Showing papers on "Esterase published in 1992"
TL;DR: The Escherichia coli ferric enterobactin esterase gene (fes) was cloned into the vector pGEM3Z under the control of the T7 gene 10 promoter and overexpressed to approximately 15% of the total cellular protein, exhibiting a 4-fold greater activity on the free ligand.
147 citations
TL;DR: In waste water, esterase and dehydrogenase activities were found to correlate with microbial abundance measured as colony forming units of heterotrophic bacteria, suggesting that microbial populations were different, or had different physiological properties, in the two types of sludge.
139 citations
TL;DR: Synergistic effects were observed between the esterase and other xylan-degrading enzymes in hydrolyzing the ferulic and, to a lesser extent, the ϱ-coumaric acid esters from both wheat straw xylan and the grass cell walls.
93 citations
TL;DR: It appears that two distinct haplotypes with an esterase B gene coding an enzyme with identical electrophoretic mobility have been amplified, which are named in mosquitoes from France and Cyprus B4 and B5, respectively.
Abstract: Two new highly active esterases were detected by starch electrophoretic studies in Culex pipiens mosquitoes from the area of Montpellier (France) and from Cyprus. We demonstrate here that both the French and the Cyprus esterases B are overproduced due to amplification of the coding gene. The production of the esterase B is approximately 50- and 500-fold higher in mosquitoes from France and Cyprus, respectively, than in susceptible insects, whereas the number of gene copies is about 25 and 250. Differences of about 7- and 95-fold were also found in the degree of chlorpyrifos resistance. RFLP comparison of the amplified region containing the esterase B gene revealed large differences between French and Cyprus mosquitoes. It thus appears that two distinct haplotypes with an esterase B gene coding an enzyme with identical electrophoretic mobility have been amplified. We therefore named the haplotypes in mosquitoes from France and Cyprus B4 and B5, respectively. The estimated genetic distance between these two haplotypes is not smaller than those observed in all pair comparisons of other known esterase B haplotypes. These results are discussed in the context of amplification phenomena.
91 citations
TL;DR: In this article, the Spanish Serrano dry-cured ham was subjected to post-mortem pork muscle and adipose tissue assays and the results showed that the recovered esterase activity was around 50% of the original activity at the end of the drycuring process.
Abstract: Lipase and esterase activities in post-mortem pork muscle and adipose tissue were assayed. Acid lipases showed optimal activity in the presence of 0.8 mg bovine serum albumin (BSA)/ml and 0.05% (by vol.) Triton X-100, while neutral/basic lipases required 5 mg BSA/ml and no addition of Triton X-100. All lipases had an optimal temperature of 37° C except neutral muscle lipase, which was optimally active at 45° C. A wider range of optimal temperatures of 30–45 and 15–45° C was found for muscle acid esterase activity and neutral esterase activity, respectively. In adipose tissue, higher temperatures of 60° C and 45–75° C were found for maximal acid esterase and neutral esterase activities, respectively. Lipolytic and esterolytic activity assays in muscle and adipose tissue were conducted at four different stages in the processing of Spanish Serrano dry-cured ham. Recovered activities of muscle enzymes were more than 40% of the original activity, even at the end of the drycuring process. In adipose tissue, recovered esterase activity was also around 50% of the original activity at the end of the process, while lipolytic activity was significant only during the post-salting stage.
89 citations
87 citations
TL;DR: It was demonstrated that the esterase substrate p-nitrophenylbutyrate is hydrolyzed by phenmedipham hydrolase, indicating that the gene for phenylcarbamate degradation (pcd) is plasmid encoded.
