Showing papers on "Esterase published in 2003"
TL;DR: The results indicated that the insecticide resistance observed in the field strain was due to multiple resistance mechanisms, including increased detoxification of these insecticides by microsomal oxidases, glutathione S -transferases, hydrolases and reductases, and target site insensitivity such as insensitive acetylcholinesterase.
197 citations
TL;DR: A genomic DNA library was made from the alkaliphilic cellulase-producing Bacillus agaradhaerans in order to prove the technologies for gene isolation prior to using them with samples of DNA isolated directly from environmental samples.
Abstract: A genomic DNA library was made from the alkaliphilic cellulase-producing Bacillus agaradhaerans in order to prove our technologies for gene isolation prior to using them with samples of DNA isolated directly from environmental samples. Clones expressing a cellulase activity were identified and sequenced. A new cellulase gene was identified. Genomic DNA libraries were then made from DNA isolated directly from the Kenyan soda lakes, Lake Elmenteita and Crater Lake. Crater Lake clones expressing a cellulase activity and Lake Elmenteita clones expressing a lipase/esterase activity were identified and sequenced. These were encoded by novel genes as judged by DNA sequence comparisons. Genomic DNA libraries were also made from laboratory enrichment cultures of Lake Nakuru and Lake Elmenteita samples. Selective enrichment cultures were grown in the presence of carboxymethylcellulose (CMC) and olive oil. A number of new cellulase and lipase/esterase genes were discovered in these libraries. Cellulase-positive clones from Lake Nakuru were isolated at a frequency of 1 in 15,000 from a library made from a CMC enrichment as compared to 1 in 60,000 from a minimal medium enrichment. Esterase/lipase-positive clones from Lake Elmenteita were isolated with a frequency of 1 in 30,000 from a library made from an olive-oil enrichment as compared to 1 in 100,000 from an environmental library.
144 citations
TL;DR: Determination of k(cat)/K(m) revealed that the enzyme hydrolysed methyl sinapinate 6, 21 and 40 times more efficiently than methyl ferulate, methyl coumarate and methyl caffeate, respectively, indicating a synergistic interaction between FAE-II and S. thermophile xylanase.
126 citations
TL;DR: The enzyme was able to hydrolyze both soluble and insoluble emulsified substrates and was classified as a lipase, expressing some esterase activity as well, and the most advantageous method for immobilization was found to be ionic binding to DEAE Cellulose.
Abstract: Extracellular thermostable lipase produced by the thermophilic Bacillus stearothermophilus MC 7 was purified to 19.25-fold with 10.2% recovery. The molecular weight of the purified enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was shown to be 62 500 Da. The purified enzyme expressed maximum activity at 75-80 OC and its half life was 30min at 70 OC. The K,,, and V,,,,, were calculated to be, respectively, 0.33 mM and 188 pMmin-' mg-' withp-nitrophenyl palmitate @NPP) as a substrate. Enzyme activity was inhibited by divalent ions of heavy metals, thiol and serine inhibitors, whereas calcium ion stimulated its activity. The most advantageous method for immobilization was found to be ionic binding to DEAE Cellulose. The enzyme was able to hydrolyze both soluble and insoluble emulsified substrates and was classified as a lipase, expressing some esterase activity as well. 0 2003 Elsevier Science B.V. All rights reserved.
120 citations
TL;DR: The substrate specificity of selected enzymes classified under Carbohydrate Esterase family 4 (CE4) has been examined and the activities of all enzymes examined were significantly increased in the presence of Co(2+) when chitinous substrates were employed and inactive towards cell wall peptidoglycan.
118 citations
TL;DR: The interaction of both enantiomers with the substrate binding sites of acetylcholinesterases and p‐nitrobenzyl esterase from Bacillus subtilis was modeled and correlated to experimental enantioselectivity, and enantiopreference and ranking by enantiOSElectivity could be predicted by the model.
