recombinant (re-) AuFaeAA126C-N152C was increased by 6°C compared to that of re-AuFaeA. The thermal inactivation half-lives of re-AuFaeAA126C-N152C at 55 and 60°C were 188 and 40 min, which were 12.5- and 10-folds longer than those of re-AuFaeA. The catalytic efficiency (kcat/Km) of re-AuFaeAA126C-N152C was similar to that of re-AuFaeA. Additionally, after elimination of each native disulfide bridge in AuFaeA, a great decrease in expression level and at least 10°C decrease in thermal stability of recombinant AuEaeA variants were also observed.

  2. Production of cellulolytic enzymes containing cinnamic acid esterase from Schizophyllum commune.

    PubMed

    Tsujiyama, Sho-ichi; Ueno, Hitomi

    2011-01-01

    To develop enzyme preparations capable of digesting plant biomass, we examined the production of cinnamic acid esterase as well as cellulolytic and xylanolytic enzymes in cultures of Schizophyllum commune. The cinnamic acid esterase was produced in the cultures containing solid cellulosic substrates, with production being enhanced by delignifying the wood powder. This indicates that these esterases are produced by cellulose, despite their substrates being phenolic compounds. Cellulolytic and xylanolytic enzymes, with the exception of α-arabinofuranosidase, were also produced in cultures containing cellulosic substances. These results show that enzyme preparation can have high activity of cinnamic acid esterase and cellulolytic and xylanolytic enzymes when S. commune is incubated in the presence of cellulose. These enzyme preparations will be useful for digesting plant biomass and for releasing cinnamic acid derivatives from plant cell walls.

  3. Deacetylation of sialic acid by esterases potentiates pneumococcal neuraminidase activity for mucin utilization, colonization and virulence

    PubMed Central

    Kahya, Hasan F.; Andrew, Peter W.

    2017-01-01

    Pneumococcal neuraminidase is a key enzyme for sequential deglycosylation of host glycans, and plays an important role in host survival, colonization, and pathogenesis of infections caused by Streptococcus pneumoniae. One of the factors that can affect the activity of neuraminidase is the amount and position of acetylation present in its substrate sialic acid. We hypothesised that pneumococcal esterases potentiate neuraminidase activity by removing acetylation from sialic acid, and that will have a major effect on pneumococcal survival on mucin, colonization, and virulence. These hypotheses were tested using isogenic mutants and recombinant esterases in microbiological, biochemical and in vivo assays. We found that pneumococcal esterase activity is encoded by at least four genes, SPD_0534 (EstA) was found to be responsible for the main esterase activity, and the pneumococcal esterases are specific for short acyl chains. Assay of esterase activity by using natural substrates showed that both the Axe and EstA esterases could use acetylated xylan and Bovine Sub-maxillary Mucin (BSM), a highly acetylated substrate, but only EstA was active against tributyrin (triglyceride). Incubation of BSM with either Axe or EstA led to the acetate release in a time and concentration dependent manner, and pre-treatment of BSM with either enzyme increased sialic acid release on subsequent exposure to neuraminidase A. qRT-PCR results showed that the expression level of estA and axe increased when exposed to BSM and in respiratory tissues. Mutation of estA alone or in combination with nanA (codes for neuraminidase A), or the replacement of its putative serine active site to alanine, reduced the pneumococcal ability to utilise BSM as a sole carbon source, sialic acid release, colonization, and virulence in a mouse model of pneumococcal pneumonia. PMID:28257499

  4. Release of short chain fatty acids from cream lipids by commercial lipases and esterases.

    PubMed

    Saerens, K; Descamps, D; Dewettinck, K

    2008-02-01

    Lipases and esterases are frequently used in dairy production processes to enhance the buttery flavour of the end product. Short chain fatty acids, and especially butanoic acid, play a key role in this and different enzymes with specificity towards short chain fatty acids are commercially available as potent flavouring tools. We have compared six lipases/esterases associated with buttery flavour production. Although specificity to short chain fatty acids was ascribed to each enzyme, clear differences in free fatty acid profiles were found when these enzymes were applied on cream. Candida cylindraceae lipase was the most useful enzyme for buttery flavour production in cream with the highest yield of free fatty acids (57 g oleic acid 100 g(-1) fat), no release of long chain fatty acids and specificity towards butanoic acid.

  5. 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

  6. GLYCOENGINEERING OF ESTERASE ACTIVITY THROUGH METABOLIC FLUX-BASED MODULATION OF SIALIC ACID.

    PubMed

    Mathew, Mohit; Tan, Elaine; Labonte, Jason W; Shah, Shivam; Saeui, Christopher T; Liu, Lingshu; Bhattacharya, Rahul; Bovonratwet, Patawut; Gray, Jeffrey J; Yarema, Kevin

    2017-02-20

    This report describes the metabolic glycoengineering (MGE) of intracellular esterase activity in human colon cancer (LS174T) and Chinese hamster ovary (CHO) cells. In silico analysis of the carboxylesterases CES1 and CES2 suggested that these enzymes are modified with sialylated N-glycans, which are proposed to stabilize the active multimeric forms of these enzymes. This premise was supported by treating cells with butanolylated ManNAc to increase sialylation, which in turn increased esterase activity. By contrast, hexosamine analogs not targeted to sialic acid biosynthesis (e.g., butanoylated GlcNAc or GalNAc) had minimal impact. Measurement of mRNA and protein confirmed that esterase activity was controlled through glycosylation and not through transcription or translation. Azide-modified ManNAc analogs widely used in MGE also enhanced esterase activity and provided a way to enrich targeted "glycoengineered" proteins (such as CES2), thereby providing unambiguous evidence that the compounds were converted to sialosides and installed into the glycan structures of esterases as intended. Overall, this study provides a pioneering example of the modulation of intracellular enzyme activity through MGE, which expands the value of this technology from its current status as a labeling strategy and modulator of cell surface biological events.

  7. Baby Fae: a beastly business.

    PubMed

    Kushner, T; Belliotti, R

    1985-12-01

    The Baby Fae experiment has highlighted the growing trend in medicine of using animal parts in the treatment of humans. This paper raises the question of the logical and moral justification for these current practices and their proposed expansion. We argue that the Cognitive Capacity Principle establishes morally justified necessary and sufficient conditions for the use of non-human animals in medical treatments and research. Some alternative sources for medical uses are explored as well as some possible programmes for their implementation.

  8. 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.

  9. 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.

  10. 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

  11. A colorimetric assay for the determination of acetyl xylan esterase or cephalosporin C acetyl esterase activities using 7-amino cephalosporanic acid, cephalosporin C, or acetylated xylan as substrate.

    PubMed

    Martínez-Martínez, Irene; Montoro-García, Silvia; Lozada-Ramírez, José Daniel; Sánchez-Ferrer, Alvaro; García-Carmona, Francisco

    2007-10-15

    A bromothymol blue-based colorimetric assay has been devised to screen for acetyl xylan esterase or cephalosporin C (CPC) deacetylase activities using 7-amino cephalosporanic acid (7-ACA), CPC, or acetylated xylan as substrate. These enzymes are not screened with their natural substrates because of the tedious procedures available previously. Acetyl xylan esterase from Bacillus pumilus CECT 5072 was cloned, expressed in Escherichia coli Rosetta (DE3), and characterized using this assay. Similar K(M) values for 7-ACA and CPC were obtained when compared with those described using HPLC methods. The assay is easy to perform and can be carried out in robotic high-throughput colorimetric devices normally used in directed evolution experiments. The assay allowed us to detect improvements in activity at a minimum of twofold with a very low coefficient of variance in 96-well plates. This method is significantly faster and more convenient to use than are known HPLC and pH-stat procedures.

  12. Biocatalytic Resolution of Rac-α-Ethyl-2-Oxo-Pyrrolidineacetic Acid Methyl Ester by Immobilized Recombinant Bacillus cereus Esterase.

    PubMed

    Zheng, Jian-Yong; Liu, Yin-Yan; Luo, Wei-Feng; Zheng, Ren-Chao; Ying, Xiang-Xian; Wang, Zhao

    2016-04-01

    A new esterase-producing strain (Bacillus cereus WZZ001) which exhibiting high hydrolytic activity and excellent enantioselectivity on rac-α-ethyl-2-oxo-pyrrolidineacetic acid methyl ester (R, S-1) has been isolated from soil sample by our laboratory. In this study, the stereoselective hydrolysis of (R, S-1) was performed using the recombinant Bacillus cereus esterase which expressed in Escherichia coli BL21 (DE3). Under the optimized conditions of pH 8.0, 35 °C, and concentration of substrate 400 mM, a successful enzymatic resolution was achieved with an e.e. s of 99.5 % and conversion of 49 %. Immobilization considerably increased the reusability of the recombinant esterase; the immobilized enzyme showed excellent reusability during 6 cycles of repeated 2 h reactions at 35 °C. Thereby, it makes the recombinant B. cereus esterase a usable biocatalyst for industrial application.

  13. Characterization and purification of a bacterial chlorogenic acid esterase detected during the extraction of chlorogenic acid from arbuscular mycorrhizal tomato roots.

    PubMed

    Negrel, Jonathan; Javelle, Francine; Morandi, Dominique; Lucchi, Géraldine

    2016-12-01

    A Gram-negative bacterium able to grow using chlorogenic acid (5-caffeoylquinic acid) as sole carbon source has been isolated from the roots of tomato plants inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis. An intracellular esterase exhibiting very high affinity (Km = 2 μM) for chlorogenic acid has been extracted and purified by FPLC from the chlorogenate-grown cultures of this bacterium. The molecular mass of the purified esterase determined by SDS-PAGE was 61 kDa and its isoelectric point determined by chromatofocusing was 7.75. The esterase hydrolysed chlorogenic acid analogues (caffeoylshikimate, and the 4- and 3-caffeoylquinic acid isomers), feruloyl esterases substrates (methyl caffeate and methyl ferulate), and even caffeoyl-CoA in vitro but all of them were less active than chlorogenic acid, demonstrating that the esterase is a genuine chlorogenic acid esterase. It was also induced when the bacterial strain was cultured in the presence of hydroxycinnamic acids (caffeic, p-coumaric or ferulic acid) as sole carbon source, but not in the presence of simple phenolics such as catechol or protocatechuic acid, nor in the presence of organic acids such as succinic or quinic acids. The purified esterase was remarkably stable in the presence of methanol, rapid formation of methyl caffeate occurring when its activity was measured in aqueous solutions containing 10-60% methanol. Our results therefore show that this bacterial chlorogenase can catalyse the transesterification reaction previously detected during the methanolic extraction of chlorogenic acid from arbuscular mycorrhizal tomato roots. Data are presented suggesting that colonisation by Rhizophagus irregularis could increase chlorogenic acid exudation from tomato roots, especially in nutrient-deprived plants, and thus favour the growth of chlorogenate-metabolizing bacteria on the root surface or in the mycorhizosphere.

  14. Development and validation of functional CAPS markers for the FAE genes in Brassica juncea and their use in marker-assisted selection

    PubMed Central

    Saini, Navinder; Singh, Naveen; Kumar, Anil; Vihan, Nitika; Yadav, Sangita; Vasudev, Sujata; Yadava, D.K.

    2016-01-01

    Low erucic acid is a major breeding target to improve the edible oil quality in Brassica juncea. The single nucleotide polymorphism (SNP) in fatty acid elongase 1 (FAE1.1 and FAE1.2) gene was exploited to expedite the breeding program. The paralogs of FAE1 gene were sequenced from low erucic acid genotype Pusa Mustard 30 and SNPs were identified through homologous alignment with sequence downloaded from NCBI GenBank. Two SNPs in FAE1.1 at position 591 and 1265 and one in FAE1.2 at 237 were found polymorphic among low and high erucic acid genotypes. These SNPs either create or change the recognition site of restriction enzymes. Transition of a single nucleotide at position 591 and 1265 in FAE1.1, and at position 237 in FAE1.2, leads to a change in the recognition site of Hpy99I, BglII and MnlI restriction enzymes, respectively. Two CAPS markers for FAE1.1 and one for FAE1.2 were developed to differentiate low and high erucic acid genotypes. The efficiency of these CAPS markers was found 100 per cent when validated in Brassica juncea, and B. nigra genotypes and used in back-cross breeding. These CAPS markers will facilitate in marker-assisted selection for improvement of oil quality in Brassica juncea. PMID:28163599

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

    PubMed

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

    2010-04-01

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

  16. 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.

  17. Biochemical Properties of Two Cinnamoyl Esterases Purified from a Lactobacillus johnsonii Strain Isolated from Stool Samples of Diabetes-Resistant Rats▿

    PubMed Central

    Lai, Kin Kwan; Lorca, Graciela L.; Gonzalez, Claudio F.

    2009-01-01

    Cinnamic acids (i.e., ferulic and caffeic acids) that are esterified to the vegetable cell walls should be enzymatically released to be absorbed in a mammal's intestines. A low dosage of ferulic acid in rodent diets stimulates insulin production and alleviates symptoms caused by diabetes (M. Sri Balasubashini, R. Rukkumani, and V. P. Menon, Acta Diabetol. 40:118-122, 2003). Several lactic acid bacteria are able to display ferulic acid esterase (FAE) activity, suggesting that their probiotic activity could be, in part, mediated by the slow release of ferulic acid. In the present work, we describe the isolation of one strain identified as being Lactobacillus johnsonii that displayed strong FAE activity in stool samples from diabetes-resistant biobreeding rats. These animals are genetically susceptible to becoming diabetic but do not develop the disease. By using genomic analysis coupled to protein purification and catalytic screening, we were able to purify two proteins with FAE activity. The enzymes displayed 42% sequence identity and a broad range of substrate preferences. High affinities and catalytic efficiencies toward aromatic compounds such as ethyl ferulate (Km = 20 to 60 μM) and chlorogenic acid (Km = 10 to 50 μM) were observed. The strain isolated herein as well as the enzymes studied could be potentially useful for the formulation of probiotics to ameliorate diabetes symptoms. PMID:19502437

  18. Metabolism of parabens (4-hydroxybenzoic acid esters) by hepatic esterases and UDP-glucuronosyltransferases in man.

    PubMed

    Abbas, Suzanne; Greige-Gerges, Hélène; Karam, Nancy; Piet, Marie-Hélène; Netter, Patrick; Magdalou, Jacques

    2010-01-01

    Parabens (alkyl esters of 4-hydroxybenzoic acid) are widely used as preservatives in drugs, cosmetic products, and foodstuffs. Safety concerns have recently increased due to the potential health risks associated to exposure to large amounts of these substances. Biotransformation of parabens mainly includes hydrolysis of the ester bond and glucuronidation reactions. The hydrolysis and glucuronidation of a series of six parabens differing by the nature of the alkyl group were investigated in human liver microsomes and plasma, and the major human UDP-glucuronosyltransferase (UGT) isoforms involved in the reaction were identified. Methyl- and ethylparaben were stable in human plasma, with 95% of the initial concentration remaining after 24 h. On the other hand, propyl-, butyl- and benzylparaben concentrations decreased by 50% under similar conditions. In contrast, rapid hydrolysis was measured with human liver microsomes depending on the alkyl chain length, with t(1/2) varying from 22 min for methylparaben to 87 min for butylparaben. All parabens were actively glucuronidated by liver microsomes, in comparison to 4-hydroxybenzoic acid. They were mainly substrates of human recombinant UGT1A1, UGT1A8, UGT1A9, UGT2B7, UGT2B15 and UGT2B17. In conclusion, the parabens were readily metabolized in human liver through esterase hydrolysis and glucuronidation by several UGT isoforms. These results suggest that these parabens do not accumulate in human tissue.

  19. Factors affecting ferulic acid release from Brewer's spent grain by Fusarium oxysporum enzymatic system.

    PubMed

    Xiros, Charilaos; Moukouli, Maria; Topakas, Evangelos; Christakopoulos, Paul

    2009-12-01

    In this study, the factors affecting ferulic acid (FA) release from Brewer's spent grain (BSG), by the crude enzyme extract of Fusarium oxysporum were investigated. In order to evaluate the importance of the multienzyme preparation on FA release, the synergistic action of feruloyl esterase (FAE, FoFaeC-12213) and xylanase (Trichoderma longibrachiatum M3) monoenzymes was studied. More than double amount of FA release (1 mg g(-1) dry BSG) was observed during hydrolytic reactions by the crude enzyme extract compared to hydrolysis by the monoenzymes (0.37 mg g(-1) dry BSG). The protease content of the crude extract and the inhibitory effect of FA as an end-product were also evaluated concerning their effect on FA release. The protease treatment prior to hydrolysis by monoenzymes enhanced FA release about 100%, while, for the first time in literature, FA in solution found to have a significant inhibitory effect on FAE activity and on total FA release.

  20. An eleven amino acid residue deletion expands the substrate specificity of acetyl xylan esterase II (AXE II) from Penicillium purpurogenum

    NASA Astrophysics Data System (ADS)

    Colombres, Marcela; Garate, José A.; Lagos, Carlos F.; Araya-Secchi, Raúl; Norambuena, Patricia; Quiroz, Soledad; Larrondo, Luis; Pérez-Acle, Tomas; Eyzaguirre, Jaime

    2008-01-01

    The soft-rot fungus Penicillium purpurogenum secretes to the culture medium a variety of enzymes related to xylan biodegradation, among them three acetyl xylan esterases (AXE I, II and III). AXE II has 207 amino acids; it belongs to family 5 of the carbohydrate esterases and its structure has been determined by X-ray crystallography at 0.9 Å resolution (PDB 1G66). The enzyme possesses the α/β hydrolase fold and the catalytic triad typical of serine esterases (Ser90, His187 and Asp175). AXE II can hydrolyze esters of a large variety of alcohols, but it is restricted to short chain fatty acids. An analysis of its three-dimensional structure shows that a loop that covers the active site may be responsible for this strict specificity. Cutinase, an enzyme that hydrolyzes esters of long chain fatty acids and shows a structure similar to AXE II, lacks this loop. In order to generate an AXE II with this broader specificity, the preparation of a mutant lacking residues involving this loop (Gly104 to Ala114) was proposed. A set of molecular simulation experiments based on a comparative model of the mutant enzyme predicted a stable structure. Using site-directed mutagenesis, the loop's residues have been eliminated from the AXE II cDNA. The mutant protein has been expressed in Aspergillus nidulans A722 and Pichia pastoris, and it is active towards a range of fatty acid esters of up to at least 14 carbons. The availability of an esterase with broader specificity may have biotechnological applications for the synthesis of sugar esters.

  1. Nucleophilic and general acid catalysis at physiological pH by a designed miniature esterase.

    PubMed

    Nicoll, Andrew J; Allemann, Rudolf K

    2004-08-07

    A 31-residue peptide (Art-Est) was designed to catalyse the hydrolysis of p-nitrophenyl esters through histidine catalysis on the solvent exposed face of the alpha-helix of bovine pancreatic polypeptide. NMR spectroscopy indicated that Art-Est adopted a stable 3-dimensional structure in solution. Art-Est was an efficient catalyst with second order rate constants of up to 0.050 M(-1) s(-1). The activity of Art-Est was a consequence of the increased nucleophilicity of His-22, which had a reduced pK(a) value of 5.5 as a consequence of its interaction with His-18 and the positively charged Arg-25 and Arg-26. Mass spectrometry and NMR spectroscopy confirmed that the Art-Est catalysed hydrolysis of p-nitrophenyl esters proceeded through an acyl-enzyme intermediate. A solvent kinetic isotope effect of 1.8 indicated that the transition state preceding the acyl intermediate was stabilised through interaction with the protonated side-chain of His-18 and indicated a reaction mechanism similar to that generally observed for natural esterases. The involvement in the reaction of two histidine residues with different pK(a) values led to a bell-shaped dependence of the reaction rate on the pH of the solution. The catalytic behaviour of Art-Est indicated that designed miniature enzymes can act in a transparent mechanism based fashion with enzyme-like behaviour through the interplay of several amino acid residues.

  2. Increased blood plasma hydrolysis of acetylsalicylic acid in type 2 diabetic patients: a role of plasma esterases.

    PubMed

    Gresner, Peter; Dolník, Martin; Waczulíková, Iveta; Bryszewska, Maria; Sikurová, Libusa; Watala, Cezary

    2006-02-01

    Hydrolysis of acetylsalicylic acid (ASA, aspirin), an antiplatelet drug commonly used in the prevention of stroke and myocardial infarction, seems to play a crucial role in its pharmacological action. Thirty-eight healthy volunteers and 38 type 2 diabetic patients were enrolled to test the hypothesis that the enhanced plasma degradation and lowered bioavailability of ASA in diabetic patients is associated with the attenuation of platelet response. Aspirin esterase activities were tested at pH 7.4 and 5.5. A significantly higher overall aspirin esterase activity was noted at pH 7.4 in the diabetic patients (P<0.003), corresponding to faster ASA hydrolysis (P<0.006). This increased activity was attributable to butyrylcholinesterase and probably to albumin, because it was effectively inhibited by eserine and 4-bis-nitrophenyl phosphate (P<0.01). No significant differences between control and diabetic subjects were found at pH 5.5 in either enzymatic activities or ASA hydrolysis rates. The enhanced plasma ASA degradation in diabetic subjects was significantly associated with the refractoriness of blood platelets to ASA (P<0.05) and modulated by plasma cholesterol (P<0.01). No direct effects of plasma pH or albumin were observed. In conclusion, higher aspirin esterase activity contributes to the lowered response of diabetic platelets to ASA-mediated antiplatelet therapy.

  3. 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.

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

    SciTech Connect

    Lagartera, Laura; González, Ana; Stelter, Meike; García, Pedro; Kahn, Richard; Menéndez, Margarita; Hermoso, Juan A.

    2005-02-01

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

  5. Heterologous production of a feruloyl esterase from Pleurotus sapidus synthesizing feruloyl-saccharide esters.

    PubMed

    Kelle, Sebastian; Nieter, Annabel; Krings, Ulrich; Zelena, Katerina; Linke, Diana; Berger, Ralf G

    2016-11-01

    The feruloyl esterase (FAE) gene EST1 from the basidiomycete Pleurotus sapidus was heterologously expressed in Escherichia coli and Pichia pastoris. Catalytically active recombinant Est1 was secreted using P. pastoris as a host. For expression in P. pastoris, the expression vector pPIC9K was applied. The EST1 gene was cloned with an N-terminal α-mating factor pre-pro sequence and expressed under the control of a methanol inducible alcohol oxidase 1 promotor. Est1 was purified to homogeneity using ion exchange and hydrophobic interaction chromatography. The recombinant Est1 showed optima at pH 5.0 and 50 °C, and released ferulic acid from saccharide esters and from the natural substrate destarched wheat bran. Substrate specificity profile and descriptor-based analysis demonstrated unique properties, showing that Est1 did not fit into the current FAE classification model. Transferuloylation synthesis of feruloyl-saccharide esters was proven for mono- and disaccharides.

  6. 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

  7. Ferulic acid release and 4-vinylguaiacol formation during brewing and fermentation: indications for feruloyl esterase activity in Saccharomyces cerevisiae.

    PubMed

    Coghe, Stefan; Benoot, Koen; Delvaux, Filip; Vanderhaegen, Bart; Delvaux, Freddy R

    2004-02-11

    The release of ferulic acid and the subsequent thermal or enzymatic decarboxylation to 4-vinylguaiacol are inherent to the beer production process. Phenolic, medicinal, or clove-like flavors originating from 4-vinylguaiacol frequently occur in beer made with wheat or wheat malt. To evaluate the release of ferulic acid and the transformation to 4-vinylguaiacol, beer was brewed with different proportions of barley malt, wheat, and wheat malt. Ferulic acid as well as 4-vinylguaiacol levels were determined by HPLC at several stages of the beer production process. During brewing, ferulic acid was released at the initial mashing phase, whereas moderate levels of 4-vinylguaiacol were formed by wort boiling. Higher levels of the phenolic flavor compound were produced during fermentations with brewery yeast strains of the Pof(+) phenotype. In beer made with barley malt, ferulic acid was mainly released during the brewing process. Conversely, 60-90% of ferulic acid in wheat or wheat malt beer was hydrolyzed during fermentation, causing higher 4-vinylguaiacol levels in these beers. As cereal enzymes are most likely inactivated during wort boiling, the additional release of ferulic acid during fermentation suggests the activity of feruloyl esterases produced by brewer's yeast.

  8. 12-((5-Iodo-4-azido-2-hydroxybenzoyl)amino)dodecanoic acid: Biological recognition by cholesterol esterase and acyl-CoA:cholesterol O-acyltransferase

    SciTech Connect

    Kinnunen, P.M.; Klopf, F.H.; Bastiani, C.A.; Gelfman, C.M.; Lange, L.G. )

    1990-02-13

    Potential probes of protein cholesterol and fatty acid binding sites, namely, 12-((5-iodo-4-azido-2-hydroxybenzoyl)amino)dodecanoate (IFA) and its coenzyme A (IFA:CoA) and cholesteryl (IFA:CEA) esters, were synthesized. These radioactive, photoreactive lipid analogues were recognized as substrates and inhibitors of acyl-CoA;cholesterol O-acyltransferase (ACAT) and cholesterol esterase, neutral lipid binding enzymes which are key elements in the regulation of cellular cholesterol metabolism. In the dark, IFA reversibly inhibited cholesteryl ({sup 14}C)oleate hydrolysis by purified bovine pancreatic cholesterol esterase with an apparent K{sub i} of 150 {mu}M. Cholesterol esterase inhibition by IFA became irreversible after photolysis with UV light and oleic acid provided 50% protection against inactivation. Incubation of homogeneous bovine pancreatic cholesterol esterase with IFA:CEA resulted in its hydrolysis to IFA and cholesterol, indicating recognition of IFA:CEA as a substrate by cholesterol esterase. The coenzyme A ester, IFA:CoA, was a reversible inhibitor of microsomal ACAT activity under dark conditions, and photolysis resulted in irreversible inhibition of enzyme activity with 87% efficiency. IFA:CoA was also recognized as a substrate by both liver and aortic microsomal ACATs, with resultant synthesis of {sup 125}IFA:CEA. IFA and its derivatives, IFA:CEA and IFA:CoA, are thus inhibitors and substrates for cholesterol esterase and ACAT. Biological recognition of these photoaffinity lipid analogues will facilitate the identification and structural analysis of hitherto uncharacterized protein lipid binding sites.

  9. Rapid method for identification of macrophages in suspension by acid alpha-naphthyl acetate esterase activity.

    PubMed

    Ennist, D L; Jones, K H

    1983-07-01

    A supravital staining procedure for the identification of macrophages in cell suspension using a modification of a standard cytochemical assay for alpha-naphthyl acetate esterase (ANAE) activity is described. Macrophages are stained an intense red-brown after 5 min incubation in a buffer using ANAE as the substrate and hexazonium pararosaniline as the coupler for the azo dye. There is close agreement in the number of ANAE-positive cells found and the number of macrophages identified in smears by morphological criteria, by phagocytosis, and by the presence of Fc receptors. Therefore, this stain provides a quick, inexpensive method to estimate the number of macrophages present in suspensions of lymphocytic tissues from rats and mice.

  10. 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.

  11. 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.

  12. [On the problem of histochemical demonstration of esterase activity by the thiolacetic acid method in the acellular slime mold Physarum confertum (author's translation)].

    PubMed

    Dierkes, U

    1977-01-01

    The application of the histochemical thiolacetic acid method on plasmodia of the acellular slime mold Physarum confertum leads to the formation of lead sulfide deposits at the outer cytoplasmic surface and its invaginations. The reaction cannot be reduced by esterase- and cholin/acetylcholinesterase inhibitors. Successive application of lead and sulfide in the absence of substrate results in a lead sulfide deposit at the same sites indicating that the underlying reaction is based on an artificial adsorption of ions at the surface of the plasmodium. This finding means that the thiolacetic acid method is not suited for the demonstration of a surface-associated esterase/cholinesterase activity in slime molds. Based on the ion adsorption property of the surface of plasmodia a simple method is developed for the "in toto" demonstration of the plasmamembrane-invagination-system in aceullar slime molds.

  13. 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.

  14. Generation of fatty acids by an acyl esterase in the bioluminescent system of Photobacterium phosphoreum

    SciTech Connect

    Carey, L.M.; Rodriguez, A.; Meighen, E.

    1984-08-25

    The fatty acid reductase complex from Photobacterium phosphoreum has been discovered to have a long chain ester hydrolase activity associated with the 34K protein component of the complex. This protein has been resolved from the other components (50K and 58K) of the fatty acid reductase complex with a purity of > 95% and found to catalyze the transfer of acyl groups from acyl-CoA primarily to thiol acceptors with a low level of transfer to glycerol and water. Addition of the 50K protein of the complex caused a dramatic change in specificity increasing the transfer to oxygen acceptors. The acyl-CoA hydrolase activity increased almost 10-fold, and hence free fatty acids can be generated by the 34K protein when it is present in the fatty acid reductase complex. Hydrolysis of acyl-S-mercaptoethanol and acyl-1-glycerol and the ATP-dependent reduction of the released fatty acids to aldehyde for the luminescent reaction were also demonstrated for the reconstituted fatty acid reductase complex, raising the possibility that the immediate source of fatty acids for this reaction in vivo could be the membrane lipids and/or the fatty acid synthetase system.

  15. Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, selected for the biotransformation of ferulic acid to vanillin, are also able to produce cell wall polysaccharide-degrading enzymes and feruloyl esterases.

    PubMed

    Bonnina; Brunel; Gouy; Lesage-Meessen; Asther; Thibault

    2001-01-02

    The filamentous fungal strains Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, previously selected for the bioconversion of ferulic acid to vanillic acid and vanillin respectively, were grown on sugar beet pulp. A large spectrum of polysaccharide-degrading enzymes was produced by A. niger and very few levels of feruloyl esterases were found. In contrast, P. cinnabarinus culture filtrate contained low amount of polysaccharide-degrading enzymes and no feruloyl esterases. In order to enhance feruloyl esterases in A. niger cultures, feruloylated oligosaccharide-rich fractions were prepared from sugar beet pulp or cereal bran and used as carbon sources. Number of polysaccharide-degrading enzymes were induced. Feruloyl esterases were much higher in maize bran-based medium than in sugar beet pulp-based medium, demonstrating the ability of carbon sources originating from maize to induce the synthesis of feruloyl esterases. Thus, A. niger I-1472 could be interesting to release ferulic acid from sugar beet pulp or maize bran.

  16. Analysis of fae and fhcD genes in Mono Lake, California.

    PubMed

    Nercessian, Olivier; Kalyuzhnaya, Marina G; Joye, Samantha B; Lidstrom, Mary E; Chistoserdova, Ludmila

    2005-12-01

    Genes for two enzymes of the tetrahydromethanopterin-linked C(1) transfer pathway (fae and fhcD) were detected in hypersaline, hyperalkaline Mono Lake (California), via PCR amplification and analysis. Low diversity for fae and fhcD was noted, in contrast to the diversity previously detected in a freshwater lake, Lake Washington (Washington).

  17. A third-generation esterase inoculant alters fermentation pattern and improves aerobic stability of barley silage and the efficiency of body weight gain of growing feedlot cattle.

    PubMed

    Addah, W; Baah, J; Okine, E K; McAllister, T A

    2012-05-01

    This study investigated the effects of a mixed bacterial inoculant possessing ferulic acid esterase (FAE) activity on silage fermentation characteristics, aerobic stability, and growth performance of growing feedlot steers. Whole-crop barley (Hordeum vulgare L.) forage (35% DM) was chopped and ensiled without a silage inoculant (UN) or with a mixed bacterial culture containing 1.0 × 10(11) cfu/g of Lactobacillus buchneri LN4017 that produces FAE, 2.0 × 10(10) cfu/g of Lactobacillus plantarum LP7109, and 1.0 × 10(10) cfu/g of Lactobacillus casei LC3200 at a combined rate of 1.3 × 10(5) cfu/g of fresh forage (IN) in mini and Ag-Bag (Ag-Bag Int. Ltd., Warrenton, OR) silos. Silages from the mini silos were assessed for the effect of inoculation on fermentation characteristics and aerobic stability, whereas silages from Ag-Bags were used to formulate 2 barley silage-based total mixed rations (UN and IN) that were fed to growing feedlot steers for 112 d. The IN silage exhibited a homolactic fermentation during the first 7 d of ensiling as reflected by an increased (P ≤ 0.02) lactic acid concentration and an accelerated rate (P < 0.01) of pH decline. Thereafter, fermentation of IN silage became more heterolactic, resulting in greater concentrations of acetic acid (P < 0.01) and pH (P < 0.01) but less (P < 0.01) lactic acid than UN silage. Inoculation did not affect DM losses (P = 0.52) from mini silos. The IN silage remained stable during 21 d, but temperature and yeasts counts in the UN silage increased after 5 d of aerobic exposure. Growing steers fed the IN silage diet had superior (P = 0.03) feed conversion efficiency compared with those fed UN silage. Inoculation of whole-crop barley silage with a mixed culture of homolactic lactic acid-producing bacteria and FAE-producing L. buchneri at ensiling changed fermentation from a homolactic to a heterolactic form during ensiling and improved aerobic stability of the silage and efficiency of BW gain of growing feedlot

  18. 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

  19. 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 ...

  20. Leukocyte esterase urine test

    MedlinePlus

    ... the urine. This may mean you have a urinary tract infection . If this test is positive, the urine should ... Results Mean An abnormal result indicates a possible urinary tract infection. Alternative Names WBC esterase Images Male urinary system ...

  1. 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.

  2. Crystallization of the FaeE chaperone of Escherichia coli F4 fimbriae

    PubMed Central

    Van Molle, Inge; Buts, Lieven; Coppens, Fanny; Qiang, Liu; Wyns, Lode; Loris, Remy; Bouckaert, Julie; De Greve, Henri

    2005-01-01

    F4 (formerly K88) fimbriae from enterotoxigenic Escherichia coli are assembled via the FaeE/FaeD chaperone/usher pathway. The chaperone FaeE crystallizes in three crystal forms, all belonging to space group C2. Crystals of form 1 diffract to 2.3 Å and have unit-cell parameters a = 195.7, b = 78.5, c = 184.6 Å, β = 102.2°. X-ray data for crystal form 2 were collected to 2.7 Å using an SeMet variant of FaeE. The crystals have unit-cell parameters a = 136.4, b = 75.7, c = 69.4 Å, β = 92.8°. Crystals of form 3 were formed in a solution containing the FaeE–FaeG complex and diffract to 2.8 Å. Unit-cell parameters are a = 109.7, b = 78.6, c = 87.8 Å, β = 96.4°. PMID:16511060

  3. 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.

  4. Identification of Genes Encoding Microbial Glucuronoyl Esterases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    One type of covalent linkages connecting lignin and hemicellulose in plant cell walls is the ester linkage between 4-0-methyl-D-glucuronic acid of glucuronoxylan and lignin alcohols. An enzyme that could hydrolyze such linkages, named glucuronoyl esterase, occurs in the cellulolytic system of the w...

  5. Organophosphates and monocyte esterase deficiency.

    PubMed Central

    McClean, E; Mackey, H; Markey, G M; Morris, T C

    1995-01-01

    AIMS--To examine the possibility that monocyte esterase deficiency (MED) could be caused by exposure to organophosphates. METHODS--Pseudocholinesterase, paraoxonase and arylesterase activities were measured in the serum and acetylcholinesterase activity was measured in the red cells of a group of monocyte esterase deficient subjects and compared with the enzyme activities of a control group of monocyte esterase positive subjects. RESULTS--No significant difference was found between the enzyme activities of the monocyte esterase deficient group and the control group for any of the esterases investigated. CONCLUSION--Current or recent exposure to organophosphorus is not the cause of MED. PMID:7560207

  6. Phenol esterase activity of porcine skin.

    PubMed

    Laszlo, Joseph A; Smith, Leslie J; Evans, Kervin O; Compton, David L

    2015-01-01

    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 with octadecanol, glycerol, and dioleoylglycerol. These phenolic derivatives were treated in taurodeoxycholate microemulsion and unilamellar liposomes with ex vivo porcine skin and an aqueous extract of the skin. Extracted esterases hydrolyzed the microemulsions at rates in the order: tyrosyl lipoate > tyrosyl decanoate > hydroxytyrosyl lipoate > hydroxytyrosyl decanoate. The tyrosyl decanoate was subject to comparatively little hydrolysis (10-30% after 24h) when incorporated into liposomes, while hydroxytyrosyl decanoate in liposomes was not hydrolyzed at all by the skin extract. Ferulate esters were not hydrolyzed by the extract in aqueous buffer, microemulsion, nor liposomes. Tyrosyl decanoate applied topically to skin explants in microemulsion were readily hydrolyzed within 4h, while hydrolysis was minimal when applied in liposomes. These findings indicate that porcine skin displays a general esterase activity toward medium-chain esters of tyrosol and hydroxytyrosol, which can be moderated by the physiochemical properties of the lipid vehicle, but no feruloyl esterase activity.

  7. Improved bioavailability of dietary phenolic acids in whole grain barley and oat groat following fermentation with probiotic Lactobacillus acidophilus , Lactobacillus johnsonii , and Lactobacillus reuteri.

    PubMed

    Hole, Anastasia S; Rud, Ida; Grimmer, Stine; Sigl, Stefanie; Narvhus, Judith; Sahlstrøm, Stefan

    2012-06-27

    The aim of this study was to improve the bioavailability of the dietary phenolic acids in flours from whole grain barley and oat groat following fermentation with lactic acid bacteria (LAB) exhibiting high feruloyl esterase activity (FAE). The highest increase of free phenolic acids was observed after fermentation with three probiotic strains, Lactobacillus johnsonii LA1, Lactobacillus reuteri SD2112, and Lactobacillus acidophilus LA-5, with maximum increases from 2.55 to 69.91 μg g(-1) DM and from 4.13 to 109.42 μg g(-1) DM in whole grain barley and oat groat, respectively. Interestingly, higher amounts of bound phenolic acids were detected after both water treatment and LAB fermentation in whole grain barley, indicating higher bioaccessibility, whereas some decrease was detected in oat groat. To conclude, cereal fermentation with specific probiotic strains can lead to significant increase of free phenolic acids, thereby improving their bioavailability.

  8. Influence of Randomly Inserted Feruloyl Esterase A on β-Glucosidase Activity in Trichoderma reesei.

    PubMed

    Hou, YunHua; Pan, Yang; Yan, MengJie; He, Huan; Yang, QinZheng; Zhong, YaoHua

    2017-02-24

    As a well-known industrial fungus for cellulase production, the strain RUT-C30 of Trichoderma reesei was selected to produce the feruloyl esterase A (FAEA) by a random integration protocol. The strong promoter of cellobiohydrolase 1 (cbh1) gene was used to drive the expression of FAEA. Using double-joint PCR protocol, Pcbh1-faeA-TtrpC expression cassette was successfully constructed and co-transformed into RUT C30 strain of T. reesei. One transformant with high feruloyl esterase yield (3.44 ± 0.16 IU/mL) was obtained through plate screening and named TrfaeA1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of fermentation supernatant from transformant TrfaeA1 showed a distinct protein band appearing at the position of about 34 kDa, indicating that faeA gene has been successfully expressed in T. reesei. Compared with that in original RUT C30 strain, β-glucosidase production in transformant TrfaeA1 was significantly increased by about 86.4%, reaching 63.2 IU/mL due to the random insertion of faeA. Moreover, the total secretion protein and filter paper activities of the transformant TrfaeA1 were also improved by up to 5.5 and 4.3%, respectively. The present results indicated that the random insertion strategy could be an effective and feasible method to improve and optimize the cellulase system of filamentous fungi.

  9. 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

  10. Evaluating Prodrug Strategies for Esterase-Triggered Release of Alcohols

    PubMed Central

    Perez, Christian; Daniel, Kevin B.

    2013-01-01

    Prodrugs are effective tools in overcoming drawbacks typically associated with drug formulation and delivery. Those employing esterase-triggered functional groups are frequently utilized to mask polar carboxylic acids and phenols, increasing drug-like properties such as lipophilicity. Herein we detail a comprehensive assessment for strategies that effectively release hydroxyl and phenolic moieties in the presence of an esterase. Matrix metalloproteinases (MMPs) serve as our proof-of-concept target. Three distinct ester-responsive protecting groups are incorporated into MMP proinhibitors containing hydroxyl moieties. Analytical evaluation of the proinhibitors demonstrates that the use of a benzyl ether group appended to the esterase trigger leads to considerably faster kinetics of conversion and enhanced aqueous stability when compared to more conventional approaches where the trigger is directly attached to the inhibitor. Biological assays confirm that all protecting groups effectively cleave in the presence of esterase to generate the active inhibitor. PMID:23929690

  11. Production of feruloyl esterases and xylanases by Talaromyces stipitatus and Humicola grisea var. thermoidea on industrial food processing by-products.

    PubMed

    Mandalari, G; Bisignano, G; Lo Curto, R B; Waldron, K W; Faulds, C B

    2008-07-01

    Feruloyl esterase (FAE) and xylanase activities were detected in culture supernatants from Humicola grisea var. thermoidea and Talaromyces stipitatus grown on brewers' spent grain (BSG) and wheat bran (WB), two agro-industrial by-products. Maximum activities were detected from cultures of H. grisea grown at 150 rpm, with 16.9 U/ml and 9.1 U/ml of xylanase activity on BSG and WB, respectively. Maximum FAE activity was 0.47 U/ml and 0.33 U/ml on BSG and WB, respectively. Analysis of residual cell wall material after microbial growth shows the preferential solubilisation of arabinoxylan and cellulose, two main polysaccharides present in BSG and WB. The production of low-cost cell-wall-deconstructing enzymes on agro-industrial by-products could lead to the production of low-cost enzymes for use in the valorisation of food processing wastes.

  12. Pseudo-esterase Activity of Human Albumin

    PubMed Central

    Lockridge, Oksana; Xue, Weihua; Gaydess, Andrea; Grigoryan, Hasmik; Ding, Shi-Jian; Schopfer, Lawrence M.; Hinrichs, Steven H.; Masson, Patrick

    2008-01-01

    Human albumin is thought to hydrolyze esters because multiple equivalents of product are formed for each equivalent of albumin. Esterase activity with p-nitrophenyl acetate has been attributed to turnover at tyrosine 411. However, p-nitrophenyl acetate creates multiple, stable, acetylated adducts, a property contrary to turnover. Our goal was to identify residues that become acetylated by p-nitrophenyl acetate and determine the relationship between stable adduct formation and turnover. Fatty acid-free human albumin was treated with 0.5 mm p-nitrophenyl acetate for 5 min to 2 weeks, or with 10 mm p-nitrophenyl acetate for 48 h to 2 weeks. Aliquots were digested with pepsin, trypsin, or GluC and analyzed by mass spectrometry to identify labeled residues. Only Tyr-411 was acetylated within the first 5 min of reaction with 0.5 mm p-nitrophenyl acetate. After 0.5–6 h there was partial acetylation of 16–17 residues including Asp-1, Lys-4, Lys-12, Tyr-411, Lys-413, and Lys-414. Treatment with 10 mm p-nitrophenyl acetate resulted in acetylation of 59 lysines, 10 serines, 8 threonines, 4 tyrosines, and Asp-1. When Tyr-411 was blocked with diisopropylfluorophosphate or chlorpyrifos oxon, albumin had normal esterase activity with β-naphthyl acetate as visualized on a nondenaturing gel. However, after 82 residues had been acetylated, esterase activity was almost completely inhibited. The half-life for deacetylation of Tyr-411 at pH 8.0, 22 °C was 61 ± 4 h. Acetylated lysines formed adducts that were even more stable. In conclusion, the pseudo-esterase activity of albumin is the result of irreversible acetylation of 82 residues and is not the result of turnover. PMID:18577514

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. Three multidomain esterases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 that carry divergent dockerin sequences.

    PubMed

    Aurilia, V; Martin, J C; McCrae, S I; Scott, K P; Rincon, M T; Flint, H J

    2000-06-01

    Three enzymes carrying esterase domains have been identified in the rumen cellulolytic anaerobe Ruminococcus flavefaciens 17. The newly characterized CesA gene product (768 amino acids) includes an N-terminal acetylesterase domain and an unidentified C-terminal domain, while the previously characterized XynB enzyme (781 amino acids) includes an internal acetylesterase domain in addition to its N-terminal xylanase catalytic domain. A third gene, xynE, is predicted to encode a multidomain enzyme of 792 amino acids including a family 11 xylanase domain and a C-terminal esterase domain. The esterase domains from CesA and XynB share significant sequence identity (44%) and belong to carbohydrate esterase family 3; both domains are shown here to be capable of deacetylating acetylated xylans, but no evidence was found for ferulic acid esterase activity. The esterase domain of XynE, however, shares 42% amino acid identity with a family 1 phenolic acid esterase domain identified from Clostridum thermocellum XynZ. XynB, XynE and CesA all contain dockerin-like regions in addition to their catalytic domains, suggesting that these enzymes form part of a cellulosome-like multienzyme complex. The dockerin sequences of CesA and XynE differ significantly from those previously described in R. flavefaciens polysaccharidases, including XynB, suggesting that they might represent distinct dockerin specificities.