Abstract: Arthrobacter oxydans P52 isolated from soil samples was found to degrade the phenylcarbamate herbicides phenmedipham and desmedipham cometabolically by hydrolyzing their central carbamate linkages. The phenylcarbamate hydrolase (phenmedipham hydrolase) responsible for the degradative reaction was purified to homogeneity. The enzyme was shown to be a monomer with a molecular weight of 55,000. A 41-kb wild-type plasmid (pHP52) was identified in A. oxydans P52, but not in a derivative of this strain that had spontaneously lost the ability to hydrolyze phenylcarbamates, indicating that the gene for phenylcarbamate degradation (pcd) is plasmid encoded. Determination of two partial amino acid sequences allowed the localization of the coding sequence of the pcd gene on a 3.3-kb PstI restriction fragment within pHP52 DNA by hybridization with synthetic oligonucleotides. The phenylcarbamate hydrolase was functionally expressed in Escherichia coli under control of the lacZ promoter after the 3.3-kb PstI fragment was subcloned into the vector pUC19. A stretch of 1,864 bases within the cloned Pst fragment was sequenced. Sequence analysis revealed an open reading frame of 1,479 bases containing the amino acid partial sequences determined for the purified enzyme. Sequence comparisons revealed significant homology between the pcd gene product and the amino acid sequences of esterases of eukaryotic origin. Subsequently, it was demonstrated that the esterase substrate p-nitrophenylbutyrate is hydrolyzed by phenmedipham hydrolase.
77 citations
76 citations
TL;DR: Forty strains of bacilli were isolated from flat sour evaporated milk and were characterized as Bacillus stearothermophilus (5 strains), Bacillus licheniformis (10 strains, Bacillus coagulans (15 strains).
75 citations
TL;DR: It is demonstrated here that simple isoprenylated cysteine derivatives, such as L-AFCM, L-AGGCM, and ebelactone B all inhibit the demethylation of the endogenous ROS substrates, showing that the same enzymatic activity is involved in the processing of the synthetic and physiological substrates.
Abstract: Proteins from eucaryotic cells which have a carboxyl-terminal CAAX motif are posttranslationally modified by isoprenylation. The pathway involves the linkage of an all-trans-farnesyl (C15) or an all-trans-geranylgeranyl (C20) moiety to the cysteine residue followed by proteolysis which generates the modified cysteine as the carboxyl-terminal residue. Carboxylmethylation of the modified cysteine residue completes the pathway. This latter methylation reaction is the only potentially reversible reaction in the pathway and thus of possible regulatory significance. A specific esterase is required to reverse the methylation. It is demonstrated here that simple isoprenylated cysteine derivatives, such as N-acetyl-S-farnesyl-L-cysteine methyl ester (L-AFCM) and N-acetyl-S-geranylgeranyl-L-cysteine methyl ester (L-AGGCM), are substrates for a rod outer segment (ROS) membrane esterase activity. The KM and Vmax values for L-AFCM and L-AGGCM are 186 microM and 2.2 nmol mg-1 min-1 and 435 microM and 4.8 nmol mg-1 min-1, respectively. The enzyme(s) is stereoselective rather than stereospecific because D-AFCM is enzymatically hydrolyzed with KM and Vmax values of 157 microM and 0.46 nmol mg-1 min-1, respectively. The enzyme(s) does not process N-acetyl-L-cysteine methyl ester, demonstrating that the isoprenyl moiety is required for substrate activity. Ebelactone B is a potent mechanism-based inactivator of the enzyme with a KI = 42 microM and a kinh = 3.7 x 10(-3) s-1. Importantly, L-AFCM, L-AGGCM, and ebelactone B all inhibit the demethylation of the endogenous ROS substrates, showing that the same enzymatic activity is involved in the processing of the synthetic and physiological substrates.
74 citations
TL;DR: The 4-acyloxybenzyl phosphoesters provide the first example of a protecting group which will enable the bioactivation of phosphonate prodrugs at rates appropriate to biological systems as discussed by the authors.