Abstract: Carboxylesterases containing the sequence motif GGGX catalyze hydrolysis of esters of chiral tertiary alcohols, albeit at only low to moderate enantioselectivity towards three model substrates (linalyl acetate, methyl-1-pentin-1-yl acetate, 2-phenyl-3-butin-2-yl acetate). In order to understand the molecular mechanism of enantiorecognition and to improve enantioselectivity towards this interesting substrate class, the interaction of both enantiomers with the substrate binding sites of acetylcholinesterases and p-nitrobenzyl esterase from Bacillus subtilis was modeled and correlated to experimental enantioselectivity. For all substrate-enzyme pairs, enantiopreference and ranking by enantioselectivity could be predicted by the model. In p-nitrobenzyl esterase, one of the key residues in determining enantioselectivity was G105: exchange of this residue by alanine led to a six-fold increase of enantioselectivity (E=19) towards 2-phenyl-3-butin-2-yl acetate. However, the effect of this mutation is personalized: towards the substrate linalyl acetate, the same mutant had a reversed enantiopreference. Thus, depending on the substrate structure, the same mutant had either increased enantioselectivity or opposite enantiopreference compared to wild type enzyme.
112 citations
TL;DR: The findings suggested that protein binding on the surfaces was saturated even though enzyme degradation did not achieve saturation on any of the surfaces, which highlights a need for further study in this area.
109 citations
93 citations
TL;DR: Surprisingly, lipase 2 is a more efficient esterase than lipase 3, showing a twofold specific activity against cholesteryl [1-14C]oleate in the authors' experimental conditions.
91 citations
TL;DR: The structure of an active site mutant in complex with the reaction product, acetate, reveals details of the putative oxyanion binding site, and suggests that substrates bind predominantly through non-specific contacts with protein hydrophobic residues.
85 citations
TL;DR: It is suggested that ferulic acid may be located in regions of low substitution on arabinoxylans while the 5,5′ diferulate moiety is located in more branched regions of the xylan chain.
TL;DR: The pure feruloyl esterase transforms both 2-O and 5-O arabinofuranosidase-linked ferulate equally well and shows high activity on the substrates but reveals only low activity on p-nitrophenyl-butyrate.
Abstract: The lignocellulolytic fungus Aureobasidium pullulans NRRL Y 2311-1 produces feruloyl esterase activity when grown on birchwood xylan. Feruloyl esterase was purified from culture supernatant by ultrafiltration and anion-exchange, hydrophobic interaction, and gel filtration chromatography. The pure enzyme is a monomer with an estimated molecular mass of 210 kDa in both native and denatured forms and has an apparent degree of glycosylation of 48%. The enzyme has a pI of 6.5, and maximum activity is observed at pH 6.7 and 60°C. Specific activities for methyl ferulate, methyl p-coumarate, methyl sinapate, and methyl caffeate are 21.6, 35.3, 12.9, and 30.4 μmol/min/mg, respectively. The pure feruloyl esterase transforms both 2-O and 5-O arabinofuranosidase-linked ferulate equally well and also shows high activity on the substrates 4-O-trans-feruloyl-xylopyranoside, O-{5-O-[(E)-feruloyl]-α-l-arabinofuranosyl}-(1,3)-O-β-d-xylopyranosyl-(1,4)-d-xylopyranose, and p-nitrophenyl-acetate but reveals only low activity on p-nitrophenyl-butyrate. The catalytic efficiency (kcat/Km) of the enzyme was highest on methyl p-coumarate of all the substrates tested. Sequencing revealed the following eight N-terminal amino acids: AVYTLDGD.
TL;DR: Mixtures containing apoemulsan and either the catalytically active soluble form of the recombinant esterase isolated from cell extracts or the solubilized inactiveform of the enzyme recovered from the inclusion bodies formed stable oil-water emulsions with very hydrophobic substrates such as hexadecane under conditions in which emulsan itself was ineffective.