  18. 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.

  19. 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...

  20. 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.

  1. 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.

  2. Novel Family of Carbohydrate Esterases, Based on Identification of the Hypocrea jecorina Acetyl Esterase Gene▿ †

    PubMed Central

    Li, Xin-Liang; Skory, Christopher D.; Cotta, Michael A.; Puchart, Vladimir; Biely, Peter

    2008-01-01

    Plant cell walls have been shown to contain acetyl groups in hemicelluloses and pectin. The gene aes1, encoding the acetyl esterase (Aes1) of Hypocrea jecorina, was identified by amino-terminal sequencing, peptide mass spectrometry, and genomic sequence analyses. The coded polypeptide had 348 amino acid residues with the first 19 serving as a secretion signal peptide. The calculated molecular mass and isoelectric point of the secreted enzyme were 37,088 Da and pH 5.89, respectively. No significant homology was found between the predicated Aes1 and carbohydrate esterases of known families, but putative aes1 orthologs were found in genomes of many fungi and bacteria that produce cell wall-degrading enzymes. The aes1 transcript levels were high when the fungal cells were induced with sophorose, cellulose, oat spelt xylan, lactose, and arabinose. The recombinant Aes1 produced by H. jecorina transformed with aes1 under the cellobiohydrolase I promoter displayed properties similar to those reported for the native enzyme. The enzyme hydrolyzed acetate ester bond specifically. Using 4-nitrophenyl acetate as substrate, the activity of the recombinant enzyme was enhanced by d-xylose, d-glucose, cellobiose, d-galactose, and xylooligosaccharides but not by arabinose, mannose, or lactose. With the use of 4-nitrophenyl-β-d-xylopyranoside monoacetate as substrate in a β-xylosidase-coupled assay, Aes1 hydrolyzed positions 3 and 4 with the same efficiency while the H. jecorina acetylxylan esterase 1 exclusively deacetylated the position 2 acetyl group. Aes1 was capable of transacetylating methylxyloside in aqueous solution. The data presented demonstrate that Aes1 and other homologous microbial proteins may represent a new family of esterases for lignocellulose biodegradation. PMID:18978092

  3. Transport of mistletoe lectin by M cells in human intestinal follicle-associated epithelium (FAE) in vitro.

    PubMed

    Lyu, Su-Yun; Park, Won-Bong

    2008-12-01

    Purified mistletoe lectins are known to have cytotoxic and stimulating activities in the immune system. Mistletoe extract has been given subcutaneously because of its unstablity and poor absorption in the Gastrointestinal (GI) tract. A hallmark of M cells is their capacity to internalize material from the lumen and to transfer it efficiently to the underlying lymphoid cells. Although lectins are the prime candidates for oral vaccine delivery, the mechanisms whereby lectins are taken up, transported by M cells, and affect underlying immune cells remain poorly understood. In this study, uptake mechanism of Korean mistletoe lectin (Viscum album L. var. coloratum aggulutinin, VCA) across the human FAE (follicle associated epithelium) was investigated. An inverted FAE model of co-culture was obtained by a co-culture system of Caco-2 cells and human Raji B lymphocytes, and VCA transport across the in vitro model of human FAE was investigated. There was a greater transport of VCA across FAE monolayer cells than that of Caco-2 monolayer cells. These observations will be useful to assess the transport of other orally administered material in the GI tract.

  4. 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…

  5. 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.

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

    PubMed

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

    1979-07-01

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

  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.

  8. 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.

  9. [Phosphonate esterase activity in human serum (author's transl)].

    PubMed

    Labadie, M; Laplaud, P M; Lachatre, G; Breton, J C

    1980-02-01

    A simple methodology for the spectrophotometric assay of phosphonate esterase activity in human serum samples is described, featuring incubation at 30 degrees C in a medium containing p-nitrophenol and phenyl-phosphonic acid ester. Reproducibility of the method as well a mean values in normal patients vs age and sex are reported. Serum activity appears to be increased almost exclusively during pregnancy or administration of estrogenic drugs (as oral contraceptives or in prostate neoplasms).

  10. A stereoselective esterase from Bacillus megaterium: purification, gene cloning, expression and catalytic properties.

    PubMed

    Zheng, Jian-Yong; Wang, Jian; Zhou, Sha-Sha; Li, Xiao-Jun; Ying, Xiang-Xian; Wang, Zhao

    2015-10-27

    Esterases (EC 3.1.1.X) have been used as biocatalysts due to their good stability, high chemo-, regio- and stereoselectivity. In our previous studies, Bacillus megaterium WZ009 harboring esterase displayed the unique capability to convert (S)-4-Chloro-3-hydroxyethylbutyrate (CHBE) in the racemate to (S)-3-hydroxy-γ-butyrolactone (HL) through stereoselective hydrolysis, dechlorination, and lactonization. The remaining (R)-CHBE and formed (S)-HL could be obtained in a one-pot enzymatic reaction. An esterase from B. megaterium WZ009 was purified and was found to have 466 encoded amino acids and an apparent molecular mass of 55 kDa. The purified esterase exhibited maximal activity at a temperature of 25 °C and at a pH of 11.5 towards 100 mM CHBE. When the stereoselective biocatalysis of rac-CHBE was performed using the recombinant Escherichia coli BL21 (DH3) cells harboring the esterase, the catalytic activity increased by 20-fold compared with the original strain B. megaterium WZ009. With the addition of activated carbon (62 g/L) in the reaction system, the conversion was increased from 39% to 45% at a substrate concentration of 750 mM. Another remarkable advantage is that both of the obtained residual (R)-CHBE and the formed (S)-HL had high optical purities (e.e.s > 99.9%, e.e.p > 99.9%), thereby making this esterase a usable biocatalyst for industrial application.

  11. Bacterial cinnamoyl esterase activity screening for the production of a novel functional food product.

    PubMed

    Guglielmetti, Simone; De Noni, Ivano; Caracciolo, Federica; Molinari, Francesco; Parini, Carlo; Mora, Diego

    2008-02-01

    Lactobacillus helveticus MIMLh5 was selected for its strong cinnamoyl esterase activity on chlorogenic acid and employed for the preparation of a food product containing a high concentration of free caffeic acid. The novel food product was demonstrated to display high total antioxidant power and potential probiotic properties.

  12. Bacterial Cinnamoyl Esterase Activity Screening for the Production of a Novel Functional Food Product▿ †

    PubMed Central

    Guglielmetti, Simone; De Noni, Ivano; Caracciolo, Federica; Molinari, Francesco; Parini, Carlo; Mora, Diego

    2008-01-01

    Lactobacillus helveticus MIMLh5 was selected for its strong cinnamoyl esterase activity on chlorogenic acid and employed for the preparation of a food product containing a high concentration of free caffeic acid. The novel food product was demonstrated to display high total antioxidant power and potential probiotic properties. PMID:18165367

  13. Esterase isozymes of the hen's oviduct.

    PubMed

    Grunder, A A; Holland, K G

    1977-11-01

    Esterase isozymes of magnum, isthmus and uterus of three strains of Single Comb White Leghorn hens were examined by zone electrophoresis on starch gels. Although three regions (I, II and III) of esterase activity were observed, the electrophoretic system was optimized to characterize the pattern of up to five zones of esterase activity that were identified in Region I. These esterases were classified as aliesterases based on reactions in the presence of various substrates and inhibitors. No genetic polymorphisms were observed for these isozymes. However, two of these isozymes were perceived to have an electrophoretic mobility slightly faster in patterns of the magnum of layers than in the isthmus, uterus, and magnum of non-layers. It was shown that egg albumen was present in relatively high quantities in the magnum of layers and that egg albumen, when added to supernatant preparations of isthmus, uterus and magnum of non-layers, caused the faster electrophoretic mobility of these two esterase isozymes. No relation between specific gravity of eggs laid by hens and presence of various Region I esterase isozymes could be detected.

  14. Mycobacteriocins produced by rapidly growing mycobacteria are Tween-hydrolyzing esterases.

    PubMed Central

    Saito, H; Tomioka, H; Watanabe, T; Yoneyama, T

    1983-01-01

    Smegmatocin, a protein produced by Mycobacterium smegmatis ATCC 14468, was found to have an esterase activity, hydrolyzing Tween 80, polyoxyethylene sorbitan monooleate, added to the assay medium for various "bacteriocins" from mycobacteria. Because M. diernhoferi ATCC 19340 (indicator strain for smegmatocin) is highly susceptible to oleic acid and smegmatocin requires Tween 80 for manifestation of its anti-M. diernhoferi activity, it is likely that smegmatocin-mediated antimicrobial action is caused by oleic acid generated by hydrolysis of Tween 80 by the inherent esterase action of smegmatocin. Other mycobacteriocins from rapidly growing mycobacteria also have inherent esterase activity against Tween 80 and require Tween 80 for expression of antimycobacterial action. Smegmatocin was found to hydrolyze various polyoxyethylene (sorbitan) fatty acyl esters but not sorbitan monooleate and glyceryl esters. Images PMID:6826523

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

    PubMed Central

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

    2000-01-01

    The gene encoding the major intracellular tributyrin esterase of Lactococcus lactis was cloned using degenerate DNA probes based on 19 known N-terminal amino acid residues of the purified enzyme. The gene, named estA, was sequenced and found to encode a protein of 258 amino acid residues. The transcription start site was mapped 233 nucleotides upstream of the start codon, and a canonical promoter sequence was identified. The deduced amino acid sequence of the estA product contained the typical GXSXG motif found in most lipases and esterases. The protein was overproduced up to 170-fold in L. lactis by use of the nisin-controlled expression system recently developed for lactic acid bacteria. The estA gene was inactivated by chromosomal integration of a temperature-sensitive integration vector. This resulted in the complete loss of esterase activity, which could then be recovered after complementation of the constructed esterase-deficient strain with the wild-type estA gene. This confirms that EstA is the main enzyme responsible for esterase activity in L. lactis. Purified recombinant enzyme showed a preference for short-chain acyl esters, surprisingly also including phospholipids. Medium- and long-acyl-chain lipids were also hydrolyzed, albeit less efficiently. Intermediate characteristics between esterases and lipases make intracellular lactococcal EstA difficult to classify in either of these two groups of esterolytic enzymes. We suggest that, in vivo, EstA could be involved in (phospho)lipid metabolism or cellular detoxification or both, as its sequence showed significant similarity to S-formylglutathione hydrolase (FGH) of Paracoccus denitrificans and human EstD (or FGH), which are part of a universal formaldehyde detoxification pathway. PMID:10742212

  16. Distinction between 'A'-esterases and arylesterases. Implications for esterase classification.

    PubMed Central

    Mackness, M I; Thompson, H M; Hardy, A R; Walker, C H

    1987-01-01

    'A'-esterase activities (substrates paraoxon and pirimiphos-methyloxon) and arylesterase activities (substrate phenyl acetate) were assayed in the sera of 14 species of birds representing seven different orders and 11 species of mammal representing five different orders. Ten species of birds had no detectable 'A'-esterase, and the remaining four species only low activity, yet all birds showed considerable arylesterase activity (16.8-99.3 mumol/min per ml of serum). Ten species of mammal showed both 'A'- and 'aryl'-esterase activities. In humans, gel filtration of serum completely separated peaks representing paraoxonase and arylesterase activities. Thus, in both birds and humans, serum enzymes exist that express arylesterase activity but not 'A'-esterase activity. These findings suggest that a distinction should be made between these two types of esterase in future classifications. PMID:2822017

  17. Drug-induced Fanconi syndrome associated with fumaric acid esters treatment for psoriasis: a case series

    PubMed Central

    Balak, Deepak M.W.; Bouwes Bavinck, Jan Nico; de Vries, Aiko P.J.; Hartman, Jenny; Neumann, Hendrik A. Martino; Zietse, Robert; Thio, Hok Bing

    2016-01-01

    Background Fumaric acid esters (FAEs), an oral immunomodulating treatment for psoriasis and multiple sclerosis, have been anecdotally associated with proximal renal tubular dysfunction due to a drug-induced Fanconi syndrome. Few data are available on clinical outcomes of FAE-induced Fanconi syndrome. Methods Descriptive case series with two cases of Fanconi syndrome associated with FAE treatment diagnosed at two Dutch university nephrology departments, three cases reported at the Dutch and German national pharmacovigilance databases and six previously reported cases. Results All 11 cases involved female patients with psoriasis. The median age at the time of onset was 38 years [interquartile range (IQR) 37–46]. Patients received long-term FAEs treatment with a median treatment duration of 60 months (IQR 28–111). Laboratory tests were typically significant for low serum levels of phosphate and uric acid, while urinalysis showed glycosuria and proteinuria. Eight (73%) patients had developed a hypophosphataemic osteomalacia and three (27%) had pathological bone fractures. All patients discontinued FAEs, while four (36%) patients were treated with supplementation of phosphate and/or vitamin D. Five (45%) patients had persisting symptoms despite FAEs discontinuation. Conclusions FAEs treatment can cause drug-induced Fanconi syndrome, but the association has been reported infrequently. Female patients with psoriasis treated long term with FAEs seem to be particularly at risk. Physicians treating patients with FAEs should be vigilant and monitor for the potential occurrence of Fanconi syndrome. Measurement of the urinary albumin:total protein ratio is a suggested screening tool for tubular proteinuria in Fanconi syndrome. PMID:26798466

  18. 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.

  19. 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

  20. Characterization of a novel cold active and salt tolerant esterase from Zunongwangia profunda.

    PubMed

    Rahman, Mohammad Asadur; Culsum, Umma; Tang, Wenhao; Zhang, Shao Wei; Wu, Gaobing; Liu, Ziduo

    2016-04-01

    A novel cold active esterase, EstLiu was cloned from the marine bacterium Zunongwangia profunda, overexpressed in E. coli BL21 (DE3) and purified by glutathione-S transferase (GST) affinity chromatography. The mature esterase EstLiu sequence encodes a protein of 273 amino acids residues, with a predicted molecular weight of 30KDa and containing the classical pentapeptidase motif from position 156 to 160 with the catalytic triad Ser158-Asp211-His243. Although, EstLiu showed 64% similarity with the hypothetical esterase from Chryseobacterium sp. StRB126 (WP_045498424), phylogenetic analysis showed it had no similarity with any of the established family of lipases/esterases, suggesting that it could be considered as a new family. The purified enzyme showed broad substrate specificity with the highest hydrolytic activity against p-nitrophenyl butyrate (C4). EstLiu showed remarkable activity (75%) at 0°Cand the optimal activity at pH 8.0 and 30°C with good thermostability and quickened inactivation above 60°C. EstLiu retained 81, 103, 67 and 78% of its original activity at 50% (v/v) in ethanol, isopropanol, DMSO and ethylene glycol, respectively. In the presence of Tween 20, Tween 80 and Triton X-100, EstLiu showed 88, 100 and 117% of relative activity. It is also co-factor independent. The high activity at low temperature and desirable stability in organic solvents and salts of this novel family esterase represents a good evidence of novel biocatalyst. Overall, this novel enzyme showed better activity than previously reported esterases in extreme reaction conditions and could promote the reaction in both aqueous and non-aqueous conditions, indicating its great potential for industrial applications.

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

    PubMed

    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.

  2. Xylella fastidiosa esterase rather than hydroxynitrile lyase.

    PubMed

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

    2015-03-02

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

  3. An Inserted α/β Subdomain Shapes the Catalytic Pocket of Lactobacillus johnsonii Cinnamoyl Esterase

    PubMed Central

    Vu, Clara; Xu, Xiaohui; Cui, Hong; Molloy, Sara; Savchenko, Alexei; Yakunin, Alexander; Gonzalez, Claudio F.

    2011-01-01

    Background Microbial enzymes produced in the gastrointestinal tract are primarily responsible for the release and biochemical transformation of absorbable bioactive monophenols. In the present work we described the crystal structure of LJ0536, a serine cinnamoyl esterase produced by the probiotic bacterium Lactobacillus johnsonii N6.2. Methodology/Principal Findings We crystallized LJ0536 in the apo form and in three substrate-bound complexes. The structure showed a canonical α/β fold characteristic of esterases, and the enzyme is dimeric. Two classical serine esterase motifs (GlyXSerXGly) can be recognized from the amino acid sequence, and the structure revealed that the catalytic triad of the enzyme is formed by Ser106, His225, and Asp197, while the other motif is non-functional. In all substrate-bound complexes, the aromatic acyl group of the ester compound was bound in the deepest part of the catalytic pocket. The binding pocket also contained an unoccupied area that could accommodate larger ligands. The structure revealed a prominent inserted α/β subdomain of 54 amino acids, from which multiple contacts to the aromatic acyl groups of the substrates are made. Inserts of this size are seen in other esterases, but the secondary structure topology of this subdomain of LJ0536 is unique to this enzyme and its closest homolog (Est1E) in the Protein Databank. Conclusions The binding mechanism characterized (involving the inserted α/β subdomain) clearly differentiates LJ0536 from enzymes with similar activity of a fungal origin. The structural features herein described together with the activity profile of LJ0536 suggest that this enzyme should be clustered in a new group of bacterial cinnamoyl esterases. PMID:21876742

  4. Purification and characterization of novel extracellular cholesterol esterase from Acinetobacter sp.

    PubMed

    Du, Liangjun; Huo, Ying; Ge, Fanglan; Yu, Jiajun; Li, Wei; Cheng, Guiying; Yong, Bin; Zeng, Lihuang; Huang, Min

    2010-12-01

    CHE4-1, a bacterial strain that belongs to the genus Acinetobacter and expresses high level of inducible extracellular cholesterol esterase (CHE), was isolated from feces of carnivore Panthera pardus var. The cholesterol esterase of the strain CHE4-1 was purified by ultrafiltration followed with DEAE-Sepharose FF chromatography and Phenyl-Sepharose CL-4B chromatography, and then by Sephadex G-50 gel filtration. Different from other known microbial cholesterol esterase, the purified CHE from CHE4-1 strain is a monomer with molecular weight of 6.5 kD and has high activity to both long-chain and short-chain cholesterol ester. Enzymatic activity was enhanced in the presence of metal ion Ca(2+), Zn(2+) and boracic acid, and was not significantly affected by several detergents including sodium cholate, Triton X100 and Tween-80. The enzyme was found to be stable during long-term aqueous storage at 4 °C, indicating its potential as a clinical diagnostic reagent. To the best of our knowledge, this is the first report regarding purification and characterization of CHE from Acinetobacter sp. The results demonstrated that this particular CHE is a novel cholesterol esterase.

  5. Esterase detoxification of acetylcholinesterase inhibitors by ...

    EPA Pesticide Factsheets

    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 factors underlying age-related sensitivity differences. We used an in vitro system to measure detoxification 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 PONs, measuring activity of both esterases) or EGTA (to inhibit PONs, thereby measuring CaE activity). Inhibitory concentrations of aldicarb, chlorpyrifos oxon, malaoxon, methamidophos, oxamyl, paraoxon, and methyl paraoxon were incubated with liver from adult male rat or one of 20 commercially provided human (11-83 years of age) liver samples. Detoxification was the difference in inhibition produced by the pesticide alone or in combination with liver plus Ca+2 or EGTA. Generally, rat liver produced more detoxification than did the human samples. There were large detoxification differences, which were not correlated with age or sex, across human samples for some pesticides (especially malaoxon, chlorpyrifos oxon) but not for others (e.g., aldicarb, methamidophos). Chlorpyrifos oxon was detoxified only in the presence of Ca+2 in both rat and human livers. Detoxification of pa

  6. 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

  7. A comparative study on esterases from three species of Raillietina.

    PubMed

    Balasubramanian, M P; Dhandayuthapani, S; Nellaiappan, K; Ramalingam, K

    1984-06-01

    The multiplicity of soluble esterases in Raillietina tetragona, R. echinobothrida and R. cesticillus was studied by use of slab polyacrylamide gel electrophoresis. Five fractions of esterase activity were observed in R. tetragona, seven in R. echinobothrida and three in R. cesticillus. The various fractions of esterase activity of closely related species of Raillietina showed differential behaviour towards various chemicals. Based on the inhibitory effect of inhibitors p-CMB, EDTA, malathion, silver nitrate and eserine sulphate, the various esterases have been classified into arylesterase, carboxylesterase, acetylesterase and cholinesterase.

  8. Production and characterization of a tributyrin esterase from Lactobacillus plantarum suitable for cheese lipolysis.

    PubMed

    Esteban-Torres, M; Mancheño, J M; de las Rivas, B; Muñoz, R

    2014-11-01

    Lactobacillus plantarum is a lactic acid bacterium that can be found during cheese ripening. Lipolysis of milk triacylglycerols to free fatty acids during cheese ripening has fundamental consequences on cheese flavor. In the present study, the gene lp_1760, encoding a putative esterase or lipase, was cloned and expressed in Escherichia coli BL21 (DE3) and the overproduced Lp_1760 protein was biochemically characterized. Lp_1760 hydrolyzed p-nitrophenyl esters of fatty acids from C2 to C16, with a preference for p-nitrophenyl butyrate. On triglycerides, Lp_1760 showed higher activity on tributyrin than on triacetin. Although optimal conditions for activity were 45°C and pH 7, Lp_1760 retains activity under conditions commonly found during cheese making and ripening. The Lp_1760 showed more than 50% activity at 5°C and exhibited thermal stability at high temperatures. Enzymatic activity was strongly inhibited by sodium dodecyl sulfate and phenylmethylsulfonyl fluoride. The Lp_1760 tributyrin esterase showed high activity in the presence of NaCl, lactic acid, and calcium chloride. The results suggest that Lp_1760 might be a useful tributyrin esterase to be used in cheese manufacturing.

  9. 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

  10. 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.

  11. Novel Family of Carbohydrate Esterases, Based on Identification of the Hypocrea jecorina Acetyl Esterase Gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant cell walls have been shown to contain acetyl groups in hemicelluloses and pectin. The gene, ae1, encoding the acetyl esterase (Ae1) of Hypocrea jecorina was identified by amino terminal sequencing, peptide mass spectrometry, and genomic sequence analyses. The coded polypeptide had 348 amino ...

  12. Phenyl valerate esterase activity of human butyrylcholinesterase.

    PubMed

    Mangas, Iris; Vilanova, Eugenio; Estévez, Jorge

    2017-03-15

    Phenyl valerate is used for detecting and measuring neuropathy target esterase (NTE) and has been used for discriminating esterases as potential target in hen model of organophosphorus delayed neuropathy. In previous studies we observed that phenyl valerate esterase (PVase) activity of an enzymatic fraction in chicken brain might be due to a butyrylcholinesterase protein (BuChE), and it was suggested that this enzymatic fraction could be related to the potentiation/promotion phenomenon of the organophosphate-induced delayed neuropathy (OPIDN). In this work, PVase activity of purified human butyrylcholinesterase (hBuChE) is demonstrated and confirms the novel observation that a relationship of BuChE with PVase activities is also relevant for humans, as is, therefore the potential role in toxicity for humans. The KM and catalytic constant (kcat) were estimated as 0.52/0.72 µM and 45,900/49,200 min(-1) respectively. Furthermore, this work studies the inhibition by preincubation of PVase and cholinesterase activities of hBuChE with irreversible inhibitors (mipafox, iso-OMPA or PMSF), showing that these inhibitors interact similarly in both activities with similar second-order inhibition constants. Acethylthiocholine and phenyl valerate partly inhibit PVase and cholinesterase activities, respectively. All these observations suggest that both activities occur in the same active center. The interaction with a reversible inhibitor (ethopropazine) showed that the cholinesterase activity was more sensitive than the PVase activity, showing that the sensitivity for this reversible inhibitor is affected by the nature of the substrate. The present work definitively establishes the capacity of BuChE to hydrolyze the carboxylester phenyl valerate using a purified enzyme (hBuChE). Therefore, BuChE should be considered in the research of organophosphorus targets of toxicity related with PVase proteins.

  13. Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases.

    PubMed

    Adesioye, Fiyinfoluwa A; Makhalanyane, Thulani P; Biely, Peter; Cowan, Don A

    2016-11-01

    Acetyl xylan esterases (AcXEs), also termed xylan deacetylases, are broad specificity Carbohydrate-Active Enzymes (CAZymes) that hydrolyse ester bonds to liberate acetic acid from acetylated hemicellulose (typically polymeric xylan and xylooligosaccharides). They belong to eight families within the Carbohydrate Esterase (CE) class of the CAZy database. AcXE classification is largely based on sequence-dependent phylogenetic relationships, supported in some instances with substrate specificity data. However, some sequence-based predictions of AcXE-encoding gene identity have proved to be functionally incorrect. Such ambiguities can lead to mis-assignment of genes and enzymes during sequence data-mining, reinforcing the necessity for the experimental confirmation of the functional properties of putative AcXE-encoding gene products. Although one-third of all characterized CEs within CAZy families 1-7 and 16 are AcXEs, there is a need to expand the sequence database in order to strengthen the link between AcXE gene sequence and specificity. Currently, most AcXEs are derived from a limited range of (mostly microbial) sources and have been identified via culture-based bioprospecting methods, restricting current knowledge of AcXEs to data from relatively few microbial species. More recently, the successful identification of AcXEs via genome and metagenome mining has emphasised the huge potential of culture-independent bioprospecting strategies. We note, however, that the functional metagenomics approach is still hampered by screening bottlenecks. The most relevant recent reviews of AcXEs have focused primarily on the biochemical and functional properties of these enzymes. In this review, we focus on AcXE phylogeny, classification and the future of metagenomic bioprospecting for novel AcXEs.

  14. 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...

  15. 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.

  16. Characterisation of a cold adapted esterase and mutants from a psychotolerant Pseudomonas sp. strain.

    PubMed

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

    2016-07-13

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

  17. 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)).

  18. 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

  19. 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.

  20. Juvenile hormone esterase: biochemistry and structure

    PubMed Central

    Kamita, Shizuo G.; Hammock, Bruce D.

    2013-01-01

    Synopsis Normal insect development requires a precisely timed, precipitous drop in hemolymph juvenile hormone (JH) titer. This drop occurs through a coordinated halt in JH biosynthesis and increase in JH metabolism. In many species, JH esterase (JHE) is critical for metabolism of the resonance-stabilized methyl ester of JH. JHE metabolizes JH with a high kcat/KM ratio that results primarily from an exceptionally low KM. Here we review the biochemistry and structure of authentic and recombinant JHEs from six insect orders, and present updated diagnostic criteria that help to distinguish JHEs from other carboxylesterases. The use of a JHE-encoding gene to improve the insecticidal efficacy of biopesticides is also discussed. PMID:23543805

  1. 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.

  2. Metagenomic mining for thermostable esterolytic enzymes uncovers a new family of bacterial esterases

    PubMed Central

    Zarafeta, Dimitra; Moschidi, Danai; Ladoukakis, Efthymios; Gavrilov, Sergey; Chrysina, Evangelia D.; Chatziioannou, Aristotelis; Kublanov, Ilya; Skretas, Georgios; Kolisis, Fragiskos N.

    2016-01-01

    Biocatalysts exerting activity against ester bonds have a broad range of applications in modern biotechnology. Here, we have identified a new esterolytic enzyme by screening a metagenomic sample collected from a hot spring in Kamchatka, Russia. Biochemical characterization of the new esterase, termed EstDZ2, revealed that it is highly active against medium chain fatty acid esters at temperatures between 25 and 60 °C and at pH values 7–8. The new enzyme is moderately thermostable with a half-life of more than six hours at 60 °C, but exhibits exquisite stability against high concentrations of organic solvents. Phylogenetic analysis indicated that EstDZ2 is likely an Acetothermia enzyme that belongs to a new family of bacterial esterases, for which we propose the index XV. One distinctive feature of this new family, is the presence of a conserved GHSAG catalytic motif. Multiple sequence alignment, coupled with computational modelling of the three-dimensional structure of EstDZ2, revealed that the enzyme lacks the largest part of the “cap” domain, whose extended structure is characteristic for the closely related Family IV esterases. Thus, EstDZ2 appears to be distinct from known related esterolytic enzymes, both in terms of sequence characteristics, as well as in terms of three-dimensional structure. PMID:27991516

  3. Conversion of a Rhizopus chinensis lipase into an esterase by lid swapping.

    PubMed

    Yu, Xiao-Wei; Zhu, Shan-Shan; Xiao, Rong; Xu, Yan

    2014-06-01

    In an effort to explore the feasibility of converting a lipase into an esterase by modifying the lid region, we designed and characterized two novel Rhizopus chinensis lipase variants by lid swapping. The substrate specificity of an R. chinensis lipase was successfully modified toward water-soluble substrates, that is, turned into an esterase, by replacing the hydrophobic lid with a hydrophilic lid from ferulic acid esterase from Aspergillus niger Meanwhile, as a comparison, the lid of R. chinensis lipase was replaced by a hydrophobic lid from Rhizomucor miehei lipase, which did not alter its substrate specificity but led to a 5.4-fold higher catalytic efficiency (k*cat/K*m) toward p-nitrophenyl laurate. Based on the analysis of structure-function relationships, it suggests that the amphipathic nature of the lid is very important for the substrate specificity. This study provides new insight into the structural basis of lipase specificities and a way to tune the substrate preference of lipases.

  4. A key esterase required for the mineralization of quizalofop-p-ethyl by a natural consortium of Rhodococcus sp. JT-3 and Brevundimonas sp. JT-9.

    PubMed

    Zhang, Hui; Li, Mengya; Li, Jie; Wang, Guangli; Li, Feng; Xu, Dayong; Liu, Yuan; Xiong, Minghua

    2017-04-05

    A natural consortium, named L1, of Rhodococcus sp. JT-3 and Brevundimonas sp. JT-9 was obtained from quizalofop-p-ethyl (QE) polluted soil. The consortium was able to use QE as a sole carbon source for growth and degraded 100mgL(-1) of QE in 60h. Strain JT-3 initiated the catabolism of QE to quizalofop acid (QA), which was used by strain JT-9 as carbon source for growth and to simultaneously feed strain JT-3. A novel esterase EstS-JT, which was responsible for the transformation of QE to QA and essential for the mineralization of QE by the consortium, was cloned from strain JT-3. EstS-JT showed low amino acid identity to other reported esterases from esterase family VIII and represents a new member of this family. The deduced amino acid sequence contained the esterase family VIII conserved motifs S-X-X-K, YSV and WAG. The purified recombinant EstS-JT displayed maximal esterase activity at 35°C and pH 7.5. An inhibitor assay, site-directed mutagenesis and 3D modeling analysis revealed that S64, K67 and Y175 were essential for catalysis and probably comprised the catalytic center of EstS-JT. Additionally, EstS-JT had broad substrate specificity and was capable of hydrolyzing p-nitrophenyl esters (C2-C8) and various AOPP herbicides.

  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. 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.

  7. Characterization of patatin esterase activity in AOT-isooctane reverse micelles.

    PubMed

    Jiménez, M; Escribano, J; Gandía-Herrero, F; Chazarra, S; Cabanes, J; García-Carmona, F; Pérez-Gilabert, M

    2002-01-01

    Patatin is a family of glycoproteins that accounts for 30-40% of the total soluble protein in potato (Solanum tuberosum L.) tubers. This protein has been reported not only to serve as a storage protein but also to exhibit lipid acyl hydrolase (LAH) activity. In this study patatin is characterized in AOT-isooctane reverse micelles. The influence on the enzymatic activity of characteristic parameters of reverse micelles, w(o) (= H(2)O/AOT), and the percentage of H(2)O, theta, were investigated. The results obtained show that patatin esterase activity varies with w(o) but remains constant throughout the range of theta values studied. The variation with w(o) showed that the activity follows an S-shaped behavior pattern, reaching a maximum at about w(o) = 20 for 2% H(2)O. Patatin esterase activity was compared with p-nitrophenyl (PNP) fatty acid esters of different chain lengths. The activity was much higher for PNP-caprylate. The pH optimum was 6.0, different from the value obtained when patatin esterase activity was measured in mixed micelle systems. The optimal temperature was 35 degrees C, above which the activity decreased to almost zero. The kinetic parameters were also evaluated (K(m) = 10 mM, V(m) = 158 microM/min, V(m)/K(m) = 15.8 x 10(-3) min(-1)). This paper shows the suitability of reverse micelles for measuring patatin esterase activity, since it allows the study of the enzyme in similar conditions to that prevailing in vivo.

  8. 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

  9. 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

  10. Esterase mutation is a mechanism of resistance to antimalarial compounds

    PubMed Central

    Istvan, Eva S.; Mallari, Jeremy P.; Corey, Victoria C.; Dharia, Neekesh V.; Marshall, Garland R.; Winzeler, Elizabeth A.; Goldberg, Daniel E.

    2017-01-01

    Pepstatin is a potent peptidyl inhibitor of various malarial aspartic proteases, and also has parasiticidal activity. Activity of pepstatin against cultured Plasmodium falciparum is highly variable depending on the commercial source. Here we identify a minor contaminant (pepstatin butyl ester) as the active anti-parasitic principle. We synthesize a series of derivatives and characterize an analogue (pepstatin hexyl ester) with low nanomolar activity. By selecting resistant parasite mutants, we find that a parasite esterase, PfPARE (P. falciparum Prodrug Activation and Resistance Esterase) is required for activation of esterified pepstatin. Parasites with esterase mutations are resistant to pepstatin esters and to an open source antimalarial compound, MMV011438. Recombinant PfPARE hydrolyses pepstatin esters and de-esterifies MMV011438. We conclude that (1) pepstatin is a potent but poorly bioavailable antimalarial; (2) PfPARE is a functional esterase that is capable of activating prodrugs; (3) Mutations in PfPARE constitute a mechanism of antimalarial resistance. PMID:28106035

  11. 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

  12. Hydrolysis of synthetic polyesters by Clostridium botulinum esterases.

    PubMed

    Perz, Veronika; Baumschlager, Armin; Bleymaier, Klaus; Zitzenbacher, Sabine; Hromic, Altijana; Steinkellner, Georg; Pairitsch, Andris; Łyskowski, Andrzej; Gruber, Karl; Sinkel, Carsten; Küper, Ulf; Ribitsch, Doris; Guebitz, Georg M

    2016-05-01

    Two novel esterases from the anaerobe Clostridium botulinum ATCC 3502 (Cbotu_EstA and Cbotu_EstB) were expressed in Escherichia coli BL21-Gold(DE3) and were found to hydrolyze the polyester poly(butylene adipate-co-butylene terephthalate) (PBAT). The active site residues (triad Ser, Asp, His) are present in both enzymes at the same location only with some amino acid variations near the active site at the surrounding of aspartate. Yet, Cbotu_EstA showed higher kcat values on para-nitrophenyl butyrate and para-nitrophenyl acetate and was considerably more active (sixfold) on PBAT. The entrance to the active site of the modeled Cbotu_EstB appears more narrowed compared to the crystal structure of Cbotu_EstA and the N-terminus is shorter which could explain its lower activity on PBAT. The Cbotu_EstA crystal structure consists of two regions that may act as movable cap domains and a zinc metal binding site.

  13. Juvenile hormone (JH) esterase of the mosquito Culex quinquefasciatus is not a target of the JH analog insecticide methoprene.

    PubMed

    Kamita, Shizuo G; Samra, Aman I; Liu, Jun-Yan; Cornel, Anthony J; Hammock, Bruce D

    2011-01-01

    Juvenile hormones (JHs) are essential sesquiterpenes that control insect development and reproduction. JH analog (JHA) insecticides such as methoprene are compounds that mimic the structure and/or biological activity of JH. In this study we obtained a full-length cDNA, cqjhe, from the southern house mosquito Culex quinquefasciatus that encodes CqJHE, an esterase that selectively metabolizes JH. Unlike other recombinant esterases that have been identified from dipteran insects, CqJHE hydrolyzed JH with specificity constant (k(cat)/K(M) ratio) and V(max) values that are common among JH esterases (JHEs). CqJHE showed picomolar sensitivity to OTFP, a JHE-selective inhibitor, but more than 1000-fold lower sensitivity to DFP, a general esterase inhibitor. To our surprise, CqJHE did not metabolize the isopropyl ester of methoprene even when 25 pmol of methoprene was incubated with an amount of CqJHE that was sufficient to hydrolyze 7,200 pmol of JH to JH acid under the same assay conditions. In competition assays in which both JH and methoprene were available to CqJHE, methoprene did not show any inhibitory effects on the JH hydrolysis rate even when methoprene was present in the assay at a 10-fold higher concentration relative to JH. Our findings indicated that JHE is not a molecular target of methoprene. Our findings also do not support the hypothesis that methoprene functions in part by inhibiting the action of JHE.

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

    PubMed Central

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

    2013-01-01

    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

  15. Functional Characterization of a Marine Bacillus Esterase and its Utilization in the Stereo-Selective Production of D-Methyl Lactate.

    PubMed

    Huang, Jinlong; Zhang, Yun; Hu, Yunfeng

    2016-12-01

    Chiral lactic acid and its ester derivatives are crucial building blocks and platforms in the generation of high value-added drugs, fine chemicals and functional materials. Optically pure D-lactic acid and its ester derivatives cannot be directly generated from fermentation and are quite expensive. Herein, we identified, heterologously expressed and functionally characterized one Bacillus esterase BSE01701 from the deep sea of the Indian Ocean. Esterase BSE01701 could enzymatically resolve inexpensive racemic methyl lactate and generate chiral D-methyl lactate. The enantiomeric excess of desired chiral D-methyl lactate and the substrate conversion could reach over 99 % and 60 %, respectively, after process optimization. Notably, the addition of 60 % (v/v) organic co-solvent heptane could greatly improve both the enantiomeric excess of D-methyl lactate and the conversion. BSE01701 was a very promising marine microbial esterase in the generation of chiral chemicals in industry.

  16. 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.

  17. 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.

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

    PubMed

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

    2013-12-01

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

  19. 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.

  20. Extracellular and intracellular esterase processing of SCFA-hexosamine analogs: implications for metabolic glycoengineering and drug delivery.

    PubMed

    Mathew, Mohit P; Tan, Elaine; Shah, Shivam; Bhattacharya, Rahul; Adam Meledeo, M; Huang, Jun; Espinoza, Freddy A; Yarema, Kevin J

    2012-11-15

    This report provides a synopsis of the esterase processing of short chain fatty acid (SCFA)-derivatized hexosamine analogs used in metabolic glycoengineering by demonstrating that the extracellular hydrolysis of these compounds is comparatively slow (e.g., with a t(1/2) of ∼4 h to several days) in normal cell culture as well as in high serum concentrations intended to mimic in vivo conditions. Structure-activity relationship (SAR) analysis of common sugar analogs revealed that O-acetylated and N-azido ManNAc derivatives were more refractory against extracellular inactivation by FBS than their butanoylated counterparts consistent with in silico docking simulations of Ac(4)ManNAc and Bu(4)ManNAc to human carboxylesterase 1 (hCE1). By contrast, all analogs tested supported increased intracellular sialic acid production within 2h establishing that esterase processing once the analogs are taken up by cells is not rate limiting.

  1. Cell-bound lipase and esterase of Brevibacterium linens.

    PubMed

    Sorhaug, T; Ordal, Z J

    1974-03-01

    The activities of glycerol ester hydrolase, lipase (EC 3.1.1.3) and carboxylesterase, and esterase (EC 3.1.1.1) were determined for whole cell preparations of Brevibacterium linens by using the pH-stat assay. The culture growth liquors were inactive against the three substrates, tributyrin emulsion, triacetin, and methyl butyrate. Cells washed in water had less activity than cells washed in 5% NaCl; the ratio of activities was close to 1:2 for all strains using tributyrin emulsion as the substrate. For the esterase substrates, this relationship varied widely and was strain dependent. The ability to hydrolyze the two esterase substrates varied independently of the level of lipase activity.

  2. 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.

  3. New insights on molecular interactions of organophosphorus pesticides with esterases.

    PubMed

    Mangas, Iris; Estevez, Jorge; Vilanova, Eugenio; França, Tanos Celmar Costa

    2017-02-01

    Organophosphorus compounds (OPs) are a large and diverse class of chemicals mainly used as pesticides and chemical weapons. People may be exposed to OPs in several occasions, which can produce several distinct neurotoxic effects depending on the dose, frequency of exposure, type of OP, and the host factors that influence susceptibility and sensitivity. These neurotoxic effects are mainly due to the interaction with enzyme targets involved in toxicological or detoxication pathways. In this work, the toxicological relevance of known OPs targets is reviewed. The main enzyme targets of OPs have been identified among the serine hydrolase protein family, some of them decades ago (e.g. AChE, BuChE, NTE and carboxylesterases), others more recently (e.g. lysophospholipase, arylformidase and KIA1363) and others which are not molecularly identified yet (e.g. phenylvalerate esterases). Members of this family are characterized by displaying serine hydrolase activity, containing a conserved serine hydrolase motif and having an alpha-beta hydrolase fold. Improvement in Xray-crystallography and in silico methods have generated new data of the interactions between OPs and esterases and have established new methods to study new inhibitors and reactivators of cholinesterases. Mass spectrometry for AChE, BChE and APH have characterized the active site serine adducts with OPs being useful to detect biomarkers of OPs exposure and inhibitory and postinhibitory reactions of esterases and OPs. The purpose of this review is focus specifically on the interaction of OP with esterases, mainly with type B-esterases, which are able to hydrolyze carboxylesters but inhibited by OPs by covalent phosphorylation on the serine or tyrosine residue in the active sites. Other related esterases in some cases with no-irreversible effect are also discussed. The understanding of the multiple molecular interactions is the basis we are proposing for a multi-target approach for understanding the

  4. The apeE Gene of Salmonella typhimurium Encodes an Outer Membrane Esterase Not Present in Escherichia coli

    PubMed Central

    Carinato, Maria E.; Collin-Osdoby, Patricia; Yang, Xioming; Knox, Tina M.; Conlin, Christopher A.; Miller, Charles G.

    1998-01-01

    Salmonella typhimurium apeR mutations lead to overproduction of an outer membrane-associated N-acetyl phenylalanine β-naphthyl ester-cleaving esterase that is encoded by the apeE gene (P. Collin-Osdoby and C. G. Miller, Mol. Gen. Genet. 243:674–680, 1994). This paper reports the cloning and nucleotide sequencing of the S. typhimurium apeE gene as well as some properties of the esterase that it encodes. The predicted product of apeE is a 69.9-kDa protein which is processed to a 67-kDa species by removal of a signal peptide. The predicted amino acid sequence of ApeE indicates that it is a member of the GDSL family of serine esterases/lipases. It is most similar to a lipase excreted by the entomopathogenic bacterium Photorhabdus luminescens. The Salmonella esterase catalyzes the hydrolysis of a variety of fatty acid naphthyl esters and of C6 to C16 fatty acid p-nitrophenyl esters but will not hydrolyze peptide bonds. A rapid diagnostic test reported to be useful in distinguishing Salmonella spp. from related organisms makes use of the ability of Salmonella to hydrolyze the chromogenic ester substrate methyl umbelliferyl caprylate. We report that the apeE gene product is the enzyme in Salmonella uniquely responsible for the hydrolysis of this substrate. Southern blot analysis indicates that Escherichia coli K-12 does not contain a close analog of apeE, and it appears that the apeE gene is contained in a region of DNA present in Salmonella but not in E. coli. PMID:9657991

  5. Cloning and characterization of a novel thermostable esterase from Bacillus gelatini KACC 12197.

    PubMed

    Kim, Jinyeong; Deng, Lili; Hong, Eunsoo; Ryu, Yeonwoo

    2015-12-01

    A novel gene encoding a thermostable esterase (designated as Est-gela) was isolated from the moderate thermophile Bacillus gelatini KACC 12197. The open reading frame of this gene (1170 bp) encodes 389 amino acid residues, and the molecular weight of Est-gela is approximately 42 kDa. The protein sequence of Est-gela shows similarity with β-lactamases and esterases (⩽ 43%). Est-gela contains the Ser-X-X-Lys conserved sequence (Ser58-Met59-Thr60-Lys61) and belongs to family VIII of esterases. We overexpressed Est-gela in Escherichia coli XL1-blue and purified this protein using a His tag. Est-gela showed a strong enzymatic activity toward p-nitrophenyl esters with short acyl chains (⩽ C4) and the strongest activity toward p-nitrophenyl butyrate. Est-gela showed an enhanced enzymatic activity at 65-75 °C and retained more than 90% of the activity after incubation at 65 °C for 180 min. These results indicated that Est-gela was thermostable. In addition, Est-gela showed the maximal activity at pH 10. We also evaluated the effects of surfactants and organic solvents. Surfactants were more effective at improving the enzymatic activity than were organic solvents. Finally, Est-gela hydrolyzed (R,S)-ketoprofen ethyl ester (Kcat/Km = 5.0 ± 0.2 s(-1) mM(-1), mean ± standard error) with enantioselectivity toward (S)-ketoprofen ethyl ester rather than (R)-ketoprofen ethyl ester.