Abstract: The di(4-acetoxybenzyl) ester of methylphosphonate 4(X = H, R = Me) and the di(4-acyloxybenzyl) esters of methoxycarbonylmethylphosphonate 4(X = MeO2C, R = Me, Et, Pr, Pri, Bu or But) were prepared from the appropriate benzyl alcohol and phosphonic dichloride. At pD 8.0 and 37 °C, both series of compounds hydrolyse with half-lives greater than 24 h to the corresponding mono(4-acyloxybenzyl) esters 5(X = H or MeO2C, R = Me, Et, Pr, Pri Bu or But) which were prepared by treatment of the di(4-acyloxybenzyl) esters 4 with sodium or lithium iodide. The mono(4-acyloxybenzyl) esters 5(X = H, R = Me) and 5(X = MeO2C, R = Me, Et, Pr, Pri or But) undergo chemical hydrolysis to methylphosphonate 6(X = H), and methoxycarbonylmethylphosphonate 6(X = MeO2C) respectively, together with 4-hydroxybenzyl alcohol and the appropriate acylate anion. The rates of hydrolysis of the mono(4-acyloxybenzyl) esters decrease as the length and steric bulk of the acyl group increases, with half-lives ranging from ∼ 150 h for the acetyl analogues to 2240 h for the pivaloyl derivative. The hydrolyses of the di- and mono-(4-acyloxybenzyl) esters were catalysed by porcine liver carboxyesterase (PLCE), and in all cases the acylate anion was formed. The rate of enzymatic hydrolysis was most rapid for the 4-butanoyloxybenzyl and 4-isobutanoyloxybenzyl analogues. The methoxycarbonyl ester of the phosphonoacetate analogues was not cleaved by PLCE. The methylphosphonate generated from the reaction of 4(X = H, R = Me) in the presence of esterase and H218O, did not contain 18O attached directly to phosphorus. These results suggest that both the chemical and enzymatic hydrolyses of the mono(4-acyloxybenzyl) esters and the PLCE-catalysed hydrolyses of the di(4-acyloxybenzyl) esters proceed via hydrolysis of the acyl group to give the acylate anion and the unstable 4-hydroxybenzyl esters. The electron-donating 4-hydroxy group facilitates the cleavage of the benzyl-oxygen bond with the formation of the 4-hydroxybenzyl carbonium ion 9, which readily reacts either with water or the phosphate buffer. The 4-acyloxybenzyl phosphoesters provide the first example of a protecting group which will enable the bioactivation of phosphonate prodrugs at rates appropriate to biological systems.
TL;DR: Activating effects were observed at polyamine concentrations that have been shown to exist in the plant cell wall in vivo, suggesting that polyamines may act as efficient regulators of the cell-wall pH via the control of the electrostatic cell- wall potential.
Abstract: The kinetic study of the de-esterification of natural pectin by soya bean or orange pectin methyl esterase shows that the rate of the reaction is highly controlled by the presence of polyamines. The reaction rate versus the polyamine concentration is a bell-shaped curve similar to that which is obtained when the concentration of salts is varied in the reaction mixture. However polyamines, in particular the largest ones, are more efficient than salts. The results may be interpreted by assuming that polyamines mainly interact with the negative charges of the pectic substrate which condition the binding of the pectin methyl esterase. Activating effects were observed at polyamine concentrations that have been shown to exist in the plant cell wall in vivo. Thus, polyamines may act as efficient regulators of the cell-wall pH via the control of the electrostatic cell-wall potential. If such is the case, they might have a role in all regulatory mechanisms in which cell-wall enzymes are involved.
TL;DR: Thermus sp.
Abstract: Thermus sp. strain Rt41A produces an extracellular thermostable alkaline proteinase. The enzyme has a high isoelectric point (10.25-10.5) which can be exploited in purification by using cation-exchange chromatography. The proteinase was purified to homogeneity and has a molecular mass of 32.5 kDa by SDS/PAGE. It is a glycoprotein, containing 0.7% carbohydrate as glucose equivalents, and has four half-cystine residues present as two disulphide bonds. Maximum proteolytic activity was observed at pH 8.0 against azocasein and greater than 75% of this activity was retained in the pH range 7.0-10.0. Substrate inhibition was observed with casein and azocasein. The enzyme was stable in the pH range 5.0-10.0 and maximum activity, in a 10-min assay, was observed at 90 degrees C with 5 mM CaCl2 present. No loss of activity was observed after 24 h at 70 degrees C and the half-lives at 80 degrees C and 90 degrees C were 13.5 h and 20 min, respectively. Removal of Ca2+ reduced the temperature for maximum proteolytic activity against azocasein to 60 degrees C and the half-life at 70 degrees C was 2.85 min. The enzyme was stable at low and high ionic strength and in the presence of denaturing reagents and organic solvents. Rt41A proteinase cleaved a number of synthetic amino acid p-nitrophenol esters, the kinetic data indicating that small aliphatic or aromatic amino acids were the preferred residue at the P1 position. The kinetic data for the hydrolysis of a number of peptide p-nitroanilide substrates are also reported. Primary cleavage of the oxidized insulin B chain occurred at sites where the P1' amino acid was aromatic. Minor cleavage sites (24 h incubation) were for amino acids with aliphatic side chains at the P1' position. The esterase and insulin cleavage data indicate the specificity is similar for both the P1 and P1' sites.