Abstract: The oil-degrading microorganism Acinetobacter venetianus RAG-1 produces an extracellular polyanionic, heteropolysaccharide bioemulsifier termed emulsan. Emulsan forms and stabilizes oil-water emulsions with a variety of hydrophobic substrates. Removal of the protein fraction yields a product, apoemulsan, which exhibits much lower emulsifying activity on hydrophobic substrates such as n-hexadecane. One of the key proteins associated with the emulsan complex is a cell surface esterase. The esterase (molecular mass, 34.5 kDa) was cloned and overexpressed in Escherichia coli BL21(DE3) behind the phage T7 promoter with the His tag system. After overexpression, about 80 to 90% of the protein was found in inclusion bodies. The overexpressed esterase was recovered from the inclusion bodies by solubilization with deoxycholate and, after slow dialysis, was purified by metal chelation affinity chromatography. Mixtures containing apoemulsan and either the catalytically active soluble form of the recombinant esterase isolated from cell extracts or the solubilized inactive form of the enzyme recovered from the inclusion bodies formed stable oil-water emulsions with very hydrophobic substrates such as hexadecane under conditions in which emulsan itself was ineffective. Similarly, a series of esterase-defective mutants were generated by site-directed mutagenesis, cloned, and overexpressed in E. coli. Mutant proteins defective in catalytic activity as well as others apparently affected in protein conformation were also active in enhancing the apoemulsan-mediated emulsifying activity. Other proteins, including a His-tagged overexpressed esterase from the related organism Acinetobacter calcoaceticus BD4, showed no enhancement.
TL;DR: The aim is to test the anaerobic fungus, Piromyces sp.
Abstract: S . S . P A U L , D . N . K A M R A , V . R . B . S A S T R Y , N . P . S A H U A N D A . K U M A R . 2003. Aims: To test the anaerobic fungus, Piromyces sp. FNG5, for its tolerance to phenolic monomers released in the rumen by degradation of lignocellulosic poor-quality feeds. Methods and Results: Effects of phenolic monomers on biomass and fibrolytic enzyme activities of a pure culture of lignocellulolytic anaerobic fungus (Piromyces sp. FNG5) isolated from faeces of wild nil gai (blue bull, Baselophus tragocamelus) were evaluated. There was a reduction in fungal biomass at 1 mM M concentration of catechol with complete inhibition at 10 mM. p-Coumaric acid caused a reduction in biomass at 10 mM and no growth was observed above 20 mM concentration. The fungal isolate could tolerate up to 5 mM of ferulic acid without any reduction in biomass level, and was able to grow to some extent up to the highest level of ferulic acid tested (20 mM). Vanillic acid had no effect on biomass of the fungus even up to 50 mM M level. The phenolic monomers varied in their potential to inhibit the secretion of carboxymethyl cellulase, xylanase, b-glucosidase and acetyl esterase activities with catechol being the most inhibitory and vanillic acid being the least inhibitory. After 14 days of incubation, 38AE49‐65AE14% p-Coumaric acid, 65AE22‐74AE10% ferulic acid and 34AE13‐66AE78% vanillic acid disappeared from the medium under anaerobic conditions. Conclusions, Significance and Impact of the Study: It is concluded that the anaerobic fungus Piromyces sp. FNG5 is tolerant to phenolic monomers and has ability to degrade them. Therefore, such anaerobic fungi may play an important role in fibre degradation in the rumen.
TL;DR: The recombinant protein cloned into Escherichia coli showed both esterase activity and acylamino acid-releasing enzyme (AARE) activities and was extremely stable and protein concentration-dependent.
Abstract: The gene APE1547 of the aerobic thermophilic Aeropyrum pernix K1 encoding 582 amino acid residues was cloned into Escherichia coli. BL21 (DE3) by using vector pET11a with a T7 promoter. An alignment of similarity analysis of APE1547 with protein sequences from A. pernix K1 databank revealed that it showed a lipase motif and low homology with the known thermophilic esterases. However, it had a high degree homology with several acyl amino acid-releasing enzymes. After purified by ion exchange chromatography and gel filtration chromatography, the recombinant protein showed both esterase activity and acylamino acid-releasing enzyme (AARE) activities. The optimum of temperature and pH of the esterase activity are 90 °C and 8.0, respectively. The recombinant protein showed the hydrolytic activity for a wide range of substrates, such as p-nitrophenyl alkanoate esters of varying alkyl chain lengths, pNA-labelled amino acid and peptide. The highest activity was observed for the substrate p-nitrophenyl caprylate. The recombinant enzyme was extremely stable and protein concentration-dependent. Its half-life at 90 °C was over 160 h. at the concentration of 2.14 mg/ml, which renders this new esterase very attractive for biotechnological applications.
TL;DR: The HerE complex reveals the structural basis for the broad scope inhibition of serine hydrolases by As(V)-containing organic compounds and provides the first example of an As-containing compound in a serine esterase active site and the first examples of covalent modification of Serine by arsenic.