  6. Structural insights into the substrate specificity of two esterases from the thermophilic Rhizomucor miehei

    PubMed Central

    Yang, Shaoqing; Qin, Zhen; Duan, Xiaojie; Yan, Qiaojuan; Jiang, Zhengqiang

    2015-01-01

    Two hormone-sensitive lipase (HSL) family esterases (RmEstA and RmEstB) from the thermophilic fungus Rhizomucor miehei, exhibiting distinct substrate specificity, have been recently reported to show great potential in industrial applications. In this study, the crystal structures of RmEstA and RmEstB were determined at 2.15 Å and 2.43 Å resolutions, respectively. The structures of RmEstA and RmEstB showed two distinctive domains, a catalytic domain and a cap domain, with the classical α/β-hydrolase fold. Catalytic triads consisting of residues Ser161, Asp262, and His292 in RmEstA, and Ser164, Asp261, and His291 in RmEstB were found in the respective canonical positions. Structural comparison of RmEstA and RmEstB revealed that their distinct substrate specificity might be attributed to their different substrate-binding pockets. The aromatic amino acids Phe222 and Trp92, located in the center of the substrate-binding pocket of RmEstB, blocked this pocket, thus narrowing its catalytic range for substrates (C2–C8). Two mutants (F222A and W92F in RmEstB) showing higher catalytic activity toward long-chain substrates further confirmed the hypothesized interference. This is the first report of HSL family esterase structures from filamentous fungi.jlr The information on structure-function relationships could open important avenues of exploration for further industrial applications of esterases. PMID:26108223

  7. In vitro modifications of rat NTE and other esterases by chemicals which induce delayed neurotoxicity in vivo.

    PubMed

    Blasco, R; Moreno, E; Sanz, P; Repetto, M

    1990-10-01

    A rat in vitro model has been developed which permits direct study of the biochemical mechanisms involved in delayed neurotoxicity induced by any chemical compound, not only organophosphates. Using rat brain homogenate, a parallel study on the activity of neurotoxic esterase (NTE) and total esterases (TE) compared the action of metamidophos, which is known to induce delayed neurotoxicity, and the synthetic fatty acid anilides, oleylanilide and linoleylanilide. Inhibition in the activity of NTE and TE, unrelated to the concentration and the incubation time assayed, was caused by metamidophos, while the anilides showed a 2-phase concentration-time dependent behaviour. This confirmed the results we previously obtained in vivo. In both cases the appearance of delayed neuropathy was related to modification of NTE activity. We concluded that phosphorylation of the enzyme may not be the only biochemical requirement for the development of delayed neurotoxicity syndromes in which modification of NTE is produced.

  8. Endophytic fungi producing of esterases: evaluation in vitro of the enzymatic activity using pH indicator.

    PubMed

    Lisboa, Helen Cristina Fávero; Biasetto, Carolina Rabal; de Medeiros, João Batista; Âraújo, Angela 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.

  9. 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

  10. 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.

  11. 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.

  12. A Novel Alkaliphilic Bacillus Esterase Belongs to the 13th Bacterial Lipolytic Enzyme Family

    PubMed Central

    Rao, Lang; Xue, Yanfen; Zheng, Yingying; Lu, Jian R.; Ma, Yanhe

    2013-01-01

    Background Microbial derived lipolytic hydrolysts are an important class of biocatalysts because of their huge abundance and ability to display bioactivities under extreme conditions. In spite of recent advances, our understanding of these enzymes remains rudimentary. The aim of our research is to advance our understanding by seeking for more unusual lipid hydrolysts and revealing their molecular structure and bioactivities. Methodology/Principal Findings Bacillus. pseudofirmus OF4 is an extreme alkaliphile with tolerance of pH up to 11. In this work we successfully undertook a heterologous expression of a gene estof4 from the alkaliphilic B. pseudofirmus sp OF4. The recombinant protein called EstOF4 was purified into a homologous product by Ni-NTA affinity and gel filtration. The purified EstOF4 was active as dimer with the molecular weight of 64 KDa. It hydrolyzed a wide range of substrates including p-nitrophenyl esters (C2–C12) and triglycerides (C2–C6). Its optimal performance occurred at pH 8.5 and 50°C towards p-nitrophenyl caproate and triacetin. Sequence alignment revealed that EstOF4 shared 71% identity to esterase Est30 from Geobacillus stearothermophilus with a typical lipase pentapeptide motif G91LS93LG95. A structural model developed from homology modeling revealed that EstOF4 possessed a typical esterase 6α/7β hydrolase fold and a cap domain. Site-directed mutagenesis and inhibition studies confirmed the putative catalytic triad Ser93, Asp190 and His220. Conclusion EstOF4 is a new bacterial esterase with a preference to short chain ester substrates. With a high sequence identity towards esterase Est30 and several others, EstOF4 was classified into the same bacterial lipolytic family, Family XIII. All the members in this family originate from the same bacterial genus, bacillus and display optimal activities from neutral pH to alkaline conditions with short and middle chain length substrates. However, with roughly 70% sequence identity, these

  13. Identification and Characterization of a Novel Salt-Tolerant Esterase from the Deep-Sea Sediment of the South China Sea.

    PubMed

    Zhang, Yi; Hao, Jie; Zhang, Yan-Qi; Chen, Xiu-Lan; Xie, Bin-Bin; Shi, Mei; Zhou, Bai-Cheng; Zhang, Yu-Zhong; Li, Ping-Yi

    2017-01-01

    Marine esterases play an important role in marine organic carbon degradation and cycling. Halotolerant esterases from the sea may have good potentials in industrial processes requiring high salts. Although a large number of marine esterases have been characterized, reports on halotolerant esterases are only a few. Here, a fosmid library containing 7,200 clones was constructed from a deep-sea sediment sample from the South China Sea. A gene H8 encoding an esterase was identified from this library by functional screening and expressed in Escherichia coli. Phylogenetic analysis showed that H8 is a new member of family V of bacterial lipolytic enzymes. H8 could effectively hydrolyze short-chain monoesters (C4-C10), with the highest activity toward p-nitrophenyl hexanoate. The optimal temperature and pH for H8 activity were 35°C and pH 10.0, respectively. H8 had high salt tolerance, remaining stable in 4.5 M NaCl, which suggests that H8 is well adapted to the marine saline environment and that H8 may have industrial potentials. Unlike reported halophilic/halotolerant enzymes with high acidic/basic residue ratios and low pI values, H8 contains a large number of basic residues, leading to its high basic/acidic residue ratio and high predicted pI (9.09). Moreover, more than 10 homologous sequences with similar basic/acidic residue ratios and predicted pI values were found in database, suggesting that H8 and its homologs represent a new group of halotolerant esterases. We also investigated the role of basic residues in H8 halotolerance by site-directed mutation. Mutation of Arg195, Arg203 or Arg236 to acidic Glu significantly decreased the activity and/or stability of H8 under high salts, suggesting that these basic residues play a role in the salt tolerance of H8. These results shed light on marine bacterial esterases and halotolerant enzymes.

  14. Identification and Characterization of a Novel Salt-Tolerant Esterase from the Deep-Sea Sediment of the South China Sea

    PubMed Central

    Zhang, Yi; Hao, Jie; Zhang, Yan-Qi; Chen, Xiu-Lan; Xie, Bin-Bin; Shi, Mei; Zhou, Bai-Cheng; Zhang, Yu-Zhong; Li, Ping-Yi

    2017-01-01

    Marine esterases play an important role in marine organic carbon degradation and cycling. Halotolerant esterases from the sea may have good potentials in industrial processes requiring high salts. Although a large number of marine esterases have been characterized, reports on halotolerant esterases are only a few. Here, a fosmid library containing 7,200 clones was constructed from a deep-sea sediment sample from the South China Sea. A gene H8 encoding an esterase was identified from this library by functional screening and expressed in Escherichia coli. Phylogenetic analysis showed that H8 is a new member of family V of bacterial lipolytic enzymes. H8 could effectively hydrolyze short-chain monoesters (C4–C10), with the highest activity toward p-nitrophenyl hexanoate. The optimal temperature and pH for H8 activity were 35°C and pH 10.0, respectively. H8 had high salt tolerance, remaining stable in 4.5 M NaCl, which suggests that H8 is well adapted to the marine saline environment and that H8 may have industrial potentials. Unlike reported halophilic/halotolerant enzymes with high acidic/basic residue ratios and low pI values, H8 contains a large number of basic residues, leading to its high basic/acidic residue ratio and high predicted pI (9.09). Moreover, more than 10 homologous sequences with similar basic/acidic residue ratios and predicted pI values were found in database, suggesting that H8 and its homologs represent a new group of halotolerant esterases. We also investigated the role of basic residues in H8 halotolerance by site-directed mutation. Mutation of Arg195, Arg203 or Arg236 to acidic Glu significantly decreased the activity and/or stability of H8 under high salts, suggesting that these basic residues play a role in the salt tolerance of H8. These results shed light on marine bacterial esterases and halotolerant enzymes. PMID:28386249

  15. Amino acid alignment of cholinesterases, esterases, lipases, and related proteins

    SciTech Connect

    Gentry, M.K.; Doctor, B.P.

    1995-12-31

    The alignments previously published (Gentry Doctor, 1991; Cygler et al., 1993), nine and 32 sequences respectively, have been further expanded by the addition of 22 newly-found sequences. References and protein sequences were found by searching on the term acetylcholinesterase using the software package Entrez, an integrated citation and sequence retrieval system (National Center for Biotechnology Information, NLM, Bethesda, MD).

  16. 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

  17. An Esterase from Anaerobic Clostridium hathewayi Can Hydrolyze Aliphatic-Aromatic Polyesters.

    PubMed

    Perz, Veronika; Hromic, Altijana; Baumschlager, Armin; Steinkellner, Georg; Pavkov-Keller, Tea; Gruber, Karl; Bleymaier, Klaus; Zitzenbacher, Sabine; Zankel, Armin; Mayrhofer, Claudia; Sinkel, Carsten; Kueper, Ulf; Schlegel, Katharina; Ribitsch, Doris; Guebitz, Georg M

    2016-03-15

    Recently, a variety of biodegradable polymers have been developed as alternatives to recalcitrant materials. Although many studies on polyester biodegradability have focused on aerobic environments, there is much less known on biodegradation of polyesters in natural and artificial anaerobic habitats. Consequently, the potential of anaerobic biogas sludge to hydrolyze the synthetic compostable polyester PBAT (poly(butylene adipate-co-butylene terephthalate) was evaluated in this study. On the basis of reverse-phase high-performance liquid chromatography (RP-HPLC) analysis, accumulation of terephthalic acid (Ta) was observed in all anaerobic batches within the first 14 days. Thereafter, a decline of Ta was observed, which occurred presumably due to consumption by the microbial population. The esterase Chath_Est1 from the anaerobic risk 1 strain Clostridium hathewayi DSM-13479 was found to hydrolyze PBAT. Detailed characterization of this esterase including elucidation of the crystal structure was performed. The crystal structure indicates that Chath_Est1 belongs to the α/β-hydrolases family. This study gives a clear hint that also micro-organisms in anaerobic habitats can degrade manmade PBAT.

  18. 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.

  19. 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

  20. 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.

  1. Screening, purification, and characterization of a novel organic solvent-tolerant esterase, Lip2, from Monascus purpureus strain M7.

    PubMed

    Kang, Li-Jing; Meng, Zi-Tong; Hu, Chen; Zhang, Yan; Guo, Hai-Lun; Li, Qing; Li, Mu

    2017-03-01

    Organic solvent-tolerant esterases are proven to be excellent biocatalysts in chemical and pharmaceutical industries. A novel organic solvent-tolerant esterase gene, lip2, was isolated from filamentous fungi Monascus purpureus M7. The sequence analysis suggested that lip2 has a conserved "GDSL" motif near the active center. The multiple-sequence alignment and phylogenetic analysis revealed that Lip2 displayed two unique amino-acid sequence motifs that clearly separate it from any other previously described lipase family. After incubation in 20% methanol and ethanol for 3 h, the Lip2 displayed 190 and 180% residual activities, respectively. It retained 99-110% relative activity in 20% (v/v) hydrophilic organic solvents after incubation for 1 day. This esterase showed optimal activity at 40 °C and retained about 70% maximal activity at 60 °C. The enzyme also displayed more than 50% residual activity over a range of pH 5-11. In the presence of most of metal ions or additives, Lip2 retained most of the activity. These unique properties of Lip2 make it a promising as biocatalyst for industrial processes.

  2. 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

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

    PubMed Central

    Agger, Jane W.; Busk, Peter K.; Pilgaard, Bo; Meyer, Anne S.; Lange, Lene

    2017-01-01

    Glucuronoyl esterases are a novel type of enzymes believed to catalyze the hydrolysis of ester linkages between lignin and glucuronoxylan in lignocellulosic biomass, linkages known as lignin carbohydrate complexes. These complexes contribute to the recalcitrance of lignocellulose. Glucuronoyl esterases are a part of the microbial machinery for lignocellulose degradation and coupling their role to the occurrence of lignin carbohydrate complexes in biomass is a desired research goal. Glucuronoyl esterases have been assigned to CAZymes family 15 of carbohydrate esterases, but only few examples of characterized enzymes exist and the exact activity is still uncertain. Here peptide pattern recognition is used as a bioinformatic tool to identify and group new CE15 proteins that are likely to have glucuronoyl esterase activity. 1024 CE15-like sequences were drawn from GenBank and grouped into 24 groups. Phylogenetic analysis of these groups made it possible to pinpoint groups of putative fungal and bacterial glucuronoyl esterases and their sequence variation. Moreover, a number of groups included previously undescribed CE15-like sequences that are distinct from the glucuronoyl esterases and may possibly have different esterase activity. Hence, the CE15 family is likely to comprise other enzyme functions than glucuronoyl esterase alone. Gene annotation in a variety of fungal and bacterial microorganisms showed that coprophilic fungi are rich and diverse sources of CE15 proteins. Combined with the lifestyle and habitat of coprophilic fungi, they are predicted to be excellent candidates for finding new glucuronoyl esterase genes. PMID:28293230

  4. Spruce budworm (Choristoneura fumiferana) juvenile hormone esterase: hormonal regulation, developmental expression and cDNA cloning.

    PubMed

    Feng, Q L; Ladd, T R; Tomkins, B L; Sundaram, M; Sohi, S S; Retnakaran, A; Davey, K G; Palli, S R

    1999-02-25

    We have used the differential display of mRNAs technique to identify Choristoneura fumiferana genes that are induced by juvenile hormone I (JH I). Of the six PCR products identified, one bound to a 2.8-kb mRNA from CF-203 cells whose abundance increased when the cells were grown in the presence of JH I. The same 2.8-kb mRNA decreased to undetectable levels when the CF-203 cells were grown in the presence of 20-hydroxyecdysone (20E). The PCR fragment probe also detected a 2.8-kb mRNA in the C. fumiferana larval tissues. This 2.8-kb mRNA was present on the first day of the first, third, fourth, fifth and sixth larval and pupal stadia, but was conspicuously absent on the first day of the second larval stadium, as well as during the intermolt periods of the first to fifth instar larval stages. In the sixth instar larvae the 2.8-kb mRNA was detected in the fat body, epidermis and midgut during the intermolt period. The PCR fragment was used as a probe to screen a cDNA library. The deduced amino acid sequence of this 2.8-kb cDNA clone showed similarity with the deduced amino acid sequences of Heliothis virescens juvenile hormone esterases (HvJHE). The deduced amino acid sequence of the cDNA clone contained all five functional motifs that are present in most of esterases, proteases and lipases. The cDNA clone was expressed in the baculovirus expression system, producing a protein that showed JHE activity.

  5. 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.

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

    PubMed Central

    Loughna, S; Bennett, P; Gau, G; Nicolaides, K; Blunt, S; Moore, G

    1993-01-01

    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. Images Figure 1 Figure 2 Figure 3 PMID:8213811

  7. 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.

  8. Penicillium purpurogenum produces a family 1 acetyl xylan esterase containing a carbohydrate-binding module: characterization of the protein and its gene.

    PubMed

    Gordillo, Felipe; Caputo, Valentina; Peirano, Alessandra; Chavez, Renato; Van Beeumen, Jozef; Vandenberghe, Isabel; Claeyssens, Marc; Bull, Paulina; Ravanal, María Cristina; Eyzaguirre, Jaime

    2006-10-01

    At least three acetyl xylan esterases (AXE I, II and III) are secreted by Penicillium purpurogenum. This publication describes more detailed work on AXE I and its gene. AXE I binds cellulose but not xylan; it is glycosylated and inactivated by phenylmethylsulphonyl fluoride, showing that it is a serine esterase. The axe1 gene presents an open reading frame of 1278 bp, including two introns of 68 and 61 bp; it codes for a signal peptide of 31 residues and a mature protein of 351 amino acids (molecular weight 36,693). AXE I has a modular structure: a catalytic module at the amino terminus belonging to family 1 of the carbohydrate esterases, a linker rich in serines and threonines, and a family 1 carboxy terminal carbohydrate binding module (CBM). The CBM is similar to that of AXE from Trichoderma reesei, (with a family 5 catalytic module) indicating that the genes for catalytic modules and CBMs have evolved separately, and that they have been linked by gene fusion. The promoter sequence of axe1 contains several putative sequences for binding of gene expression regulators also found in other family 1 esterase gene promoters. It is proposed that AXE I and II act in succession in xylan degradation; first, xylan is attacked by AXE I and other xylanases possessing CBMs (which facilitate binding to lignocellulose), followed by other enzymes acting mainly on soluble substrates.

  9. 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

  10. Mechanism and diversity of the erythromycin esterase family of enzymes.

    PubMed

    Morar, Mariya; Pengelly, Kate; Koteva, Kalinka; Wright, Gerard D

    2012-02-28

    Macrolide antibiotics such as azithromycin and erythromycin are mainstays of modern antibacterial chemotherapy, and like all antibiotics, they are vulnerable to resistance. One mechanism of macrolide resistance is via drug inactivation: enzymatic hydrolysis of the macrolactone ring catalyzed by erythromycin esterases, EreA and EreB. A genomic enzymology approach was taken to gain insight into the catalytic mechanisms and origins of Ere enzymes. Our analysis reveals that erythromycin esterases comprise a separate group in the hydrolase superfamily, which includes homologues of uncharacterized function found on the chromosome of Bacillus cereus, Bcr135 and Bcr136, whose three-dimensional structures have been determined. Biochemical characterization of Bcr136 confirms that it is an esterase that is, however, unable to inactivate macrolides. Using steady-state kinetics, homology-based structure modeling, site-directed mutagenesis, solvent isotope effect studies, pH, and inhibitor profiling performed in various combinations for EreA, EreB, and Bcr136 enzymes, we identified the active site and gained insight into some catalytic features of this novel enzyme superfamily. We rule out the possibility of a Ser/Thr nucleophile and show that one histidine, H46 (EreB numbering), is essential for catalytic function. This residue is proposed to serve as a general base in activation of a water molecule as the reaction nucleophile. Furthermore, we show that EreA, EreB, and Bcr136 are distinct, with only EreA inhibited by chelating agents and hypothesized to contain a noncatalytic metal. Detailed characterization of these esterases allows for a direct comparison of the resistance determinants, EreA and EreB, with their prototype, Bcr136, and for the discussion of their potential connections.

  11. 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.

  12. The catalytic triad of the influenza C virus glycoprotein HEF esterase: characterization by site-directed mutagenesis and functional analysis.

    PubMed

    Pleschka, S; Klenk, H D; Herrler, G

    1995-10-01

    Influenza C virus is able to inactivate its own cellular receptors by virtue of a sialate 9-O-acetylesterase that releases the acetyl residue at position C-9 of N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2). The receptor-destroying enzyme activity is a function of the surface glycoprotein HEF and this esterase belongs to the class of serine hydrolases. In their active site, these enzymes contain a catalytic triad made up of a serine, a histidine and an aspartic acid residue. Sequence comparison with other serine esterases has indicated that, in addition to serine-71 (S71), the amino acids histidine-368 or -369 (H368/369) and aspartic acid 261 (D261) are the most likely candidates to form the catalytic triad of the influenza C virus glycoprotein. By site-directed mutagenesis, mutants were generated in which alanine substituted for either of these amino acids. Using a phagemid expression vector, pSP1D-HEF the HEF gene was expressed in both COS 7 and MDCK I cells. The glycoprotein was obtained in a functional form only in the latter cells, as indicated by its transport to the cell surface and measurable enzyme activity. The low level of expression could be increased by stimulating the NF-KB-binding activity of the cytomegalovirus immediate-early promoter/enhancer element of the vector. The esterase activity of the mutant proteins was compared with that of the wild-type glycoprotein. With Neu5,9Ac2 as the substrate, the esterase specific activities of the S71/A mutant and the H368,369/A mutant were reduced by more than 90%. In the case of the D261/A mutant the specific activity was reduced by 64%. From this data we conclude that S71, H368/369 and D261 are likely to represent the catalytic triad of the influenza C virus glycoprotein HEF. In addition, N280 is proposed to stabilize the oxyanion of the presumptive transition state intermediate formed by the enzyme-substrate complex.

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

    PubMed Central

    Horsfall, Louise E.; Wardrope, Caroline; Togneri, Peter D.; Marles-Wright, Jon; Rosser, Susan J.

    2016-01-01

    Proteins in the serine esterase family are widely distributed in bacterial phyla and display activity against a range of biologically produced and chemically synthesized esters. A serine esterase from the psychrophilic bacterium Pseudoalteromonas arctica with a C-terminal OsmC-like domain was recently characterized; here we report on the identification and characterization of further putative esterases with OsmC-like domains constituting a new esterase family that is found in a variety of bacterial species from different environmental niches. All of these proteins contained the Ser-Asp-His motif common to serine esterases and a highly conserved pentapeptide nucleophilic elbow motif. We produced these proteins heterologously in Escherichia coli and demonstrated their activity against a range of esterase substrates. Two of the esterases characterized have activity of over two orders of magnitude higher than other members of the family, and are active over a wide temperature range. We determined the crystal structure of the esterase domain of the protein from Rhodothermus marinus and show that it conforms to the classical α/β hydrolase fold with an extended ‘lid’ region, which occludes the active site of the protein in the crystal. The expansion of characterized members of the esterase family and demonstration of activity over a wide-range of temperatures could be of use in biotechnological applications such as the pharmaceutical, detergent, bioremediation and dairy industries. PMID:27851780

  14. Isozymic variations in specific and nonspecific esterase and its thermostability in silkworm, Bombyx mori L.

    PubMed

    Patnaik, Bharat Bhusan; Biswas, Tapati Datta; Nayak, Sandeepta Kumar; Saha, A K; Majumdar, M K

    2012-09-01

    Esterase isozymic variations were documented in the haemolymph of developed multivoltine and bivoltine silkworm breeds during unfavorable seed crop seasons of May - September using á- and â- napthylacetate separately to identify specific and nonspecific esterase having thermotolerant potentiality. Variations existed in the isozyme pattern with three bands (Est-2, 3 and 4) in pure Nistari race and other developed multivoltine and bivoltine breeds. Est-2 and Est-3 were non-specific esterases as they were observed when both á- and â-napthylacetate was used as substrates separately. Est-4 band was observed only with á-napthylacetate as substrate and was therefore confirmed to be specific á-esterase band in the haemolymph of silkworm, Bombyx mori L. Zymograms showed that the non-specific esterase band (Est-3) with R1 of 0.43 and specific á-esterase band (Est-4) with R(f) of 0.32 predominately withstood a temperature of 70 +/- 2 degrees C for a duration of 10 min and were confirmed as thermostable esterases in haemolymph of silkworm, Bombyx mori L. This also categorized the presence of thermostable esterases in developed multivoltine and bivoltine breeds of silkworm, even though the qualitative activity was more in the former than the latter. The qualitative presence of thermostable esterases and their activity could be adopted as an indicative biochemical marker in relation to thermotolerance in silkworm.

  15. The effect of liver esterases and temperature on remifentanil degradation in vitro.

    PubMed

    Piazza, Ornella; Cascone, Sara; Sessa, Linda; De Robertis, Edoardo; Lamberti, Gaetano

    2016-08-20

    Remifentanil is a potent opioid metabolized by serum and tissue esterases; it is routinely administered to patients with liver failure as anaesthetic and analgo-sedative without variation in doses, even if prolonged clinical effects and respiratory depression have been observed in these patients. The aim of this study was to determine remifentanil enzymatic degradation kinetics bearing in mind the effect of liver esterases in order to trace a more accurate pharmacokinetic profile of the drug. Solution samples were taken over time and analysed to measure remifentanil concentration by HPLC. We reproduced the physiological settings, varying temperature and pH in vitro and evaluated the kinetics of degradation of remifentanil in the presence of Rhizopus Oryzae esterases, equine liver esterases and porcine liver esterases. Remifentanil kinetics of degradation was accelerated by porcine liver esterases. Remifentanil in vitro half-life decreases with increasing temperatures in the presence of porcine liver esterases. A drug model simulation considering the effect of temperature in the presence of liver esterases was developed. Remifentanil in vitro half-life decreases with increasing temperatures when porcine liver esterases are present. In this paper we propose a model for describing remifentanil degradation kinetics at various temperatures.

  16. Purification, characterization, and molecular cloning of organic-solvent-tolerant cholesterol esterase from cyclohexane-tolerant Burkholderia cepacia strain ST-200.

    PubMed

    Takeda, Yasuhiko; Aono, Rikizo; Doukyu, Noriyuki

    2006-08-01

    Extracellular cholesterol esterase of Burkholderia cepacia strain ST-200 was purified from the culture supernatant. Its molecular mass was 37 kDa. The enzyme was stable at pH 5.5-12 and active at pH 5.5-6, showing optimal activity at pH 7.0 at 45 degrees C. Relative to the commercially available cholesterol esterases, the purified enzyme was highly stable in the presence of various water-miscible organic solvents. The enzyme preferentially hydrolyzed long-chain fatty acid esters of cholesterol, except for that of cholesteryl palmitate. The enzyme exhibited lipolytic activity toward various p-nitrophenyl esters. The hydrolysis rate of p-nitrophenyl caprylate was enhanced 3.5- to 7.2-fold in the presence of 5-20% (vol/vol) water-miscible organic solvents relative to that in the absence of organic solvents. The structural gene encoding the cholesterol esterase was cloned and sequenced. The primary translation product was predicted to be 365 amino acid residues. The mature product is composed of 325 amino acid residues. The amino acid sequence of the product showed the highest similarity to the lipase LipA (87%) from B. cepacia DSM3959.

  17. Biochemical and Domain Analyses of FSUAxe6B, a Modular Acetyl Xylan Esterase, Identify a Unique Carbohydrate Binding Module in Fibrobacter succinogenes S85▿ †

    PubMed Central

    Yoshida, Shosuke; Mackie, Roderick I.; Cann, Isaac K. O.

    2010-01-01

    Acetyl xylan esterase (EC 3.1.1.72) is a member of a set of enzymes required to depolymerize hemicellulose, especially xylan that is composed of a main chain of β-1,4-linked xylopyranoside residues decorated with acetyl side groups. Fibrobacter succinogenes S85 Axe6B (FSUAxe6B) is an acetyl xylan esterase encoded in the genome of this rumen bacterium. The enzyme is a modular protein comprised of an esterase domain, a carbohydrate-binding module, and a region of unknown function. Sequences that are homologous to the region of unknown function are paralogously distributed, thus far, only in F. succinogenes. Therefore, the sequences were designated Fibrobacter succinogenes-specific paralogous module 1 (FPm-1). The FPm-1s are associated with at least 24 polypeptides in the genome of F. succinogenes S85. A bioinformatics search showed that most of the FPm-1-appended polypeptides are putative carbohydrate-active enzymes, suggesting a potential role in carbohydrate metabolism. Truncational analysis of FSUAxe6B, together with catalytic and substrate binding studies, has allowed us to delineate the functional modules in the polypeptide. The N-terminal half of FSUAxe6B harbors the activity that cleaves side chain acetyl groups from xylan-like substrates, and the binding of insoluble xylan was determined to originate from FPm-1. Site-directed mutagenesis studies of highly conserved active-site residues in the esterase domain suggested that the esterase activity is derived from a tetrad composed of Ser44, His273, Glu194, and Asp270, with both Glu194 and Asp270 functioning as helper acids, instead of a single carboxylate residue proposed to initiate catalysis. PMID:19897648

  18. Effects of fumaric acid esters on blood-brain barrier tight junction proteins.

    PubMed

    Bénardais, Karelle; Pul, Refik; Singh, Vikramjeet; Skripuletz, Thomas; Lee, De-Hyung; Linker, Ralf A; Gudi, Viktoria; Stangel, Martin

    2013-10-25

    The blood-brain barrier (BBB) is composed of a network of tight junctions (TJ) which interconnect cerebral endothelial cells (EC). Alterations in the TJ proteins are common in inflammatory diseases of the central nervous system (CNS) like multiple sclerosis (MS). Modulation of the BBB could thus represent a therapeutic mechanism. One pathway to modulate BBB integrity could be the induction of nuclear-factor (erythroid derived 2) related factor-2 (Nrf2) mediated oxidative stress responses which are targeted by fumaric acid esters (FAE). Here we analyze effects of FAE on the expression of TJ proteins in the human cerebral endothelial cell line hCMEC/D3 and experimental autoimmune encephalomyelitis (EAE). We show that dimethylfumarate (DMF) and its primary metabolite monomethylfumarate (MMF) induce the expression of the Nrf2/NQO1 pathway in endothelial cells. Neither MMF nor DMF had a consistent modulatory effect on the expression of TJ molecules in hCMEC/D3 cells. Tumor necrosis factor (TNFα)-induced downregulation of TJ proteins was at least partially reversed by treatment with FAE. However, DMF had no effect on claudin-5 expression in EAE, despite its effect on the clinical score and infiltration of immune cells. These data suggest that the modulation of the BBB is not a major mechanism of action of FAE in inflammatory demyelinating diseases of the CNS.

  19. A novel esterase gene cloned from a metagenomic library from neritic sediments of the South China Sea

    PubMed Central

    2011-01-01

    Background Marine microbes are a large and diverse group, which are exposed to a wide variety of pressure, temperature, salinity, nutrient availability and other environmental conditions. They provide a huge potential source of novel enzymes with unique properties that may be useful in industry and biotechnology. To explore the lipolytic genetic resources in the South China Sea, 23 sediment samples were collected in the depth < 100 m marine areas. Results A metagenomic library of South China Sea sediments assemblage in plasmid vector containing about 194 Mb of community DNA was prepared. Screening of a part of the unamplified library resulted in isolation of 15 unique lipolytic clones with the ability to hydrolyze tributyrin. A positive recombinant clone (pNLE1), containing a novel esterase (Est_p1), was successfully expressed in E. coli and purified. In a series of assays, Est_p1 displayed maximal activity at pH 8.57, 40°C, with ρ-Nitrophenyl butyrate (C4) as substrate. Compared to other metagenomic esterases, Est_p1 played a notable role in specificity for substrate C4 (kcat/Km value 11,500 S-1m M-1) and showed no inhibited by phenylmethylsulfonyl fluoride, suggested that the substrate binding pocket was suitable for substrate C4 and the serine active-site residue was buried at the bottom of substrate binding pocket which sheltered by a lid structure. Conclusions Esterase, which specificity towards short chain fatty acids, especially butanoic acid, is commercially available as potent flavoring tools. According the outstanding activity and specificity for substrate C4, Est_p1 has potential application in flavor industries requiring hydrolysis of short chain esters. PMID:22067554

  20. Esterase activity of BSA-ZnO nanoparticle complex

    NASA Astrophysics Data System (ADS)

    Bhogale, A.; Nair, A.; Patel, N.; Miotello, A.; Kothari, D. C.

    2014-04-01

    The effect of Zinc Oxide Nanoparticles (ZnO NPs) on functional properties of Bovine Serum Albumin (BSA) protein was studied. ZnO NPs were synthesized with average size of ˜7.5 nm as obtained from TEM analysis. The catalytic conversion of p-nitrophenylacetate (PNPA) to p-nitrophenol in the presence of BSA attached with ZnO NPs was examined by UV-Vis spectroscopy at room temperature. The result suggests that esterase activity of BSA is significantly enhanced (6 times) due to the ground state BSA-ZnO complex formation.

  1. 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...

  2. 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...

  3. 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...

  4. 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...

  5. 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...

  6. 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.

  7. A Comparison of Multiple Esterases as Biomarkers of Organophosphate Exposure and Effect in Two Earthworm Species

    PubMed Central

    Schneider, Ashley; Stoskopf, Michael K.

    2011-01-01

    Two different earthworm species, Eisenia fetida and Lumbricus terrestris, were exposed to 5 μg/cm2 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

  8. 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

  9. Enzymatic degradation of lignin-carbohydrate complexes (LCCs): model studies using a fungal glucuronoyl esterase from Cerrena unicolor.

    PubMed

    d'Errico, Clotilde; Jørgensen, Jonas O; Krogh, Kristian B R M; Spodsberg, Nikolaj; Madsen, Robert; Monrad, Rune Nygaard

    2015-05-01

    Lignin-carbohydrate complexes (LCCs) are believed to influence the recalcitrance of lignocellulosic plant material preventing optimal utilization of biomass in e.g. forestry, feed and biofuel applications. The recently emerged carbohydrate esterase (CE) 15 family of glucuronoyl esterases (GEs) has been proposed to degrade ester LCC bonds between glucuronic acids in xylans and lignin alcohols thereby potentially improving delignification of lignocellulosic biomass when applied in conjunction with other cellulases, hemicellulases and oxidoreductases. Herein, we report the synthesis of four new GE model substrates comprising α- and ɣ-arylalkyl esters representative of the lignin part of naturally occurring ester LCCs as well as the cloning and purification of a novel GE from Cerrena unicolor (CuGE). Together with a known GE from Schizophyllum commune (ScGE), CuGE was biochemically characterized by means of Michaelis-Menten kinetics with respect to substrate specificity using the synthesized compounds. For both enzymes, a strong preference for 4-O-methyl glucuronoyl esters rather than unsubstituted glucuronoyl esters was observed. Moreover, we found that α-arylalkyl esters of methyl α-D-glucuronic acid are more easily cleaved by GEs than their corresponding ɣ-arylalkyl esters. Furthermore, our results suggest a preference of CuGE for glucuronoyl esters of bulky alcohols supporting the suggested biological action of GEs on LCCs. The synthesis of relevant GE model substrates presented here may provide a valuable tool for the screening, selection and development of industrially relevant GEs for delignification of biomass.

  10. 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.

  11. 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

  12. Hemagglutinin-esterase-fusion (HEF) protein of influenza C virus.

    PubMed

    Wang, Mingyang; Veit, Michael

    2016-01-01

    Influenza C virus, a member of the Orthomyxoviridae family, causes flu-like disease but typically only with mild symptoms. Humans are the main reservoir of the virus, but it also infects pigs and dogs. Very recently, influenza C-like viruses were isolated from pigs and cattle that differ from classical influenza C virus and might constitute a new influenza virus genus. Influenza C virus is unique since it contains only one spike protein, the hemagglutinin-esterase-fusion glycoprotein HEF that possesses receptor binding, receptor destroying and membrane fusion activities, thus combining the functions of Hemagglutinin (HA) and Neuraminidase (NA) of influenza A and B viruses. Here we briefly review the epidemiology and pathology of the virus and the morphology of virus particles and their genome. The main focus is on the structure of the HEF protein as well as on its co- and post-translational modification, such as N-glycosylation, disulfide bond formation, S-acylation and proteolytic cleavage into HEF1 and HEF2 subunits. Finally, we describe the functions of HEF: receptor binding, esterase activity and membrane fusion.

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

    PubMed

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

    2011-11-01

    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 His(6)-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 His(6)-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.

  14. Screening and its potential application of lipolytic activity from a marine environment: characterization of a novel esterase from Yarrowia lipolytica CL180.

    PubMed

    Kim, Jun-Tae; Kang, Sung Gyun; Woo, Jung-Hee; Lee, Jung-Hyun; Jeong, Byeong Chul; Kim, Sang-Jin

    2007-03-01

    To develop an enantioselective lipase/esterase hydrolyzing racemic ofloxacin ester to levofloxacin, samples were collected from a variety of marine environments such as cold sea, hydrothermal vent area, sediment, tidal flat area, arctic sea, marine organisms, and so on. Microorganisms were isolated by plating on an enrichment medium with simultaneous detection of lipolytic activities and screened for the hydrolysis of ofloxacin ester. Three candidates among isolates were selected, and one of them, identified as Yarrowia lipolytica CL180, hydrolyzed preferentially S-enantiomer of racemic ofloxacin ester. The lipase/esterase gene (yli180) was cloned by screening a genomic library. The sequence analysis revealed an open reading frame consisting of 1,431 bp that encoded a protein of 476 amino acids with a molecular mass of 53 kDa. The yli180 gene was expressed in Escherichia coli and purified to homogeneity. The optimum activity of the recombinant protein (rYli180) occurred at pH 7.5 and 35 degrees C, respectively. rYli180 preferentially hydrolyzed p-nitrophenyl esters of fatty acids with short chain lengths of < or =10 carbon atoms. This study represents a novel esterase of type B1 carboxylesterase/lipase family from a marine isolate, showing a potential usage as a biocatalyst because of enantioselectivity toward racemic ofloxacin ester.

  15. 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.

  16. 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

  17. Expression and characterization of a new esterase with GCSAG motif from a permafrost metagenomic library.

    PubMed

    Petrovskaya, Lada E; Novototskaya-Vlasova, Ksenia A; Spirina, Elena V; Durdenko, Ekaterina V; Lomakina, Galina Yu; Zavialova, Maria G; Nikolaev, Evgeny N; Rivkina, Elizaveta M

    2016-05-01

    As a result of construction and screening of a metagenomic library prepared from a permafrost-derived microcosm, we have isolated a novel gene coding for a putative lipolytic enzyme that belongs to the hormone-sensitive lipase family. It encodes a polypeptide of 343 amino acid residues whose amino acid sequence displays maximum likelihood with uncharacterized proteins from Sphingomonas species. A putative catalytic serine residue of PMGL2 resides in a new variant of a recently discovered GTSAG sequence in which a Thr residue is replaced by a Cys residue (GCSAG). The recombinant PMGL2 was produced in Escherichia coli cells and purified by Ni-affinity chromatography. The resulting protein preferably utilizes short-chain p-nitrophenyl esters (C4 and C8) and therefore is an esterase. It possesses maximum activity at 45°C in slightly alkaline conditions and has limited thermostability at higher temperatures. Activity of PMGL2 is stimulated in the presence of 0.25-1.5 M NaCl indicating the good salt tolerance of the new enzyme. Mass spectrometric analysis demonstrated that N-terminal methionine in PMGL2 is processed and cysteine residues do not form a disulfide bond. The results of the study demonstrate the significance of the permafrost environment as a unique genetic reservoir and its potential for metagenomic exploration.

  18. 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.

  19. Characterization of a novel enzyme-Starmerella bombicola lactone esterase (SBLE)-responsible for sophorolipid lactonization.

    PubMed

    Ciesielska, Katarzyna; Roelants, Sophie L K W; Van Bogaert, Inge N A; De Waele, Stijn; Vandenberghe, Isabel; Groeneboer, Sara; Soetaert, Wim; Devreese, Bart

    2016-11-01

    We recently discovered a novel enzyme in the exoproteome of Starmerella bombicola, which is structurally related to Candida antarctica lipase A. A knockout strain for this enzyme does no longer produce lactonic sophorolipids, prompting us to believe that this protein is the missing S. bombicola lactone esterase (SBLE). SBLE catalyzes a rather unusual reaction, i.e., an intramolecular esterification (lactonization) of acidic sophorolipids in an aqueous environment, which raised questions about its activity and mode of action. Here, we report the heterologous production of this enzyme in Pichia pastoris and its purification in a two-step strategy. Purified recombinant SBLE (rSBLE) was used to perform HPLC and liquid chromatography mass spectrometry (LCMS)-based assays with different sophorolipid mixtures. We experimentally confirmed that SBLE is able to perform ring closure of acetylated acidic sophorolipids. This substrate was selected for rSBLE kinetic studies to estimate the apparent values of K m . We established that rSBLE displays optimal activity in the pH range of 3.5 to 6 and has an optimal temperature in the range of 20 to 50 °C. Additionally, we generated a rSBLE mutant through site-directed mutagenesis of Ser194 in the predicted active site pocket and show that this mutant is lacking the ability to lactonize sophorolipids. We therefore propose that SBLE operates via the common serine hydrolase mechanism in which the catalytic serine residue is assisted by a His/Asp pair.

  20. The first description of a hormone-sensitive lipase from a basidiomycete: Structural insights and biochemical characterization revealed Bjerkandera adusta BaEstB as a novel esterase.

    PubMed

    Sánchez-Carbente, María Del Rayo; Batista-García, Ramón Alberto; Sánchez-Reyes, Ayixón; Escudero-Garcia, Angela; Morales-Herrera, Catalina; Cuervo-Soto, Laura I; French-Pacheco, Leidys; Fernández-Silva, Arline; Amero, Carlos; Castillo, Edmundo; Folch-Mallol, Jorge Luis

    2017-03-01

    The heterologous expression and characterization of a Hormone-Sensitive Lipases (HSL) esterase (BaEstB) from the Basidiomycete fungus Bjerkandera adusta is reported for the first time. According to structural analysis, amino acid similarities and conservation of particular motifs, it was established that this enzyme belongs to the (HSL) family. The cDNA sequence consisted of 969 nucleotides, while the gene comprised 1133, including three introns of 57, 50, and 57 nucleotides. Through three-dimensional modeling and phylogenetic analysis, we conclude that BaEstB is an ortholog of the previously described RmEstB-HSL from the phylogenetically distant fungus Rhizomucor miehei. The purified BaEstB was characterized in terms of its specificity for the hydrolysis of different acyl substrates confirming its low lipolytic activity and a noticeable esterase activity. The biochemical characterization of BaEstB, the DLS analysis and the kinetic parameters determination revealed this enzyme as a true esterase, preferentially found in a dimeric state, displaying activity under alkaline conditions and relative low temperature (pH = 10, 20°C). Our data suggest that BaEstB is more active on substrates with short acyl chains and bulky aromatic moieties. Phylogenetic data allow us to suggest that a number of fungal hypothetical proteins could belong to the HSL family.

  1. 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.

  2. 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.

  3. MECHANISMS OF ACTIVATION OF C'1 ESTERASE IN HEREDITARY ANGIONEUROTIC EDEMA PLASMA IN VITRO

    PubMed Central

    Donaldson, Virginia H.

    1968-01-01

    The generation of C'1 esterase activity in siliconed plasma obtained from individuals with hereditary angioneurotic edema in remission tends to occur spontaneously, but can be hastened during its incubation with preparations of activated Hageman factor. This effect of activated Hageman factor could not be shown during its incubation with normal siliconed plasma, nor could consumption of normal serum inhibition of C'1 esterase be clearly shown. Soy bean trypsin inhibitor and heparin could impair this enhanced generation of C'1 esterase but neither inhibits the esterolytic function of C'1 esterase once formed. Trasylol was less effective in blocking this effect of activated Hageman factor. While the mechanism of the effect of activated Hageman factor upon C'1 activation remains obscure, it is apparent that some intermediate steps, possibly involving a kinin-forming system of plasma, may play a role. PMID:5299945

  4. 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...

  5. 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.

  6. A novel protein from mung bean hypocotyl cell walls with acetyl esterase activity.

    PubMed

    Bordenave, M; Goldberg, R; Huet, J C; Pernollet, J C

    1995-01-01

    An acetyl esterase was purified from cell walls isolated from mung bean hypocotyls. The purified enzyme had an apparent Mr of 43,300 and an apparent pI > 9. It rapidly deesterified triacetin and p-nitrophenylacetate and slowly released acetate from beet and flax pectins, the deesterification rate being increased by previous demethylation of the pectins. No significant peptide sequence identity between the acetyl esterase and any known protein could be found in protein data bases.

  7. Esterase in Imported Fire Ants, Solenopsis invicta and S. richteri (Hymenoptera: Formicidae): Activity, Kinetics and Variation

    PubMed Central

    Chen, J.; Rashid, T.; Feng, G.