TL;DR: Streptomyces strains screened for ability to produce enzymes (cutinases) capable of hydrolyzing the insoluble plant biopolyester cutin were confirmed to producing enzymes with cutin-degrading activity ( cutinases).
Abstract: Forty-fiveStreptomyces strains, including representatives of the plant pathogensS. acidiscabies, S. scabies, andS. ipomoea, were screened for ability to produce enzymes (cutinases) capable of hydrolyzing the insoluble plant biopolyester cutin. Initially, all strains were tested for production of extracellular esterase in liquid shake (250 rpm) cultures at room temperature in defined (glycerol-asparagine) or complex (tryptone-yeast extract with or without addition of mannitol) broth media supplemented with either tomato or apple cutin. Esterase activity was determined by a spectrophotometric assay utilizing the model substratep-nitrophenyl butyrate. Of the five strains exhibiting highest esterase activity, four (S. acidiscabies ATCC 49003,S. “scabies” ATCC 15485 and IMRU 3018, andS. badius ATCC 19888) were confirmed to produce enzymes with cutin-degrading activity (cutinases). Confirmation of extracellular cutinase production was accomplished by use of a new high-performance liquid chromatography method for separation and quantification of released cutin monomers. Monomer identification was confirmed by GC/MS analyses. Cutinase production was induced 2- to 17-fold by inclusion of cutin in the media. To our knowledge this constitutes the first report of cutinase production byStreptomyces spp. other thanS. scabies.
TL;DR: It is shown here that juvenile hormone (JH) hydrolytic activity is not associated with either EST20 in D. melanogaster or the p-esterase inD. virilis, which has been claimed to be JHE.
TL;DR: An alpha-l-arabinofuranosidase was purified from the cytoplasm of Butyrivibrio fibrisolvens GS113 and apparently played a major role in the assimilation of arabinose-containing xylooligosaccharides generated from xylosidase, phenolic esterase, xylanase, and other enzymatic activities on xylans.
Abstract: An alpha-l-arabinofuranosidase (EC 3.2.1.55) was purified from the cytoplasm of Butyrivibrio fibrisolvens GS113. The native enzyme had an apparent molecular mass of 240 kDa and was composed of eight polypeptide subunits of 31 kDa. The enzyme displayed an isoelectric point of 6.0, a pH optimum of 6.0 to 6.5, a pH stability of 4.0 to 8.0, and a temperature optimum of 45 degrees C and was stable to 55 degrees C. The K(m) and V(max) for p-nitrophenyl-alpha-l-arabinofuranoside were 0.7 mM and 109 mumol/min/mg of protein, respectively. The enzyme was specific for the furanoside configuration and also readily cleaved methylumbelliferyl-alpha-l-arabinofuranoside but had no activity on a variety of other nitrophenyl- or methylumbelliferyl glycosides. When the enzyme was incubated with cellulose, carboxymethyl cellulose, or arabinogalactan, no release of sugars was found. Arabinose was found as the hydrolysis product of oatspelt xylan, corn endosperm xylan, or beet arabinan. No activity was detected when either coumaric or ferulic acid ester linked to arabinoxylobiose was used as substrates, but arabinoxylobiose was degraded to arabinose and xylobiose. Since B. fibrisolvens GS113 possesses essentially no extracellular arabinofuranosidase activity, the major role of the purified enzyme is apparently in the assimilation of arabinose-containing xylooligosaccharides generated from xylosidase, phenolic esterase, xylanase, and other enzymatic activities on xylans.
TL;DR: Two hundred thirty-two nonfilamentous bacterial strains, including saprophytes, plant pathogens, and opportunistic plant and human pathogens, were screened for the ability to produce cutinases (cutin-degrading esterases) and significant induction (10-fold or above) of esterase activity by addition of cutin to nutrient broth-yeast extract medium was demonstrated.