TL;DR: The present investigation was carried out for increasing the yield of tannase of Aspergillus niger and the physico‐chemical characterization of this enzyme, and tannic acid was the best substrate for three substrates tested, followed by methyl gallate and propyl gallate.
Abstract: The present investigation was carried out for increasing the yield of tannase of Aspergillus niger and the physico-chemical characterization of this enzyme. the extraction of enzyme protein. However, extraction of fungal pigments and proteins was observed to have high pH dependence, and maximum enzyme extraction was obtained at pH 5.5. The two-step purification protocol gave 51-fold purified enzyme with a yield of 20%. The total tannase activity was made up of nearly equal activity of esterase and depsidase. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of purified tannase protein indicated it to be made up of two polypeptides of molecular weight 102 and 83 kDa. Based on the Michaelis-Menten constant (Km) of tannase for three substrates tested, tannic acid was the best substrate with Km of 2.8 x 10(-4) M, followed by methyl gallate and propyl gallate. The inhibition was maximum for CaCl2 (58%) whereas EDTA had no modulatory effect on tannase activity. The inhibitor binding constant (KI) of CaCl2 was 5.9 x 10(-4) M Homogenization and detergent pretreatments did not have any remarkable effect on and the inhibition was of noncompetitive type.
TL;DR: The 3.9 kb chromosomal DNA was cloned from Serratia marcescens Sr41, which confers on Escherichia coli cells a phenotype of clear halo formation on tributyrin agar plates, and one ORF was demonstrated to encode a 28 kDa protein of 255 amino acids related to esterase activity.
TL;DR: The toxic effects of the final degradation products were investigated, using various recombinant bioluminescent bacteria and, as a result, the degradation products from yeast esterase were shown to contain a toxic hazard, causing oxidative stress and damage to protein synthesis.
Abstract: The efficiency of two lypolytic enzymes (fungal cutinase, yeast esterase) in the degradation of di-(2-ethylhexyl)-phthalate (DEHP) was investigated. The DEHP-degradation rate of fungal cutinase was surprisingly high, i.e. almost 70% of the initial DEHP (500 mg/l) was decomposed within 2.5 h and nearly 50% of the degraded DEHP disappeared within the initial 15 min. With the yeast esterase, despite the same concentration, more than 85% of the DEHP remained even after 3 days of treatment. During the enzymatic degradation of DEHP, several DEHP-derived compounds were detected and time-course changes in composition were also monitored. During degradation with fungal cutinase, most DEHP was converted into 1,3-isobenzofurandione (IBF) by diester hydrolysis. In the degradation by yeast esterase, two organic chemicals were produced from DEHP: IBF and an unidentified compound (X). The final chemical composition after 3 days was significantly dependent on the enzyme used. Fungal cutinase produced IBF as a major degradation compound. However, in the DEHP degradation by yeast esterase, compound X was produced in abundance in addition to IBF. The toxic effects of the final degradation products were investigated, using various recombinant bioluminescent bacteria and, as a result, the degradation products from yeast esterase were shown to contain a toxic hazard, causing oxidative stress and damage to protein synthesis.
TL;DR: It is demonstrated that additional amino acids at the N-terminus of the FAEC sequence do not influence the catalytic capacity of the enzyme, and that the nature of the signal sequence has a limited effect on the yield of the secreted enzyme.
TL;DR: It is interesting that it is never detected silencing of amplified FE4 genes, possibly because they are at multiple loci and therefore less likely to be subject to synchronous control, but it does suggest that the resistant phenotype in M. persicae is under epigenetic control.
Abstract: Insecticide treatment of the aphid Myzus persicae (Sulzer) has led to the evolution of several insecticide resistance mechanisms, including the detoxification of insecticides by elevated esterases. This results from amplification of one of two closely related esterase genes (E4 or FE4) with up to 80 copies in the most resistant aphids. The amplified E4 genes are at a single site linked to a chromosomal translocation and resistance can be unstable. Individuals within a clone lose their elevated esterase and resistant phenotype, a good example of'clonal variation'. This loss of esterase is accompanied by a loss of the corresponding mRNA but the amplified genes are retained with no detectable sequence differences. However, the expressed E4 genes contain 5-methylcytosine, which is lost at the same time as the genes are turned off. This is in direct contrast with vertebrate genes where DNA methylation causes gene silencing, but it does suggest that the resistant phenotype in M. persicae is under epigenetic control. One hypothesis is that 5-methylcytosine in E4 genes facilitates expression by preventing the production of incorrectly initiated transcripts. It is interesting that we have never detected silencing of amplified FE4 genes, possibly because they are at multiple loci and therefore less likely to be subject to synchronous control.