    2014-01-01

    Solenopsis invicta and Solenopsis richteri are two closely related invasive ants native to South America. Despite their similarity in biology and behavior, S. invicta is a more successful invasive species. Toxic tolerance has been found to be important to the success of some invasive species. Esterases play a crucial role in toxic tolerance of insects. Hence, we hypothesized that the more invasive S. invicta would have a higher esterase activity than S. richteri. Esterase activities were measured for workers and male and female alates of both ant species using α-naphthyl acetate and β-naphthyl acetate as substrates. Esterase activities in S. invicta were always significantly higher than those in S. richteri supporting our hypothesis. In S. invicta, male alates had the highest esterase activities followed by workers then female alates for both substrates. In S. richetri, for α-naphthyl acetate, male alates had the highest activity followed by female alates then workers, while for β-naphthyl acetate, female alates had the highest activity followed by male alates then workers. For workers, S. richteri showed significantly higher levels of variation about the mean esterase activity than S. invicta. However, S. invicta showed significantly higher levels of variation in both female and male alates. PMID:25408118

  8. 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.

  9. Molecular basis for the behavioral effects of the odorant degrading enzyme Esterase 6 in Drosophila

    PubMed Central

    Younus, Faisal; Fraser, Nicholas J.; Coppin, Chris W.; Liu, Jian-Wei; Correy, Galen J.; Chertemps, Thomas; Pandey, Gunjan; Maïbèche, Martine; Jackson, Colin J.; Oakeshott, John G.

    2017-01-01

    Previous electrophysiological and behavioural studies implicate esterase 6 in the processing of the pheromone cis-vaccenyl acetate and various food odorants that affect aggregation and reproductive behaviours. Here we show esterase 6 has relatively high activity against many of the short-mid chain food esters, but negligible activity against cis-vaccenyl acetate. The crystal structure of esterase 6 confirms its substrate-binding site can accommodate many short-mid chain food esters but not cis-vaccenyl acetate. Immunohistochemical assays show esterase 6 is expressed in non-neuronal cells in the third antennal segment that could be accessory or epidermal cells surrounding numerous olfactory sensilla, including basiconics involved in food odorant detection. Esterase 6 is also produced in trichoid sensilla, but not in the same cell types as the cis-vaccenyl acetate binding protein LUSH. Our data support a model in which esterase 6 acts as a direct odorant degrading enzyme for many bioactive food esters, but not cis-vaccenyl acetate. PMID:28393888

  10. 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

  11. 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

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

    PubMed Central

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

    2010-01-01

    Summary 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 (Tm) of the mutants over the native BcEST that has a Tm of 50°C was outperformed by TtEST, a naturally occurring homologue with a Tm 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

  13. 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.

  14. Characterization of a Novel Esterase Rv0045c from Mycobacterium tuberculosis

    PubMed Central

    Xu, Lipeng; Liu, Zhongyuan; Xu, Kehui; Li, Shentao; Wen, Tingyi; Liu, Siguo; Pang, Hai

    2010-01-01

    Background It was proposed that there are at least 250 enzymes in M. tuberculosis involved in lipid metabolism. Rv0045c was predicted to be a hydrolase by amino acid sequence similarity, although its precise biochemical characterization and function remained to be defined. Methodology/Principal Findings We expressed the Rv0045c protein to high levels in E. coli and purified the protein to high purity. We confirmed that the prepared protein was the Rv0045c protein by mass spectrometry analysis. Circular dichroism spectroscopy analysis showed that the protein possessed abundant β-sheet secondary structure, and confirmed that its conformation was stable in the range pH 6.0–10.0 and at temperatures ≤40°C. Enzyme activity analysis indicated that the Rv0045c protein could efficiently hydrolyze short chain p-nitrophenyl esters (C2–C8), and its suitable substrate was p-nitrophenyl caproate (C6) with optimal catalytic conditions of 39°C and pH 8.0. Conclusions/Significance Our results demonstrated that the Rv0045c protein is a novel esterase. These experiments will be helpful in understanding ester/lipid metabolism related to M. tuberculosis. PMID:20957207

  15. Esterase 22 and beta-glucuronidase hydrolyze retinoids in mouse liver.

    PubMed

    Schreiber, Renate; Taschler, Ulrike; Wolinski, Heimo; Seper, Andrea; Tamegger, Stefanie N; Graf, Maria; Kohlwein, Sepp D; Haemmerle, Guenter; Zimmermann, Robert; Zechner, Rudolf; Lass, Achim

    2009-12-01

    Excess dietary vitamin A is esterified with fatty acids and stored in the form of retinyl ester (RE) predominantly in the liver. According to the requirements of the body, liver RE stores are hydrolyzed and retinol is delivered to peripheral tissues. The controlled mobilization of retinol ensures a constant supply of the body with the vitamin. Currently, the enzymes catalyzing liver RE hydrolysis are unknown. In this study, we identified mouse esterase 22 (Es22) as potent RE hydrolase highly expressed in the liver, particularly in hepatocytes. The enzyme is located exclusively at the endoplasmic reticulum (ER), implying that it is not involved in the mobilization of RE present in cytosolic lipid droplets. Nevertheless, cell culture experiments revealed that overexpression of Es22 attenuated the formation of cellular RE stores, presumably by counteracting retinol esterification at the ER. Es22 was previously shown to form a complex with beta-glucuronidase (Gus). Our studies revealed that Gus colocalizes with Es22 at the ER but does not affect its RE hydrolase activity. Interestingly, however, Gus was capable of hydrolyzing the naturally occurring vitamin A metabolite retinoyl beta-glucuronide. In conclusion, our observations implicate that both Es22 and Gus play a role in liver retinoid metabolism.

  16. Hypocholesterolaemic mechanism of bitter melon aqueous extracts via inhibition of pancreatic cholesterol esterase and reduction of cholesterol micellar solubility.

    PubMed

    Su, Jianhui; Wang, Hongxin; Ma, Chaoyang; Liu, Chengxiang; Gao, Chuanzhong; Nie, Rongjing; Tanver Rahman, Md Ramim

    2016-01-01

    This study investigated the hypocholesterolaemic effects of bitter melon aqueous extracts (BMAE) in vitro, the inhibitory effects of BMAE on pancreatic cholesterol esterase (CEase) and incorporation of cholesterol into micelles were investigated. BMAE decreased the in vitro micellar solubility of cholesterol in a dose-dependent manner. The conformation of CEase was investigated by means of circular dichroism (CD) and fluorescence. The result revealed the decrease of α-helix contents, increase of β-sheet and exposure of aromatic amino acid residuals. The incorporation of cholesterol into micelles was inhibited by BMAE. A complex was observed by transmission electron microscopy (TEM), which indicated interaction between cholesterol and BMAE. The result revealed that BMAE can play a role in decreased intestinal cholesterol absorption via inhibition of CEase, and of micelle formation.

  17. Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins.

    PubMed Central

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

    1993-01-01

    Based on the recently determined X-ray structures of Torpedo californica acetylcholinesterase and Geotrichum candidum lipase and on their three-dimensional superposition, an improved alignment of a collection of 32 related amino acid sequences of other esterases, lipases, and related proteins was obtained. On the basis of this alignment, 24 residues are found to be invariant in 29 sequences of hydrolytic enzymes, and an additional 49 are well conserved. The conservation in the three remaining sequences is somewhat lower. The conserved residues include the active site, disulfide bridges, salt bridges, and residues in the core of the proteins. Most invariant residues are located at the edges of secondary structural elements. A clear structural basis for the preservation of many of these residues can be determined from comparison of the two X-ray structures. PMID:8453375

  18. Leukocyte esterase-nitrite and bioluminescence assays as urine screens.

    PubMed Central

    Males, B M; Bartholomew, W R; Amsterdam, D

    1985-01-01

    The 1-min leukocyte esterase (LE)-nitrite test (Chemstrip 9; Biodynamics, Division of Boehringer Mannheim Biochemicals, Indianapolis, Ind.) and a bioluminescence assay (Monolight centrifugation method; Analytical Luminescence Laboratory, Inc., San Diego, Calif.) were tested for their efficacy as urine screens among 453 patients at a tertiary-care teaching hospital. Both methods had the capacity to exclude significant bacteriuria (greater than or equal to 10(5) CFU/ml) when compared with the results of conventional culture methods, with predictive values of 99 and 93%, respectively, for a negative test. Bioluminescence was the more accurate nonculture method used. Sensitivity and specificity values were 97 and 71%, respectively, for bioluminescence, 82 and 60%, respectively, for LE with nitrite, and 72 and 64%, respectively, for LE without nitrite. At reduced levels of bacteriuria less than 10(5) CFU/ml), the sensitivities of LE-nitrite and bioluminescence were decreased but comparable. The addition of protein and blood test results in the Chemstrip 9, along with LE-nitrite as bacteriuria indicators, were unsatisfactory because of the large numbers of false-positive results attributed to protein and blood determinations. LE activity as detected by the LE test was a poor predictor of significant bacteriuria in both male and female patients. The sensitivity (71%) and specificity (57%) of the LE test in male patients were significantly lower than those previously reported and varied with the patient population studied. PMID:3935662

  19. 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.

  20. Function of Phe-259 and Thr-314 within the Substrate Binding Pocket of the Juvenile Hormone Esterase of Manduca sexta†

    PubMed Central

    Kamita, Shizuo G.; Wogulis, Mark D.; Law, Christopher S.; Morisseau, Christophe; Tanaka, Hiromasa; Huang, Huazhang; Wilson, David K.; Hammock, Bruce D.

    2013-01-01

    Juvenile hormone (JH) is a key insect developmental hormone that is found at low nanomolar levels in larval insects. The methyl ester of JH is hydrolyzed in many insects by an esterase that shows high specificity for JH. We have previously determined a crystal structure of the JH esterase (JHE) of the tobacco hornworm Manduca sexta (MsJHE) [Wogulis, M., Wheelock, C. E., Kamita, S. G., Hinton, A. C., Whetstone, P. A., Hammock, B. D., and Wilson, D. K. (2006) Biochemistry 45, 4045-4057]. Our molecular modeling indicates that JH fits very tightly within the substrate binding pocket of MsJHE. This tight fit places two non-catalytic amino acid residues, Phe-259 and Thr-314, within the appropriate distance and geometry to potentially interact with the α,β-unsaturated ester and epoxide, respectively, of JH. These residues are highly conserved in numerous biologically active JHEs. Kinetic analyses of mutants of Phe-259 or Thr-314 indicate that these residues contribute to the low KM that MsJHE shows for JH. This low KM, however, comes at the cost of reduced substrate turnover. Neither nucleophilic attack of the resonance stabilized ester by the catalytic serine nor the availability of a water molecule for attack of the acyl-enzyme intermediate appear to be a rate-determining step in the hydrolysis of JH by MsJHE. We hypothesize that the release of the JH acid metabolite from the substrate binding pocket limits the catalytic cycle. Our findings also demonstrate that chemical bond strength does not necessarily correlate with how reactive the bond will be to metabolism. PMID:20307057

  1. 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.

  2. 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

  3. 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.

  4. Esterase profiles of organophosphorus compounds in vitro predict their behavior in vivo.

    PubMed

    Makhaeva, Galina F; Rudakova, Elena V; Serebryakova, Olga G; Aksinenko, Alexey Yu; Lushchekina, Sofya V; Bachurin, Sergey O; Richardson, Rudy J

    2016-11-25

    We studied 4 serine esterases (EOHs) that are associated with the following consequences from their inhibition by organophosphorus compounds (OPCs): 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, which we hypothesize can serve as a predictor of its overall physiological effects. To test this hypothesis, we selected 3 OPCs known from previous work on reference enzymes to span a wide range of esterase profiles, neuropathic potential, and acute cholinergic toxicity. For each compound, we determined in vitro IC50 and in vivo ED50 values for inhibition of AChE, BChE, CaE, and NTE in mouse brain and blood. The results showed good correlations between in vitro and in vivo measures of potency and selectivity except for brain CaE, a tissue-specific isoform of the enzyme that was less sensitive to the test compounds than expected. Thus, this synthesis of new and previously published results indicates that the concept of the esterase profile of OPCs is useful for the prediction of therapeutic and toxic effects in vivo.

  5. Organophosphate and Pyrethroid Hydrolase Activities of Mutant Esterases from the Cotton Bollworm Helicoverpa armigera

    PubMed Central

    Li, Yongqiang; Farnsworth, Claire A.; Coppin, Chris W.; Teese, Mark G.; Liu, Jian-Wei; Scott, Colin; Zhang, Xing; Russell, Robyn J.; Oakeshott, John G.

    2013-01-01

    Two mutations have been found in five closely related insect esterases (from four higher Diptera and a hymenopteran) which each confer organophosphate (OP) hydrolase activity on the enzyme and OP resistance on the insect. One mutation converts a Glycine to an Aspartate, and the other converts a Tryptophan to a Leucine in the enzymes’ active site. One of the dipteran enzymes with the Leucine mutation also shows enhanced activity against pyrethroids. Introduction of the two mutations in vitro into eight esterases from six other widely separated insect groups has also been reported to increase substantially the OP hydrolase activity of most of them. These data suggest that the two mutations could contribute to OP, and possibly pyrethroid, resistance in a variety of insects. We therefore introduced them in vitro into eight Helicoverpa armigera esterases from a clade that has already been implicated in OP and pyrethroid resistance. We found that they do not generally enhance either OP or pyrethroid hydrolysis in these esterases but the Aspartate mutation did increase OP hydrolysis in one enzyme by about 14 fold and the Leucine mutation caused a 4–6 fold increase in activity (more in one case) of another three against some of the most insecticidal isomers of fenvalerate and cypermethrin. The Aspartate enzyme and one of the Leucine enzymes occur in regions of the H. armigera esterase isozyme profile that have been previously implicated in OP and pyrethroid resistance, respectively. PMID:24204917

  6. Molecular and kinetic evidence for allelic variants of esterase Estbeta1 in the mosquito Culex quinquefasciatus.

    PubMed

    Small, G J; Karunaratne, S H; Chadee, D D; Hemingway, J

    1999-07-01

    Elevated esterase Estbeta1 was purified from larvae of newly isolated strains of the mosquito Culex quinquefasciatus from Colombia (COL) and Trinidad (TRI) with resistance to organophosphate (OP) insecticides. Insecticide interactions were compared with those of elevated Estbeta1(2) from the OP-resistant Habana strain and the non-elevated Estbeta1(3) from the susceptible PelSS strain. On the basis of insecticide binding efficiency, all elevated Estbeta1 esterases were readily distinguishable. Differences between the EcoRI restriction fragment patterns of the amplified estbeta1 gene in COL and TRI strains compared with each other, and between amplified estbeta1(1), estbeta1(2) and the non-amplified estbeta1(3), suggest differences in their nucleotide sequence. Considering their variable insecticide binding efficiencies, these genetic differences would imply that, in contrast to estalpha2 and estbeta2, amplification of estbeta1 has occurred several times independently. Generally, the elevated Estbeta1s were more reactive with insecticides than the non-elevated Estbeta1(3). This supports the hypothesis that the elevated esterase-based mechanism confers resistance through amplification of alleles coding for esterases which have a greater specificity for the insecticides they sequester than the esterases coded by their non-amplified counterparts.

  7. 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.

  8. 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.

  9. Reaction mechanism for cocaine esterase-catalyzed hydrolyses of (+)- and (-)-cocaine: unexpected common rate-determining step.

    PubMed

    Liu, Junjun; Zhao, Xinyun; Yang, Wenchao; Zhan, Chang-Guo

    2011-05-05

    First-principles quantum mechanical/molecular mechanical free energy calculations have been performed to examine the catalytic mechanism for cocaine esterase (CocE)-catalyzed hydrolysis of (+)-cocaine in comparison with CocE-catalyzed hydrolysis of (-)-cocaine. It has been shown that the acylation of (+)-cocaine consists of nucleophilic attack of the hydroxyl group of Ser117 on the carbonyl carbon of (+)-cocaine benzoyl ester and the dissociation of (+)-cocaine benzoyl ester. The first reaction step of deacylation of (+)-cocaine, which is identical to that of (-)-cocaine, is rate-determining, indicating that CocE-catalyzed hydrolyses of (+)- and (-)-cocaine have a common rate-determining step. The computational results predict that the catalytic rate constant of CocE against (+)-cocaine should be the same as that of CocE against (-)-cocaine, in contrast with the remarkable difference between human butyrylcholinesterase-catalyzed hydrolyses of (+)- and (-)-cocaine. The prediction has been confirmed by experimental kinetic analysis on CocE-catalyzed hydrolysis of (+)-cocaine in comparison with CocE-catalyzed hydrolysis of (-)-cocaine. The determined common rate-determining step indicates that rational design of a high-activity mutant of CocE should be focused on the first reaction step of the deacylation. Furthermore, the obtained mechanistic insights into the detailed differences in the acylation between the (+)- and (-)-cocaine hydrolyses provide indirect clues for rational design of amino acid mutations that could more favorably stabilize the rate-determining transition state in the deacylation and, thus, improve the catalytic activity of CocE. This study provides a valuable mechanistic base for rational design of an improved esterase for therapeutic treatment of cocaine abuse.

  10. Novel Feruloyl Esterase from Lactobacillus fermentum NRRL B-1932 and Analysis of the Recombinant Enzyme Produced in Escherichia coli

    PubMed Central

    Bischoff, Kenneth M.; Anderson, Amber M.; Rich, Joseph O.

    2016-01-01

    ABSTRACT A total of 33 Lactobacillus strains were screened for feruloyl esterase (FE) activity using agar plates containing ethyl ferulate as the sole carbon source, and Lactobacillus fermentum NRRL B-1932 demonstrated the strongest FE activity among a dozen species showing a clearing zone on the opaque plate containing ethyl ferulate. FE activities were monitored using high-performance liquid chromatography with an acetonitrile-trifluoroacetic acid gradient. To produce sufficient purified FE from L. fermentum strain NRRL B-1932 (LfFE), the cDNA encoding LfFE (Lffae) was amplified and cloned by using available closely related genome sequences and overexpressed in Escherichia coli. A 29.6-kDa LfFE protein was detected from the protein extract of E. coli BL21(pLysS) carrying pET28bLffae upon IPTG (isopropyl-β-d-thiogalactopyranoside) induction. The recombinant LfFE containing a polyhistidine tag was purified by nickel-nitrilotriacetic acid affinity resin. The purified LfFE showed strong activities against several artificial substrates, including p-nitrophenyl acetate and 4-methylumbelliferyl p-trimethylammoniocinnamate chloride. The optimum pH and temperature of the recombinant LfFE were around 6.5 and 37°C, respectively, as determined using either crude or purified recombinant LfFE. This study will be essential for the production of the LfFE in E. coli on a larger scale that could not be readily achieved by L. fermentum fermentation. IMPORTANCE The production of feruloyl esterase (FE) from Lactobacillus fermentum NRRL B-1932 reported in this study will have immense potential commercial applications not only in biofuel production but also in pharmaceutical, polymer, oleo chemical, cosmetic additive, and detergent industries, as well as human health-related applications, including food flavoring, functional foods, probiotic agents, preventive medicine, and animal feed. Given the essential role FE plays in the production of hydroxycinnamic acids and ferulic acid

  11. 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.

  12. Role of an esterase in flavor volatile variation within the tomato clade.

    PubMed

    Goulet, Charles; Mageroy, Melissa H; Lam, Nghi B; Floystad, Abbye; Tieman, Denise M; Klee, Harry J

    2012-11-13

    Tomato flavor is dependent upon a complex mixture of volatiles including multiple acetate esters. Red-fruited species of the tomato clade accumulate a relatively low content of acetate esters in comparison with the green-fruited species. We show that the difference in volatile ester content between the red- and green-fruited species is associated with insertion of a retrotransposon adjacent to the most enzymatically active member of a family of esterases. This insertion causes higher expression of the esterase, resulting in the reduced levels of multiple esters that are negatively correlated with human preferences for tomato. The insertion was evolutionarily fixed in the red-fruited species, suggesting that high expression of the esterase and consequent low ester content may provide an adaptive advantage in the ancestor of the red-fruited species. These results illustrate at a molecular level how closely related species exhibit major differences in volatile production by altering a volatile-associated catabolic activity.

  13. Properties of phenyl valerate esterase activities from chicken serum are comparable with soluble esterases of peripheral nerves in relation with organophosphorus compounds inhibition.

    PubMed

    Garcia-Pérez, Adolfo G; Barril, José; Estévez, Jorge; Vilanova, Eugenio

    2003-04-30

    Chicken serum, the usual in vivo animal for testing organophosphorus delayed neuropathy, has long been reported not to contain a homologous activity of the neuronal neuropathy target esterase (NTE) activity when it is assayed according to standard methods as the phenyl valerate esterase (PVase) activity, which is resistant to paraoxon and sensitive to mipafox. However, a PVase activity (1000-1500 nmol/min/ml) can be measured in serum that is extremely sensitive to both paraoxon, a non-neuropathic organophosphorus compound and mipafox, a model neuropathy inducer. The inhibition was time progressive in both cases, suggesting a covalent phosphorilating reaction. The fixed time inhibition curves suggest at least two sensitive components. The IC50 for 30 min, at 37 degrees C are 6 and 51 nM for paraoxon and 4 and 110 nM for mipafox, for every sensitive component. When paraoxon was removed from a serum sample pretreated with the inhibitor, the paraoxon sensitive PVase activity was recovered, in spite of showing a time progressive inhibition suggesting that hydrolytic dephosphorylating reaction recovered at a significant rate. The reactivation of the phosphorylated enzyme could explain that the time progressive inhibitions curves for long time with paraoxon tend to reach a plateau depending on the inhibition concentration. However, with mipafox, the curve approached the same maximal inhibitions at all concentrations as expected for a permanent covalent irreversible phosphorylation, which is coherent with the observations that the activity remained inhibited after removing the inhibitor. Data of serum esterases described in this paper showed similar properties to those previously reported for peripheral nerve soluble phenylvalerate esterase: (1) extremely high sensitivity to paraoxon and mipafox; (2) time progressive kinetic with two sensitive components; (3) recovery of activity after removal of paraoxon; and (4) permanent inhibition with mipafox. These properties of

  14. 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.

  15. Effects of bis homoallylic and homoallylic hydroxyl substitution on the olefinic 13C resonance shifts in fatty acid methyl esters.

    PubMed

    Pfeffer, P E; Sonnet, P E; Schwartz, D P; Osman, S F; Weisleder, D

    1992-04-01

    Substitution of a hydroxyl group at the bis homoallylic position (OH group located three carbons away from the olefinic carbon) in C18 unsaturated fatty acid esters (FAE) induces a 0.73 +/- 0.05 ppm upfield and a 0.73 +/- 0.06 ppm downfield shift on the delta and epsilon olefinic 13C resonances relative to the unsubstituted FAE, respectively. If the hydroxyl group is located on the carboxyl side of the double bond of the bis homoallylic hydroxy fatty acid esters (BHAHFA), the olefinic resonances are uniformly shifted apart by [formula: see text] where delta delta dbu represents the absolute value of the double bond resonance separation in the unsubstituted FAE and 1.46 ppm is the sum of the absolute values of the delta and epsilon shift parameters. With hydroxyl substitution on the terminal methyl side of the double bond, the olefinic shift separation is equal to [formula: see text] In homoallylic (OH group located two carbons away from the olefinic carbon) substituted FAE the gamma and delta induced hydroxyl shifts for the cis double bond resonances are +3.08 and -4.63 ppm, respectively while the trans double bond parameters are +4.06 and -4.18 ppm, respectively. The double bond resonance separation in homoallylic hydroxy fatty acid esters (HAHFA) can be calculated from the formula [formula: see text] for cis and [formula: see text] for the trans case when the OH substitution is on the carboxyl side of the double bond. Conversely, when the OH resides on the terminal methyl side, the double bond shift separations for cis and trans isomers are [formula: see text] and [formula: see text] respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. 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.

  17. A first continuous 4-aminoantipyrine (4-AAP)-based screening system for directed esterase evolution.

    PubMed

    Lülsdorf, Nina; Vojcic, Ljubica; Hellmuth, Hendrik; Weber, Thomas T; Mußmann, Nina; Martinez, Ronny; Schwaneberg, Ulrich

    2015-06-01

    Esterases hydrolyze ester bonds with an often high stereoselectivity as well as regioselectivity and are therefore industrially employed in the synthesis of pharmaceuticals, in food processing, and in laundry detergents. Continuous screening systems based on p-nitrophenyl- (e.g., p-nitrophenyl acetate) or umbelliferyl-esters are commonly used in directed esterase evolution campaigns. Ongoing challenges in directed esterase evolution are screening formats which offer a broad substrate spectrum, especially for complex aromatic substrates. In this report, a novel continuous high throughput screening system for indirect monitoring of esterolytic activity was developed and validated by detection of phenols employing phenyl benzoate as substrate and p-nitrobenzyl esterase (pNBEBL from Bacillus licheniformis) as catalyst. The released phenol directly reacts with 4-aminoantipyrine yielding the red compound 1,5-dimethyl-4-(4-oxo-cyclohexa-2,5-dienylidenamino)-2-phenyl-1,2-dihydro-pyrazol-3-one. In this continuous B. licheniformis esterase activity detection system (cBLE-4AAP), the product formation is followed through an increase in absorbance at 509 nm. The cBLE-4AAP screening system was optimized in 96-well microtiter plate format in respect to standard deviation (5 %), linear detection range (15 to 250 μM), lower detection limit (15 μM), and pH (7.4 to 10.4). The cBLE-4AAP screening system was validated by screening a random epPCR pNBEBL mutagenesis library (2000 clones) for improved esterase activity at elevated temperatures. Finally, the variant T3 (Ser378Pro) was identified which nearly retains its specific activity at room temperature (WT 1036 U/mg and T3 929 U/mg) and shows compared to WT a 4.7-fold improved residual activity after thermal treatment (30 min incubation at 69.4 °C; WT 170 U/mg to T3 804 U/mg).

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

    PubMed

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

    2015-01-01

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

  19. 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

  20. Structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution.

    PubMed

    Zeng, Qinghong; Langereis, Martijn A; van Vliet, Arno L W; Huizinga, Eric G; de Groot, Raoul J

    2008-07-01

    The hemagglutinin-esterases (HEs) are a family of viral envelope glycoproteins that mediate reversible attachment to O-acetylated sialic acids by acting both as lectins and as receptor-destroying enzymes (RDEs). Related HEs occur in influenza C, toro-, and coronaviruses, apparently as a result of relatively recent lateral gene transfer events. Here, we report the crystal structure of a coronavirus (CoV) HE in complex with its receptor. We show that CoV HE arose from an influenza C-like HE fusion protein (HEF). In the process, HE was transformed from a trimer into a dimer, whereas remnants of the fusion domain were adapted to establish novel monomer-monomer contacts. Whereas the structural design of the RDE-acetylesterase domain remained unaltered, the HE receptor-binding domain underwent remodeling to such extent that the ligand is now bound in opposite orientation. This is surprising, because the architecture of the HEF site was preserved in influenza A HA over a much larger evolutionary distance, a switch in receptor specificity and extensive antigenic variation notwithstanding. Apparently, HA and HEF are under more stringent selective constraints than HE, limiting their exploration of alternative binding-site topologies. We attribute the plasticity of the CoV HE receptor-binding site to evolutionary flexibility conferred by functional redundancy between HE and its companion spike protein S. Our findings offer unique insights into the structural and functional consequences of independent protein evolution after interviral gene exchange and open potential avenues to broad-spectrum antiviral drug design.

  1. 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

  2. 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.

  3. Cocaine esterase: interactions with cocaine and immune responses in mice.

    PubMed

    Ko, Mei-Chuan; Bowen, Luvina D; Narasimhan, Diwahar; Berlin, Aaron A; Lukacs, Nicholas W; Sunahara, Roger K; Cooper, Ziva D; Woods, James H

    2007-02-01

    Cocaine esterase (CocE) is the most efficient protein catalyst for the hydrolysis of cocaine characterized to date. The aim of this study was to investigate the in vivo potency of CocE in blocking cocaine-induced toxicity in the mouse and to assess CocE's potential immunogenicity. Cocaine toxicity was quantified by measuring the occurrence of convulsions and lethality. Intravenous administration of CocE (0.1-1 mg) 1 min before cocaine administration produced dose-dependent rightward shifts of the dose-response curve for cocaine toxicity. More important, i.v. CocE (0.1-1 mg), given 1 min after the occurrence of cocaine-induced convulsions, shortened the recovery time after the convulsions and saved the mice from subsequent death. Effects of repeated exposures to CocE were evaluated by measuring anti-CocE antibody titers and the protective effects of i.v. CocE (0.32 mg) against toxicity elicited by i.p. cocaine (320 mg/kg) (i.e., 0-17% occurrence of convulsions and lethality). CocE retained its potency against cocaine toxicity in mice after a single prior CocE exposure (0.1-1 mg), and these mice did not show an immune response. CocE retained similar effectiveness in mice after three prior CocE exposures (0.1-1 mg/week for 3 weeks), although these mice displayed 10-fold higher antibody titers. CocE partially lost effectiveness (i.e., 33-50% occurrence of convulsions and lethality) in mice with four prior exposures to CocE (0.1-1 mg/2 week for four times), and these mice displayed approximately 100-fold higher antibody titers. These results suggest that CocE produces robust protection and reversal of cocaine toxicity, indicating CocE's therapeutic potential for acute cocaine toxicity. Repeated CocE exposures may increase its immunogenicity and partially reduce its protective ability.

  4. Mechanisms of Oxidative Damage in Multiple Sclerosis and Neurodegenerative Diseases: Therapeutic Modulation via Fumaric Acid Esters

    PubMed Central

    Lee, De-Hyung; Gold, Ralf; Linker, Ralf A.

    2012-01-01

    Oxidative stress plays a crucial role in many neurodegenerative conditions such as Alzheimer’s disease, amyotrophic lateral sclerosis and Parkinson’s as well as Huntington’s disease. Inflammation and oxidative stress are also thought to promote tissue damage in multiple sclerosis (MS). Recent data point at an important role of anti-oxidative pathways for tissue protection in chronic-progressive MS, particularly involving the transcription factor nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2). Thus, novel therapeutics enhancing cellular resistance to free radicals could prove useful for MS treatment. Here, fumaric acid esters (FAE) are a new, orally available treatment option which had already been tested in phase II/III MS trials demonstrating beneficial effects on relapse rates and magnetic resonance imaging markers. In vitro, application of dimethylfumarate (DMF) leads to stabilization of Nrf2, activation of Nrf2-dependent transcriptional activity and abundant synthesis of detoxifying proteins. Furthermore, application of FAE involves direct modification of the inhibitor of Nrf2, Kelch-like ECH-associated protein 1. On cellular levels, the application of FAE enhances neuronal survival and protects astrocytes against oxidative stress. Increased levels of Nrf2 are detected in the central nervous system of DMF treated mice suffering from experimental autoimmune encephalomyelitis (EAE), an animal model of MS. In EAE, DMF ameliorates the disease course and improves preservation of myelin, axons and neurons. Finally, Nrf2 is also up-regulated in the spinal cord of autopsy specimens from untreated patients with MS, probably as part of a naturally occurring anti-oxidative response. In summary, oxidative stress and anti-oxidative pathways are important players in MS pathophysiology and constitute a promising target for future MS therapies like FAE. PMID:23109883

  5. Green synthesis of β-sitostanol esters catalyzed by the versatile lipase/sterol esterase from Ophiostoma piceae.

    PubMed

    Molina-Gutiérrez, María; Hakalin, Neumara L S; Rodríguez-Sanchez, Leonor; Prieto, Alicia; Martínez, María Jesús

    2017-04-15

    β-sitostanol esters, used as dietary complement for decreasing cholesterol absorption, have been synthesized at 28°C via direct esterification or transesterification catalyzed by the versatile lipase/sterol esterase from the ascomycete fungus O. piceae. Direct esterification was conducted in biphasic isooctane: water systems containing 10mM β-sitostanol and lauric or oleic acid as acyl donors, reaching 90% esterification in 3h with the recombinant enzyme. The use of molar excesses of the free fatty acids did not improve direct esterification rate, and the enzyme did not convert one of the two fatty acids preferentially when both were simultaneously available. On the other hand, solvent-free transesterification was an extremely efficient mechanism to synthesize β-sitostanyl oleate, yielding virtually full conversion of up to 80mM β-sitostanol in 2h. This process may represent a promising green alternative to the current chemical synthesis of these esters of unquestionable nutraceutical value.

  6. 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.

  7. 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

  8. 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...

  9. 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.

  10. 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...

  11. 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

  12. 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...

  13. Negative correlation between phospholipase and esterase activity produced by Fusarium isolates

    PubMed Central

    Ishida, K.; Alviano, D.S.; Silva, B.G.; Guerra, C.R.; Costa, A.S.; Nucci, M.; Alviano, C.S.; Rozental, S.

    2012-01-01

    Fusarium species have emerged as one of the more outstanding groups of clinically important filamentous fungi, causing localized and life-threatening invasive infections with high morbidity and mortality. The ability to produce different types of hydrolytic enzymes is thought to be an important virulence mechanism of fungal pathogens and could be associated with the environment of the microorganism. Here, we have measured the production of two distinct lipolytic enzymes, phospholipase and esterase, by sixteen Fusarium isolates recovered from the hospital environment, immunocompromised patients' blood cultures, foot interdigital space scrapings from immunocompromised patients, and foot interdigital space scrapings from immunocompetent patients (4 isolates each). Fourteen of these 16 isolates were identified as Fusarium solani species complex (FSSC) and two were identified as F. oxysporum species complex (FOSC). Some relevant genus characteristics were visualized by light and electron microscopy such as curved and multicelled macroconidia with 3 or 4 septa, microconidia, phialides, and abundant chlamydospores. All Fusarium isolates were able to produce esterase and phospholipase under the experimental conditions. However, a negative correlation was observed between these two enzymes, indicating that a Fusarium isolate with high phospholipase activity has low esterase activity and vice versa. In addition, Fusarium isolated from clinical material produced more phospholipases, while environmental strains produced more esterases. These observations may be correlated with the different types of substrates that these fungi need to degrade during their nutrition processes. PMID:22415116

  14. 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.

  15. Detoxication of paraoxon by rat liver homogenate and serum carboxylesterases and A-esterases.

    PubMed

    Tang, J; Chambers, J E

    1999-01-01

    Paraoxon, the active metabolite of parathion, can be detoxified through a noncatalytic pathway by carboxylesterases and a catalytic pathway by calcium-dependent A-esterases, producing p-nitrophenol as a common metabolite. The detoxication patterns of carboxylesterases and A-esterases were investigated in vitro in the present study with a high tissue concentration (75 mg/mL rat liver homogenate or 50% rat serum solution) to more closely reflect enzyme concentrations in intact tissues. A final paraoxon concentration of 3.75 microM was used to incubate with liver homogenates or serum solutions for 5 seconds or 3, 5, 15, or 25 minutes; also 0.625, 1.25, 2.5, 3.125, 3.75, or 5.0 microM paraoxon (final concentration) was incubated with liver homogenates or serum solutions for 15 minutes. Phenyl saligenin cyclic phosphate and EDTA were used to inhibit carboxylesterases and A-esterases, respectively. Significant amounts of p-nitrophenol were generated with or without either inhibitor during a 15 minute incubation with paraoxon from low (0.625 microM) to high (5.0 microM) concentrations. The amount of p-nitrophenol generated via carboxylesterase phosphorylation was greater than via A-esterase-mediated hydrolysis in the initial period of incubation or when incubating with a low concentration of paraoxon. Plateau shape curves of p-nitrophenol concentration versus time or paraoxon concentration indicated that carboxylesterase phosphorylation was saturable. When incubated for long time intervals or with high concentrations of paraoxon, more p-nitrophenol was generated via A-esterase-mediated hydrolysis than from carboxylesterase phosphorylation. The ratio of paraoxon concentration to tissue amount used in in vitro assays of this study was equivalent to dosing a rat with toxicologically relevant dosages. These in vitro data suggest that both carboxylesterases and A-esterases detoxify paraoxon in vivo; carboxylesterases may be an important mode of paraoxon detoxication in initial

  16. Biochemical Diversity of Carboxyl Esterases and Lipases from Lake Arreo (Spain): a Metagenomic Approach

    PubMed Central

    Martínez-Martínez, Mónica; Alcaide, María; Tchigvintsev, Anatoli; Reva, Oleg; Polaina, Julio; Bargiela, Rafael; Guazzaroni, María-Eugenia; Chicote, Álvaro; Canet, Albert; Valero, Francisco; Rico Eguizabal, Eugenio; Guerrero, María del Carmen; Yakunin, Alexander F.

    2013-01-01

    The esterases and lipases from the α/β hydrolase superfamily exhibit an enormous sequence diversity, fold plasticity, and activities. Here, we present the comprehensive sequence and biochemical analyses of seven distinct esterases and lipases from the metagenome of Lake Arreo, an evaporite karstic lake in Spain (42°46′N, 2°59′W; altitude, 655 m). Together with oligonucleotide usage patterns and BLASTP analysis, our study of esterases/lipases mined from Lake Arreo suggests that its sediment contains moderately halophilic and cold-adapted proteobacteria containing DNA fragments of distantly related plasmids or chromosomal genomic islands of plasmid and phage origins. This metagenome encodes esterases/lipases with broad substrate profiles (tested over a set of 101 structurally diverse esters) and habitat-specific characteristics, as they exhibit maximal activity at alkaline pH (8.0 to 8.5) and temperature of 16 to 40°C, and they are stimulated (1.5 to 2.2 times) by chloride ions (0.1 to 1.2 M), reflecting an adaptation to environmental conditions. Our work provides further insights into the potential significance of the Lake Arreo esterases/lipases for biotechnology processes (i.e., production of enantiomers and sugar esters), because these enzymes are salt tolerant and are active at low temperatures and against a broad range of substrates. As an example, the ability of a single protein to hydrolyze triacylglycerols, (non)halogenated alkyl and aryl esters, cinnamoyl and carbohydrate esters, lactones, and chiral epoxides to a similar extent was demonstrated. PMID:23542620

  17. Crystal Structure and Functional Characterization of an Esterase (EaEST) from Exiguobacterium antarcticum

    PubMed Central

    Kim, Boo-Young; Yoo, Wanki; Ryu, Bum Han; Kim, Han-Woo; Shin, Seung Chul; Kim, Sunghwan; Park, Hyun; Kim, T. Doohun; Lee, Jun Hyuck

    2017-01-01

    A novel microbial esterase, EaEST, from a psychrophilic bacterium Exiguobacterium antarcticum B7, was identified and characterized. To our knowledge, this is the first report describing structural analysis and biochemical characterization of an esterase isolated from the genus Exiguobacterium. Crystal structure of EaEST, determined at a resolution of 1.9 Å, showed that the enzyme has a canonical α/β hydrolase fold with an α-helical cap domain and a catalytic triad consisting of Ser96, Asp220, and His248. Interestingly, the active site of the structure of EaEST is occupied by a peracetate molecule, which is the product of perhydrolysis of acetate. This result suggests that EaEST may have perhydrolase activity. The activity assay showed that EaEST has significant perhydrolase and esterase activity with respect to short-chain p-nitrophenyl esters (≤C8), naphthyl derivatives, phenyl acetate, and glyceryl tributyrate. However, the S96A single mutant had low esterase and perhydrolase activity. Moreover, the L27A mutant showed low levels of protein expression and solubility as well as preference for different substrates. On conducting an enantioselectivity analysis using R- and S-methyl-3-hydroxy-2-methylpropionate, a preference for R-enantiomers was observed. Surprisingly, immobilized EaEST was found to not only retain 200% of its initial activity after incubation for 1 h at 80°C, but also retained more than 60% of its initial activity after 20 cycles of reutilization. This research will serve as basis for future engineering of this esterase for biotechnological and industrial applications. PMID:28125606

  18. 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

  19. 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).

  20. Phenolic Biotransformations during Conversion of Ferulic Acid to Vanillin by Lactic Acid Bacteria

    PubMed Central

    Kaur, Baljinder; Kumar, Balvir

    2013-01-01

    Vanillin is widely used as food additive and as a masking agent in various pharmaceutical formulations. Ferulic acid is an important precursor of vanillin that is available in abundance in cell walls of cereals like wheat, corn, and rice. Phenolic biotransformations can occur during growth of lactic acid bacteria (LAB), and their production can be made feasible using specialized LAB strains that have been reported to produce ferulic acid esterases. The present study aimed at screening a panel of LAB isolates for their ability to release phenolics from agrowaste materials like rice bran and their biotransformation to industrially important compounds such as ferulic acid, 4-ethyl phenol, vanillic acid, vanillin, and vanillyl alcohol. Bacterial isolates were evaluated using ferulic acid esterase, ferulic acid decarboxylase, and vanillin dehydrogenase assays. This work highlights the importance of lactic acid bacteria in phenolic biotransformations for the development of food grade flavours and additives. PMID:24066293

  1. Polypeptide having acetyl xylan esterase activity and uses thereof

    SciTech Connect

    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.

  2. Comparative study of human intestinal and hepatic esterases as related to enzymatic properties and hydrolizing activity for ester-type drugs.

    PubMed

    Inoue, M; Morikawa, M; Tsuboi, M; Ito, Y; Sugiura, M

    1980-08-01

    In attempts to determine the exact role of intestinal esterase in the body, we purified esterases from human intestinal mucosa and liver, and compared the enzymatic properties and substrate specificities with those of purified esterases. Esterase from human liver was purified 58-fold, by treatment with butanol, DE-52 and DEAE Sephadex A-50 column chromatographies, Sephadex G-200 gel filtration, and isoelectric focusing. The purified preparation showed a single band by polyacylamide gel electrophoresis. The molecular weights of intestinal and hepatic esterases were determined to be 53,000-55,000 and 180,000, respectively, by gel filtration on Sephadex G-200. The activity of the purified intestinal and hepatic esterases was strongly inhibited by diethyl-p-nitrophenyl phosphate and diisopropyl fluorophosphate, and was not inhibited by eserine sulfate and p-chloromercuribenzoate. Moreover, the purified esterases hydrolyzed ester-type drugs such as aspirin, clofibrate, indanyl carbenicillin and procaine. Hepatic esterase had properties similar to those of intestinal esterase with respect to the sensitivity to organophosphate and the substrate specificity. However, the two purified esterases differed in properties such as molecular weight, isoelectric point, thermostability and optimal pH.

  3. Acute promyelocytic leukemia, hypogranular variant, with uncharacteristic staining with chloroacetate esterase.

    PubMed

    Dunphy, C H; Polski, J M; Johns, G; Evans, H L; Gardner, L J

    2001-06-01

    A diagnosis of the hypogranular variant of acute promyelocytic leukemia (APLv) may be difficult to establish based on cytomorphology alone. However, the great majority of cases have a classical immunophenotype by flow cytometric immunophenotyping (FCI) (CD13+, CD33+, dim CD64+, HLA-DR-, and CD34-) and a classical enzyme cytochemical (EC) staining pattern. [intensely staining with myeloperoxidase, Sudan Black B, and chloroacetate esterase (CAE) and negative with alpha'-naphthyl acetate and butyrate esterases]. Although the immunophenotype of APLv by FCI has varied in the literature (HLA-DR +/- and CD34 +/-), the EC staining pattern has remained constant. We report a case of APLv with characteristic cytomorphology, compatible FCI data (CD13+, CD33+, dim CD64+, HLA-DR +/-, and CD34-), chromosomal detection of t(15; 17), and molecular detection of the PML/RAR alpha fusion gene; however, staining of the leukemic cells with CAE was quite uncharacteristic. We describe our findings.

  4. Heterozygosity of the sheep: Polymorphism of 'malic enzyme', isocitrate dehydrogenase (NADP+), catalase and esterase.

    PubMed

    Baker, C M; Manwell, C

    1977-04-01

    In contrast to other reports, it is found that the sheep has approximately as much enzyme variation as man. Most of the genetically interpretable enzyme variation in heart, liver, kidney and muscle from 52 sheep (Merinos or Merino crosses) is in the NADP-dependent dehydrogenases [two 'malic enzymes' and the supernatant isocitrate dehydrogenase (NADP+)] and in the esterases. Ten different loci for NAD-dependent dehydrogenases are electrophoretically monomorphic, as are five different NADH diaphorases from heart muscle and 15 different major proteins from skeletal muscle. It is highly statistically significant that NADP-dependent dehydrogenases and esterases are polymorphic but representatives of several other major classes of enzymes are not. The physiological significance of this polymorphism may be related to the role of these enzymes in growth and detoxication, sheep having been selected by man for faster growth, of wool or of carcass, and for grazing a wide variety of plants.