Abstract: Two hundred thirty-two nonfilamentous bacterial strains, including saprophytes, plant pathogens, and opportunistic plant and human pathogens, were screened for the ability to produce cutinases (cutin-degrading esterases). Initially, esterase activity of culture filtrates of strains grown in nutrient broth-yeast extract medium supplemented with 0.4% apple or tomato cutin was determined by a spectrophotometric assay utilizing the model substrate p-nitrophenyl butyrate. The culture filtrates of the 10 Pseudomonas aeruginosa strains tested exhibited the highest esterase activity, with values of >500 nmol/min/ml. Of these 10 strains, 3 (K799, 1499A, and DAR41352) demonstrated significant induction (10-fold or above) of esterase activity by addition of cutin to nutrient broth-yeast extract medium. The ability of culture filtrates of the three strains to cause release of apple cutin monomers was confirmed by a novel high-performance liquid chromatography technique. Monomer identification was confirmed by gas chromatography-mass spectroscopy analyses. Addition of the nonionic detergent n-octylglucoside stimulated cutinase activity of culture filtrates from strains K799 and DAR41352, but not that of filtrates from strain 1499A. Time course studies in nutrient broth-yeast extract medium supplemented with apple cutin indicated maximal levels of cutinase in the culture fluids after cultures entered stationary phase. Incubation temperatures below the optimal temperature for growth (37°C) led to maximal production of cutinase.
TL;DR: The model building and the molecular dynamics simulation of thrombin Salakta suggest that the Glu-466----Ala substitution would change the proper conformation around the substrate binding site containing Trp-468, which is a unique surface loop on theThrombin molecule.
Abstract: Structural studies on a hereditary abnormal prothrombin, prothrombin Salakta, have been performed to identify the difference responsible for its reduced fibrinogen clotting activity and its reduced esterase activity. Amino acid composition and sequence analyses of a peptide isolated from a lysylendopeptidase digest of the abnormal thrombin indicated that Glu-466 had been replaced by Ala. This amino acid substitution can result from a single nucleotide change in the codon for Glu-466 (GAG----GCG). The model building and the molecular dynamics simulation of thrombin Salakta suggest that the Glu-466----Ala substitution would change the proper conformation around the substrate binding site containing Trp-468, which is a unique surface loop on the thrombin molecule. This is the experimental and theoretical evidence supporting the role of the surface loop containing Trp-468 for the proper conformation of the substrate binding site.
TL;DR: Thirty-six single-ascospore isolates of Leptosphaeria maculans were obtained from eight stubbles of diseased Brassica napus plants collected from two locations in France, and factorial correspondence analysis of soluble protein profiles showed that one individual clearly departed from the main cluster.
TL;DR: Certain strains of Staphylococcus aureus produce an enzyme capable of inactivating the bactericidal fatty acids produced in staphylitis abscesses by esterification to various alcohols, which appears to be the preferred substrate for cholesterol.
Abstract: Certain strains of Staphylococcus aureus produce an enzyme capable of inactivating the bactericidal fatty acids produced in staphylococcal abscesses by esterification to various alcohols. The enzyme, called FAME (fatty acid modifying enzyme), has a pH optimum between 5.5 and 6.0 and a temperature optimum of about 40 degrees C. Enzyme activity is not affected by edetic acid or by the presence or absence of sodium and potassium ions. Although FAME can utilise methanol, ethanol, 1-propanol, 2-propanol, 1-butanol or cholesterol as substrates, cholesterol appears to be the preferred substrate. FAME esterifies without being an esterase operating in reverse. Strains capable of producing the enzyme can synthesise it in trypticase soy broth and in a chemically defined medium, but not necessarily in equal amounts. FAME production is correlated with the ability of a strain to grow and survive within the tissues.
TL;DR: Insecticide resistant Myzus persicae (Sulzer) employ increased esterase activity to detoxify insecticides by hydrolysis and sequestration.