TL;DR: Investigation of esterase-lipase activity in the digestive gland of redclaw crayfish Cherax quadricarinatus showed that the optimum enzyme activity occurred between 35 and 40 degrees C, with 100 mM NaCl at pH 8.5.
Abstract: Investigation of esterase–lipase activity in the digestive gland of redclaw crayfish Cherax quadricarinatus showed that the optimum enzyme activity occurred between 35 and 40 °C, with 100 mM NaCl at pH 8.5. Heavy metals completely inhibited and calcium ions partially inhibited enzyme activity. The enzyme activity diminished as the length of the fatty acid chain of substrates increased. Molecular masses for four isozymes were 43, 46, 63 and 118 kDa, respectively, as determined by PAGE.
TL;DR: The ability of Streptococcus thermophilus ST1 and 19 other dairy lactic acid bacteria to synthesize esters was investigated in an aqueous environment through a transferase reaction (alcoholysis) in which fatty acyl groups from glycerides were transferred to alcohols.
Abstract: The ability of Streptococcus thermophilus ST1 and 19 other dairy lactic acid bacteria (LAB) to synthesize esters was investigated in an aqueous environment. These LAB were able to synthesize esters from alcohols and glycerides via a transferase reaction (alcoholysis) in which fatty acyl groups from glycerides were transferred to alcohols. S. thermophilus ST1 was active on tributyrin and on di- or monoglycerides of up to C10 with ethanol as the acyl acceptor. This strain was also active on a diglyceride of C6 and monoglyceride of C8 with 2-phenyl ethanol as the acyl acceptor. Alcoholysis occurred preferentially over hydrolysis. S. thermophilus ST1 had an apparent K
m value of 250 mM for ethanol and an apparent K
m value of 1.3 mM for tributyrin, measured against whole cells. Around 80% of both the transferase activity and the esterase activity were detected in the cell-free extract (CFE) of strain ST1. Both activities in the CFEs of five LAB tested were, to a similar degree, enhanced slightly by growth in the presence of ethanol and tributyrin. Using tributyrin and ethanol as substrates, the transferase activities ranged over 0.006–1.37 units/mg cell dry weight among the LAB tested and were both species- and strain-dependent.
TL;DR: Two mutations in carboxylesterase E3 that enable it to hydrolyse the phosphate ester of various organophosphates, plus the carboxlyester in the leaving group in the case of malathion are introduced into cloned genes encoding E3 and EST23 and expressed in vitro with the baculovirus system.
TL;DR: To develop new esterase substrates, a series of α-cyanoesters that yield fluorescent products upon hydrolysis was evaluated for use in carboxylesterase assays, demonstrating the utility of these new substrates as both general and pyrethroid-selective reporters of ester enzyme activity.
TL;DR: The substrate selectivity of esterases, aw, and enzyme type may be important factors in the development of fruity flavors, as evidenced by results in this model system simulating Parmesan cheese ripening conditions.
Journal Article•
TL;DR: Results suggest that temephos resistance could be developed in Ae.
Abstract: Aedes aegypti, at the larval stage, has been subjected to the temephos selection in laboratory. The level of temephos resistance was detected in a microplate by biochemical assay using WHO bioassay technique. The major enzyme-based resistance mechanisms involved in temephos resistance include elevated nonspecific esterase, oxidase and insensitive acetylcholinesterase. After 19 generations of temephos selection, the selected group showed resistance ratios of 4.64 and 16.92, when compared with a non-selected group and the WHO susceptible strain, respectively. The two seperated forms, type form and the pale form of Ae. aegypti showed low levels of resistance to temephos after 19 generations of selection, with resistance ratios of 4.82 and 4.07 for the type form and the pale form, respectively; when compared with the non-selected strain, 17.58 and 14.84, when compared with the WHO susceptible strain. This showed that the type form could develop higher level resistance than the pale form. The esterase inhibitor (S,S,S-tributyl phosphorotrithioate, DEF) or synergist implicated detoxifying esterase in all the temephos selected groups and the presence of elevated esterase were confirmed by biochemical assay. There were significant differences in elevated esterase activity between the temephos selected groups and the non-selected group. However no significant difference between the type form and the pale form was found. Besides the elevated esterase, there was no change in monooxygenase activity and no evidence of insensitive acetylcholinesterease for all temephos selected groups. These results suggest that temephos resistance could be developed in Ae. aegypti under selection pressure and that the main mechanism is based only on esterase detoxification.