  5. 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.

  6. 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

    2014-09-05

    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.

  7. Is Esterase-P Encoded by a Cryptic Pseudogene in Drosophila Melanogaster?

    PubMed Central

    Balakirev, E. S.; Ayala, F. J.

    1996-01-01

    We have amplified and sequenced the gene encoding Esterase-P (Est-P) in 10 strains of Drosophila melanogaster. Three premature termination codons occur in the coding region of the gene in two strains. This observation, together with other indirect evidence, leads us to propose that Est-P may be a pseudogene in D. melanogaster. Est-P would be a ``cryptic'' pseudogene, in the sense that it retains intact the coding sequence (without stop codons and other alterations usually observed in pseudogenes) in most D. melanogaster strains. We conjecture that the β-esterase cluster may consist in other Drosophila species of functional and nonfunctional genes. We also conjecture that the rarity of detected pseudogenes in Drosophila may be due to the difficulty of discovering them, because most of them are cryptic. PMID:8978040

  8. Esterase-Sensitive Prodrugs with Tunable Release Rates and Direct Generation of Hydrogen Sulfide.

    PubMed

    Zheng, Yueqin; Yu, Bingchen; Ji, Kaili; Pan, Zhixiang; Chittavong, Vayou; Wang, Binghe

    2016-03-24

    Prodrugs that release hydrogen sulfide upon esterase-mediated cleavage of an ester group followed by lactonization are described herein. By modifying the ester group and thus its susceptibility to esterase, and structural features critical to the lactonization rate, H2 S release rates can be tuned. Such prodrugs directly release hydrogen sulfide without the involvement of perthiol species, which are commonly encountered with existing H2 S donors. Additionally, such prodrugs can easily be conjugated to another non-steroidal anti-inflammatory agent, leading to easy synthesis of hybrid prodrugs. As a biological validation of the H2 S prodrugs, the anti-inflammatory effects of one such prodrug were examined by studying its ability to inhibit LPS-induced TNF-α production in RAW 264.7 cells. This type of H2 S prodrugs shows great potential as both research tools and therapeutic agents.

  9. Characterization and affinity purification of juvenile hormone esterase from Bombyx mori.

    PubMed

    Shiotsuki, T; Bonning, B C; Hirai, M; Kikuchi, K; Hammock, B D

    2000-08-01

    Juvenile hormone esterase (JHE) from hemolymph of the silkworm moth Bombyx mori was characterized for substrate specificity and inhibitor sensitivity. B. mori JHE hydrolyzed the juvenile hormone surrogate substrate methyl n-heptylthioacetothioate (HEPTAT) more efficiently than p-nitrophenyl acetate and 1-naphthyl acetate substrates widely used to assay total carboxylesterase activity. B. mori JHE was sensitive to 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP), which was developed as a selective inhibitor for lepidopteran JHE, and relatively insensitive to diisopropyl fluorophosphate (DFP), an inhibitor of serine esterases but not of all JHEs. Affinity purification with a trifluoromethyl ketone ligand was more efficient for purification of B. mori JHE than DEAE ion exchange chromatography.

  10. The role of stearate attachment to the hemagglutinin-esterase-fusion glycoprotein HEF of influenza C virus.

    PubMed

    Wang, Mingyang; Ludwig, Kai; Böttcher, Christoph; Veit, Michael

    2016-05-01

    The only spike of influenza C virus, the hemagglutinin-esterase-fusion glycoprotein (HEF) combines receptor binding, receptor hydrolysis and membrane fusion activities. Like other hemagglutinating glycoproteins of influenza viruses HEF is S-acylated, but only with stearic acid at a single cysteine located at the cytosol-facing end of the transmembrane region. Previous studies established the essential role of S-acylation of hemagglutinin for replication of influenza A and B virus by affecting budding and/or membrane fusion, but the function of acylation of HEF was hitherto not investigated. Using reverse genetics we rescued a virus containing non-stearoylated HEF, which was stable during serial passage and showed no competitive fitness defect, but the growth rate of the mutant virus was reduced by one log. Deacylation of HEF does neither affect the kinetics of its plasma membrane transport nor the protein composition of virus particles. Cryo-electron microscopy showed that the shape of viral particles and the hexagonal array of spikes typical for influenza C virus were not influenced by this mutation indicating that virus budding was not disturbed. However, the extent and kinetics of haemolysis were reduced in mutant virus at 37°C, but not at 33°C, the optimal temperature for virus growth, suggesting that non-acylated HEF has a defect in membrane fusion under suboptimal conditions.

  11. Factor IX Amagasaki: A new mutation in the catalytic domain resulting in the loss of both coagulant and esterase activities

    SciTech Connect

    Miyata, Toshiyuki; Iwanaga, Sadaaki ); Sakai, Toshiyuki; Sugimoto, Mitsuhiko; Naka, Hiroyuki; Yamamoto, Kazukuni; Yoshioka, Akira; Fukui, Hiromu ); Mitsui, Kotoko; Kamiya, Kensyu; Umeyama, Hideaki )

    1991-11-26

    Factor IX Amagasaki (AMG) is a naturally occurring mutant of factor IX having essentially no coagulant activity, even though normal levels of antigen are detected in plasma. Factor IX AMG was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Factor IX AMG was cleaved normally by factor VIIa-tissue factor complex, yielding a two-chain factor IXa. Amino acid composition and sequence analysis of one of the tryptic peptides isolated from factor IX AMG revealed that Gly-311 had been replaced by Glu. The authors identified a one-base substitution of guanine to adenine in exon VIII by amplifying exon VIII using the polymerase chain reaction method and sequencing the product. This base mutation also supported the replacement of Gly-311 by Glu. In the purified system, factor IXa AMG did not activate for factor X in the presence of factor VIII, phospholipids, and Ca{sup 2+}, and no esterase activity toward Z-Arg-p-nitrobenzyl ester was observed. The model building of the serine protease domain of factor IXa suggests that the Gly-311 {yields} Glu exchange would disrupt the specific conformational state in the active site environment, resulting in the substrate binding site not forming properly. This is the first report to show the experimental evidence for importance of a highly conserved Gly-142 (chymotrypsinogen numbering) located in the catalytic site of mammalian serine proteases so far known.

  12. Intrinsic temperature sensitivity of influenza C virus hemagglutinin-esterase-fusion protein.

    PubMed

    Takashita, Emi; Muraki, Yasushi; Sugawara, Kanetsu; Asao, Hironobu; Nishimura, Hidekazu; Suzuki, Koji; Tsuji, Takashi; Hongo, Seiji; Ohara, Yoshiro; Ohara, Yoshihiro; Kawaoka, Yoshihiro; Ozawa, Makoto; Matsuzaki, Yoko

    2012-12-01

    Influenza C virus replicates more efficiently at 33°C than at 37°C. To determine whether hemagglutinin-esterase-fusion protein (HEF), a surface glycoprotein of influenza C virus, is a restricting factor for this temperature sensitivity, we analyzed the biological and biochemical properties of HEF at 33°C and 37°C. We found that HEF exhibits intrinsic temperature sensitivities for surface expression and fusion activity.

  13. Total cholesterol, high density lipoprotein cholesterol and choline esterase in overseas and Japanese university students.

    PubMed

    Nakamura, S

    1985-04-01

    Serum lipids were studied in 97 overseas and 282 Japanese university students. As compared with Japanese, serum total cholesterol levels were low and high density lipoprotein/total cholesterol ratio was high in the overseas students, especially in Chinese and Korean students. 30-39-year-old Chinese students, moreover, showed elevated high density lipoprotein levels. Choline esterase levels were significantly lower in 30-39-year-old Chinese and Korean students than in Japanese and Taiwanese.

  14. [Methods of increasing the activity of extracellular esterase, beta-fructofuranosidase and proteases of wine yeast].

    PubMed

    Abdurazakova, S Kh; Salomov, Kh T

    1975-01-01

    Upon regular fermentation changes in the activity of the enzymes esterase, beta-fructofuranosidase and protease of the yeast Saccharomyces mini of the Parkent I race were examined. The maximum activity of the enzymes occurred in the stationary phase of the yeast growth. An increase in the activity of the above enzymes was shown possible during a prolonged stabilization of the stationary conditions in the process of a continuous chemostat cultivation of wine yeast.

  15. Esterase Activated Carbonyl Sulfide/Hydrogen Sulfide (H2S) Donors.

    PubMed

    Chauhan, Preeti; Bora, Prerona; Ravikumar, Govindan; Jos, Swetha; Chakrapani, Harinath

    2017-01-06

    Hydrogen sulfide (H2S) is a mediator of a number of cellular processes, and modulating cellular levels of this gas has emerged as an important therapeutic area. Localized generation of H2S is thus very useful but highly challenging. Here, we report pivaloyloxymethyl-based carbonothioates and carbamothioates that are activated by the enzyme, esterase, to generate carbonyl sulfide (COS), which is hydrolyzed to H2S.

  16. Purification of neuropathy target esterase from avian brain after prelabelling with [3H]diisopropyl phosphorofluoridate.

    PubMed

    Rüffer-Turner, M E; Read, D J; Johnson, M K

    1992-01-01

    Neuropathy target esterase from hen brains was radiolabelled at the active site with [3H]diisopropyl phosphorofluoridate. The labelled protein was purified by differential centrifugation and Nonidet P40 solubilization, detergent phase partitioning, anion exchange, and preparative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The volatilizable counts assay and analytical SDS-PAGE were used to monitor the protein. The 150-kDa subunit polypeptide appears as a single band on analytical SDS-PAGE.

  17. Structural role of a conserved active site cis proline in the Thermotoga maritima acetyl esterase from the carbohydrate esterase family 7.

    PubMed

    Singh, Mrityunjay K; Manoj, Narayanan

    2017-04-01

    A conserved cis proline residue located in the active site of Thermotoga maritima acetyl esterase (TmAcE) from the carbohydrate esterase family 7 (CE7) has been substituted by alanine. The residue was known to play a crucial role in determining the catalytic properties of the enzyme. To elucidate the structural role of the residue, the crystal structure of the Pro228Ala variant (TmAcEP228A ) was determined at 2.1 Å resolution. The replacement does not affect the overall secondary, tertiary, and quaternary structures and moderately decreases the thermal stability. However, the wild type cis conformation of the 227-228 peptide bond adopts a trans conformation in the variant. Other conformational changes in the tertiary structure are restricted to residues 222-226, preceding this peptide bond and are located away from the active site. Overall, the results suggest that the conserved proline residue is responsible for the cis conformation of the peptide and shapes the geometry of the active site. Elimination of the pyrrolidine ring results in the loss of van der Waals and hydrophobic interactions with both the alcohol and acyl moeities of the ester substrate, leading to significant impairment of the activity and perturbation of substrate specificity. Furthermore, a cis-to-trans conformational change arising out of residue changes at this position may be associated with the evolution of divergent activity, specificity, and stability properties of members constituting the CE7 family. Proteins 2017; 85:694-708. © 2016 Wiley Periodicals, Inc.

  18. 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

  19. Structural and functional profile of the carbohydrate esterase gene complement in Phytophthora infestans.

    PubMed

    Ospina-Giraldo, Manuel D; McWalters, Jessica; Seyer, Lauren

    2010-12-01

    The plant cell cuticle is the first obstacle for penetration of the host by plant pathogens. To breach this barrier, most pathogenic fungi employ a complex assortment of cell wall-degrading enzymes including carbohydrate esterases, glycoside hydrolases, and polysaccharide lyases. We characterized the full complement of carbohydrate esterase-coding genes in three Phytophthora species and analyzed the expression of cutinase in vitro and in planta; we also determined the cutinase allele distribution in multiple isolates of P. infestans. Our investigations revealed that there are 49, 21, and 37 esterase homologs in the P. infestans, P. ramorum, and P. sojae genomes, respectively, with a considerable number predicted to be extracellular. Four cutinase gene copies were found in both the P. infestans and P. ramorum genomes, while 16 copies were found in P. sojae. Transcriptional analyses of cutinase in P. infestans revealed that its expression level during infection is significantly upregulated at all time points compared to that of the same gene in mycelium grown in vitro. Expression achieves maximum values at 15 hpi, declining at subsequent time points. These results may suggest, therefore, that cutinase most likely plays a role in P. infestans pathogenicity.

  20. B-esterase determination and organophosphate insecticide inhibitory effects in JEG-3 trophoblasts.

    PubMed

    Espinoza, Marlon; Rivero Osimani, Valeria; Sánchez, Victoria; Rosenbaum, Enrique; Guiñazú, Natalia

    2016-04-01

    The placenta and trophoblasts express several B-esterases. This family includes acethylcholinesterase (AChE), carboxylesterase (CES) and butyrylcholinesterase (BChE), which are important targets of organophosphate insecticide (OP) toxicity. To better understand OP effects on trophoblasts, B-esterase basal activity and kinetic behavior were studied in JEG-3 choriocarcinoma cell cultures. Effects of the OP azinphos-methyl (Am) and chlorpyrifos (Cp) on cellular enzyme activity were also evaluated. JEG-3 cells showed measurable activity levels of AChE and CES, while BChE was undetected. Recorded Km for AChE and CES were 0.33 and 0.26 mM respectively. Native gel electrophoresis and RT-PCR analysis demonstrated CES1 and CES2 isoform expression. Cells exposed for 4 and 24 h to the OP Am or Cp, showed a differential CES and AChE inhibition profiles. Am inhibited CES and AChE at 4 h treatment while Cp showed the highest inhibition profile at 24 h. Interestingly, both insecticides differentially affected CES1 and CES2 activities. Results demonstrated that JEG-3 trophoblasts express AChE, CES1 and CES2. B-esterase enzymes were inhibited by in vitro OP exposure, indicating that JEG-3 cells metabolization capabilities include phase I enzymes, able to bioactivate OP. In addition, since CES enzymes are important for medicinal drug activation/deactivation, OP exposure may interfere with trophoblast CES metabolization, probably being relevant in a co-exposure scenario during pregnancy.

  1. Isolation and Characterization of a Novel Cold-Adapted Esterase, MtEst45, from Microbulbifer thermotolerans DAU221.

    PubMed

    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 K m and V max values were 0.0998 mM and 550 μmol/min/mg of protein, respectively. MtEst45 was strongly inhibited by Hg(2+), Zn(2+), and Cu(2+) ions; by phenylmethanesulfonyl fluoride; and by β-mercaptoethanol. Ca(2+) 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.

  2. 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

  3. 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.

  4. 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

  5. Allergenicity of Hev b 13, a major esterase allergen in natural rubber latex (Hevea brasiliensis) allergy, does not only depend on its carbohydrate moiety.

    PubMed

    Rougé, Pierre; Culerrier, Raphaël; Campistron, Marion; Granier, Claude; Bienvenu, Françoise; Bienvenu, Jacques; Didier, Alain; Barre, Annick

    2010-01-01

    The three-dimensional model built for the major latex allergen Hev b 13 consists of the typical organization of plant esterases made of a central bundle of five parallel beta-strands surrounded by five alpha-helices associated to two shorter alpha-helical segments. Up to 12 sets of sequential IgE-binding peptides were identified in SPOT experiments along the amino acid sequence of Hev b 13. They correspond in fact to eight IgE-binding epitopic stretches exposed on the surface of the allergen. With the exception of epitope #5, all other epitopes contain charged residues. Epitope #8 contains the 3rd putative N-glycosylation site of Hev b 13 and should consist of a glycotope, whereas all other identified IgE-binding areas occur outside the two remaining putative N-glycosylation sites. Accordingly, the allergenicity of Hev b 13 does not primarily depends on its carbohydrate moiety.

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

    PubMed

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

    2015-06-01

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

  8. A Tripartite Fusion, FaeG-FedF-LT192A2: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 Central

    Ruan, Xiaosai; Liu, Mei; Casey, Thomas A.; Zhang, Weiping

    2011-01-01

    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 (LT192) A2 and B subunits for a tripartite adhesin-adhesin-toxoid fusion (FaeG-FedF-LT192A2: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-LT192A2: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-LT192A2: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

  9. 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

  10. 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.

  11. An integron cassette encoding erythromycin esterase, ere(A), from Providencia stuartii.

    PubMed

    Plante, Isabelle; Centrón, Daniela; Roy, Paul H

    2003-04-01

    We have mapped the variable region of the two class 1 integrons found in the multiresistant strain Providencia stuartii 1723. Integron 1 contains a new arrangement of gene cassettes, aacA4-aadB-aadA1, conferring resistance to all aminoglycosides used for clinical treatment. Integron 2 contains a variant of the gene cassette ere(A), coding for an erythromycin esterase, whose nucleotide sequence shares 93.7% DNA identity with ere(A) from Escherichia coli BM2195 plasmid pIP1100.

  12. Effects on operant learning and brain acetylcholine esterase activity in rats following chronic inorganic arsenic intake.

    PubMed

    Nagaraja, T N; Desiraju, T

    1994-05-01

    1. Very young and adult Wistar rats were given As5+, 5 mg arsenic kg-1 body weight day-1 (sodium arsenate). 2. Operant learning was tested in a Skinner box at the end of exposure and, in the case of developing animals, also after a recovery period. 3. Acetylcholine esterase (AChE) activity was estimated in discrete brain regions of these animals. 4. The animals exposed to arsenic took longer to acquire the learned behaviour and to extinguish the operant. AChE activity was inhibited in some regions of the brain.

  13. Substrate-Preference Polymorphism at an Esterase Locus of DROSOPHILA MOJAVENSIS

    PubMed Central

    Zouros, E.; van Delden, W.

    1982-01-01

    In a larval esterase of Drosophila mojavensis there are alleles whose products preferentially hydrolyze α-naphthyl esters, whereas the majority of the alleles hydrolyze preferentially β-naphthyl esters. In a collection of laboratory stocks α alleles have a frequency of 15%. Three different mobilities of α alleles were discovered, suggesting a polymorphism rather than a single mutation event. If substrate-preference polymorphisms are common among "multiple-substrate" enzymes (category II of Gillespie and Langley 1974), allozyme variation at these enzyme loci may well be maintained by balancing selection. PMID:7106559

  14. Balance of activities of alcohol acetyltransferase and esterase in Saccharomyces cerevisiae is important for production of isoamyl acetate.

    PubMed

    Fukuda, K; Yamamoto, N; Kiyokawa, Y; Yanagiuchi, T; Wakai, Y; Kitamoto, K; Inoue, Y; Kimura, A

    1998-10-01

    Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in Saccharomyces cerevisiae and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and used these strains in small-scale sake brewing. Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity. Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash.

  15. Balance of Activities of Alcohol Acetyltransferase and Esterase in Saccharomyces cerevisiae Is Important for Production of Isoamyl Acetate

    PubMed Central

    Fukuda, Kiyoshi; Yamamoto, Nagi; Kiyokawa, Yoshifumi; Yanagiuchi, Toshiyasu; Wakai, Yoshinori; Kitamoto, Katsuhiko; Inoue, Yoshiharu; Kimura, Akira

    1998-01-01

    Isoamyl acetate is synthesized from isoamyl alcohol and acetyl coenzyme A by alcohol acetyltransferase (AATFase) in Saccharomyces cerevisiae and is hydrolyzed by esterases at the same time. We hypothesized that the balance of both enzyme activities was important for optimum production of isoamyl acetate in sake brewing. To test this hypothesis, we constructed yeast strains with different numbers of copies of the AATFase gene (ATF1) and the isoamyl acetate-hydrolyzing esterase gene (IAH1) and used these strains in small-scale sake brewing. Fermentation profiles as well as components of the resulting sake were largely alike; however, the amount of isoamyl acetate in the sake increased with an increasing ratio of AATFase/Iah1p esterase activity. Therefore, we conclude that the balance of these two enzyme activities is important for isoamyl acetate accumulation in sake mash. PMID:9758847

  16. 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.

  17. Benzoyl-L-arginine methyl ester (BAME)-esterase activity in human plasma during the gravidic-puerperal cycle.

    PubMed

    Salles Meirelles, R

    1977-01-01

    Benzoyl-L-arginine methyl ester (BAME)-esterase activity of plasma was measured in women going through the gravidic-puerperal cycle and compared with plasma of non-pregnant women. Plasma from women in the 36th to 40th week of pregnancy hydrolyzes BAME two times more rapidly than that from non-pregnant women. During pregnancy, BAME-esterase activity in plasma increases progressively up to the 40th week, decreases during labor, and after delivery reaches the same level as in non-pregnant women. The BAME-esterase activity of plasma was affected by the storage temperature, with differences demonstrable between -20 and -4 C and between pregnant and non-pregnant women.

  18. 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.

  19. Effects of an esterase-producing inoculant on fermentation, aerobic stability, and neutral detergent fiber digestibility of corn silage.

    PubMed

    Kang, T W; Adesogan, A T; Kim, S C; Lee, S S

    2009-02-01

    This experiment evaluated effects of an inoculant containing esterase-producing bacteria on fermentation, aerobic stability, in situ dry matter digestibility (DMD), and neutral detergent fiber (NDF) digestibility (NDFD) of corn silage. Two corn hybrids grown on adjacent fields [Croplan Genetics 851RR2 (CS1) and Vigoro 61R36 (CS2)] were harvested at approximately 39% dry matter. Each forage was conserved in quadruplicate in 20-L mini silos with or without application of an inoculant at a level to achieve 1.0 x 10(4) cfu/g of Lactobacillus casei and 1.0 x 10(5) cfu/g of Lactobacillus buchneri. After 110 d of ensiling, silos were opened and silages were analyzed for chemical composition, fermentation indices, microbial counts, and aerobic stability. In situ DMD, 24-h and 48-h DMD, and NDFD were measured by incubating ground (6-mm) samples in triplicate in each of 2 lactating, fistulated dairy cows fed a corn silage-based diet. Inoculation decreased concentrations of total fermentation acids and lactate, as well as lactate to acetate ratio, and increased propionate concentration compared with the uninoculated control in CS1 but not CS2. Inoculation tended to decrease yeast counts of CS1 but increased yeast counts and tended to increase the mold counts of CS2. Consequently, inoculation improved the aerobic stability of CS1 by 57.3 h (98%) but decreased that of CS2 by 20.5 h (20%). Inoculation also increased the potentially degradable fraction of CS1 and the total degradable fraction, 24-h and 48-h DMD, and 48-h NDFD of CS2. Inoculation of CS1 modified the fermentation, improved the aerobic stability, and increased the potentially degradable DM fraction. Inoculation of CS2 did not affect fermentation, but decreased the aerobic stability and increased the total degradable DM fraction, 24-h and 48-h DMD, and 48-h NDFD.

  20. 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

  1. Engineering of a thermostable esterase Est816 to improve its quorum-quenching activity and the underlying structural basis

    PubMed Central

    Liu, Xiwen; Cao, Li-chuang; Fan, Xin-jiong; Liu, Yu-huan; Xie, Wei

    2016-01-01

    N-acyl-homoserine lactones (AHLs) are small diffusible molecules called autoinducers that mediate cell-to-cell communications. Enzymatic degradation of AHLs is a promising bio-control strategy known as quorum-quenching. To improve the quorum-quenching activity of a thermostable esterase Est816, which had been previously cloned, we have engineered the enzyme by random mutagenesis. One of the mutants M2 with double amino acid substitutions (A216V/K238N) showed 3-fold improvement on catalytic efficiency. Based on the crystal structure determined at 2.64 Å, rational design of M2 was conducted, giving rise to the mutant M3 (A216V/K238N/L122A). The kcat/KM value of the mutant M3 is 21.6-fold higher than that of Est816. Furthermore, activity assays demonstrated that M3 reached 99% conversion of 10-μM N-octanoyl-DL-homoserine lactone (C8-HSL) to N-octanoyl- DL-homoserine (C8-Hse) in 20 min, in contrast to the 8 h required by wild type Est816. The dramatic activity enhancement may be attributed to the increased hydrophobic interactions with the lactone ring by the mutation A216V, and the reduced steric clashes between the long side chain of L122 and the aliphatic tail of HSL by the mutation L122A, according to the crystal structure. This study sheds lights on the activity-structure relationship of AHL-lactonases, and may provide useful information in engineering AHL-degrading enzymes. PMID:27909291

  2. Engineering of a thermostable esterase Est816 to improve its quorum-quenching activity and the underlying structural basis.

    PubMed

    Liu, Xiwen; Cao, Li-Chuang; Fan, Xin-Jiong; Liu, Yu-Huan; Xie, Wei

    2016-12-02

    N-acyl-homoserine lactones (AHLs) are small diffusible molecules called autoinducers that mediate cell-to-cell communications. Enzymatic degradation of AHLs is a promising bio-control strategy known as quorum-quenching. To improve the quorum-quenching activity of a thermostable esterase Est816, which had been previously cloned, we have engineered the enzyme by random mutagenesis. One of the mutants M2 with double amino acid substitutions (A216V/K238N) showed 3-fold improvement on catalytic efficiency. Based on the crystal structure determined at 2.64 Å, rational design of M2 was conducted, giving rise to the mutant M3 (A216V/K238N/L122A). The kcat/KM value of the mutant M3 is 21.6-fold higher than that of Est816. Furthermore, activity assays demonstrated that M3 reached 99% conversion of 10-μM N-octanoyl-DL-homoserine lactone (C8-HSL) to N-octanoyl- DL-homoserine (C8-Hse) in 20 min, in contrast to the 8 h required by wild type Est816. The dramatic activity enhancement may be attributed to the increased hydrophobic interactions with the lactone ring by the mutation A216V, and the reduced steric clashes between the long side chain of L122 and the aliphatic tail of HSL by the mutation L122A, according to the crystal structure. This study sheds lights on the activity-structure relationship of AHL-lactonases, and may provide useful information in engineering AHL-degrading enzymes.

  3. 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.

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

    PubMed Central

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

    2008-01-01

    The catalytic domain of the glucuronoyl esterase from 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 P212121 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. PMID:18391420

  5. 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.

  6. 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%.

  7. Kinetic mechanism of the detoxification of the organophosphate paraoxon by human serum A-esterase.

    PubMed

    Vitarius, J A; Sultatos, L G

    1994-01-01

    The mammalian detoxification of certain organophosphates, such as paraoxon [O,O-diethyl (p-nitrophenyl) phosphate], is catalyzed by the enzyme A-esterase. In this study, incubations of human serum in 50 mM glycine buffer (pH 10.5) with paraoxon resulted in the nonlinear production of p-nitrophenol, characterized by a rapid initial phase for the first several minutes of the incubation, followed by a second, slower phase in which the velocity approached constancy. Production of p-nitrophenol could be accurately fitted to the velocity equation for an Ordered Uni Bi kinetic mechanism with initial-burst activity, yielding estimates of appk2, appk3, and appE, for 10 human subjects. Increasing calcium concentration in the incubation resulted in increases in appk3 and appE, without affecting appk2. Conversely, addition of 1 M sodium chloride decreased the appk3 and appE, but did not alter appk2. And finally, a continuous system computer model was constructed based on the differential equations descriptive of an Ordered Uni Bi kinetic mechanism. This model accurately simulated production of p-nitrophenol from human serum, providing further support that A-esterase hydrolyzes paraoxon by an Ordered Uni Bi kinetic mechanism with initial-burst activity.

  8. 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

  9. Diet quality determines lipase gene expression and lipase/esterase activity in Daphnia pulex

    PubMed Central

    Schwarzenberger, Anke; Wacker, Alexander

    2017-01-01

    ABSTRACT We studied the short- (12 h) and long-term (144 h) response of Daphnia pulex lipases to quality shifts in diets consisting of different mixtures of the green alga Scenedesmus with the cyanobacterium Synechococcus, two species with contrasting lipid compositions. The lipase/esterase activity in both the gut and the body tissues had fast responses to the diet shift and increased with higher dietary contributions of Synechococcus. When screening the Daphnia genome for TAG lipases, we discovered a large gene-family expansion of these enzymes. We used a subset of eight genes for mRNA expression analyses and distinguished between influences of time and diet on the observed gene expression patterns. We identified five diet-responsive lipases of which three showed a sophisticated short- and long-term pattern of expression in response to small changes in food-quality. Furthermore, the gene expression of one of the lipases was strongly correlated to lipase/esterase activity in the gut suggesting its potentially major role in digestion. These findings demonstrate that the lipid-related enzymatic machinery of D. pulex is finely tuned to diet and might constitute an important mechanism of physiological adaptation in nutritionally complex environments. PMID:28069588

  10. Use of esterase activities for the detection of chemical neurotoxic agents.

    PubMed

    Manco, Giuseppe; Nucci, Roberto; Febbraio, Ferdinando

    2009-01-01

    The quest for a quick and easy detection of the neurotoxin levels in the environment has fostered the search for systems alternative to currently employed analytical methods such as spectrophotometer, gas-liquid chromatography, thin-layer chromatography, and more recently mass spectrometry. These drawbacks lead to intense research efforts to develop biosensor devices for the determination of these compounds. In this review, we present an overview of the actual development of research in neurotoxin detection by using enzymatic biosensors based on esterase activity, in particular cholinesterases, and carboxylesterases. Detection by enzymatic activity could be carried out measuring the hydrolysis products or the residual enzymatic activity after inhibition, using a transducer system that makes possible the correlation between the determined activity and the analyte concentration. Several transducer systems were adopted for the neurotoxins identification using esterases, including electrochemical, optical, conductimetric and piezoelectric procedures. The differences in the used transducer determine the final sensitivity and specificity of the biosensor. Moreover, a brief description of immobilization procedure, that is an important step in the biosensor development and could affect the final characteristic of biosensor (sensibility, stability, response time and reproducibility), was accomplished. Final considerations on advantages and problems, related to actual development of these technologies, and its prospective were discussed.

  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

    2015-01-27

    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.

  14. Correlation between esterase electrophoretic polymorphism and virulence-associated traits in extra-intestinal invasive strains of Escherichia coli.

    PubMed Central

    Goullet, P.; Picard, B.; Contrepois, M.; De Rycke, J.; Barnouin, J.

    1994-01-01

    The electrophoretic variations of carboxylesterase B and of esterases A, C and I, the presence of mannose resistant haemagglutinin, alpha-haemolysin, cytotoxic necrotizing factor type 1 (CNF1) and certain O antigens were compared in 150 strains of Escherichia coli responsible for extra-intestinal infections. Electrophoretic mobilities of outer membrane proteins (OMP) were also studied for strains belonging to O4, O6, O7, O8 and O75 serogroups. Fast migrating allozymes of carboxylesterase B (pattern B1) were correlated with slow migrating allozymes of esterase C, serogroups O7 and O8, lack of virulence factor, and particular OMP patterns, whereas slow migrating allozymes of carboxylesterase B (pattern B2) were correlated with fast migrating allozymes of esterase C, serogroups O2, O4, O6, O18 and O75, virulence factor production, and distinct OMP patterns. Allozymes of esterases A and I were not clearly correlated with the distribution of virulence factors. The pattern B2 was more strongly associated with CNF1 than with alpha-haemolysin and mannose resistant haemagglutinin. These results substantiate the view that the electrophoretic pattern B2 of carboxylesterase B identified most of the highly pathogenic strains implicated in extra-intestinal infection of humans. Images Fig. 2 PMID:7509755

  15. 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...

  16. Identification and functional characterization of esterases in Euschistus heros (Hemiptera, Pentatomidae) and their relationship with thiamethoxam and lambda-cyhalothrin.

    PubMed

    Hegeto, L A; Ronqui, L; Lapenta, A S; Albuquerque, F A

    2015-09-22

    The brown stink bug Euschistus heros is the most abundant species of the soybean-sucking bugs, and causes large economic losses. Applying different chemical groups of organosynthetic insecticides for its control increases the potential for resistance. Esterases are a group of enzymes that play a variety of roles in insects, and some of them are related to the metabolism of xenobiotics. The aim of this study was to analyze the esterase isoenzyme system of this species and investigate its response to Engeo™ Pleno (thiamethoxam and lambda-cyhalothrin), which is the most widely used pesticide in soybean crops. Two strains were analyzed: the EB strain, which had been free of insecticides for several generations; and the MA strain, which was collected in a location exposed to agrochemicals. By analyzing the polyacrylamide gel electrophoresis profile, seven different esterases in adults and nymphs of both strains were found. Eight gene loci were responsible for the synthesis of these enzymes. The differences in esterases between the two strains and enzyme changes in insects exposed to Engeo™ Pleno suggest that EST-2 and EST-4 are related to the metabolism of the agrochemical used and are mechanisms of resistance.

  17. Overexpression of Drosophila juvenile hormone esterase binding protein results in anti-JH effects and reduced pheromone abundance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The titer of juvenile hormone (JH), which has wide ranging physiological effects in insects, is regulated in part by JH esterase (JHE). We show that overexpression in Drosophila melanogaster of the JHE binding protein, DmP29 results in a series of apparent anti-JH effects. We hypothesize that DmP29 ...

  18. 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...

  19. 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.

  20. Role of bifidobacteria in the hydrolysis of chlorogenic acid.

    PubMed

    Raimondi, Stefano; Anighoro, Andrew; Quartieri, Andrea; Amaretti, Alberto; Tomás-Barberán, Francisco A; Rastelli, Giulio; Rossi, Maddalena

    2015-02-01

    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.

  1. 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

  2. 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

  3. Paraben esters: review of recent studies of endocrine toxicity, absorption, esterase and human exposure, and discussion of potential human health risks.

    PubMed

    Darbre, Philippa D; Harvey, Philip W

    2008-07-01

    This toxicology update reviews research over the past four years since publication in 2004 of the first measurement of intact esters of p-hydroxybenzoic acid (parabens) in human breast cancer tissues, and the suggestion that their presence in the human body might originate from topical application of bodycare cosmetics. The presence of intact paraben esters in human body tissues has now been confirmed by independent measurements in human urine, and the ability of parabens to penetrate human skin intact without breakdown by esterases and to be absorbed systemically has been demonstrated through studies not only in vitro but also in vivo using healthy human subjects. Using a wide variety of assay systems in vitro and in vivo, the oestrogen agonist properties of parabens together with their common metabolite (p-hydroxybenzoic acid) have been extensively documented, and, in addition, the parabens have now also been shown to possess androgen antagonist activity, to act as inhibitors of sulfotransferase enzymes and to possess genotoxic activity. With the continued use of parabens in the majority of bodycare cosmetics, there is a need to carry out detailed evaluation of the potential for parabens, together with other oestrogenic and genotoxic co-formulants of bodycare cosmetics, to increase female breast cancer incidence, to interfere with male reproductive functions and to influence development of malignant melanoma which has also recently been shown to be influenced by oestrogenic stimulation.

  4. Molecular cloning and developmental expression of the gene encoding juvenile hormone esterase in the yellow-spotted longicorn beetle, Psacothea hilaris.

    PubMed

    Munyiri, Florence N; Ishikawa, Yukio

    2007-05-01

    Juvenile hormone (JH) plays a key role in the regulation of growth, development, diapause and reproduction in insects. The regulation of JH titers in the insect body is therefore crucial throughout postembryonic development. One of the major pathways of JH metabolism is degradation by a highly selective enzyme, juvenile hormone esterase (JHE). We obtained a full-length cDNA encoding JHE in Psacothea hilaris (PhJHE). The complete PhJHE cDNA sequence is comprised of 1989 bp with an open reading frame of 1785 bp encoding 595 amino acid residues. The deduced protein sequence of PhJHE showed high homology with the Tenebrio molitor JHE (50% amino acid identity) and moderate homology with the Drosophila melanogaster JHE (34%). The PhJHE transcript was expressed mainly in the fat body. PhJHE transcript levels were low until day 3 of the 5th (final) larval instar, then steadily increased reaching a peak on day 13 (the prepupa stage), coinciding well with the peak hemolymph enzyme activity level. Sustained starvation of larvae after a period of feeding stimulated the expression of PhJHE mRNA while feeding the larvae with glucose downregulated its expression. These results are discussed with reference to the induction of precocious metamorphosis in this beetle by starvation.

  5. Purification and characterization of a novel cholesterol esterase from Pseudomonas aeruginosa, with its application to cleaning lipid-stained contact lenses.

    PubMed

    Sugihara, Akio; Shimada, Yuji; Nomura, Atsuo; Terai, Tadamasa; Imayasu, Masaki; Nagai, Yusuke; Nagao, Toshihiro; Watanabe, Yomi; Tominaga, Yoshio

    2002-11-01

    With the aim of developing a new cholesterol esterase for eliminating lipids on used contact lenses, microorganisms were screened for the enzyme activity. A Pseudomonas aeruginosa isolated from soil was found to produce a desirable enzyme. The enzyme had an isoelectric point of 3.2, and molecular mass of 58 kDa. The optimal temperature was around 53 degrees C at pH 7.0, and the optimal pH was from 5.5 to 9.5. The enzyme was stable between pH 5 and 10 for 19 h at 25 degrees C, and retained its activity up to 53 degrees C on 30 min of incubation at pH 7.0. The rates of hydrolysis of cholesteryl esters of different fatty acids were in the following order: linoleate > oleate > stearate > palmitate > caprylate > myristate > laurate, caprate > caproate > butyrate, acetate. Addition of (tauro)cholate to a final concentration of 100 mM markedly promoted the hydrolysis of triglycerides of short-, medium-, and long-chain fatty acids. When used with taurocholate, the enzyme acted as an effective cleaner for contact lenses stained with lipids consisting of cholesteryl oleate, tripalmitin, and stearyl stearate.

  6. Identification of catalytically essential residues in Escherichia coli esterase by site-directed mutagenesis.

    PubMed

    Haruki, M; Oohashi, Y; Mizuguchi, S; Matsuo, Y; Morikawa, M; Kanaya, S

    1999-07-09

    Escherichia coli esterase (EcE) is a member of the hormone-sensitive lipase family. We have analyzed the roles of the conserved residues in this enzyme (His103, Glu128, Gly163, Asp164, Ser165, Gly167, Asp262, Asp266 and His292) by site-directed mutagenesis. Among them, Gly163, Asp164, Ser165, and Gly167 are the components of a G-D/E-S-A-G motif. We showed that Ser165, Asp262, and His292 are the active-site residues of the enzyme. We also showed that none of the other residues, except for Asp164, is critical for the enzymatic activity. The mutation of Asp164 to Ala dramatically reduced the catalytic efficiency of the enzyme by the factor of 10(4) without seriously affecting the substrate binding. This residue is probably structurally important to make the conformation of the active-site functional.

  7. Nucleotide sequence of the gene ereA encoding the erythromycin esterase in Escherichia coli.

    PubMed

    Ounissi, H; Courvalin, P

    1985-01-01

    We have cloned and determined the nucleotide sequence of the gene ereA of plasmid pIP1100 which confers high-level resistance to erythromycin (Em) in Escherichia coli. The gene was defined by initiation and termination codons and by in vitro insertion-inactivation into an open reading frame (ORF) of 1032 bp corresponding to a product with an Mr of 37 765. However, the enzyme, an Em esterase, displayed an apparent Mr of 43 000 upon electrophoresis of a minicell extract on the SDS-polyacrylamide gels. The G + C content (50.5%) of the gene ereA and the preferential codon usage in its ORF suggest that this resistance determinant should be indigenous to E. coli.

  8. Substrate specificity of xenobiotic metabolizing esterases in the liver of two catfish species

    SciTech Connect

    Jaiswal, R.G.; Huang, T.L.; Obih, P.O.

    1994-12-31

    The preliminary studies were conducted on the characterization of substrate specificity in the liver microsomes and cytosol of two catfish species, Ictalurus punctatus and Ictalurus natalie. A series of five esters of p-nitrophenol were used as calorimetric substrates to assay the carboxylesterases. The substrate specificity of liver microsomal and cytosolic carboxylesterases were remarkably different from each other. The valerate ester of p-nitrophenol was most rapidly hydrolyzed by the microsomal carboxylesterases, whereas the prioponate ester was the best substrate for cytosolic carboxylesterases. The Ictalurus natalie catfish species were obtained from the Devil Swamp site of the Mississippi River Basin which is known to be heavily contaminated with toxic and hazardous industrial wastes. These results will be discussed in relation to the responses of xenobiotic metabolizing esterases to environmental pollutants and their possible use as biomarkers.

  9. 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.

  10. [Influence of UV-light on erythrocyte membrane structure and catalytic behaviour of membrane acetylcholine esterase].

    PubMed

    Volotovskiĭ, I D; Sheĭko, L M; Konev, S V

    1976-01-01

    UV-light is shown to induce the structural transitions in the erythrocyte membrane described by S-shape curves in plots of the structural response versus the irradiation dose. In contrast to the free acetylcholine esterase (AChE) UV-light acts on the membrane enzyme as a mixed inhibitor (simultaneous change in Vmax and Km). The modification of the environment structure of residual enzyme is suggested to be the main reason of this phenomenon. The effect is under the control of membrane integrity and disappears after its desintegration. Membrane AChE treated ultrasonically both prior to and after irradiation is inactivated without a Km change. The data obtained show the influence of erythrocyte membrane structure on the catalytic behaviour of membrane-bound AChE.

  11. 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.

  12. Effects of three reputed carboxylesterase inhibitors upon rat serum esterase activity.

    PubMed

    Chambers, J P; Hartgraves, S L; Murphy, M R; Wayner, M J; Kumar, N; Valdes, J J

    1991-01-01

    Rats have very high endogenous levels of serum carboxylesterase (CAE) compared to primates. This difference accounts for the lower sensitivity of rats to toxic organophosphates, which interact with CAE instead of the more critical acetylcholinesterase. Pretreatment of rats with CAE inhibitors potentiates the effects of organophosphates. In this study, the effects of three putative CAE inhibitors, 2-(o-Cresyl)-4H-1:3:2-benzodioxaphosphorin-2-oxide (CBDP), bis-p-nitrophenyl-phosphate (BNPP), and tetraisopropyl pyrophosphoramide (Iso-OMPA), on the hydrolysis of several commercially available substrates were determined. Respective kinetic constants Km and Vmax were derived and effects of inhibitors compared using saturating amounts of substrate. Data presented here indicate significant differences in substrate affinity (Km), reactivity (Vmax), as well as effects of inhibitors. CBDP inhibits hydrolysis of specific naphthyl and paranitrophenyl esters at relatively low concentrations (1-10 microM). In contrast, significantly higher concentrations (mM) of BNPP and Iso-OMPA were required for inhibition of serum esterase activity. Of the inhibitors tested, Iso-OMPA in general exhibited the smallest inhibitory effect on ester hydrolysis. Although inhibition of hydrolysis of specific paranitrophenyl and naphthyl esters occurred in the presence of similar amounts of CBDP, the degree of inhibition differed significantly (50-75% vs. greater than 90%, respectively). These data suggest that there exists in rat serum, a pool of naphthyl ester esterase activity that is very sensitive ex vivo (greater than 90% inhibition) to CBDP and may be very useful in validating a rodent model for soman toxicity.

  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.

  14. Fundamental reaction mechanism and free energy profile for (-)-cocaine hydrolysis catalyzed by cocaine esterase.

    PubMed

    Liu, Junjun; Hamza, Adel; Zhan, Chang-Guo

    2009-08-26

    The fundamental reaction mechanism of cocaine esterase (CocE)-catalyzed hydrolysis of (-)-cocaine and the corresponding free energy profile have been studied by performing pseudobond first-principles quantum mechanical/molecular mechanical free energy (QM/MM-FE) calculations. On the basis of the QM/MM-FE results, the entire hydrolysis reaction consists of four reaction steps, including the nucleophilic attack on the carbonyl carbon of (-)-cocaine benzoyl ester by the hydroxyl group of Ser117, dissociation of (-)-cocaine benzoyl ester, nucleophilic attack on the carbonyl carbon of (-)-cocaine benzoyl ester by water, and finally dissociation between the (-)-cocaine benzoyl group and Ser117 of CocE. The third reaction step involving the nucleophilic attack of a water molecule was found to be rate-determining, which is remarkably different from (-)-cocaine hydrolysis catalyzed by wild-type butyrylcholinesterase (BChE; where the formation of the prereactive BChE-(-)-cocaine complex is rate-determining) or its mutants containing Tyr332Gly or Tyr332Ala mutation (where the first chemical reaction step is rate-determining). Besides, the role of Asp259 in the catalytic triad of CocE does not follow the general concept of the "charge-relay system" for all serine esterases. The free energy barrier calculated for the rate-determining step of CocE-catalyzed hydrolysis of (-)-cocaine is 17.9 kcal/mol, which is in good agreement with the experimentally derived activation free energy of 16.2 kcal/mol. In the present study, where many sodium ions are present, the effects of counterions are found to be significant in determining the free energy barrier. The finding of the significant effects of counterions on the free energy barrier may also be valuable in guiding future mechanistic studies on other charged enzymes.