Abstract: Insecticide resistant Myzus persicae (Sulzer) employ increased esterase activity to detoxify insecticides by hydrolysis and sequestration. The amount of esterase, and hence resistance, in individual aphids can be determined by measuring either overall naphthyl acetate hydrolysis in crude homogenates, or the specific enzyme responsible (E4 or FE4) after electrophoresis or immunological isolation. The ability of a total esterase assay, done in microplates, to discriminate between susceptible (S) aphids and resistant variants (R1, R2) with different amounts of E4/FE4, was compared with the resolving power of the more elaborate immunoassay technique. The immunoassay gave the better discrimination between variants, resolving them all with greater than 95% confidence, with particularly good separation of R1, from S. The microplate assay using crude homogenates, although a poorer discriminator, identified most of the very resistant (R2) aphids, and provided a robust and widely accessible method for broadly representing the resistance of field populations.
TL;DR: Results of the reaction in the presence of excess N-CBZ-D(L)-AlaNP over HSA indicated the existence of one strong reactive site on HSA, and the effects of the reversible binding of the site-specific drug and the chemical modification by site- specific reagents on the HSA activity showed that the reactive site is the R site located near tyrosine-411 residue of HSA.
Abstract: The reactions of human serum albumin (HSA) with optically active amino acid p-nitrophenyl esters (substrate, S) were examined kinetically at 25°C. The rate data were analyzed in terms of a mechanism involving 1 : 1 complexing (S·HSA) between S and HSA. The dissociation constant (KS in M) and the catalytic rate constant (k2 in S-1) of S·HSA were determined. Among ten substrates examined, the reactions with N-carbobenzoxy-D(L)-alanine p-nitrophenyl esters (N-CBZ-D(L)-AlaNP) were most accelerated by HSA. Results of the reaction in the presence of excess N-CBZ-D(L)-AlaNP over HSA indicated the existence of one strong reactive site on HSA. The effects of the reversible binding of the site-specific drug and the chemical modification by site-specific reagents on the HSA activity showed that the reactive site towards N-CBZ-D(L)-AlaNP is the R site located near tyrosine-411 residue of HSA.
TL;DR: The enzyme was proven to be a metalloprotease, as shown by the inhibitory effects exerted by EDTA and o-phenanthroline; furthermore, dialysis against EDTA led to a complete loss of activity, which could be restored by addition of Zn2+ in the micromolar range, and, to a lesser extent, by Co2+.
Abstract: A carboxypeptidase was purified to electrophoretic homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular masses assessed by SDS/PAGE and gel filtration were 42 kDa and 170 kDa, respectively, which points to a tetrameric structure for the molecule. An isoelectric point of 5.9 was also determined. The enzyme was proven to be a metalloprotease, as shown by the inhibitory effects exerted by EDTA and o-phenanthroline; furthermore, dialysis against EDTA led to a complete loss of activity, which could be restored by addition of Zn2+ in the micromolar range, and, to a lesser extent, by Co2+. The enzyme was endowed with a broad substrate specificity, as shown by its ability to release basic, acidic and aromatic amino acids from the respective benzoylglycylated and benzyloxycarbonylated amino acids. An esterase activity of the carboxypeptidase was also demonstrated on different esterified amino acids and dipeptides blocked at the N-terminus. The enzyme displayed broad pH optima ranging over 5.5-7.0, or 5.5-9.0, when using an acidic or a basic benzyloxycarbonylated amino acid, respectively. With regard to thermostability, it was proven to be completely stable on incubation for 15 min at 85°C. Furthermore, thanks to its relatively low activation energy, i.e. 31.0 kJ/mol, it was still significantly active at room temperature. At 40°C, the enzyme could withstand 0.1% SDS and different organic solvents: particularly ethanol up to 99%. Amino acid and N-terminal sequence analyses did not evidence any similarity to carboxypeptidases A nor thermolysin. A weak similarity was only found with bovine carboxypeptidase B.
TL;DR: It is found that tissue factor greatly potentiates the factor VIIa-catalyzed hydrolysis of Z-Arg-ONb, and the gamma-carboxyglutamic acid domain is probably required as a potent determinant for interactions with tissue factor, even in the absence of phospholipids in the reaction mixture.
TL;DR: Comparison of kinetic constants for esters demonstrates that p-nitrophenyl butyrate is the worst substrate of all esters tested, suggesting that the active site topography is uniquely unfavorable for p- nitrogen-based esters, which is a good substrate for butyraldehyde.