TL;DR: The in vivo anticonvulsant and anesthetic activities of prolinate 6a, intraperitoneally administered in water solution, showed that is a water-soluble propofol prodrug candidate for developing formulations useful for parenteral administration.
TL;DR: The electrophoretic assays revealed that aphid feeding elicited a significant increase in superoxide dismutase activity, which served as the control of glutathione S‐transferase activity assays, which suggested that D. noxia feeding imposes not only toxic, but also oxidative stresses on the cereals.
Abstract: Activities of the detoxification enzymes esterase, glutathione S-transferase, and of superoxide dismutase in aphids and aphid-infested cereal leaves were assayed using polyacrylamide gel electrophoresis and a spectrophotometer to elucidate the enzymatic mechanisms of aphid resistance in cereal plants. A chlorosis-eliciting Russian wheat aphid, Diuraphis noxia (Mordvilko), and non-chlorosis-eliciting bird cherry-oat aphid, Rhopalosiphum padi (L.), and four cereals were used in this study. The four cereal genotypes were ‘Arapahoe’ (susceptible) and ‘Halt’ (resistant) wheat (Triticum aestivum L.), ‘Morex’ (susceptible) barley (Hordeum vulgare L.), and ‘Border’ (resistant) oat (Avena sativa L.). Esterase isozymes differed between the two aphid species, although glutathione S-transferase and superoxide dismutase did not. Esterase, glutathione S-transferase, and superoxide dismutase activities in either aphid species were not affected by the level of resistance of a cereal to D. noxia. The assays of cereal leaf samples showed that D. noxia feeding elicited an increase in esterase activity in all four cereal genotypes, although R. padi feeding did not. The increase of esterase activity in cereals, however, was not correlated to aphid resistance in the cereals. The time-series assays of aphid-infested cereal leaves showed that D. noxia-infested Morex barley had a significant increase in esterase activity on all sampling dates (3, 6, and 9 days) in comparison with either uninfested or R. padi-infested barley. No difference in glutathione S-transferase activity was detected among either aphid infestations or sampling dates. The electrophoretic assays, however, revealed that aphid feeding elicited a significant increase in superoxide dismutase activity, which served as the control of glutathione S-transferase activity assays. The increase in esterase and superoxide dismutase activities suggested that D. noxia feeding imposes not only toxic, but also oxidative stresses on the cereals. The ramification of using these enzyme activity data to understand the etiology of D. noxia-elicited chlorosis is discussed.
TL;DR: The results for EST9 and EST23 concur with previous suggestions that the products of the alpha esterase cluster function in digestion and detoxification of xenobiotic esters, but many of the other genes in the cluster show developmental or tissue‐specific expression that seems inconsistent with such roles.
Abstract: Here we show how the 10 genes of the alpha esterase cluster of Drosophila melanogaster have diverged substantially in their expression profiles. Together with previously described sequence divergence this suggests substantial functional diversification. By peptide mass fingerprinting and in vitro gene expression we have also shown that two of the genes encode the isozymes EST9 (formerly ESTC) and EST23. EST9 is the major ‘alpha staining’ esterase in zymograms of gut tissues in feeding stages while orthologues of EST23 confer resistance to organophosphorus insecticides in other higher Diptera. The results for EST9 and EST23 concur with previous suggestions that the products of the alpha esterase cluster function in digestion and detoxification of xenobiotic esters. However, many of the other genes in the cluster show developmental or tissue-specific expression that seems inconsistent with such roles. Furthermore, there is generally poor correspondence between the mRNA expression patterns of the remaining eight genes and isozymes previously characterized by standard techniques of electrophoresis and staining, suggesting that the alpha cluster might only account for a small minority of the esterase isozyme profile.