  15. Direct imaging of ER calcium with targeted-esterase induced dye loading (TED).

    PubMed

    Samtleben, Samira; Jaepel, Juliane; Fecher, Caroline; Andreska, Thomas; Rehberg, Markus; Blum, Robert

    2013-05-07

    Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca(2+) indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca(2+) indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca(2+) indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca(2+) indicator and a hydrophilic fluorescent dye/Ca(2+) complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0.

  16. The effect of depth of centrifuged synovial fluid on leukocyte esterase test for periprosthetic joint infection.

    PubMed

    Ruangsomboon, Pakpoom; Chinprasertsuk, Sriprapa; Khejonnit, Varanya; Chareancholvanich, Keerati

    2017-03-17

    Centrifugation of aspirated synovial fluid before leukocytes esterase (LE) testing for diagnosing periprosthetic joint infection (PJI) may make blood tinged specimens interpretable. We aimed to establish the proper sampling depth of centrifuged specimens for LE testing as one diagnostic criterion and also AS-D chloroacetate esterase (CAE) staining testing as an adjunctive tool. A definite PJI knee joint group and an aseptic primary total knee arthroplasty control group were studied quasi-experimentally (N = 46). At 2000 g for 15 minutes, 3 ml of synovial fluid was centrifuged. LE strip testing and median synovial WBC count were performed at 2, 4, and 6 mm depths. CAE staining test characterized LE particles. ROC curve, area under the curve, and significant differences were determined. The proper predictive depth to diagnose PJI was sought by forward stepwise logistic regression. All fresh blood-tinged specimens had uncertain interpretations. Centrifugation increased interpretability (55% to 100%). ROC curve and area under the curve at 2, 4, and 6 mm depths were 0.822, 0.804, and 0.786, respectively. The cut point of ++ to diagnose PJI was statistically significant (p < 0.05) at all depths. P-values of forward stepwise logistic regression at 2, 4, and 6 mm were 0.001, 0.752, and 0.756, respectively. CAE staining confirmed extracellular LE release by polymorphonuclear neutrophils (PMN). A specimen at < 2 mm from the surface of centrifuged synovial fluid at a grading of ++ or more for PJI diagnosis is proper for LE testing. CAE staining testing adjunctively characterizes LE particles and cell morphology. This article is protected by copyright. All rights reserved.

  17. 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.

  18. Functional Characterization of a Robust Marine Microbial Esterase and Its Utilization in the Stereo-Selective Preparation of Ethyl (S)-3-Hydroxybutyrate.

    PubMed

    Wang, Yilong; Zhang, Yun; Hu, Yunfeng

    2016-11-01

    One novel microbial esterase PHE21 was cloned from the genome of Pseudomonas oryzihabitans HUP022 identified from the deep sea of the Western Pacific. PHE21 was heterologously expressed and functionally characterized to be a robust esterase which behaved high resistance to various metal ions, organic solvents, surfactants, and NaCl. Despite the fact that the two enantiomers of ethyl 3-hydroxybutyrate were hard to be enzymatically resolved before, we successfully resolved racemic ethyl 3-hydroxybutyrate through direct hydrolysis reactions and generated chiral ethyl (S)-3-hydroxybutyrate using esterase PHE21. After process optimization, the enantiomeric excess, the conversion rate, and the yield of desired product ethyl (S)-3-hydroxybutyrate could reach 99, 65, and 87 %, respectively. PHE21 is a novel marine microbial esterase with great potential in asymmetric synthesis as well as in other industries.

  19. 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

  20. Whole-Cell Biocatalytic Synthesis of Cinnamyl Acetate with a Novel Esterase from the DNA Library of Acinetobacter hemolyticus.

    PubMed

    Dong, Hao; Secundo, Francesco; Xue, Changhu; Mao, Xiangzhao

    2017-02-28

    Cinnamyl acetate has a wide application in the flavor and fragrance industry because of its sweet, balsamic, and floral odor. Up to now, lipases have been mainly used in enzyme-mediated synthesis of cinnamyl acetate, whereas esterases are used in only a few cases. Moreover, the use of purified enzymes is often a disadvantage, which leads to increases of the production costs. In this paper, a genomic DNA library of Acinetobacter hemolyticus was constructed, and a novel esterase (EstK1) was identified. After expression in Escherichia coli, the whole-cell catalyst of EstK1 displayed high transesterification activity to produce cinnamyl acetate in nonaqueous systems. Furthermore, under optimal conditions (vinyl acetate as acyl donor, isooctane as solvent, molar ratio 1:4, temperature 40 °C), the conversion ratio of cinnamyl alcohol could be up to 94.1% at 1 h, and it reached an even higher level (97.1%) at 2 h.

  1. 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

  2. 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

  3. Multiple resistance to pirimiphos-methyl and bifenthrin in Tribolium castaneum involves the activity of lipases, esterases, and laccase2.

    PubMed

    Julio, Alison Henrique Ferreira; Gigliolli, Adriana Aparecida Sinópolis; Cardoso, Kátia Aparecida Kern; Drosdoski, Sandro Daniel; Kulza, Rodrigo Amaral; Seixas, Flávio Augusto Vicente; Ruvolo-Takasusuki, Maria Claudia Colla; de Souza, Cristina Giatti Marques; Lapenta, Ana Silvia

    2017-05-01

    Several recent studies have elucidated the molecular mechanisms that confer insecticide resistance on insect pests. However, little is known about multiple resistance in red flour beetle (Tribolium castaneum) at molecular level. The multiple resistance is characterized as resistance to different classes of insecticides that have different target sites, and is mediated by several enzymatic systems. In this study, we investigated the biochemical and molecular mechanisms involved in multiple resistance of T. castaneum to bifenthrin (pyrethroid [Pyr]) and pirimiphos-methyl (organophosphate [Org]). We used artificial selection, biochemical and in silico approaches including structural computational biology. After five generations of artificial selection in the presence of bifenthrin (F5Pyr) or pirimiphos-methyl (F5Org), we found high levels of multiple resistance. The hierarchical enzymatic cluster revealed a pool of esterases (E), lipases (LIPs) and laccase2 (LAC2) potentially contributing to the resistance in different ways throughout development, after one or more generations in the presence of insecticides. The enzyme-insecticide interaction network indicated that E2, E3, LIP3, and LAC2 are enzymes potentially required for multiple resistance phenotype. Kinetic analysis of esterases from F5Pyr and F5Org showed that pirimiphos-methyl and specially bifenthrin promote enzyme inhibition, indicating that esterases mediate resistance by sequestering bifenthrin and pirimiphos-methyl. Our computational data were in accordance with kinetic results, indicating that bifenthrin has higher affinity at the active site of esterase than pirimiphos-methyl. We also report the capability of these insecticides to modify the development in T. castaneum. Our study provide insights into the biochemical mechanisms employed by T. castaneum to acquire multiple resistance.

  4. Experimental measurements for the effect of dilution procedure in blood esterases as animals biomarker for exposure to OP compounds.

    PubMed

    Abass, Kasim Sakran

    2014-01-01

    Organophosphate compounds can bind to carboxylesterase, which may lower the concentration of organophosphate pesticides at the target site enzyme, cholinesterase. It is unclear from the literature whether it is the carboxylesterase affinity for the organophosphate and/or the number of carboxylesterase molecules that is the dominant factor in determining the protective potential of carboxylesterase. The fundamental dilutions and kinetic effects of esterase enzyme are still poorly understood. This study aims to confirm and extend our current knowledge about the effects of dilutions on esterases activities in the blood for birds with respect to protecting the enzyme from organophosphate inhibition. There was significantly higher esterases activities in dilution 1 : 10 in the all blood samples from quail, duck, and chick compared to other dilutions (1 : 5, 1 : 15, 1 : 20, and 1 : 25) in all cases. Furthermore, our results also pointed to the importance of estimating different dilutions effects prior to using in birds as biomarker tools of environmental exposure. Concentration-inhibition curves were determined for the inhibitor in the presence of dilutions 1 : 5, 1 : 10, plus 1 : 15 (to stimulate carboxylesterase). Point estimates (concentrations calculated to produce 20, 50, and 80% inhibition) were compared across conditions and served as a measure of esterase-mediated detoxification. Results with well-known inhibitors (malathion) were in agreement with the literature, serving to support the use of this assay. Among the thiol-esters dilution 1 : 5 was observed to have the highest specificity constant (k(cat)/K(m)), and the K m and k cat values were 176 μM and 16,765 s(-1), respectively, for S-phenyl thioacetate ester, while detected in dilution 1: 15 was the lowest specificity constant (k(cat)/K(m)), and the Km and k cat values were 943 μM and 1154 s(-1), respectively, for acetylthiocholine iodide ester.

  5. A thraustochytrid diacylglycerol acyltransferase 2 with broad substrate specificity strongly increases oleic acid content in engineered Arabidopsis thaliana seeds

    PubMed Central

    Zhang, Chunyu; Iskandarov, Umidjon; Cahoon, Edgar B.

    2013-01-01

    Diacylglycerol acyltransferase (DGAT) catalyses the last step in acyl-CoA-dependent triacylglycerol (TAG) biosynthesis and is an important determinant of cellular oil content and quality. In this study, a gene, designated TaDGAT2, encoding a type 2 DGAT (DGAT2)-related enzyme was identified from the oleaginous marine protist Thraustochytrium aureum. The deduced TaDGAT2 sequence contains a ~460 amino acid domain most closely related to DGAT2s from Dictyostelium sp. (45–50% identity). Recombinant TaDGAT2 restored TAG biosynthesis to the Saccharomyces cerevisiae H1246 TAG-deficient mutant, and microsomes from the complemented mutant displayed DGAT activity with C16 and C18 saturated and unsaturated fatty acyl-CoA and diacylglycerol substrates. To examine its biotechnological potential, TaDGAT2 was expressed under control of a strong seed-specific promoter in wild-type Arabidopsis thaliana and the high linoleic acid fad3fae1 mutant. In both backgrounds, little change was detected in seed oil content, but a striking increase in oleic acid content of seeds was observed. This increase was greatest in fad3fae1 seeds, where relative amounts of oleic acid increased nearly 2-fold to >50% of total fatty acids. In addition, >2-fold increase in oleic acid levels was detected in the triacylglycerol sn-2 position and in the major seed phospholipid phosphatidylcholine. These results suggest that increased seed oleic acid content mediated by TaDGAT2 is influenced in part by the fatty acid composition of host cells and occurs not by enhancing oleic acid content at the TAG sn-3 position directly but by increasing total oleic acid levels in seeds, presumably by limiting flux through phosphatidylcholine-based desaturation reactions. PMID:23814277

  6. A thraustochytrid diacylglycerol acyltransferase 2 with broad substrate specificity strongly increases oleic acid content in engineered Arabidopsis thaliana seeds.

    PubMed

    Zhang, Chunyu; Iskandarov, Umidjon; Klotz, Elliott T; Stevens, Robyn L; Cahoon, Rebecca E; Nazarenus, Tara J; Pereira, Suzette L; Cahoon, Edgar B

    2013-08-01

    Diacylglycerol acyltransferase (DGAT) catalyses the last step in acyl-CoA-dependent triacylglycerol (TAG) biosynthesis and is an important determinant of cellular oil content and quality. In this study, a gene, designated TaDGAT2, encoding a type 2 DGAT (DGAT2)-related enzyme was identified from the oleaginous marine protist Thraustochytrium aureum. The deduced TaDGAT2 sequence contains a ~460 amino acid domain most closely related to DGAT2s from Dictyostelium sp. (45-50% identity). Recombinant TaDGAT2 restored TAG biosynthesis to the Saccharomyces cerevisiae H1246 TAG-deficient mutant, and microsomes from the complemented mutant displayed DGAT activity with C16 and C18 saturated and unsaturated fatty acyl-CoA and diacylglycerol substrates. To examine its biotechnological potential, TaDGAT2 was expressed under control of a strong seed-specific promoter in wild-type Arabidopsis thaliana and the high linoleic acid fad3fae1 mutant. In both backgrounds, little change was detected in seed oil content, but a striking increase in oleic acid content of seeds was observed. This increase was greatest in fad3fae1 seeds, where relative amounts of oleic acid increased nearly 2-fold to >50% of total fatty acids. In addition, >2-fold increase in oleic acid levels was detected in the triacylglycerol sn-2 position and in the major seed phospholipid phosphatidylcholine. These results suggest that increased seed oleic acid content mediated by TaDGAT2 is influenced in part by the fatty acid composition of host cells and occurs not by enhancing oleic acid content at the TAG sn-3 position directly but by increasing total oleic acid levels in seeds, presumably by limiting flux through phosphatidylcholine-based desaturation reactions.

  7. Protein Phosphatase Methyl-Esterase PME-1 Protects Protein Phosphatase 2A from Ubiquitin/Proteasome Degradation.

    PubMed

    Yabe, Ryotaro; Miura, Akane; Usui, Tatsuya; Mudrak, Ingrid; Ogris, Egon; Ohama, Takashi; Sato, Koichi

    2015-01-01

    Protein phosphatase 2A (PP2A) is a conserved essential enzyme that is implicated as a tumor suppressor based on its central role in phosphorylation-dependent signaling pathways. Protein phosphatase methyl esterase (PME-1) catalyzes specifically the demethylation of the C-terminal Leu309 residue of PP2A catalytic subunit (PP2Ac). It has been shown that PME-1 affects the activity of PP2A by demethylating PP2Ac, but also by directly binding to the phosphatase active site, suggesting loss of PME-1 in cells would enhance PP2A activity. However, here we show that PME-1 knockout mouse embryonic fibroblasts (MEFs) exhibit lower PP2A activity than wild type MEFs. Loss of PME-1 enhanced poly-ubiquitination of PP2Ac and shortened the half-life of PP2Ac protein resulting in reduced PP2Ac levels. Chemical inhibition of PME-1 and rescue experiments with wild type and mutated PME-1 revealed methyl-esterase activity was necessary to maintain PP2Ac protein levels. Our data demonstrate that PME-1 methyl-esterase activity protects PP2Ac from ubiquitin/proteasome degradation.

  8. Effects of high hydrostatic pressure and temperature increase on Escherichia coli spp. and pectin methyl esterase inactivation in orange juice.

    PubMed

    Torres, E F; González-M, G; Klotz, B; Rodrigo, D

    2016-03-01

    The aim of this study was to evaluate the effect of high hydrostatic pressure treatment combined with moderate processing temperatures (25 ℃-50 ℃) on the inactivation of Escherichia coli O157: H7 (ATCC 700728), E. coli K12 (ATCC 23716), and pectin methyl esterase in orange juice, using pressures of 250 to 500 MPa with times ranging between 1 and 30 min. Loss of viability of E. coli O157:H7 increased significantly as pressure and treatment time increased, achieving a 6.5 log cycle reduction at 400 MPa for 3 min at 25 ℃ of treatment. With regard to the inactivation of pectin methyl esterase, the greatest reduction obtained was 90.05 ± 0.01% at 50 ℃ and 500 MPa of pressure for 15 min; therefore, the pectin methyl esterase enzyme was highly resistant to the treatments by high hydrostatic pressure. The results obtained in this study showed a synergistic effect between the high pressure and moderate temperatures in inactivating E. coli cells.

  9. Nebulized C1-Esterase Inhibitor does not Reduce Pulmonary Complement Activation in Rats with Severe Streptococcus Pneumoniae Pneumonia.

    PubMed

    de Beer, Friso; Lagrand, Wim; Glas, Gerie J; Beurskens, Charlotte J P; van Mierlo, Gerard; Wouters, Diana; Zeerleder, Sacha; Roelofs, Joris J T H; Juffermans, Nicole P; Horn, Janneke; Schultz, Marcus J

    2016-12-01

    Complement activation plays an important role in the pathogenesis of pneumonia. We hypothesized that inhibition of the complement system in the lungs by repeated treatment with nebulized plasma-derived human C1-esterase inhibitor reduces pulmonary complement activation and subsequently attenuates lung injury and lung inflammation. This was investigated in a rat model of severe Streptococcus pneumoniae pneumonia. Rats were intra-tracheally challenged with S. pneumoniae to induce pneumonia. Nebulized C1-esterase inhibitor or saline (control animals) was repeatedly administered to rats, 30 min before induction of pneumonia and every 6 h thereafter. Rats were sacrificed 20 or 40 h after inoculation with bacteria. Brochoalveolar lavage fluid and lung tissue were obtained for measuring levels of complement activation (C4b/c), lung injury and inflammation. Induction of pneumonia was associated with pulmonary complement activation (C4b/c at 20 h 1.24 % [0.56-2.59] and at 40 h 2.08 % [0.98-5.12], compared to 0.50 % [0.07-0.59] and 0.03 % [0.03-0.03] in the healthy control animals). The functional fraction of C1-INH was detectable in BALF, but no effect was found on pulmonary complement activation (C4b/c at 20 h 0.73 % [0.16-1.93] and at 40 h 2.38 % [0.54-4.19]). Twenty hours after inoculation, nebulized C1-esterase inhibitor treatment reduced total histology score, but this effect was no longer seen at 40 h. Nebulized C1-esterase inhibitor did not affect other markers of lung injury or lung inflammation. In this negative experimental animal study, severe S. pneumoniae pneumonia in rats is associated with pulmonary complement activation. Repeated treatment with nebulized C1-esterase inhibitor, although successfully delivered to the lungs, does not affect pulmonary complement activation, lung inflammation or lung injury.

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

    PubMed

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

    2008-03-01

    Here, we identify and characterise cholinesterase (ChE) and carboxylesterase (CbE) activities in the body tissues of the sediment dwelling worm Arenicola marina. Exposure to the organophosphorus pesticide azamethiphos yielded an in vitro IC50 of 5 microg l(-1) for propionylcholinesterase (PChE). PChE was significantly inhibited in vivo after a 10 day exposure to 100 microg l(-1) azamethiphos, equivalent to the recommended aquatic application rate (ANOVA; F=2.75, P=0.033). To determine sensitivity to environmental conditions, A. marina were exposed for 10 days to field collected sediments. PChE activity was significantly lower in worms exposed to sediments from an estuary classified to be at high risk from point source pollution by the UK Environment Agency (ANOVA; F=15.33, P<0.001). Whilst causality cannot be directly attributed from these latter exposures, they provide an important illustration of the potential utility of esterase activity as a biomarker of environmental quality in this ecologically relevant sentinel species.

  11. 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

  12. 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).

  13. 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.

  14. 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.

  15. Characterization of tyrosinase- and polyphenol esterase-catalyzed end products using selected phenolic substrates.

    PubMed

    Madani, W; Kermasha, S; Versari, A

    1999-06-01

    The oxidative end products that result from the biocatalysis of tyrosinase (PPO) and/or a polyphenol esterase (PPE) extract have been investigated simultaneously in model systems containing selected phenolic compounds as substrates. The spectrophotometric scanning of brown color, formed in the presence of both PPO and PPE, showed a decrease in the absorbance compared to that obtained with PPO only. Graphical analyses of the iterative spectra of oxidized phenolic end products by PPO confirmed the presence of, at least, three kinetically related absorbing species. HPLC analyses of the end products, obtained by the biocatalysis of PPE or PPO activity, indicated the presence of two main groups of compounds: colored ones of lambda(max) at 294-324 nm and colorless products of lambda(max) at 264-290 nm. PPE produced both compounds when selected substrates were used as substrates, whereas PPO produced only one type of oxidation product. However, when both enzymes were incubated together, the nature of the end products was similar to that obtained with PPE only.

  16. 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.

  17. Evolutionary Endocrinology of Juvenile Hormone Esterase in Gryllus Assimilis: Direct and Correlated Responses to Selection

    PubMed Central

    Zera, A. J.; Zhang, C.

    1995-01-01

    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. PMID:8582618

  18. Are PECTIN ESTERASE INHIBITOR Genes Involved in Mediating Resistance to Rhynchosporium commune in Barley?

    PubMed

    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.

  19. 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

  20. 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.

  1. Rational Design of a Red-Emissive Fluorophore with AIE and ESIPT Characteristics and Its Application in Light-Up Sensing of Esterase.

    PubMed

    Peng, Lu; Xu, Shidang; Zheng, Xiaokun; Cheng, Xiamin; Zhang, Ruoyu; Liu, Jie; Liu, Bin; Tong, Aijun

    2017-03-07

    The development of red fluorophores with efficient solid-state emission is still challenging. Herein, a red fluorophore 1 with aggregation-induced emission (AIE) and excited-state intramolecular proton transfer (ESIPT) characteristics is rationally designed and facilely synthesized by attaching an electron-donor diethylamine and an electron-acceptor maleonitrile group to salicyladazine. In contrast to many red fluorophores which undergo serious aggregation-caused quenching (ACQ), compound 1 emits bright red fluorescence (λem = 650 nm, ΦF = 24.3%) in the solid state with a large Stokes shift of 174 nm. Interestingly, control compounds 2 and 3, which have similar structures as 1, exhibit obvious aggregation-caused quenching (ACQ) characteristics. The difference in the crystal structures of 1, 2, and 3 reveals that the interplanar spacing among molecules plays a decisive role in realizing the AIE characteristics of 1. Moreover, when the hydroxyl group of 1 was substituted by an esterase reactive acetoxyl, a fluorescence light-up probe 4 was developed for sensing of esterase based on the selective reaction between 4 and esterase to generate the AIE and ESIPT active molecule 1. The linear range for in vitro quantification of esterase is 0.01-0.15 U/mL with a detection limit of 0.005 U/mL. Probe 4 was also successfully applied to image esterase in mitochondria of living cells.

  2. Amperometric determination of serum total cholesterol with nanoparticles of cholesterol esterase and cholesterol oxidase.

    PubMed

    Aggarwal, V; Malik, J; Prashant, A; Jaiwal, P K; Pundir, C S

    2016-05-01

    We describe the preparation of glutaraldehyde cross-linked and functionalized cholesterol esterase nanoparticles (ChENPs) and cholesterol oxidase nanoparticles (ChOxNPs) aggregates and their co-immobilization onto Au electrode for improved amperometric determination of serum total cholesterol. Transmission electron microscope (TEM) images of ChENPs and ChOxNPs showed their spherical shape and average size of 35.40 and 56.97 nm, respectively. Scanning electron microscope (SEM) studies of Au electrode confirmed the co-immobilization of enzyme nanoparticles (ENPs). The biosensor exhibited optimal response at pH 5.5 and 40°C within 5 s when polarized at +0.25 V versus Ag/AgCl. The working/linear range of the biosensor was 10-700 mg/dl for cholesterol. The sensor showed high sensitivity and measured total cholesterol as low as 0.1 mg/dl. The biosensor was evaluated and employed for total cholesterol determination in sera of apparently healthy and diseased persons. The analytical recovery of added cholesterol was 90%, whereas the within-batch and between-batch coefficients of variation (CVs) were less than 2% and less than 3%. There was a good correlation (r = 0.99) between serum cholesterol values as measured by the standard enzymic colorimetric method and the current method. The initial activity of ENPs/working electrode was reduced by 50% during its regular use (200 times) over a period of 60 days when stored dry at 4°C.

  3. 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.

  4. Acetylcholine esterase inhibitors in effluents from oil production platforms in the North Sea.

    PubMed

    Holth, T F; Tollefsen, K E

    2012-05-15

    Inhibition of acetylcholine esterase (AChE) activity is a biomarker for the exposure to neurotoxic compounds such as organophosphates and is intimately associated with the toxicity of several pesticides. In the present study, the AChE inhibiting potential of organic extracts of production water (produced water) from oil and gas production platforms in the Norwegian sector of the North Sea was determined in an in vitro bioassay based on commercially available purified AChE from the electric organ of Electrophorus electricus (L.). The results from the studies show that produced water contains a combination of AChE inhibiting compounds and compounds stimulating AChE enzymatic activity. The AChE inhibition was predominantly caused by unidentified aromatic compounds in the oil/particulate fraction of produced water, whereas polar compounds in both the water soluble and oil/particulate fraction of produced water caused an apparent stimulation of AChE activity. Substrate saturation studies with fixed concentrations of produced water extracts confirmed that the inhibition occurred in a non-destructive and competitive manner. The concentrations of AChE inhibitors (7.9-453 ng paraoxon-equivalents L⁻¹, 2.2-178 μg dichlorvos-equivalents L⁻¹) were in many cases found to be several orders of magnitude higher than background levels. The findings demonstrate that produced water contains potentially neurotoxic compounds and suggest that further laboratory studies with fish or field studies in the vicinity of oil production facilities are highly warranted.

  5. 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.

  6. 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

  7. Preliminary crystallographic analysis of a double mutant of the acetyl xylo-oligosaccharide esterase Axe2 in its dimeric form.

    PubMed

    Lansky, Shifra; Alalouf, Onit; Salama, Rachel; Dvir, Hay; Shoham, Yuval; Shoham, Gil

    2014-04-01

    Xylans are polymeric sugars constituting a significant part of the plant cell wall. They are usually substituted with acetyl side groups attached at positions 2 or 3 of the xylose backbone units. Acetylxylan esterases are part of the hemicellulolytic system of many microorganisms which utilize plant biomass for growth. These enzymes hydrolyze the ester linkages of the xylan acetyl groups and thus improve the accessibility of main-chain-hydrolyzing enzymes and their ability to break down the sugar backbone units. The acetylxylan esterases are therefore critically important for those microorganisms and as such could be used for a wide range of biotechnological applications. The structure of an acetylxylan esterase (Axe2) isolated from the thermophilic bacterium Geobacillus stearothermophilus T6 has been determined, and it has been demonstrated that the wild-type enzyme is present as a unique torus-shaped octamer in the crystal and in solution. In order to understand the functional origin of this unique oligomeric structure, a series of rational noncatalytic, site-specific mutations have been made on Axe2. Some of these mutations led to a different dimeric form of the protein, which showed a significant reduction in catalytic activity. One of these double mutants, Axe2-Y184F-W190P, has recently been overexpressed, purified and crystallized. The best crystals obtained belonged to the orthorhombic space group P212121, with unit-cell parameters a = 71.1, b = 106.0, c = 378.6 Å. A full diffraction data set to 2.3 Å resolution has been collected from a flash-cooled crystal of this type at 100 K using synchrotron radiation. This data set is currently being used for the three-dimensional structure analysis of the Axe2-Y184F-W190P mutant in its dimeric form.

  8. Cyst fluid NB/70K concentration and leukocyte esterase: two new markers for differentiating pancreatic serous tumors from pseudocysts.

    PubMed

    Yong, W H; Southern, J F; Pins, M R; Warshaw, A L; Compton, C C; Lewandrowski, K B

    1995-05-01

    Cystic lesions of the pancreas include inflammatory pseudocysts, serous cystadenomas, and mucinous tumors, some of which are malignant. Preoperative clinical and radiological parameters are unreliable and may result in incorrect diagnosis and inappropriate treatment. Cyst fluid analysis for cytology, viscosity, carcino-embryonic antigen, CA 72-4, and CA 15-3 will distinguish mucinous from nonmucinous lesions and usually help in determining malignancy. Currently, there is no reliable method to differentiate inflammatory pseudocysts from serous cystadenomas. This distinction is important because the treatment of these two lesions is different; pseudocysts are either observed or drained, whereas serous tumors are usually resected. The tumor marker NB/70K was measured in aspirated cyst fluid from 13 inflammatory pseudocysts and 11 serous cystadenomas by a commercial immunoassay. Leukocyte esterase was measured using Chemstrip SG urine test strips and amylase and lipase on a routine chemistry analyzer. The cyst fluid NB/70K concentration was significantly higher in pseudocysts (mean, 555 U/ml; range, 42-1,926 U/ml) than in serous cystadenomas (mean, 12 U/ml; range 0-130 U/ml) and this difference was significant (p < 0.0002). Leukocyte esterase was detected in 7 of 11 pseudocysts but was absent in 10 of 10 serous tumors (p = 0.002). Amylase and lipase values were generally higher in pseudocysts but these markers were unreliable due to marked outliers. Cyst fluid NB/70K and leukocyte esterase are promising markers to help differentiate pseudocysts from serous tumors on percutaneous aspirates. When combined with previously reported cyst fluid parameters (amylase, lipase, cytology, and amylase isoenzymes), these two cystic lesions can be reliably distinguished.

  9. Crystal structure and characterization of esterase Est25 mutants reveal improved enantioselectivity toward (S)-ketoprofen ethyl ester.

    PubMed

    Kim, Jinyeong; Seok, Seung-Hyeon; Hong, Eunsoo; Yoo, Tae Hyeon; Seo, Min-Duk; Ryu, Yeonwoo

    2017-03-01

    Esterases comprise a group of enzymes that catalyze the cleavage and synthesis of ester bonds. They are important in biotechnological applications owing to their enantioselectivity, regioselectivity, broad substrate specificity, and the fact that they do not require cofactors. In a previous study, we isolated the esterase Est25 from a metagenomic library. Est25 showed catalytic activity toward the (R,S)-ketoprofen ethyl ester but had low enantioselectivity toward the (S)-ketoprofen ethyl ester. Because (S)-ketoprofen has stronger anti-inflammatory effects and fewer side effects than (R)-ketoprofen, enantioselectivity of this esterase is important. In this study, we generated Est25 mutants with improved enantioselectivity toward the (S)-ketoprofen ethyl ester; improved enantioselectivity of mutants was established by analysis of their crystal structures. The enantioselectivity of mutants was influenced by substitution of Phe72 and Leu255. Substituting these residues changed the size of the binding pocket and the entrance hole that leads to the active site. The enantioselectivity of Est25 (E = 1.1 ± 0.0) was improved in the mutants F72G (E = 1.9 ± 0.2), L255W (E = 16.1 ± 1.1), and F72G/L255W (E = 60.1 ± 0.5). Finally, characterization of Est25 mutants was performed by determining the optimum reaction conditions, thermostability, effect of additives, and substrate specificity after substituting Phe72 and Leu255.

  10. Characterization and Low-Resolution Structure of an Extremely Thermostable Esterase of Potential Biotechnological Interest from Pyrococcus furiosus.

    PubMed

    Mandelli, F; Gonçalves, T A; Gandin, C A; Oliveira, A C P; Oliveira Neto, M; Squina, F M

    2016-11-01

    Enzymes isolated from extremophiles often exhibit superior performance and potential industrial applications. There are several advantages performing biocatalysis at elevated temperatures, including enhanced reaction rates, increased substrate solubility and decreased risks of contamination. Furthermore, thermophilic enzymes usually exhibit high resistance against many organic solvents and detergents, and are also more resistant to proteolytic attack. In this study, we subcloned and characterized an esterase from the hyperthermophilic archaeon Pyrococcus furiosus (Pf_Est) that exhibits optimal activity around 80 °C using naphthol-derived substrates and p-nitrophenyl palmitate (pNPP). According to the circular dichroism spectra, the secondary structure of P. furiosus esterase, which is predominantly formed by a β-sheet structure, is very stable, even after incubation at 120°C. We performed SAXS to determine the low-resolution structure of Pf_Est, which is monomeric in solution at 80 °C and has a molecular weight of 28 kDa. The Km and V max values for this esterase acting on pNPP were 0.53 mmol/L and 6.5 × 10(-3) U, respectively. Pf_Est was most active in the immiscible solvents and retained more than 50 % in miscible solvents. Moreover, Pf_Est possesses transesterification capacity, presenting better results when isobutanol was used as an acyl acceptor (2.69 ± 0.14 × 10(-2) μmol/min mg) and the highest hydrolytic activity toward olive oil among different types of oils testes in this study. Collectively, these biophysical and catalytic properties are of interest for several biotechnological applications that require harsh conditions, including high temperature and the presence of organic solvents.

  11. 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.

  12. Estrogenic and esterase-inhibiting potency in rainwater in relation to pesticide concentrations, sampling season and location.

    PubMed

    Hamers, Timo; van den Brink, Paul J; Mos, Lizzy; van der Linden, Sander C; Legler, Juliette; Koeman, Jan H; Murk, Albertinka J

    2003-01-01

    In a year-round monitoring program (1998), pesticide composition and toxic potency of the mix of pollutants present in rainwater were measured. The goal of the study was to relate atmospheric deposition of toxic potency and pesticide composition to each other and to sampling period and local agricultural activity. Rainwater was collected in 26 consecutive periods of 14 days in a background location (BACK) and in two locations representative for different agricultural practices, i.e. intensive greenhouse horticulture (HORT) and flower bulb culture (BULB). Samples were chemically analyzed for carbamate (CARB), organophosphate (OP) and organochlorine (OC) pesticides and metabolites. Esterase inhibiting potency of rainwater extracts was measured in a specially developed bio-assay with honeybee esterases and was expressed as an equivalent concentration of the model inhibitor dichlorvos. Estrogenic potency of the extracts was measured in the ER-CALUX reporter gene assay and was expressed as an equivalent concentration of estradiol. Multivariate principal component analysis (PCA) techniques proved to be valuable tools to analyze the numerous pesticide concentrations in relation to toxic potency, sampling location, and sampling season. Pesticide composition in rainwater depended much more on sampling season than on sampling location, but differences between and were mainly attributed to local differences in agricultural practice. On average, the esterase inhibiting potency exceeded the maximum permissible concentration set for dichlorvos in The Netherlands, and was significantly higher in than in and . Esterase inhibition correlated significantly with OP and CARB concentrations, as expected given the working mechanism of these insecticides. The estrogenic potency incidentally exceeded NOEC levels reported for aquatic organisms and was highest in . Although estrogenic potency of rainwater correlated with OC concentrations, the ER-CALUX responses could not be attributed to

  13. Experimental Measurements for the Effect of Dilution Procedure in Blood Esterases as Animals Biomarker for Exposure to OP Compounds

    PubMed Central

    Abass, Kasim Sakran

    2014-01-01

    Organophosphate compounds can bind to carboxylesterase, which may lower the concentration of organophosphate pesticides at the target site enzyme, cholinesterase. It is unclear from the literature whether it is the carboxylesterase affinity for the organophosphate and/or the number of carboxylesterase molecules that is the dominant factor in determining the protective potential of carboxylesterase. The fundamental dilutions and kinetic effects of esterase enzyme are still poorly understood. This study aims to confirm and extend our current knowledge about the effects of dilutions on esterases activities in the blood for birds with respect to protecting the enzyme from organophosphate inhibition. There was significantly higher esterases activities in dilution 1 : 10 in the all blood samples from quail, duck, and chick compared to other dilutions (1 : 5, 1 : 15, 1 : 20, and 1 : 25) in all cases. Furthermore, our results also pointed to the importance of estimating different dilutions effects prior to using in birds as biomarker tools of environmental exposure. Concentration-inhibition curves were determined for the inhibitor in the presence of dilutions 1 : 5, 1 : 10, plus 1 : 15 (to stimulate carboxylesterase). Point estimates (concentrations calculated to produce 20, 50, and 80% inhibition) were compared across conditions and served as a measure of esterase-mediated detoxification. Results with well-known inhibitors (malathion) were in agreement with the literature, serving to support the use of this assay. Among the thiol-esters dilution 1 : 5 was observed to have the highest specificity constant (kcat/Km), and the Km and kcat values were 176 μM and 16,765 s−1, respectively, for S-phenyl thioacetate ester, while detected in dilution 1 : 15 was the lowest specificity constant (kcat/Km), and the Km and kcat values were 943 μM and 1154 s−1, respectively, for acetylthiocholine iodide ester. PMID:24864243

  14. Inhibition of a ubiquitously expressed pectin methyl esterase in Solanum tuberosum L. affects plant growth, leaf growth polarity, and ion partitioning.

    PubMed

    Pilling, J; Willmitzer, L; Bücking, H; Fisahn, J

    2004-05-01

    Two pectin methyl esterases (PMEs; EC 3.1.1.11) from Solanum tuberosum were isolated and their expression characterised. One partial clone ( pest1) was expressed in leaves and fruit tissue, while pest2 was a functional full-length clone and was expressed ubiquitously, with a preference for aerial organs. Potato plants were transformed with a chimeric antisense construct that was designed to simultaneously inhibit pest1 and pest2 transcript accumulation; however, reduction of mRNA levels was confined to pest2. The decrease in pest2 transcript was accompanied by up to 50% inhibition of total PME activity, which was probably due to the reduction of only one PME isoform. PME inhibition affected plant development as reflected by smaller stem elongation rates of selected transformants when compared with control plants, leading to a reduction in height throughout the entire course of development. Expansion rates of young developing leaves were measured simultaneously by two displacement transducers in the direction of the leaf tip (proximal-distal axis) and in the perpendicular direction (medial-lateral axis). Significant differences in leaf growth patterns were detected between wild-type and transgenic plants. We suggest that these visual phenotypes could be correlated with modifications of ion accumulation and partitioning within the transgenic plants. The ion-binding capacities of cell walls from PME-inhibited plants were specifically modified as they preferentially bound more sodium, but less potassium and calcium. X-ray microanalysis also indicated an increase in the concentration of several ions within the leaf apoplast of transgenic plants. Moreover, quantification of the total content of major cations revealed differences specific for a given element between the leaves of PME-inhibited and wild-type plants. Reduced growth rates might also be due to effects of PME inhibition on pectin metabolism, predominantly illustrated by an accumulation of galacturonic acid

  15. Molecular cloning, characterization and expression analysis of two juvenile hormone esterase-like carboxylesterase cDNAs in Chinese mitten crab, Eriocheir sinensis.

    PubMed

    Xu, Yu; Zhao, Muzi; Deng, Yanfei; Yang, Yuanjie; Li, Xuguang; Lu, Quanping; Ge, Jiachun; Pan, Jianlin; Xu, Zhiqiang

    2017-03-01

    Precise regulation of methyl farnesoate (MF) titer is of prime importance throughout the crustacean life-cycle. Although the synthetic pathway of MF is well-documented, little is known about its degradation and recycling in crustaceans. Juvenile hormone esterase-like (JHE-like) carboxylesterase (CXE) is a key enzyme in MF degradation, thus playing a significant role in regulating the MF titer. We identified and characterized two cDNAs, Es-CXE1 and Es-CXE2, encoding JHE-like CXEs in Chinese mitten crab. Full-length cDNAs of Es-CXE1 and Es-CXE2 encode proteins composed of 584 and 597 amino acids, respectively, both of which contain a typical carboxylesterase domain. Alignment and phylogenetic analyses revealed that the Es-CXEs are highly similar to those of other crustaceans. To further validate their functions, we evaluated the mRNA expression patterns of the Es-CXEs in various tissues and in different physiological conditions. Tissue-specific expression analysis showed that the two Es-CXEs were predominantly expressed in the hepatopancreas and ovaries, which are the major tissues for MF metabolism. Es-CXE2 expression levels in the hepatopancreas and ovaries were about 100 and 25-fold higher, than the respective Es-CXE1 expressions. During ovarian rapid development stage, the global expressions of Es-CXEs were up-regulated in the hepatopancreas and down-regulated in the ovaries. After eyestalk ablation (ESA), the mRNA expressions of the two Es-CXEs were up-regulated in the hepatopancreas, further indicating their potential in degrading MF. Taken together, our results suggest that Es-CXEs, the key component of the juvenile hormone degradation pathway, may play vital roles in the development and reproduction of the Chinese mitten crab.

  16. Selective enzymatic hydrolysis of chlorogenic acid lactones in a model system and in a coffee extract. Application to reduction of coffee bitterness.

    PubMed

    Kraehenbuehl, Karin; Page-Zoerkler, Nicole; Mauroux, Olivier; Gartenmann, Karin; Blank, Imre; Bel-Rhlid, Rachid

    2017-03-01

    Chlorogenic acid lactones have been identified as key contributors to coffee bitterness. These compounds are formed during roasting by dehydration and cyclization of their precursors, the chlorogenic acids (CGAs). In the present study, we investigated an approach to decompose these lactones in a selective way without affecting the positive coffee attributes developed during roasting. A model system composed of (3-caffeoylquinic acid lactone (3-CQAL), 4- caffeoyl quinic acid lactone (4-CQAL), and 4-feruloylquinic acid lactone (4-FQAL)) was used for the screening of enzymes before treatment of the coffee extracts. Hog liver esterase (HLE) hydrolyzed chlorogenic acid lactones (CQALs, FQALs) selectively, while chlorogenate esterase hydrolyzed all chlorogenic acids (CQAs, FQAs) and their corresponding lactones (CQALs, FQALs) in a non-selective way. Enzymatically treated coffee samples were evaluated for their bitterness by a trained sensory panel and were found significantly less bitter than the untreated samples.

  17. Leukocyte Esterase as a Biomarker in the Diagnosis of Periprosthetic Joint Infection

    PubMed Central

    Wang, Chi; Li, Rui; Wang, Qi; Duan, Jinyan; Wang, Chengbin

    2017-01-01

    Background Total joint arthroplasty (TJA) has been one of the most rewarding interventions for treating patients suffering from joint disorders. However, periprosthetic joint infection (PJI) is a serious complication that frequently accompanies TJA. Our study aimed to investigate the application of the leukocyte esterase (LE) strip in the diagnosis of PJI. Material/Methods From October 2014 to July 2015, 72 patients who had undergone joint puncture after arthroplasty in our hospital were enrolled in this trial. One drop of synovial fluid from each available patient was applied to the LE strip, and the results were observed after 1–3 min. If the color turned to dark purple, we recognized this as a positive result, while other colors were regarded as negative results. Centrifugation was used when the synovial fluid was mixed with blood. The Musculoskeletal Infection Society (MSIS) definition was used as the standard reference to identify whether PJI was found in patients or not. The results of diagnosis and LE strips test were compared, and indicators reflecting diagnostic value were calculated. Correlation of the LE data with erythrocyte sedimentation rate (ESR), elevated C-reactive protein (CRP), synovial white blood cell (WBC) counts, and polymorphonuclear neutrophil (PMN) percentage was calculated. Results By MSIS criteria, 38 patients were diagnosed with PJI and 34 patients were not infected. Two types of LE strip presented the same results with sensitivity of 84.21% (95% confidence interval [CI]: 68.75~93.98%), specificity of 97.06% (95% CI: 84.67~99.93%), positive predictive value (PPV) of 96.97% (95% CI: 84.24~99.92%), and negative predictive value (NPV) of 84.62% (95% CI: 69.47~94.14%). There were one false-positive case and six false-negative cases in this trial. There is a strong correlation between LE strip and synovial fluid PMN percentage. Conclusions The sensitivity and specificity of the LE strip in the diagnosis of PJI are quite high, which means

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

    SciTech Connect

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

    2007-09-01

    A thermostable esterase (EstA) from Thermotoga maritima was cloned and purified. Crystals of EstA and its selenomethionine derivative were grown and diffract to beyond 2.6 Å resolution at 100 K using synchrotron radiation. A predicted esterase (EstA) with an unusual new domain from the hyperthermophilic bacterium Thermotoga maritima has been cloned and overexpressed in Escherichia coli. The purified protein was crystallized by the hanging-drop vapour-diffusion technique in the presence of lithium sulfate and polyethylene glycol 8000. Selenomethionine-substituted EstA crystals were obtained under the same conditions and three different-wavelength data sets were collected to 2.6 Å resolution. The crystal belongs to space group H32, with unit-cell parameters a = b = 130.2, c = 306.2 Å. There are two molecules in the asymmetric unit, with a V{sub M} of 2.9 Å{sup 3} Da{sup −1} and 58% solvent content.

  19. 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.

  20. Crystallization and preliminary crystallographic studies of LipA, a secretory lipase/esterase from Xanthomonas oryzae pv. oryzae

    SciTech Connect

    Aparna, Gudlur; Chatterjee, Avradip; Jha, Gopaljee; Sonti, Ramesh V.; Sankaranarayanan, Rajan

    2007-08-01

    The crystallization and preliminary crystallographic studies of LipA, a lipase/esterase secreted by X. oryzae pv. oryzae during its infection of rice plants, are reported. Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight, a serious disease of rice. Several enzymes that are secreted through the type II secretion system of this bacterium play an important role in the plant–microbe interaction, being important for virulence and also being able to induce potent host defence responses. One of these enzymes is a secretory lipase/esterase, LipA, which shows a very weak homology to other bacterial lipases and gives a positive tributyrin plate assay. In this study, LipA was purified from the culture supernatant of an overexpressing clone of X. oryzae pv. oryzae and two types of crystals belonging to space group C2 but with two different unit-cell parameters were obtained using the hanging-drop vapour-diffusion method. Type I crystals diffract to a maximum resolution of 1.89 Å and have unit-cell parameters a = 93.1, b = 62.3, c = 66.1 Å, β = 90.8°. Type II crystals have unit-cell parameters a = 103.6, b = 54.6, c = 66.3 Å, β = 92.6° and diffract to 1.86 Å. Solvent-content analysis shows one monomer in the asymmetric unit in both the crystal forms.

  1. Constructs of human neuropathy target esterase catalytic domain containing mutations related to motor neuron disease have altered enzymatic properties.