TL;DR: In mussel Mytilus galloprovincialis tissue-specific electrophoretic patterns of multiple molecular forms of esterases were observed and in vitro studies indicated that organophosphate and carbamate insecticides have distinct effects on different esterase isozymes.
TL;DR: An esterase gene from a lipolytic psychotroph, was cloned in Escherichia coli and its nucleotide sequence was determined, revealing an ORF encoding a polypeptide of 389 amino acid residues, consistent with that calculated from the ORF.
Abstract: Summary: An esterase gene (estA) from a lipolytic psychrotroph (Pseudomonas sp. LS107d2), was cloned in Escherichia coli and its nucleotide sequence was determined, revealing an ORF encoding a polypeptide of 389 amino acid residues, with a molecular mass of 42276 Da. Labelling of plasmid-encoded proteins with [35S)methionine, using the maxicell procedure, gave a single polypeptide of molecular mass 42 kDa, consistent with that calculated from the ORF. Colonies of E. coli cells containing estA produced a clear halo when grown on solid media containing tributyrin; no clearance was produced when cells were grown on media containing triolein. Extracts of cells containing estA also hydrolysed water-soluble nitrophenol esters, but were unable to cleave water-insoluble substrates. The preference for water-soluble substrates indicates that the gene product is an esterase.
TL;DR: In this paper, the results obtained with 3substituted cyclopropane-l,2-dicarboxylates are incompatible with the three-dimensional active-site model of PLE proposed by J.B. Jones et al.
Abstract: In order to gain a more detailed insight into the relationship between substrate structure and the stereoselectivity of the enzyme pig liver esterase (E.C. 3.1.1 .I .) a large series of mainly meso and prochiral diesters with an open chain or a cyclic structure has been studied and evaluated. Results obtained with 3substituted cyclopropane-l,2-dicarboxylates are incompatible with the three-dimensional (cubic) active-site model of PLE proposed by J.B. Jones et a/. Kinetic resolution of mesoand racemic diesters as well as of racemic monoesters has been observed. Their synthetic potential as versatile enantiomerically pure synthons for the construction of complex natural products is demonstrated by the synthesis of the pheromone endo1,3-dimethyI-2,9-dioxabicyclo[3.3.l]nonane, C(IS)-to-C(27)-Segment of rifamycin S and the C(1)to C(7)segment of 14-membered macrolide antibiotics. The efficient synthesis of biologically active compounds, either of natural or unnatural origin, frequently requires chiral synthons. Enzymes as chiral catalysts are now widely used for their preparation (ref. 1-1 l ) , because it is often rather difficult to introduce centres of chirality or perform regiospecific transformations by the application of purely "chemical methods". Especially esterases, such as pig liver esterase (PLE, E.C. 3.1.1 .I.), a serine hydrolase, have been studied extensively in recent years (ref. 7, 12-15). Stability, low costs, and the ability of hydrolyse a wide range of substrates with high stereoselectivity represent additional advantages of this enzyme which operates without the need for co-enzymes. Until now, more than one hundred different esters, mainly mesoand prochiral-diesters have been subjected to the treatment with PLE (ref. 7). To be able to fully exploit the potential of this enzyme, it is indispensable to understand the factors which are responsible for the specificity. Accordingly, we as well as other research groups have initiated investigations for securing a large number of data on hydrolyses, allowing to gain more insight into the realtionship between substrate structure and enzymic activity of PLE. Commercially available PLE preparations are mixtures of at least six isoenzymes which, however were found to exhibit essentially the same stereospecificity (ref. 16). These findings justify the attempts to rationalize the results of the hydrolyses by an active-site model which is mainly based on the measured ee values and the absolute configuration of the hydrolysis products. The following selected examples of results which were obtained by us and various other authors from mesodiesters in the malonate series appear to be very inconsistent and unpredictable with respect to the ee values and absolute configurations at first sight:
TL;DR: The isozymes of esterase, acid phosphatase and catalase were investigated electrophoretically in 44 Indian Musa cultivars belonging to the AB, AAB, ABB and AAA genomic groups and Catalase was the least polymorphic enzyme system.
Abstract: The isozymes of esterase, acid phosphatase and catalase were investigated electrophoretically in 44 Indian Musa cultivars belonging to the AB, AAB, ABB and AAA genomic groups. A high degree of poly...