    PubMed

    Hein, Nichole D; Stuckey, Jeanne A; Rainier, Shirley R; Fink, John K; Richardson, Rudy J

    2010-07-01

    Neuropathy target esterase (NTE) is a phospholipase/lysophospholipase associated with organophosphorus (OP) compound-induced delayed neurotoxicity (OPIDN). Distal degeneration of motor axons occurs in both OPIDN and the hereditary spastic paraplegias (HSPs). Recently, mutations within the esterase domain of NTE were identified in patients with a novel type of HSP (SPG39) designated NTE-related motor neuron disease (NTE-MND). Two of these mutations, arginine 890 to histidine (R890H) and methionine 1012 to valine (M1012V), were created in human recombinant NTE catalytic domain (NEST) to measure possible changes in catalytic properties. These mutated enzymes had decreased specific activities for hydrolysis of the artificial substrate, phenyl valerate. In addition, the M1012V mutant exhibited a reduced bimolecular rate constant of inhibition (k(i)) for all three inhibitors tested: mipafox, diisopropylphosphorofluoridate, and chlorpyrifos oxon. Finally, while both mutated enzymes inhibited by OP compounds exhibited altered time-dependent loss of their ability to be reactivated by nucleophiles (aging), more pronounced effects were seen with the M1012V mutant. Taken together, the results from specific activity, inhibition, and aging experiments suggest that the mutations found in association with NTE-MND have functional correlates in altered enzymological properties of NTE.

  2. 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.

  3. 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.

  4. Angiotensin-converting enzyme inhibitors-induced angioedema treated by C1 esterase inhibitor concentrate (Berinert®): about one case and review of the therapeutic arsenal.

    PubMed

    Lipski, Samuel Michael; Casimir, Georges; Vanlommel, Martine; Jeanmaire, Mathieu; Dolhen, Pierre

    2015-02-01

    C1 esterase inhibitor (Berinert®) is generally used to treat severe attack of hereditary angioedema. We describe here the case of a patient who presented with a severe angioedema induced by angiotensin-converting enzyme inhibitors (ACEIs) endangering her life. It could be successfully treated with that medicine.

  5. Using urinary leucocyte esterase tests as an indicator of infection with gonorrhoea or chlamydia in asymptomatic males in a primary health care setting.

    PubMed

    Rahman, Md Saifur; Beever, Warwick; Skov, Steven; Boffa, John

    2014-02-01

    To evaluate a leucocyte esterase test as a predictor of gonorrhoea or chlamydia in asymptomatic Aboriginal males at the Central Australian Aboriginal Congress Male Clinic (Ingkintja), first-void urine samples and clinical information were collected from consecutive asymptomatic males presenting to the Ingkintja in Alice Springs between March 2008 and December 2009. Urine was tested immediately with a leucocyte esterase test dipstick and then by polymerase chain reaction for gonorrhoea and chlamydia. Among the 292 specimens from asymptomatic males, 15.4% were positive for gonorrhoea or chlamydia. In this group, compared with polymerase chain reaction result for gonorrhoea or chlamydia, leucocyte esterase test alone and in combination with age ≤35 years showed sensitivities of 66.7% and 60%, specificities of 90.7% and 94.7%, positive predictive values of 56.6% and 67.5%, negative predictive values of 93.7% and 92.8% and the area under receiver operating characteristics curve values of 0.79 and 0.85, respectively. Leucocyte esterase tests can reasonably be used as a basis for immediate empirical treatment for gonorrhoea or chlamydia in asymptomatic central Australian Aboriginal men under 35 years of age.

  6. 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

  7. Two enzymes of a complete degradation pathway for linear alkylbenzenesulfonate (LAS) surfactants: 4-sulfoacetophenone Baeyer-Villiger monooxygenase and 4-sulfophenylacetate esterase in Comamonas testosteroni KF-1.

    PubMed

    Weiss, Michael; Denger, Karin; Huhn, Thomas; Schleheck, David

    2012-12-01

    Complete biodegradation of the surfactant linear alkylbenzenesulfonate (LAS) is accomplished by complex bacterial communities in two steps. First, all LAS congeners are degraded into about 50 sulfophenylcarboxylates (SPC), one of which is 3-(4-sulfophenyl)butyrate (3-C(4)-SPC). Second, these SPCs are mineralized. 3-C(4)-SPC is mineralized by Comamonas testosteroni KF-1 in a process involving 4-sulfoacetophenone (SAP) as a metabolite and an unknown inducible Baeyer-Villiger monooxygenase (BVMO) to yield 4-sulfophenyl acetate (SPAc) from SAP (SAPMO enzyme); hydrolysis of SPAc to 4-sulfophenol and acetate is catalyzed by an unknown inducible esterase (SPAc esterase). Transcriptional analysis showed that one of four candidate genes for BVMOs in the genome of strain KF-1, as well as an SPAc esterase candidate gene directly upstream, was inducibly transcribed during growth with 3-C(4)-SPC. The same genes were identified by enzyme purification and peptide fingerprinting-mass spectrometry when SAPMO was enriched and SPAc esterase purified to homogeneity by protein chromatography. Heterologously overproduced pure SAPMO converted SAP to SPAc and was active with phenylacetone and 4-hydroxyacetophenone but not with cyclohexanone and progesterone. SAPMO showed the highest sequence homology to the archetypal phenylacetone BVMO (57%), followed by steroid BVMO (55%) and 4-hydroxyacetophenone BVMO (30%). Finally, the two pure enzymes added sequentially, SAPMO with NADPH and SAP, and then SPAc esterase, catalyzed the conversion of SAP via SPAc to 4-sulfophenol and acetate in a 1:1:1:1 molar ratio. Hence, the first two enzymes of a complete LAS degradation pathway were identified, giving evidence for the recruitment of members of the very versatile type I BVMO and carboxylester hydrolase enzyme families for the utilization of a xenobiotic compound by bacteria.

  8. 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

  9. 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.

  10. Two Enzymes of a Complete Degradation Pathway for Linear Alkylbenzenesulfonate (LAS) Surfactants: 4-Sulfoacetophenone Baeyer-Villiger Monooxygenase and 4-Sulfophenylacetate Esterase in Comamonas testosteroni KF-1

    PubMed Central

    Weiss, Michael; Denger, Karin; Huhn, Thomas

    2012-01-01

    Complete biodegradation of the surfactant linear alkylbenzenesulfonate (LAS) is accomplished by complex bacterial communities in two steps. First, all LAS congeners are degraded into about 50 sulfophenylcarboxylates (SPC), one of which is 3-(4-sulfophenyl)butyrate (3-C4-SPC). Second, these SPCs are mineralized. 3-C4-SPC is mineralized by Comamonas testosteroni KF-1 in a process involving 4-sulfoacetophenone (SAP) as a metabolite and an unknown inducible Baeyer-Villiger monooxygenase (BVMO) to yield 4-sulfophenyl acetate (SPAc) from SAP (SAPMO enzyme); hydrolysis of SPAc to 4-sulfophenol and acetate is catalyzed by an unknown inducible esterase (SPAc esterase). Transcriptional analysis showed that one of four candidate genes for BVMOs in the genome of strain KF-1, as well as an SPAc esterase candidate gene directly upstream, was inducibly transcribed during growth with 3-C4-SPC. The same genes were identified by enzyme purification and peptide fingerprinting-mass spectrometry when SAPMO was enriched and SPAc esterase purified to homogeneity by protein chromatography. Heterologously overproduced pure SAPMO converted SAP to SPAc and was active with phenylacetone and 4-hydroxyacetophenone but not with cyclohexanone and progesterone. SAPMO showed the highest sequence homology to the archetypal phenylacetone BVMO (57%), followed by steroid BVMO (55%) and 4-hydroxyacetophenone BVMO (30%). Finally, the two pure enzymes added sequentially, SAPMO with NADPH and SAP, and then SPAc esterase, catalyzed the conversion of SAP via SPAc to 4-sulfophenol and acetate in a 1:1:1:1 molar ratio. Hence, the first two enzymes of a complete LAS degradation pathway were identified, giving evidence for the recruitment of members of the very versatile type I BVMO and carboxylester hydrolase enzyme families for the utilization of a xenobiotic compound by bacteria. PMID:23001656

  11. An Activity-Based Probe for N-Acylethanolamine Acid Amidase

    PubMed Central

    Armirotti, Andrea; Summa, Maria; Bertozzi, Fabio; Garau, Gianpiero; Bandiera, Tiziano; Piomelli, Daniele

    2015-01-01

    N-Acylethanolamine acid amidase (NAAA) is a lysosomal cysteine hydrolase involved in the degradation of saturated and monounsaturated fatty acid ethanolamides (FAEs), a family of endogenous lipid signaling molecules that includes oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). Among the reported NAAA inhibitors, α–amino–β–lactone (3–aminooxetan–2–one) derivatives have been shown to prevent FAE hydrolysis in innate-immune and neural cells and to reduce reactions to inflammatory stimuli. Recently, we disclosed two potent and selective NAAA inhibitors, the compounds ARN077 (5-phenylpentyl N-[(2S,3R)-2-methyl-4-oxo-oxetan-3-yl]carbamate) and ARN726 (4-cyclohexylbutyl-N-[(S)-2-oxoazetidin-3-yl]carbamate). The former is active in vivo by topical administration in rodent models of hyperalgesia and allodynia, while the latter exerts systemic anti-inflammatory effects in mouse models of lung inflammation. In the present study, we designed and validated a derivative of ARN726 as the first activity-based protein profiling (ABPP) probe for the in vivo detection of NAAA. The newly synthesized molecule 1 is an effective in vitro and in vivo click-chemistry activity based probe (ABP), which is able to capture the catalytically active form of NAAA in Human Embryonic Kidney 293 (HEK293) cells overexpressing human NAAA as well as in rat lung tissue. Competitive ABPP with 1 confirmed that ARN726 and ARN077 inhibit NAAA in vitro and in vivo. Compound 1 is a useful new tool to identify activated NAAA both in vitro and in vivo, and to investigate the physiological and pathological roles of this enzyme. PMID:26102511

  12. In vivo study of acetylcholine esterase in basal forebrain, amygdala, and cortex in mild to moderate Alzheimer disease.

    PubMed

    Herholz, K; Weisenbach, S; Zündorf, G; Lenz, O; Schröder, H; Bauer, B; Kalbe, E; Heiss, W-D

    2004-01-01

    It is currently unclear whether impairment of the cholinergic system is present in Alzheimer disease (AD) already at an early stage and to what extent it depends on degeneration of the nucleus basalis of Meynert (nbM). We examined acetylcholine esterase activity in vivo in the nbM, the amygdala, and cerebral neocortex. Measurements were performed in normal controls and in patients with mild to moderate AD with positron emission tomography (PET) and C-11-labeled N-methyl-4-piperidyl-acetate (MP4A) which is a specific substrate of AChE. AChE activity was reduced significantly in amygdala and cerebral cortex. In contrast, AChE activity and glucose metabolism appeared preserved or even increased in the nbM. The results support the concept that neocortical and amygdaloid functional changes of the cholinergic system are an early and leading event in AD, rather than the consequence of neurodegeneration of basal nuclei.

  13. An epididymis-specific carboxyl esterase CES5A is required for sperm capacitation and male fertility in the rat

    PubMed Central

    Ru, Yan-Fei; Xue, Hai-Min; Ni, Zi-Mei; Xia, Dong; Zhou, Yu-Chuan; Zhang, Yong-Lian

    2015-01-01

    Despite the fact that the phenomenon of capacitation was discovered over half century ago and much progress has been made in identifying sperm events involved in capacitation, few specific molecules of epididymal origin have been identified as being directly involved in this process in vivo. Previously, our group cloned and characterized a carboxyl esterase gene Ces5a in the rat epididymis. The CES5A protein is mainly expressed in the corpus and cauda epididymidis and secreted into the corresponding lumens. Here, we report the function of CES5A in sperm maturation. By local injection of Lentivirus-mediated siRNA in the CES5A-expressing region of the rat epididymis, Ces5a-knockdown animal models were created. These animals exhibited an inhibited sperm capacitation and a reduction in male fertility. These results suggest that CES5A plays an important role in sperm maturation and male fertility. PMID:25475668

  14. Effects of oxidation on the hydrolysis by cholesterol esterase of sitosteryl esters as compared to a cholesteryl ester.

    PubMed

    Julien-David, Diane; Ennahar, Saïd; Miesch, Michel; Geoffroy, Philippe; Raul, Francis; Aoude-Werner, Dalal; Lessinger, Jean-Marc; Marchioni, Eric

    2009-10-01

    Phytosteryl esters (PE) are used as ingredients in functional food to decrease plasma concentration of low density lipoprotein-cholesterol (LDL-C). Effective impairment of cholesterol absorption by PE suggests that these esters are hydrolyzed by the pancreatic cholesterol esterase (CEase, EC 3.1.1.13) and the liberated sterol may interfere with cholesterol reducing its intestinal absorption. PE-enriched foods are marketed for cooking purposes, and temperature is one of the most important factors leading to the formation of oxidation products. Very little is known about the outcome of PE oxides during the digestive process. A new analytical method based on mass spectrometric detection directly after enzymatic reaction was developed to determine in vitro the activity of CEase on PE and their oxides present in functional food. Using this method, we identified a new inhibitor of CEase: sitosteryl 9,10-dihydroxystearate, which behaves as a non-competitive inhibitor of the hydrolysis of cholesteryl oleate and sitosteryl oleate.

  15. 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.

  16. Structure and function of an acetyl xylan esterase (Est2A) from the rumen bacterium Butyrivibrio proteoclasticus.

    PubMed

    Till, Marisa; Goldstone, David C; Attwood, Graeme T; Moon, Christina D; Kelly, Willam J; Arcus, Vickery L

    2013-05-01

    Butyrivibrio proteoclasticus is a significant component of the microbial population of the rumen of dairy cattle. It is a xylan-degrading organism whose genome encodes a large number of open reading frames annotated as fiber-degrading enzymes. We have determined the three-dimensional structure of Est2A, an acetyl xylan esterase from B. proteoclasticus, at 2.1 Å resolution, along with the structure of an inactive mutant (H351A) at 2.0 Å resolution. The structure reveals two domains-a C-terminal SGNH domain and an N-terminal jelly-roll domain typical of CE2 family structures. The structures are accompanied by experimentally determined enzymatic parameters against two model substrates, para-nitrophenyl acetate and para-nitrophenyl butyrate. The suite of fiber-degrading enzymes produced by B. proteoclasticus provides a rich source of new enzymes of potential use in industrial settings.

  17. Isolation and Characterization of EstC, a New Cold-Active Esterase from Streptomyces coelicolor A3(2)

    PubMed Central

    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 (kcat/Km = 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. PMID:22396747

  18. 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.

  19. 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.

  20. Intron retention regulates the expression of pectin methyl esterase inhibitor (Pmei) genes during wheat growth and development.

    PubMed

    Rocchi, V; Janni, M; Bellincampi, D; Giardina, T; D'Ovidio, R

    2012-03-01

    Pectin is an important component of the plant cell wall and its remodelling occurs during normal plant growth or following stress responses. Pectin is secreted into the cell wall in a highly methyl-esterified form and subsequently de-methyl-esterified by pectin methyl esterase (PME), whose activity is controlled by the pectin methyl esterase inhibitor protein (PMEI). Cereal cell wall contains a low amount of pectin; nonetheless the level and pattern of pectin methyl esterification play a primary role during development or pathogen infection. Since few data are available on the role of PMEI in plant development and defence of cereal species, we isolated and characterised three Pmei genes (Tdpmei2.1, Tdpmei2.2 and Tdpmei3) and their encoded products in wheat. Sequence comparisons showed a low level of intra- and inter-specific sequence conservation of PMEIs. Tdpmei2.1 and Tdpmei2.2 share 94% identity at protein level, but only 20% identity with the product of Tdpmei3. All three Tdpmei genes code for functional inhibitors of plant PMEs and do not inhibit microbial PMEs or a plant invertase. RT-PCR analyses demonstrated, for the first time to our knowledge, that Pmei genes are regulated by intron retention. Processed and unprocessed transcripts of Tdpmei2.1 and Tdpmei2.2 accumulated in several organs, but anthers contained only mature transcripts. Tdpmei3 lacks introns and its transcript accumulated mainly in stem internodes. These findings suggest that products encoded by these Tdpmei genes control organ- or tissue-specific activity of specific PME isoforms in wheat.

  1. Relative potencies of the four stereoisomers of isomalathion for inhibition of hen brain acetylcholinesterase and neurotoxic esterase in vitro.

    PubMed

    Jianmongkol, S; Berkman, C E; Thompson, C M; Richardson, R J

    1996-08-01

    The cholinergic toxicity of malathion is exacerbated by its isomerization product, isomalathion, which inhibits detoxifying carboxylesterases as well as target acetylcholinesterase (AChE). Previous work has shown that the four stereoisomers of isomalathion, (1R, 3R), (1R, 3S), (1S, 3R), and (1S, 3S), differ in their inhibitory potencies against either rat brain or electric eel AChE. The present study examined the relative inhibitory potencies of these stereoisomers and the totally racemic mixture (1RS, 3RS) against hen brain AChE and neurotoxic esterase (NTE) to provide new data on stereoselective inhibition of neurotoxicologically significant esterases and to assess the potential of these compounds to cause organophosphorus (OP) compound-induced delayed neurotoxicity (OPIDN). The order of potencies against hen brain AChE was (1R, 3R) > (1R, 3S) > (1RS, 3RS) > (1S, 3R) > (1S, 3S), with a 15-fold difference between the strongest (ki = 388 mM-1 min-1; 20 min I50 = 89.3 nM) and weakest (ki = 25.6 mM-1 min-1; 20 min I50 = 1354 nM) inhibitors. Both asymmetric centers contributed substantially and interdependently to inhibitory potency, but the effect of changing the configuration at phosphorus alone was greater than changing the configuration at carbon alone. None of the isomalathions was an effective inhibitor of hen brain NTE (extrapolated 20 min I50 values were 1.2 to 29 mM), yielding NTE/ AChE I50 ratios (neuropathy target ratios, NTRs) of 1.5 x 10(3) to 1.5 x 10(5). NTRs of this magnitude indicate that none of the isomalathions should initiate OPIDN, even after doses greatly exceeding the LD50. Therefore, reports of OPIDN or other neuropathic sequelae associated with malathion exposures in humans cannot be explained on the basis of NTE inhibition by contaminating isomalathions.

  2. Rational design of a carboxylic esterase RhEst1 based on computational analysis of substrate binding

    DOE PAGES

    Chen, Qi; Luan, Zheng -Jiao; Yu, Hui -Lei; ...

    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

  3. 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.

  4. Some Anti-Inflammatory Agents Inhibit Esterase Activities of Human Carbonic Anhydrase Isoforms I and II: An In Vitro Study.

    PubMed

    Alım, Zuhal; Kılınç, Namık; İşgör, Mehmet M; Şengül, Bülent; Beydemir, Şükrü

    2015-10-01

    Carbonic anhydrases (CAs) are known as a drug-target enzymes. The inhibitors of the enzyme are important compounds for discovering new therapeutic agents and understanding in detail protein-drug interactions at the molecular level. For this purpose, the in vitro effects of some anti-inflammatory agents such as tenoxicam, fluorometholone acetate, and dexamethasone were investigated on esterase activity of human erythrocyte CA-I and CA-II in this study. hCA-I and hCA-II were purified by affinity chromatography with a yield of 47.25% and 87%, and a specific activity of 642.8 EU/mg proteins and 5576.9 EU/mg proteins, respectively. SDS-PAGE was performed to determine the purity of the enzymes. Inhibitory effects of the drugs on hCA-I and hCA-II were determined by spectrophotometric method. IC50 values for hCA-I and hCA-II were 0.198, 2.18, 11.7, 0.11, 17.5 and 14 μm using tenoxicam, fluorometholone acetate, and dexamethasone, respectively. For fluorometholone acetate and dexamethasone, Ki values from Lineweaver-Burk plots were obtained as 1.044 and 21.2 μm (noncompetitive) for hCA-I and 9.98 and 8.66 μm (non-competitive) for hCA-II. In conclusion, tenoxicam, fluorometholone acetate, and dexamethasone showed potent inhibitory effects on esterase activity of hCA-I and hCA-II isozymes under in vitro conditions.

  5. 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.

  6. 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.

  7. High activity of N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase and cytolytic perforin in cloned cell lines is not demonstrable in in-vivo-induced cytotoxic effector cells.

    PubMed Central

    Dennert, G; Anderson, C G; Prochazka, G

    1987-01-01

    Recent observations have suggested striking similarities between complement-mediated and cell-mediated lysis. Both pathways share the terminal insertion of channels into target membranes, and unique esterases have been postulated to participate in the activation of cytolytic effector molecules. Since killer-specific esterases and channel-forming proteins can be demonstrated in in vitro cell lines, it is important to ascertain that the described esterase and channel-forming proteins are also present in killer cells from in vivo sources. Results presented here show that killer-cell-specific N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase is induced in vitro concomitant with the sensitization of cytotoxic effector cells. In contrast, in-vivo-primed cytotoxic T cells or natural killer (NK) cells fail to express high levels of this enzyme. Assay of different cytotoxic effector cells reveals the presence of N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase in clones with T killer and NK activity, but enzyme levels do not correlate with cytolytic activity nor does inhibition of esterase activity interfere with granule-mediated cell lysis. A similar result is seen with granule-mediated cytolytic activity. Cloned NK and T killer cell lines possess granules that are able to lyse erythrocyte targets. However, T killer cells sensitized in mixed lymphocyte culture or in vivo have no detectable cytotoxic granules. Cytotoxic granules are also not detected in NK cells isolated from animals. PMID:2955414

  8. Pharmacokinetics and safety of DTS-108, a human oligopeptide bound to SN-38 with an esterase-sensitive cross-linker in patients with advanced malignancies: a Phase I study

    PubMed Central

    Coriat, Romain; Faivre, Sandrine J; Mir, Olivier; Dreyer, Chantal; Ropert, Stanislas; Bouattour, Mohammed; Desjardins, Robert; Goldwasser, François; Raymond, Eric

    2016-01-01

    Background DTS-108 is a hydrosoluble prodrug, where the SN-38 moiety is covalently linked to a 20-amino acid vector peptide by a specific esterase-sensitive cross-linker, releasing 7-ethyl-10-hydroxycampthotecin (SN-38) by esterase bond cleavage. Methods The pharmacokinetics of DTS-108, adverse events graded according to NCI-CTCv3.1, dose-limiting toxicities at cycle 1, the maximum tolerated dose (MTD), and the recommended Phase II dose (RP2D) of intravenous DTS-108 (1–2 hours) every 2 weeks were evaluated in a first-in-human Phase I study in patients with advanced/metastatic carcinomas, according to an accelerated dose escalation design. SN-38 and SN-38 glucuronide (SN-38G) levels were evaluated with fluorescence high-performance liquid chromatography (HPLC) test, then liquid chromatography–tandem mass spectrometry (LC/MS/MS) methods. Results Forty-two patients received DTS-108 across 14 dosing cohorts (range 3–416 mg/m2). At 416 mg/m2, three out of six patients had grade 4 neutropenia thereby defining the MTD and the RP2D at 313 mg/m2. Fluorescence HPLC was inaccurate to quantify DTS-108 and its metabolites (SN-38 and SN-38G). New processes and analytical LC/MS/MS methods for testing SN-38 were implemented. At a dose of 313 mg/m2, mean DTS-108, SN-38, and SN-38G area under the plasma concentration–time curve to infinity (coefficients of variation %) were 439,293 (24%), 1,992 (34%), and 4,538 (46%) h·ng/mL. Stable disease (according to Response Evaluation Criteria in Solid Tumors) was observed in nine patients. Conclusion Assessing SN-38 concentration using fluorescence HPLC is questionable since this method failed to monitor dose escalation of DTS-108, a new topoisomerase I inhibitor, due to ex vivo degradation. LC/MS/MS methods were consistent in evaluating SN-38 exposures allowing drug monitoring. The maximum tolerated dose of DTS-108 was 416 mg/m2. The RP2D for intravenous DTS-108 was 313 mg/m2 every 2 weeks in patients with advanced/metastatic solid

  9. Visualization of a substrate-induced productive conformation of the catalytic triad of the Neisseria meningitidis peptidoglycan O-acetylesterase reveals mechanistic conservation in SGNH esterase family members

    PubMed Central

    Williams, Allison H.; Veyrier, Frédéric J.; Bonis, Mathilde; Michaud, Yann; Raynal, Bertrand; Taha, Muhamed-Kheir; White, Stephen W.; Haouz, Ahmed; Boneca, Ivo G.

    2014-01-01

    Peptidoglycan O-acetylesterase (Ape1), which is required for host survival in Neisseria sp., belongs to the diverse SGNH hydrolase superfamily, which includes important viral and bacterial virulence factors. Here, multi-domain crystal structures of Ape1 with an SGNH catalytic domain and a newly identified putative peptidoglycan-detection module are reported. Enzyme catalysis was performed in Ape1 crystals and key catalytic intermediates along the SGNH esterase hydrolysis reaction pathway were visualized, revealing a substrate-induced productive conformation of the catalytic triad, a mechanistic detail that has not previously been observed. This substrate-induced productive conformation of the catalytic triad shifts the established dogma on these enzymes, generating valuable insight into the structure-based design of drugs targeting the SGNH esterase superfamily. PMID:25286847

  10. Mapping structural and functional changes in esterase-treated pectin and characterizing enzyme mode of action

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Organoleptic qualities of processed and formulated foods are primary determinates of consumer acceptability. Stabilization of milk proteins in acid dairy drinks, gelation in jams and jellies, and texture-firming ionic interactions in fruits and vegetables are product qualities mediated by structural...

  11. Characterization on malathion and permethrin resistance by bioassays and the variation of esterase activity with the life stages of the mosquito Culex quinquefasciatus.

    PubMed

    Selvi, S; Edah, M A; Nazni, W A; Lee, H L; Azahari, A H

    2007-06-01

    Larvae and adults of Culex quinquefasciatus were used for the test undertaken for malathion resistant strain (F61 - F65) and permethrin resistant strain (F54 - F58). The results showed that the LC50 for both malathion (F61 - F65) and permethrin (F54 - F58) resistant Cx. quinquefasciatus increased steadily throughout the subsequent five generations, indicating a marked development of resistance. The adult female malathion resistant strain have developed a high resistance level to malathion diagnostic dosage with a resistance ratio of 9.3 to 17.9 folds of resistance compared with the susceptible Cx. quinquefasciatus. Permethrin resistance ratio remained as 1.0 folds of resistance at every generation. It was obvious that malathion resistance developed at a higher rate in adult females compared to permethrin. Enzyme-based metabolic mechanisms of insecticide resistance were investigated based on the biochemical assay principle. From the results obtained obviously shows that there is a significant difference (p < 0.05) in esterase level in both malathion and permethrin selected strains. Female malathion selected strain has the higher level of esterase activity compared to the female permethrin selected strain at (0.8 to 1.04) alpha-Na micromol/min/mg protein versus (0.15 to 0.24) alpha-Na micromol/min/mg protein respectively. This indicated increased level of non-specific esterase is playing an important role in resistance mechanism in female malathion selected strain. Permethrin selected strain exhibited non-specific esterase activity at a very low level throughout the different life stages compared to malathion selected strain. This study suggests that life stages play a predominant role in conferring malathion and permethrin resistance in Cx. quinquefasciatus.

  12. Identification and Characterization of Carboxyl Esterases of Gill Chamber-Associated Microbiota in the Deep-Sea Shrimp Rimicaris exoculata by Using Functional Metagenomics

    PubMed Central

    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.

    2015-01-01

    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

  13. Agrobacterium tumefaciens estC, Encoding an Enzyme Containing Esterase Activity, Is Regulated by EstR, a Regulator in the MarR Family

    PubMed Central

    Giengkam, Suparat; Vattanaviboon, Paiboon

    2016-01-01

    Analysis of the A. tumefaciens genome revealed estC, which encodes an esterase located next to its transcriptional regulator estR, a regulator of esterase in the MarR family. Inactivation of estC results in a small increase in the resistance to organic hydroperoxides, whereas a high level of expression of estC from an expression vector leads to a reduction in the resistance to organic hydroperoxides and menadione. The estC gene is transcribed divergently from its regulator, estR. Expression analysis showed that only high concentrations of cumene hydroperoxide (CHP, 1 mM) induced expression of both genes in an EstR-dependent manner. The EstR protein acts as a CHP sensor and a transcriptional repressor of both genes. EstR specifically binds to the operator sites OI and OII overlapping the promoter elements of estC and estR. This binding is responsible for transcription repression of both genes. Exposure to organic hydroperoxide results in oxidation of the sensing cysteine (Cys16) residue of EstR, leading to a release of the oxidized repressor from the operator sites, thereby allowing transcription and high levels of expression of both genes. The estC is the first organic hydroperoxide-inducible esterase-encoding gene in alphaproteobacteria. PMID:28036400

  14. Production of flavour compounds from fat during cheese ripening by action of lipases and esterases.

    PubMed

    Wolf, Irma Verónica; Meinardi, Carlos Alberto; Zalazar, Carlos Antonio

    2009-01-01

    The milk fat is an essential component for the development of correct flavour in cheese. The lipolysis and catabolism of fatty acids are two biochemical events very important on flavour development of some cheese varieties. The role and characteristics of various lipolytic agents during cheese ripening is reviewed and discussed. Before starting with the specific study about formation of flavour compounds from milk fat during cheese ripening, a brief review of the technological aspects of cheese production is needed.

  15. Effect of long-term exposure to pesticides on plasma esterases from plastic greenhouse workers.

    PubMed

    Hernández, Antonio; Gómez, M Amparo; Pena, Gloria; Gil, Fernando; Rodrigo, Lourdes; Villanueva, Enrique; Pla, Antonio

    2004-07-23

    Previous reports in animals considered beta-glucuronidase activity as a novel biomarker of anticholinesterase (organophosphates and carbamates) pesticides exposure. Acid phosphatase activity was also shown to increase after organophosphates exposure. In addition, there is evidence that the paraoxonase status influences sensitivity to specific pesticides. In this study, activities of beta-glucuronidase, acid phosphatase, cholinesterase, and paraoxonase were measured in plasma from plastic greenhouse workers exposed over the long term to different pesticides, including organophosphates and carbamates, in order to evaluate the potential chronic toxicity of pesticides at occupational level. Our results show that activities of paraoxonase and cholinesterase were decreased in applicators of pesticides compared to non-applicators. Likewise, it was found that activities of beta-glucuronidase and acid phosphatase were associated with pesticide exposure in humans, and that both biochemical parameters were related to each other. Interestingly, the paraoxonase B allele (phenotyped in plasma) was associated with a higher risk of inhibition of cholinesterase activity above a 25% level, which supports the hypothesis that paraoxonase phenotypes are associated with susceptibility of humans to anticholinesterase pesticides toxicity.

  16. New structural motif for carboxylic acid perhydrolases.

    PubMed

    Yin, DeLu Tyler; Purpero, Vince M; Fujii, Ryota; Jing, Qing; Kazlauskas, Romas J

    2013-02-25

    Some serine hydrolases also catalyze a promiscuous reaction--reversible perhydrolysis of carboxylic acids to make peroxycarboxylic acids. Five X-ray crystal structures of these carboxylic acid perhydrolases show a proline in the oxyanion loop. Here, we test whether this proline is essential for high perhydrolysis activity using Pseudomonas fluorescens esterase (PFE). The L29P variant of this esterase catalyzes perhydrolysis 43-fold faster (k(cat) comparison) than the wild type. Surprisingly, saturation mutagenesis at the 29 position of PFE identified six other amino acid substitutions that increase perhydrolysis of acetic acid at least fourfold over the wild type. The best variant, L29I PFE, catalyzed perhydrolysis 83-times faster (k(cat) comparison) than wild-type PFE and twice as fast as L29P PFE. Despite the different amino acid in the oxyanion loop, L29I PFE shows a similar selectivity for hydrogen peroxide over water as L29P PFE (β(0)=170 vs. 160 M(-1)), and a similar fast formation of acetyl-enzyme (140 vs. 62 U mg(-1)). X-ray crystal structures of L29I PFE with and without bound acetate show an unusual mixture of two different oxyanion loop conformations. The type II β-turn conformation resembles the wild-type structure and is unlikely to increase perhydrolysis, but the type I β-turn conformation creates a binding site for a second acetate. Modeling suggests that a previously proposed mechanism for L29P PFE can be extended to include L29I PFE, so that an acetate accepts a hydrogen bond to promote faster formation of the acetyl-enzyme.

  17. 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

  18. In vitro functional interactions of acetylcholine esterase inhibitors and muscarinic receptor antagonists in the urinary bladder of the rat.

    PubMed

    Killi, Uday K; Wsol, Vladimir; Soukup, Ondrej; Kuca, Kamil; Winder, Michael; Tobin, Gunnar

    2014-02-01

    Obidoxime, a weak acetylcholine-esterase (AChE) inhibitor, exerts muscarinic receptor antagonism with a significant muscarinic M2 receptor selective profile. The current examinations aimed to determine the functional significance of muscarinic M2 receptors in the state of AChE inhibition, elucidating muscarinic M2 and M3 receptor interaction. In the in vitro examinations, methacholine evoked concentration-dependent bladder contractile and atrial frequency inhibitory responses. Although atropine abolished both, methoctramine (1 μmol/L) only affected the cholinergic response in the atrial preparations. However, in the presence of methoctramine, physostigmine, an AChE inhibitor, increased the basal tension of the bladder strip preparations (+68%), as well as the contractile responses to low concentrations of methacholine (< 5 μmol/L; +90-290%). In contrast to physostigmine, obidoxime alone raised the basal tension (+58%) and the responses to low concentrations of methacholine (< 5 μmol/L; +80-450%). Physostigmine concentration-dependently increased methacholine-evoked responses, similarly to obidoxime at low concentrations. However, at large concentrations (> 5 μmol/L), obidoxime, because of its unselective muscarinic receptor antagonism, inhibited the methacholine bladder responses. In conclusion, the current results show that muscarinic M2 receptors inhibit muscarinic M3 receptor-evoked contractile responses to low concentrations of acetylcholine in the synaptic cleft. The muscarinic M2 and M3 receptor crosstalk could be a counteracting mechanism in the treatment of AChE inhibition when using reactivators, such as obidoxime.

  19. 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.

  20. The effect of additives and mechanical agitation in surface modification of acrylic fibres by cutinase and esterase.

    PubMed

    Matamá, Teresa; Vaz, Filipe; Gübitz, Georg M; Cavaco-Paulo, Artur

    2006-01-01

    The surface of an acrylic fibre containing about 7% of vinyl acetate was modified using Fusarium solani pisi cutinase and a commercial esterase, Texazym PES. The effect of acrylic solvents and stabilising polyols on cutinase operational stability was studied. The half-life time of cutinase increased by 3.5-fold with the addition of 15% N,N-dimethylacetamide (DMA) and by 3-fold with 1M glycerol. The impact of additives and mechanical agitation in the protein adsorption and in the hydrolysis of vinyl acetate from acrylic fabric was investigated. The hydroxyl groups produced on the surface of the fibre were able to react specifically with Remazol Brilliant Blue R (cotton reactive dye) and to increase the colour of the acrylic-treated fabric. The best staining level was obtained with a high level of mechanical agitation and with the addition of 1% DMA. Under these conditions, the raise in the acrylic fabric colour depth was 30% for cutinase and 25% for Texazym. The crystallinity degree, determined by X-ray diffraction, was not significantly changed between control samples and samples treated with cutinase. The results showed that the outcome of the application of these enzymes depends closely on the reaction media conditions.

  1. 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

  2. Crystallization and preliminary crystallographic studies of LipA, a secretory lipase/esterase from Xanthomonas oryzae pv. oryzae.

    PubMed

    Aparna, Gudlur; Chatterjee, Avradip; Jha, Gopaljee; Sonti, Ramesh V; Sankaranarayanan, Rajan

    2007-08-01

    Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight, a serious disease of rice. Several enzymes that are secreted through the type II secretion system of this bacterium play an important role in the plant-microbe interaction, being important for virulence and also being able to induce potent host defence responses. One of these enzymes is a secretory lipase/esterase, LipA, which shows a very weak homology to other bacterial lipases and gives a positive tributyrin plate assay. In this study, LipA was purified from the culture supernatant of an overexpressing clone of X. oryzae pv. oryzae and two types of crystals belonging to space group C2 but with two different unit-cell parameters were obtained using the hanging-drop vapour-diffusion method. Type I crystals diffract to a maximum resolution of 1.89 A and have unit-cell parameters a = 93.1, b = 62.3, c = 66.1 A, beta = 90.8 degrees . Type II crystals have unit-cell parameters a = 103.6, b = 54.6, c = 66.3 A, beta = 92.6 degrees and diffract to 1.86 A. Solvent-content analysis shows one monomer in the asymmetric unit in both the crystal forms.

  3. Exploring the bioprospecting and biotechnological potential of white-rot and anaerobic Neocallimastigomycota fungi: peptidases, esterases, and lignocellulolytic enzymes.

    PubMed

    da Silva, Ronivaldo Rodrigues; Pedezzi, Rafael; Souto, Tatiane Beltramini

    2017-04-01

    Fungi constitute an invaluable natural resource for scientific research, owing to their diversity; they offer a promising alternative for bioprospecting, thus contributing to biotechnological advances. For a long time, extensive information has been exploited and fungal products have been tested as a source of natural compounds. In this context, enzyme production remains a field of interest, since it offers an efficient alternative to the hazardous processes of chemical transformations. Owing to their vast biodiversity and peculiar biochemical characteristics, two fungal categories, white-rot and anaerobic Neocallimastigomycota, have gathered considerable attention for biotechnological applications. These fungi are known for their ability to depolymerize complex molecular structures and are used in degradation of lignocellulosic biomass, improvement of animal feed digestibility, biogas and bioethanol production, and various other applications. However, there are only limited reports that describe proteolytic enzymes and esterases in these fungi and their synergistic action with lignocellulolytic enzymes on degradation of complex polymers. Thus, in this minireview, we focus on the importance of these organisms in enzyme technology, their bioprospecting, possibility of integration of their enzyme repertoire, and their prospects for future biotechnological innovation.

  4. 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

  5. Acetylcholine esterase activity and behavioral response in hypoxia induced neonatal rats: effect of glucose, oxygen and epinephrine supplementation.

    PubMed

    Chathu, Finla; Krishnakumar, Amee; Paulose, Cheramadathikudyil S

    2008-10-01

    Brain damage due to an episode of hypoxia remains a major problem in infants causing deficit in motor and sensory function. Hypoxia leads to neuronal functional failure, cerebral palsy and neuro-developmental delay with characteristic biochemical and molecular alterations resulting in permanent or transitory neurological sequelae or even death. During neonatal hypoxia, traditional resuscitation practices include the routine administration of 100% oxygen, epinephrine and glucose. In the present study, we assessed the changes in the cholinergic system by measuring the acetylcholinesterase (AChE) activity and the behavioral responses shown by hypoxia induced neonatal rats and hypoxic rats supplemented with glucose, oxygen and epinephrine using elevated plus-maze and open-field test. The acetylcholine esterase enzyme activity showed a significant decrease in cerebral cortex, whereas it increased significantly in the muscle of experimental rats when compared to control. Hypoxic rats supplemented with glucose, glucose and oxygen showed a reversal to the control status. Behavioral studies were carried out in experimental rats with elevated plus-maze test and open-field test. Hypolocomotion and anxiogenic behavioral responses were observed in all experimental rats when compared to control, hypoxic rats supplemented with glucose, glucose and oxygen. Thus, our results suggest that brain damage due to hypoxia, oxygen and epinephrine supplementation in the neonatal rats cause acetylcholine-neuromuscular-defect leading to hypolocomotion and anxiogenic behavioral response. Glucose and glucose with oxygen supplementation to hypoxic neonates protect the brain damage for a better functional status in the later life.

  6. Automated high-throughput in vitro screening of the acetylcholine esterase inhibiting potential of environmental samples, mixtures and single compounds.

    PubMed

    Froment, Jean; Thomas, Kevin V; Tollefsen, Knut Erik

    2016-08-01

    A high-throughput and automated assay for testing the presence of acetylcholine esterase (AChE) inhibiting compounds was developed, validated and applied to screen different types of environmental samples. Automation involved using the assay in 96-well plates and adapting it for the use with an automated workstation. Validation was performed by comparing the results of the automated assay with that of a previously validated and standardised assay for two known AChE inhibitors (paraoxon and dichlorvos). The results show that the assay provides similar concentration-response curves (CRCs) when run according to the manual and automated protocol. Automation of the assay resulted in a reduction in assay run time as well as in intra- and inter-assay variations. High-quality CRCs were obtained for both of the model AChE inhibitors (dichlorvos IC50=120µM and paraoxon IC50=0.56µM) when tested alone. The effect of co-exposure of an equipotent binary mixture of the two chemicals were consistent with predictions of additivity and best described by the concentration addition model for combined toxicity. Extracts of different environmental samples (landfill leachate, wastewater treatment plant effluent, and road tunnel construction run-off) were then screened for AChE inhibiting activity using the automated bioassay, with only landfill leachate shown to contain potential AChE inhibitors. Potential uses and limitations of the assay were discussed based on the present results.

  7. 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.

  8. Potato tuber pectin structure is influenced by pectin methyl esterase activity and impacts on cooked potato texture.

    PubMed

    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.

  9. 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.

  10. 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

  11. 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.

  12. Role of B-esterases in assessing toxicity of organophosphorus (chlorpyrifos, malathion) and carbamate (carbofuran) pesticides to Daphnia magna.

    PubMed

    Barata, Carlos; Solayan, Arun; Porte, Cinta

    2004-02-10

    In this study, the cladoceran Daphnia magna was exposed to two model organophosphorous and one carbamate pesticides including malathion, chlorpyrifos and carbofuran to assess acetylcholinesterase (AChE) and carboxylesterase (CbE) inhibition and recovery patterns and relate those responses with individual level effects. Our results revealed differences in enzyme inhibition and recovery patterns among the studied esterase enzymes and pesticides. CbE was more sensitive to organophosphorous than AChE, whereas both CbE and AChE showed equivalent sensitivities to the carbamate carbofuran. Recovery patterns of AChE and CbE activities following exposure to the studied pesticides were similar with 80-100% recoveries taking place 12 and 96 h after exposure to organophosphorous and carbamates pesticides, respectively. The physiological role of AChE and CbE inhibition patterns in Daphnia was examined by using organophosphorous and carbamate compounds alone and with specific inhibitors of CbE. Under exposure to organophosphorous pesticides, survival of Daphnia juveniles was impaired at AChE inhibition levels higher than 50% whereas under exposure to the carbamate carbofuran low levels of AChE inhibition affected mortality. Inhibition of CbE by 80-90% increased toxicity to organophosphorous and carbamate pesticides by up to two- and four-fold, respectively. Our results suggest that both AChE and CbE enzymes are involved in determining toxicity of Daphnia to the studied chemicals and that AChE inhibition levels higher than 50% can be considered of environmental concern to Daphnia species.

  13. Functional significance of parasitism-induced suppression of juvenile hormone esterase activity in developmentally delayed Choristoneura fumiferana larvae.

    PubMed

    Cusson, M; Laforge, M; Miller, D; Cloutier, C; Stoltz, D

    2000-03-01

    The parasitic wasp Tranosema rostrale transmits a polydnavirus (PDV) to its host, Choristoneura fumiferana, during oviposition. Last-instar C. fumiferana larvae parasitized by T. rostrale early in the stadium fail to undergo metamorphosis, and injection of the wasp's calyx fluid (CxF; contains PDV) into healthy caterpillars induces a dose-dependent delay in initiation of metamorphosis (D. Doucet and M. Cusson, 1996, Entomol. Exp. Appl. 81, 21-30). In the present work, parasitization and injection of CxF (0.5 female equivalent) on the first day of the last stadium both prevented the rise in hemolymph 20-hydroxyecdysone (20HE) titer observed between day 4 and day 7 in control and saline-injected larvae. Similarly, juvenile hormone esterase (JHE) activity was depressed following parasitization or CxF injection, whereas control larvae displayed a peak on day 4. However, neither parasitism nor injection of CxF on day 1 prevented the JH-producing glands from turning off during the first half of the last stadium. Likewise, low but clearly detectable JH titers were observed in the first hours following the molt but very low titers, at or near the detection limit of our radioimmunoassay, were seen in both control and parasitized larvae on day 4. Prothoracic glands showed no apparent sign of degeneration 4 days after injection of CxF but had significantly smaller cells than saline-injected larvae 7 days postinjection. It is not clear whether this was a direct effect of T. rostrale PDV. Thus, disruption of spruce budworm metamorphosis by T. rostrale CxF involves depression of 20HE titers but is not associated with a measurable increase in the level of JH, as shown for some other host-parasitoid systems. In view of the latter observation, we put forward three hypotheses regarding the functional significance of the observed suppression of JHE activity in developmentally arrested C. fumiferana larvae.

  14. 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.

  15. 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

  16. [Adaptation of methods for quantification of the effect of esterase, acetylcholinesterase and glutathione-S-transferase in Blattella germanica (Dictyoptera: Blattellidae)].

    PubMed

    Diaz, Cristina; Alvarez, Yudelmis; de Armas, Yaxier; Bisset, Juan A

    2006-01-01

    The resistance mechanisms of Blatella germanica, one of the most important urban plagues worldwide since it is a mechanical vector that houses a number of highly harmful viruses, fungi, helmints and bacteria were studied. There are different control methods used against Blattella germnanica, with insecticides playing the leading role. Their uncontrolled application has caused the development of insecticice resistance in this species. This paper adapted biochemical methods to detect the enzymes esterase, acetylcholinesterase and glutathine-S-transferase as posible resistance mechanisms. To this end, all the parameters that allow finding out if a strain is susceptible or resistant to each mechanism were set.

  17. Polymorphisms of the enzyme systems galactose-1-phosphate uridyltransferase (GALT) and esterase D (EsD) in the province of Cádiz, southern Spain.

    PubMed

    Gamero-Lucas, J J; Romero, J L; Vizcaya, M A; Arufe, M I

    1991-05-01

    Galactose-phosphate uridyltransferase (GALT) and esterase D (EsD) phenotypes were determined by isoelectric focusing in ultrathin-layer polyacrylamide gel (PAGIF) for 406 healthy subjects randomly chosen and residing in the province of Cádiz, in Southern Spain. The following gene frequencies were observed: for GALT, GALT1 = 0.952 970 3 and GALT2 = 0.047 029 71; for EsD, EsD1 = 0.895 320 2, EsD2 = 0.094 827 59, and EsD5 = 0.009 852 21.

  18. Morpho-functional characterization and esterase patterns of the midgut of Tribolium castaneum Herbst, 1797 (Coleoptera: Tenebrionidae) parasitized by Gregarina cuneata (Apicomplexa: Eugregarinidae).

    PubMed

    Gigliolli, Adriana A Sinópolis; Lapenta, Ana Silva; Ruvolo-Takasusuki, Maria Claudia Colla; Abrahão, Josielle; Conte, Hélio

    2015-09-01

    Tribolium castaneum (Coleoptera: Tenebrionidae) is a common pest of stored grains and byproducts and is normally infected by Gregarina cuneata (Apicomplexa: Eugregarinidae). The life cycle of this parasite includes the sporozoite, trophozoite, gamont, gametocyte, and oocyst stages, which occur between the epithelium and lumen of the host's midgut. This study aims to describe the morphofunctional alterations in the midgut and determine the esterase patterns in T. castaneum when parasitized by gregarines. To achieve this purpose, midguts of adult insects were isolated, processed, and analysed using light and electron microscopy. We determined total protein content, amylase activity, and the expression and related activities of the esterases by using polyacrylamide gel electrophoresis (PAGE). The midgut of T. castaneum is formed by digestive, regenerative, and endocrine cells. The effects of parasitism on the digestive cells are severe, because the gregarines remain attached to these cells to absorb all the nutrients they need throughout their development. In these cells, the most common alterations observed include expansion and fragmentation of the rough endoplasmic reticulum, development of the smooth endoplasmic reticulum, changes in mitochondrial cristae, cytoplasmic vacuolization, formation of myelin structures, spherites, large intercellular spaces, autophagic vesicles, expansion of the basal labyrinth, and cytoplasmic protrusions. Deposits of glycogen granules were also observed. Amylase activity was reduced in parasitized insects. Regenerative cells were found in disorganized crypts and did not differentiate into new cells, thus, compromising the restoration of the damaged epithelium. Though few morphological alterations were observed in the endocrine cells, results suggest that the synthesis and/or release of hormones might be impaired. Nine esterases (EST-1 to 9) were identified in the midgut of T. castaneum and were expressed in varying levels in response

  19. Conjugated fatty acid synthesis: residues 111 and 115 influence product partitioning of Momordica charantia conjugase.

    PubMed

    Rawat, Richa; Yu, Xiao-Hong; Sweet, Marie; Shanklin, John

    2012-05-11

    Conjugated linolenic acids (CLNs), 18:3 Δ(9,11,13), lack the methylene groups found between the double bonds of linolenic acid (18:3 Δ(9,12,15)). CLNs are produced by conjugase enzymes that are homologs of the oleate desaturases FAD2. The goal of this study was to map the domain(s) within the Momordica charantia conjugase (FADX) responsible for CLN formation. To achieve this, a series of Momordica FADX-Arabidopsis FAD2 chimeras were expressed in the Arabidopsis fad3fae1 mutant, and the transformed seeds were analyzed for the accumulation of CLN. These experiments identified helix 2 and the first histidine box as a determinant of conjugase product partitioning into punicic acid (18:3 Δ(9cis,11trans,13cis)) or α-eleostearic acid (18:3 Δ(9cis,11trans,13trans)). This was confirmed by analysis of a FADX mutant containing six substitutions in which the sequence of helix 2 and first histidine box was converted to that of FAD2. Each of the six FAD2 substitutions was individually converted back to the FADX equivalent identifying residues 111 and 115, adjacent to the first histidine box, as key determinants of conjugase product partitioning. Additionally, expression of FADX G111V and FADX G111V/D115E resulted in an approximate doubling of eleostearic acid accumulation to 20.4% and 21.2%, respectively, compared with 9.9% upon expression of the native Momordica FADX. Like the Momordica conjugase, FADX G111V and FADX D115E produced predominantly α-eleostearic acid and little punicic acid, but the FADX G111V/D115E double mutant produced approximately equal amounts of α-eleostearic acid and its isomer, punicic acid, implicating an interactive effect of residues 111 and 115 in punicic acid formation.

  20. Dual Mutation Events in the Haemagglutinin-Esterase and Fusion Protein from an Infectious Salmon Anaemia Virus HPR0 Genotype Promote Viral Fusion and Activation by an Ubiquitous Host Protease.

    PubMed

    Fourrier, Mickael; Lester, Katherine; Markussen, Turhan; Falk, Knut; Secombes, Christopher J; McBeath, Alastair; Collet, Bertrand

    2015-01-01

    In Infectious salmon anaemia virus (ISAV), deletions in the highly polymorphic region (HPR) in the near membrane domain of the haemagglutinin-esterase (HE) stalk, influence viral fusion. It is suspected that selected mutations in the associated Fusion (F) protein may also be important in regulating fusion activity. To better understand the underlying mechanisms involved in ISAV fusion, several mutated F proteins were generated from the Scottish Nevis and Norwegian SK779/06 HPR0. Co-transfection with constructs encoding HE and F were performed, fusion activity assessed by content mixing assay and the degree of proteolytic cleavage by western blot. Substitutions in Nevis F demonstrated that K276 was the most likely cleavage site in the protein. Furthermore, amino acid substitutions at three sites and two insertions, all slightly upstream of K276, increased fusion activity. Co-expression with HE harbouring a full-length HPR produced high fusion activities when trypsin and low pH were applied. In comparison, under normal culture conditions, groups containing a mutated HE with an HPR deletion were able to generate moderate fusion levels, while those with a full length HPR HE could not induce fusion. This suggested that HPR length may influence how the HE primes the F protein and promotes fusion activation by an ubiquitous host protease and/or facilitate subsequent post-cleavage refolding steps. Variations in fusion activity through accumulated mutations on surface glycoproteins have also been reported in other orthomyxoviruses and paramyxoviruses. This may in part contribute to the different virulence and tissue tropism reported for HPR0 and HPR deleted ISAV genotypes.

  1. [The distribution of free and esterified carboxyl groups within the pectin molecule after the action of pectin esterase from Aspergillus niger and oranges].

    PubMed

    Kohn, R; Dongowski, G; Bock, W

    1985-01-01

    By reaction of pectin esterase (PE) from Aspergillus niger and oranges as well as lye, with 95% esterified citrus and apple pectin we prepared series of preparations with degrees of esterification between 35 and 77%. In these partial deesterified pectins the form of distribution of the free and esterified carboxyl groups has been determined from the activity coefficient gamma Ca2+ of the calcium counterions in the solutions of the corresponding calcium pectinates, from the electrostatic free enthalpy delta (Gel/N)KCa of the ion exchange Ca2+----2K+ in these systems as well as from the relative activity of the polygalacturonase reacting with sodium pectinate. The PE from A niger hydrolyzes the esterified carboxyl groups more or less randomly, in a manner similar to the effect of lye on pectin. On the other hand PE from oranges brings about block-like groupings of free carboxyl groups in the pectin molecule. The study revealed different reaction mechanisms of the pectin deesterification by pectin esterases from Aspergillus species and higher plants.

  2. Characterization of EstB, a novel cold-active and organic solvent-tolerant esterase from marine microorganism Alcanivorax dieselolei B-5(T).

    PubMed

    Zhang, Shanshan; Wu, Guojie; Liu, Zhixiang; Shao, Zongze; Liu, Ziduo

    2014-03-01

    A novel esterase gene, estB, was cloned from the marine microorganism Alcanivorax dieselolei B-5(T) and overexpressed in E. coli DE3 (BL21). The expressed protein EstB with a predicted molecular weight of 45.1 kDa had a distinct catalytic triad (Ser(211)-Trp(353)-Gln(385)) and the classical consensus motif conserved in most lipases and esterases Gly(209)-X-Ser(211)-X-Gly(213). EstB showed very low similarity to any known proteins and displayed the highest similarity to the hypothetical protein (46%) from Rhodococcus jostii RHA1. EstB showed the optimal activity around pH 8.5 and 20 °C and was identified to be extremely cold-adaptative retaining more than 95% activity between 0 and 10 °C. The values of kinetic parameters on p-NP caproate (K m, K cat and K cat/K m) were 0.15 mM, 0.54 × 10(3) s(-1) and 3.6 × 10(3) s(-1) mM(-1), respectively. In addition, EstB showed remarkable stability in several studied organic solvents and detergents of high concentrations with the retention of more than 70% activity after treatment for 30 min. The cold activity and its tolerance towards organic solvents made it a promising biocatalyst for industrial applications under extreme conditions.

  3. Role of kdr and esterase-mediated metabolism in pyrethroid-resistant populations of Haematobia irritans irritans (Diptera: Muscidae) in Brazil.

    PubMed

    Guerrero, Felix D; Barros, A Thadeu M

    2006-09-01

    The horn fly, Haematobia irritans irritans (L.) (Diptera: Muscidae), has become a problem for Brazilian cattle producers even though its introduction into Brazil is relatively recent. Failure to control this cattle pest is becoming a concern, and horn fly populations from several ranches from the state of Mato Grosso do Sul were surveyed for pyrethroid resistance. Susceptibility bioassays revealed that cypermethrin resistance was widespread and reached high levels in horn fly populations throughout the state, with resistance factors (RFs) ranging from 50.4 to 704.8. Synergist bioassays failed to detect a major role for esterases as a pyrethroid resistance mechanism in these populations, except for the highly pyrethroid-resistant Estrela do Oeste population (RF = 704.8). The kdr sodium channel gene mutation was not detected in eight of the 13 populations, but < 7% of individuals from four populations and 50% of the flies from Estrela do Oeste exhibited this mutation. Neither the superkdr sodium channel gene mutation nor a resistance-associated gene mutation in the HialphaE7 carboxylesterase were found in any of the fly populations. Although target site insensitivity (kdr) and esterase-mediated metabolism occur in horn fly populations from Mato Grosso do Sul state, it seems that they are not the major mechanism causing pyrethroid resistance in most of these populations.

  4. The Type II Secreted Lipase/Esterase LesA is a Key Virulence Factor Required for Xylella fastidiosa Pathogenesis in Grapevines

    PubMed Central

    Nascimento, Rafael; Gouran, Hossein; Chakraborty, Sandeep; Gillespie, Hyrum W.; Almeida-Souza, Hebréia O.; Tu, Aye; Rao, Basuthkar J.; Feldstein, Paul A.; Bruening, George; Goulart, Luiz R.; Dandekar, Abhaya M.

    2016-01-01

    Pierce’s disease (PD) of grapevines is caused by Xylella fastidiosa (Xf), a xylem-limited gamma-proteobacterium that is responsible for several economically important crop diseases. The occlusion of xylem elements and interference with water transport by Xf and its associated biofilm have been posited as the main cause of PD symptom development; however, Xf virulence mechanisms have not been described. Analysis of the Xf secretome revealed a putative lipase/esterase (LesA) that was abundantly secreted in bacterial culture supernatant and was characterized as a protein ortholog of the cell wall-degrading enzyme LipA of Xanthomonas strains. LesA was secreted by Xf and associated with a biofilm filamentous network. Additional proteomic analysis revealed its abundant presence in outer membrane vesicles (OMVs). Accumulation of LesA in leaf regions associated positively with PD symptoms and inversely with bacterial titer. The lipase/esterase also elicited a hypersensitive response in grapevine. Xf lesA mutants were significantly deficient for virulence when mechanically inoculated into grapevines. We propose that Xf pathogenesis is caused by LesA secretion mediated by OMV cargos and that its release and accumulation in leaf margins leads to early stages of observed PD symptoms. PMID:26753904

  5. A new alkaliphilic cold-active esterase from the psychrophilic marine bacterium Rhodococcus sp.: functional and structural studies and biotechnological potential.

    PubMed

    De Santi, Concetta; Tedesco, Pietro; Ambrosino, Luca; Altermark, Bjørn; Willassen, Nils-Peder; de Pascale, Donatella

    2014-03-01

    The special features of cold-adapted lipolytic biocatalysts have made their use possible in several industrial applications. In fact, cold-active enzymes are known to be able to catalyze reactions at low temperatures, avoiding side reactions taking place at higher temperatures and preserving the integrity of products. A lipolytic gene was isolated from the Arctic marine bacterium Rhodococcus sp. AW25M09 and expressed in Escherichia coli as inclusion bodies. The recombinant enzyme (hereafter called RhLip) showed interesting cold-active esterase activity. The refolded purified enzyme displayed optimal activity at 30 °C and was cold-active with retention of 50% activity at 10 °C. It is worth noting that the optimal pH was 11, and the low relative activity below pH 10 revealed that RhLip was an alkaliphilic esterase. The enzyme was active toward short-chain p-nitrophenyl esters (C2-C6), displaying optimal activity with the butyrate (C4) ester. In addition, the enzyme revealed a good organic solvent and salt tolerance. These features make this an interesting enzyme for exploitation in some industrial applications.

  6. Resistance mechanisms to chlorpyrifos and F392W mutation frequencies in the acetylcholine esterase ace1 allele of field populations of the tobacco whitefly, Bemisia tabaci in China.

    PubMed

    Zhang, Ning-ning; Liu, Cai-feng; Yang, Fang; Dong, Shuang-lin; Han, Zhao-jun

    2012-01-01

    The tobacco whitefly B-biotype Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) is a worldwide pest of many crops. In China, chlorpyrifos has been used to control this insect for many years and is still being used despite the fact that some resistance has been reported. To combat resistance and maintain good control efficiency of chlorpyrifos, it is essential to understand resistance mechanisms. A chlorpyrifos resistant tobacco whitefly strain (NJ-R) and a susceptible strain (NJ-S) were derived from a field-collected population in Nanjing, China, and the resistance mechanisms were investigated. More than 30-fold resistance was achieved after selected by chlorpyrifos for 13 generations in the laboratory. However, the resistance dropped significantly to about 18-fold in only 4 generations without selection pressure. Biochemical assays indicated that increased esterase activity was responsible for this resistance, while acetylcholine esterase, glutathione S-transferase, and microsomal-O-demethylase played little or no role. F392W mutations in acel were prevalent in NJ-S and NJ-R strains and 6 field-collected populations of both B and Q-biotype from locations that cover a wide geographical area of China. These findings provide important information about tobacco whitefly chlorpyrifos resistance mechanisms and guidance to combat resistance and optimize use patterns of chlorpyrifos and other organophosphate and carbamate insecticides.

  7. Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site

    PubMed Central

    Sayer, Christopher; Finnigan, William; Isupov, Michail N.; Levisson, Mark; Kengen, Servé W. M.; van der Oost, John; Harmer, Nicholas J.; Littlechild, Jennifer A.

    2016-01-01

    A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain p-nitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions. PMID:27160974

  8. The Type II Secreted Lipase/Esterase LesA is a Key Virulence Factor Required for Xylella fastidiosa Pathogenesis in Grapevines.

    PubMed

    Nascimento, Rafael; Gouran, Hossein; Chakraborty, Sandeep; Gillespie, Hyrum W; Almeida-Souza, Hebréia O; Tu, Aye; Rao, Basuthkar J; Feldstein, Paul A; Bruening, George; Goulart, Luiz R; Dandekar, Abhaya M

    2016-01-12

    Pierce's disease (PD) of grapevines is caused by Xylella fastidiosa (Xf), a xylem-limited gamma-proteobacterium that is responsible for several economically important crop diseases. The occlusion of xylem elements and interference with water transport by Xf and its associated biofilm have been posited as the main cause of PD symptom development; however, Xf virulence mechanisms have not been described. Analysis of the Xf secretome revealed a putative lipase/esterase (LesA) that was abundantly secreted in bacterial culture supernatant and was characterized as a protein ortholog of the cell wall-degrading enzyme LipA of Xanthomonas strains. LesA was secreted by Xf and associated with a biofilm filamentous network. Additional proteomic analysis revealed its abundant presence in outer membrane vesicles (OMVs). Accumulation of LesA in leaf regions associated positively with PD symptoms and inversely with bacterial titer. The lipase/esterase also elicited a hypersensitive response in grapevine. Xf lesA mutants were significantly deficient for virulence when mechanically inoculated into grapevines. We propose that Xf pathogenesis is caused by LesA secretion mediated by OMV cargos and that its release and accumulation in leaf margins leads to early stages of observed PD symptoms.

  9. Resistance Mechanisms to Chlorpyrifos and F392W Mutation Frequencies in the Acetylcholine Esterase Ace1 Allele of Field Populations of the Tobacco Whitefly, Bemisia tabaci in China

    PubMed Central

    Zhang, Ning-ning; Liu, Cai-feng; Yang, Fang; Dong, Shuang-lin; Han, Zhao-jun

    2012-01-01

    The tobacco whitefly B-biotype Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) is a worldwide pest of many crops. In China, chlorpyrifos has been used to control this insect for many years and is still being used despite the fact that some resistance has been reported. To combat resistance and maintain good control efficiency of chlorpyrifos, it is essential to understand resistance mechanisms. A chlorpyrifos resistant tobacco whitefly strain (NJ-R) and a susceptible strain (NJ-S) were derived from a field-collected population in Nanjing, China, and the resistance mechanisms were investigated. More than 30-fold resistance was achieved after selected by chlorpyrifos for 13 generations in the laboratory. However, the resistance dropped significantly to about 18-fold in only 4 generations without selection pressure. Biochemical assays indicated that increased esterase activity was responsible for this resistance, while acetylcholine esterase, glutathione S-transferase, and microsomal-O-demethylase played little or no role. F392W mutations in acel were prevalent in NJ-S and NJ-R strains and 6 field-collected populations of both B and Q-biotype from locations that cover a wide geographical area of China. These findings provide important information about tobacco whitefly chlorpyrifos resistance mechanisms and guidance to combat resistance and optimize use patterns of chlorpyrifos and other organophosphate and carbamate insecticides. PMID:22954331

  10. Stability of caffeic acid phenethyl amide (CAPA) in rat plasma.

    PubMed

    Yang, John; Kerwin, Sean M; Bowman, Phillip D; Stavchansky, Salomon

    2012-05-01

    A validated C₁₈ reverse-phase HPLC method with UV detection at 320 nm was developed and used for the stability evaluation of caffeic acid phenethyl amide (CAPA) and caffeic acid phenethyl ester (CAPE) in rat plasma. CAPA is the amide derivative of CAPE, a naturally occurring polyphenolic compound that has been found to be active in a variety of biological pathways. CAPA has been shown to protect endothelial cells against hydrogen peroxide-induced oxidative stress to a similar degree to CAPE. CAPE has been reported to be rapidly hydrolyzed in rat plasma via esterase enzymes. CAPA is expected to display a longer half-life than CAPE by avoiding hydrolysis via plasma esterases. The stability of CAPA and CAPE in rat plasma was investigated at three temperatures. The half-lives for CAPA were found to be 41.5, 10 and 0.82 h at 25, 37 and 60 °C, respectively. The half-lives for CAPE were found to be 1.95, 0.35 and 0.13 h at 4, 25 and 37 °C, respectively. The energy of activation was found to be 22.1 kcal/mol for CAPA and 14.1 kcal/mol for CAPE. A more stable compound could potentially extend the beneficial effects of CAPE.

  11. C1 esterase inhibitor

    MedlinePlus

    ... algorithm for the diagnosis, therapy and management of hereditary angioedema. Allergy Asthma Clin Immunol . 2010;6:24. PMID: ... chap 6. Read More Cirrhosis Complement Glomerulonephritis Hepatitis Hereditary angioedema Kidney transplant Lupus nephritis Systemic lupus erythematosus Ulcerative ...

  12. Biotransformation of caffeoyl quinic acids from green coffee extracts by Lactobacillus johnsonii NCC 533

    PubMed Central

    2013-01-01

    The potential of Lactobacillus johnsonii NCC 533 to metabolize chlorogenic acids from green coffee extract was investigated. Two enzymes, an esterase and a hydroxycinnamate decarboxylase (HCD), were involved in this biotransformation. The complete hydrolysis of 5-caffeoylquinic acid (5-CQA) into caffeic acid (CA) by L. johnsonii esterase occurred during the first 16 h of reaction time. No dihydrocaffeic acid was identified in the reaction mixture. The decarboxylation of CA into 4-vinylcatechol (4-VC) started only when the maximum concentration of CA was reached (10 μmol/ml). CA was completely transformed into 4-VC after 48 h of incubation. No 4-vinylphenol or other derivatives could be identified in the reaction media. In this study we demonstrate the capability of L. johnsonii to transform chlorogenic acids from green coffee extract into 4-VC in two steps one pot reaction. Thus, the enzymatic potential of certain lactobacilli might be explored to generate flavor compounds from plant polyphenols. PMID:23692950

  13. Coexpressing Escherichia coli Cyclopropane Synthase with Sterculia foetida Lysophosphatidic Acid Acyltransferase Enhances Cyclopropane Fatty Acid Accumulation1[W][OPEN

    PubMed Central

    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

  14. Decreased Polysaccharide Feruloylation Compromises Plant Cell Wall Integrity and Increases Susceptibility to Necrotrophic Fungal Pathogens

    PubMed Central

    Reem, Nathan T.; Pogorelko, Gennady; Lionetti, Vincenzo; Chambers, Lauran; Held, Michael A.; Bellincampi, Daniela; Zabotina, Olga A.

    2016-01-01

    The complexity of cell wall composition and structure determines the strength, flexibility, and function of the primary cell wall in plants. However, the contribution of the various components to cell wall integrity (CWI) and function remains unclear. Modifications of cell wall composition can induce plant responses known as CWI control. In this study, we used transgenic expression of the fungal feruloyl esterase AnFAE to examine the effect of post-synthetic modification of Arabidopsis and Brachypodium cell walls. Transgenic Arabidopsis plants expressing AnFAE showed a significant reduction of monomeric ferulic acid, decreased amounts of wall-associated extensins, and increased susceptibility to Botrytis cinerea, compared with wild type. Transgenic Brachypodium showed reductions in monomeric and dimeric ferulic acids and increased susceptibility to Bipolaris sorokiniana. Upon infection, transgenic Arabidopsis and Brachypodium plants also showed increased expression of several defense-related genes compared with wild type. These results demonstrate a role, in both monocot and dicot plants, of polysaccharide feruloylation in plant CWI, which contributes to plant resistance to necrotrophic pathogens. PMID:27242834

  15. Testing for 'threads' and leucocyte esterase in first-void urine to exclude the diagnosis of non-specific urethritis in asymptomatic men.

    PubMed

    Pallawela, Sanjeeva N S; Sonnex, Christopher; Burdett, Julia; Cooper, Dawn; Nethercott, Katrina; Thomas, Catherina M; Goon, Peter; Webb, Hayley; Carne, Christopher

    2014-07-01

    Recent evidence suggests that asymptomatic nonspecific urethritis (NSU), which is not routinely tested for, is a clinically significant pathology.The aim of this pilot study was to determine if testing for urinary threads, leucocyte esterase (LE) or both in asymptomatic men is a good screening tool for NSU. Of the126 asymptomatic men, 8% met microscopic criteria for the diagnosis of NSU. The positive predictive value for NSU was 71% (95% confidence interval (CI): 29.3-95.5%) and the negative predictive value was 96% (95% CI: 92.8-99.5%). The absence of threads and negative LE makes urethritis highly unlikely, making urinary chlamydia (Chlamydia trachomatis) and gonorrhoea (Neisseria gonorrhoeae) testing sufficient. Incidental findings of further pathology occurred in 7%.

  16. A whole genome screening and RNA interference identify a juvenile hormone esterase-like gene of the diamondback moth, Plutella xylostella.

    PubMed

    Gu, Xiaojun; Kumar, Sunil; Kim, Eunjin; Kim, Yonggyun

    2015-09-01

    Juvenile hormone (JH) plays a crucial role in preventing precocious metamorphosis and stimulating reproduction. Thus, its hemolymph titer should be under a tight control. As a negative controller, juvenile hormone esterase (JHE) performs a rapid breakdown of residual JH in the hemolymph during last instar to induce a larval-to-pupal metamorphosis. A whole genome of the diamondback moth (DBM), Plutella xylostella, has been annotated and proposed 11 JHE candidates. Sequence analysis using conserved motifs commonly found in other JHEs proposed a putative JHE (Px004817). Px004817 (64.61 kDa, pI=5.28) exhibited a characteristic JHE expression pattern by showing high peak at the early last instar, at which JHE enzyme activity was also at a maximal level. RNA interference of Px004817 reduced JHE activity and interrupted pupal development with a significant increase of larval period. This study identifies Px004817 as a JHE-like gene of P. xylostella.

  17. Δ6-fatty acid desaturase and fatty acid elongase mRNA expression, phagocytic activity and weight-to-length relationships in channel catfish (Ictalurus punctatus) fed alternative diets with soy oil and a probiotic.

    PubMed

    Santerre, A; Téllez-Bañuelos, M C; Casas-Solís, J; Castro-Félix, P; Huízar-López, M R; Zaitseva, G P; Horta-Fernández, J L; Trujillo-García, E A; de la Mora-Sherer, D; Palafox-Luna, J A; Juárez-Carrillo, E

    2015-09-22

    A time-course feeding trial was conducted for 120 days on juvenile channel catfish (Ictalurus punctatus) to study the effects of diets differing in oil source (fish oil or soy oil) and supplementation with a commercial probiotic. Relative levels of Δ6-fatty acid desaturase (Δ6-FAD) and fatty acid elongase (FAE) expression were assessed in brain and liver tissues. Both genes showed similar expression levels in all groups studied. Fish weight-to-length relationships were evaluated using polynomial regression analyses, which identified a burst in weight and length in the channel catfish on day 105 of treatment; this increase was related to an increase in gene expression. Mid-intestinal lactic acid bacterium (LAB) count was determined according to morphological and biochemical criteria using API strips. There was no indication that intestinal LAB count was affected by the modified diets. The Cunningham glass adherence method was applied to evaluate phagocytic cell activity in peripheral blood. Reactive oxygen species (ROS) generation was assessed through the respiratory burst activity of spleen macrophages by the NBT reduction test. Probiotic-supplemented diets provided a good substrate for innate immune system function; the phagocytic index was significantly enhanced in fish fed soy oil and the probiotic, and at the end of the experimental period, ROS production increased in fish fed soy oil. The substitution of fish oil by soy oil is recommended for food formulation and will contribute to promoting sustainable aquaculture. Probiotics are also recommended for channel catfish farming as they may act as immunonutrients.

  18. Evaluation of liver and brain esterases in the spotted gar fish (Lepisosteus oculatus) as biomarkers of effect in the lower Mississippi River basin

    SciTech Connect

    Huang, T.L.; Obih, P.O.; Jaiswal, R.

    1997-05-01

    The responses of various xenobiotic metabolizing enzymes in fish models are rapidly evolving as important biomarkers for monitoring unacceptable levels of environmental contaminants. Ethoxyresorufin O-deethylase, a specific cytochrome P450-dependent monooxygenase, is often used as an indicator of polycyclic aromatic hydrocarbon pollution. Another class of enzymes which are potential biomarkers are the B-type esterases. These enzymes are sensitive to inhibition by organophosphates, and include the cholinesterases (ChE) and carboxylesterases. ChEs are further subdivided into acetylcholinesterase and butyryl cholinesterase. Among fish, AChE is predominantly localized in the brain and muscle, whereas, BuChE activity is found mainly in liver and plasma. The precise physiological role of BuChE is unknown, although it has been regarded as a marker enzyme for glial or supportive cells or other non-neuronal elements. Inhibition of ChE activity has often been associated with exposure to organophosphate and carbamate insecticides and other neurotoxic xenobiotics. Chemicals other than carbarnates and organophosphates that are environmental contaminants can also affect the activity of ChEs. Carboxylesterases represent a heterogenous group of isozymes that can catalyze the hydrolysis of a wide range of xenobiotic esters, amides and thioesters. For most CaE, their natural substrates are unknown, therefore, their physiological functions remain to be elucidated. These enzymes (CaE) occur widely in most tissues and are generally found in high levels in the liver. The purpose of this research was to evaluate the liver and brain esterases in the spotted gar fish as biomarkers of effect to multiple contaminants in the lower Mississippi River basin. 15 refs., 3 figs., 2 tabs.

  19. Flow cytometric assessment of cell structural and functional changes induced by acetic acid in the yeasts Zygosaccharomyces bailii and Saccharomyces cerevisiae.

    PubMed

    Prudêncio, C; Sansonetty, F; Côrte-Real, M

    1998-04-01

    Flow cytometry (FCM) was used with different viability dyes to assess changes in cell structure and function induced by acetic acid (AA) in populations of Zygosaccharomyces bailii (AA resistant) and Saccharomyces cerevisiae (AA sensitive). Kinetic changes in esterase activity, intracellular dye processing, and membrane integrity were monitored, and to detect those changes we used three assays involving fluorescein diacetate hydrolysis, FUN-1 processing, and propidium iodide exclusion, respectively. In S. cerevisiae, the decrease in the ability to process FUN-1 preceded the decrease in esterase activity, and there was loss of cell membrane integrity after incubation with AA. In Z. bailii, with higher AA concentrations, there was a similar decrease in the ability to process FUN-1, which also preceded the loss of cell membrane integrity. Changes in esterase activity in this yeast induced by AA treatment could not be monitored because the changes occurred independently of the presence of the acid. For control samples (untreated cells killed with 10% v/v of AA), the percentages of nonaltered cells as estimated by FCM and percentages of viable cells as estimated by colony forming unit (CFU) counts were identical. However, for cell samples treated for short periods with 3% (v/v) or less of AA, none of the dyes produced FCM results comparable to those produced by CFU counts.

  20. Fractionation of corn fiber treated by soaking in aqueous ammonia (SAA) for isolation of hemicellulose B and production of C5 sugars by enzyme hydrolysis.

    PubMed

    Nghiem, Nhuan P; Montanti, Justin; Johnston, David B; Drapcho, Caye

    2011-08-01

    A process was developed to fractionate and isolate the hemicellulose B component of corn fiber generated by corn wet milling. The process consisted of pretreatment by soaking in aqueous ammonia followed by enzymatic cellulose hydrolysis, during which the hemicellulose B was solubilized by cleavage into xylo-oligosaccharides and subsequently recovered by precipitation with ethanol. The pretreatment step resulted in high retention of major sugars and improvement of subsequent enzymatic hydrolysis. The recovered hemicellulose B was hydrolyzed by a cocktail of enzymes that consisted of β-glucosidase, pectinase, xylanase, and ferulic acid esterase (FAE). Xylanase alone was ineffective, demonstrating yields of less than 2% of xylose and arabinose. The greatest xylose and arabinose yields, 44% and 53%, respectively, were obtained by the combination of pectinase and FAE. A mass balance accounted for 87% of the initially present glucan, 91% of the xylan, and 90% of the arabinan. The developed process offered a means for production of corn fiber gum as a value-added co-product and C5 sugars, which could be converted to other valuable co-products through fermentation in a corn wet-milling biorefinery.

  1. THE CESA (CE3B) CARBOXY-TERMINAL DOMAIN OF RUMINOCOCCUS FLAVEFACIENS 17 HAS GLUCURONOYL ESTERASE ACTIVITY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several types of covalent linkages between lignin and xylan in plant cell walls have been shown. One of such linkages could be an ester bond between hydroxyl groups of lignin moieties and the carboxyl group of the 4-O-methyl-D-glucuronic acid (MeGlcA) side groups of glucuronoxylan. Enzymes capable...

  2. Wounding coordinately induces cell wall protein, cell cycle and pectin methyl esterase genes involved in tuber closing layer and wound periderm development.

    PubMed

    Neubauer, Jonathan D; Lulai, Edward C; Thompson, Asunta L; Suttle, Jeffrey C; Bolton, Melvin D

    2012-04-15

    Little is known about the coordinate induction of genes that may be involved in agriculturally important wound-healing events. In this study, wound-healing events were determined together with wound-induced expression profiles of selected cell cycle, cell wall protein, and pectin methyl esterase genes using two diverse potato genotypes and two harvests (NDTX4271-5R and Russet Burbank tubers; 2008 and 2009 harvests). By 5 d after wounding, the closing layer and a nascent phellogen had formed. Phellogen cell divisions generated phellem layers until cessation of cell division at 28 d after wounding for both genotypes and harvests. Cell cycle genes encoding epidermal growth factor binding protein (StEBP), cyclin-dependent kinase B (StCDKB) and cyclin-dependent kinase regulatory subunit (StCKS1At) were induced by 1 d after wounding; these expressions coordinated with related phellogen formation and the induction and cessation of phellem cell formation. Genes encoding the structural cell wall proteins extensin (StExt1) and extensin-like (StExtlk) were dramatically up-regulated by 1-5 d after wounding, suggesting involvement with closing layer and later phellem cell layer formation. Wounding up-regulated pectin methyl esterase genes (StPME and StPrePME); StPME expression increased during closing layer and phellem cell formation, whereas maximum expression of StPrePME occurred at 5-14 d after wounding, implicating involvement in later modifications for closing layer and phellem cell formation. The coordinate induction and expression profile of StTLRP, a gene encoding a cell wall strengthening "tyrosine-and lysine-rich protein," suggested a role in the formation of the closing layer followed by phellem cell generation and maturation. Collectively, the genes monitored were wound-inducible and their expression profiles markedly coordinated with closing layer formation and the index for phellogen layer meristematic activity during wound periderm development; results were more

  3. Probing the active sites of butyrylcholinesterase and cholesterol esterase with isomalathion: conserved stereoselective inactivation of serine hydrolases structurally related to acetylcholinesterase.

    PubMed

    Doorn, J A; Talley, T T; Thompson, C M; Richardson, R J

    2001-07-01

    Previous work has shown that acetylcholinesterase (AChE), a member of the alpha/beta-hydrolase superfamily, is stereoselectively inhibited by the four stereoisomers of isomalathion. Recent kinetic and mass spectral data demonstrated that a difference in mechanism of inactivation exists for AChE treated with (1R)- versus (1S,3S)-stereoisomers. This study sought to determine whether other alpha/beta-hydrolases are stereoselectively inhibited by isomalathion and if the difference in mechanism of AChE inactivation between (1R)- and (1S,3S)-isomers is conserved for other alpha/beta-hydrolases. Bimolecular rate constants of inhibition (k(i)) were measured for human and equine butyrylcholinesterase (HBChE and EBChE, respectively) and bovine cholesterol esterase (BCholE) with all four isomers. Isomalathion isomers inhibited these enzymes with the following order of potency: (1R,3R) > (1R,3S) > (1S,3R) > or = (1S,3S). Ratios of k(i) values for the most potent to the least potent isomer were 10.5 (HBChE), 11.9 (EBChE), and 68.6 (BCholE). Rate constants of reactivation (k(3)) were measured for enzyme inhibited by isomalathion isomers. HBChE, EBChE, and BCholE inactivated by the (1R)-isomers readily reactivated. However, enzymes modified by (1S)-isomalathions were refractory toward reactivation, and k(3) values were not significantly different from zero for HBChE and BCholE treated with the (1S,3S)-isomer. Computer-based docking experiments were performed for BCholE with (1R,3R)- and (1S,3S)-enantiomers. Calculated structures predicted a difference in primary leaving group: diethyl thiosuccinate for (1R,3R)-isomalathion and thiomethyl for the (1S,3S)-isomer. The data demonstrate that the alpha/beta-hydrolases used in this study are stereoselectively inhibited by isomalathion. Furthermore, the results suggest that the mechanistic shift demonstrated to occur for inhibition of AChE by (1R)- versus (1S,3S)-isomers is conserved for butyrylcholinesterase and cholesterol esterase.

  4. Development of ultra-high erucic acid oil in the industrial oil crop Crambe abyssinica.

    PubMed

    Li, Xueyuan; van Loo, Eibertus N; Gruber, Jens; Fan, Jing; Guan, Rui; Frentzen, Margrit; Stymne, Sten; Zhu, Li-Hua

    2012-09-01

    Erucic acid (22 : 1) is a major feedstock for the oleochemical industry. In this study, a gene stacking strategy was employed to develop transgenic Crambe abyssinica lines with increased 22 : 1 levels. Through integration of the LdLPAAT, BnFAE1 and CaFAD2-RNAi genes into the crambe genome, confirmed by Southern blot and qRT-PCR, the average levels of 18 : 1, 18 : 2 and 18 : 3 were markedly decreased and that of 22 : 1 was increased from 60% in the wild type to 73% in the best transgenic line of T4 generation. In single seeds of the same line, the 22 : 1 level could reach 76.9%, an increase of 28.0% over the wild type. The trierucin amount was positively correlated to 22 : 1 in the transgenic lines. Unlike high erucic rapeseed, the wild-type crambe contains 22 : 1 in the seed phosphatidylcholine and in the sn-2 position of triacylglycerols (5% and 8%, respectively). The transgenic line with high 22 : 1 had decreased 22 : 1 level in phosphatidylcholine, and this was negatively correlated with the 22 : 1 level at the sn-2 position of TAG. The significances of this study include (i) achieving an unprecedented level of 22 : 1 in an oil crop; (ii) disclosing mechanisms in the channelling of a triacylglycerol-specific unusual fatty acid in oil seeds; (iii) indicating potential limiting factors involved in the erucic acid biosynthesis and paving the way for further increase of this acid and (iv) development of an added value genetically modified oil crop having no risk of gene flow into feed and food crops.

  5. Structure and organization of amplicons containing the E4 esterase genes responsible for insecticide resistance in the aphid Myzus persicae (Sulzer).

    PubMed Central

    Field, L M; Devonshire, A L

    1997-01-01

    Insecticide resistance in the aphid Myzus persicae results primarily from the amplification of genes encoding the insecticide-detoxifying esterase, E4. Here we report the analysis of flanking DNA co-amplified with the E4 gene. The 5' end of this gene has an untranslated leader sequence interspersed by two introns, and the promoter region lacks TATA and CAAT boxes. The DNA breakpoint involved in the generation of the amplification is just upstream (approx. 250 bp) of the putative E4 transcription start site; thus the E4 gene is very close to the 5' end of the approx. 24 kb amplicon. PCR primers specific to the 'novel joint' generated during the amplification have been used to show that a wide range of aphid clones have the same amplicons, arranged as a series of head-to-tail direct repeats. Long-distance mapping has revealed the structure of these repeats. This has important implications for understanding both the generation of the amplified genes and the origin and spread of insecticide resistance in M. persicae. PMID:9148762

  6. Structural and functional analysis of a low-temperature-active alkaline esterase from South China Sea marine sediment microbial metagenomic library.

    PubMed

    Hu, Yongfei; Liu, Yinghui; Li, Jing; Feng, Yanbin; Lu, Na; Zhu, Baoli; Xue, Song

    2015-11-01

    A low-temperature-active alkaline esterase, Est12, from a marine sediment metagenomic fosmid library was identified. Est12 prefers short- and middle-chain p-nitrophenol esters as substrate with optimum temperature and pH value of 50 °C and 9.0, respectively, and nearly 50 % of maximum activity retained at 5 °C. The hydrolysis activity of Est12 was stable at 40 °C. Ca(2+) especially activated the activity of Est12 to about 151 % of the control. DEPC and PMSF inhibited the activity of Est12 to 34 and 25 %, respectively. In addition, Est12 was more tolerable to methanol compared to other organic solvents tested. The crystal structure of Est12 at 1.39 Å resolution showed that the cap domain which is composed of an α-helix and a flexible region resulted in a relatively wide spectrum of substrate, with p-nitrophenol caproate as the preferred one. Furthermore, the flexible cap domain and the high percentage of Gly, Ser, and Met may play important roles in the adaptation of Est12 to low temperature.

  7. Atomic insight into designed carbamate-based derivatives as acetylcholine esterase (AChE) inhibitors: a computational study by multiple molecular docking and molecular dynamics simulation.

    PubMed

    Mohammadi, Tecush; Ghayeb, Yousef

    2017-01-11

    Over 100 variants have been designed and studied, using multiple docking methods such as Autodock Vina, ArgusLab, Molegro Virtual Docker, and Hex-Cuda, to study the effect of alteration in the structure of carbamate-based acetylcholyne esterase (AChE) inhibitors. Sixteen selected systems were then subjected to 14 ns molecular dynamics (MD) simulations. Results from all the docking methods are in agreement. Variants that involved biphenyl substituents possess the most negative binding energies in the -37.64 to -39.31 kJ mol(-1) range due to their π-π interactions with AChE aromatic residues. The root mean square deviation values showed that all of these components achieved equilibration after 6 ns. Gyration radius (Rg) and solvent accessibility surface area were calculated to further investigate the AChE conformational changes in the presence of these components. MD simulation results suggested that these components might interact with AChE, possibly with no major changes in AChE secondary and tertiary structures.

  8. Cytochemical analysis of alkaline phosphatase and esterase activities and of lectin-binding and anionic sites in rat and mouse Peyer's patch M cells.

    PubMed

    Owen, R L; Bhalla, D K

    1983-10-01

    M cells in Peyer's patch follicle epithelium endocytose and transport luminal materials to intraepithelial lymphocytes. We examined (1) enzymatic characteristics of the epithelium covering mouse and rat Peyer's patches by using cytochemical techniques, (2) distribution of lectin-binding sites by peroxidase-labeled lectins, and (3) anionic site distribution by using cationized ferritin to develop a profile of M cell surface properties. Alkaline phosphatase activity resulted in deposits of dense reaction product over follicle surfaces but was markedly reduced over M cells, unlike esterase which formed equivalent or greater product over M cells. Concanavalin A, ricinus communis agglutinin, wheat germ agglutinin and peanut agglutinin reacted equally with M cells and with surrounding enterocytes over follicle surfaces. Cationized ferritin distributed in a random fashion along microvillus membranes of both M cells and enterocytes, indicating equivalent anionic site distribution. Staining for alkaline phosphatase activity provides a new approach for distinguishing M cells from enterocytes at the light microscopic level. Identical binding of lectins indicates that M cells and enterocytes share common glycoconjugates even though molecular groupings may differ. Lectin binding and anionic charge similarities of M cells and enterocytes may facilitate antigen sampling by M cells of particles and compounds that adhere to intestinal surfaces in non-Peyer's patch areas.

  9. Expression and properties of three novel fungal lipases/sterol esterases predicted in silico: comparison with other enzymes of the Candida rugosa-like family.

    PubMed

    Vaquero, María Eugenia; Prieto, Alicia; Barriuso, Jorge; Martínez, María Jesús

    2015-12-01

    Lipases from the Candida rugosa-like family are enzymes with great biotechnological interest. In a previous work, several enzymes from this family were identified by in silico mining of fungal genomes. Here, we describe the cloning, expression, and characterization of putative lipases from the genomes of Nectria haematococca, Trichoderma reesei, and Aspergillus niger and compared their catalytic properties with those of OPE, a well-characterized sterol esterase/lipase from Ophiostoma piceae. All of them hydrolyzed p-nitrophenol esters and triglyceride