A new type of aminoacyltransferase from Saccharothrix sp. AS-2 favorable for the synthesis of D-amino acid-containing peptides

A unique enzyme with some properties favorable for the synthesis of D-amino acid-containing peptides has been purified from the culture broth of Saccharothrix sp. AS-2. The purification steps included ammonium sulfate fractionation, chromatographies on CM-Toyopearl 650M and ProtEx Butyl, and sucrose density-gradient isoelectric focusing. The enzyme, consisting of four subunits of 56 kDa, showed its maximum transfer activity at around pH 8.2 and 35 degrees C, and had an isoelectric point of 5.8. The enzyme yielded homooligomers from methyl esters of D-Asp(OMe), D-Met, D-Phe, D-Trp, D-Tyr, and L-Glu(OMe), but showed no hydrolytic activity toward any of the D- or L-amino acid methyl esters tested. The homooligomers were not formed from the corresponding free amino acids. The reaction of Ac-D-Phe-OMe with DL-Ala-NH(2), DL-Leu-NH(2), DL-Phe-NH(2), or DL-Trp-NH(2) was effectively catalyzed by the enzyme, both the DD- and DL-stereoisomers of the expected N-acetyldipeptide being yielded. The resulting dipeptides remained unhydrolyzed even after 48 h incubation. Also, it showed no detectable hydrolytic activity toward casein, diastereomers of diAla, diMet, and diPhe, D-/L-amino acid amides, or D-/L-amino acid p-nitroanilides, indicating that the enzyme had no peptidase activity leading to secondary hydrolysis of the growing peptide. The enzyme activity was strongly depressed by phenylmethanesulfonyl fluoride, but not by penicillin G or ampicillin, suggesting that the protein is a serine enzyme lacking penicillin-binding ability. These observations lead us to the conclusion that the enzyme from Saccharothrix sp. AS-2 characterized in this study is a new type of aminoacyltransferase with an amino acid ester as the acyl donor, and has potential utility as a catalyst for the synthesis of D-amino acid-containing peptides.     

Partial purification and characterization of midgut leucyl aminopeptidase of Morimus funereus (Coleoptera: Cerambycidae) larvae

Exopeptidases of Morimus funereus larvae were partially purified and characterized. Specific leucyl aminopeptidase (LAP) activity was increased eight-fold by gel filtration of the crude midgut extract. The partially purified LAP had a molecular mass greater than 100 kDa with pH optima from 7.0-9.0 and no strict substrate specificity. M. funereus LAP preferentially hydrolyzed p-nitroanilides with hydrophobic amino acids in the active site, with a K(m) for leucine-p-nitroanilide of 0.21 mM. Zymogram analysis of an electropherogram obtained by native polyacrylamide gel electrophoresis revealed four enzymatically active proteinases using leucine-p-nitroanilide and methionine-p-nitroanilide as substrates and two enzymatically active proteinases using lysine-p-nitroanilide as a substrate. Although the optimal temperature of LAP activity was 40 degrees C, the enzyme was active over a broad temperature range from 2 to 60 degrees C. Among a number of inhibitors tested, heavy metals and 1,10-phenanthroline completely inhibited the enzyme, while methanol, ethanol and EGTA stimulated somewhat LAP activity.     

A novel esterase from Bacillus subtilis (RRL 1789): purification and characterization of the enzyme

An esterase (EC 3.1.1.1) produced by an isolated strain of Bacillus subtilis RRL 1789 exhibited moderate to high enantioselectivity in the kinetic resolution of several substrates like aryl carbinols, hydroxy esters, and halo esters. The enzyme named as B. subtilis esterase (BSE), was purified to >95% purity with a specific activity of 944 U/mg protein and 12% overall yield. The purified enzyme is approximately 52 kDa monomer, maximally activity at 37 degrees C and pH 8.0 and fairly stable up to 55 degrees C. The enzyme does not exhibit the phenomenon of interfacial activation with tributyrin and p-nitrophenyl butyrate beyond the saturation concentration. The enzyme showed preference for triacyglycerols and esters of p-nitrophenol with short chain fatty acid. Presence of Ca2+ ions increases the activity of enzyme by approximately 20% but its presence does not have any influence on the thermostability of the enzyme. The enzyme is not a metalloprotein and belongs to the family of serine proteases. The N-terminal amino acid sequence of BSE determined, as Met-Thr-Pro-Glu-Iso-Val-Thr-Thr-Glu-Tyr-Gly- revealed similarity with the N-terminal amino acid sequence of p-nitrobenzylesterase of B. subtilis.     

Purification and characterization of rat pancreatic fatty acid ethyl ester synthase and its structural and functional relationship to pancreatic cholesterol esterase

Formation of fatty acid ethyl esters (FAEEs, catalyzed by FAEE synthase) has been implicated in the pathogenesis of chronic pancreatitis. In previous studies, we demonstrated that FAEE synthase, purified from rat liver microsomes, is identical to rat liver carboxylesterase (pI 6.1), and structurally and functionally different than that from pancreas. In this study, we purified and characterized rat pancreatic microsomal FAEE synthase, and determined its relationship with rat pancreatic cholesterol esterase (ChE). Since most of the serine esterases express p-nitrophenyl acetate (PNPA)-hydrolyzing activity as well as synthetic activity to form fatty acid esters or amides with a wide spectrum of alcohols and amines, respectively, we used PNPA-hydrolyzing activity to monitor the purification of FAEE synthase during various chromatographic purification steps. Synthesizing activity towards FAEEs, fatty acid methyl esters, and fatty acid anilides was measured only in the pooled fractions. At each step of purification (ammonium sulfate saturation, Q Sepharose XL, and heparin-agarose column chromatographies, and high performance liquid chromatography (anion exchange and gel filtration)) synthetic as well as hydrolytic activities copurified. Using ethanol, methanol, or aniline as substrates, the ester or anilide synthesizing activity of the purified protein was found to be 8709, 13000, and 2201 nmol/h/mg protein, respectively. The purified protein displayed a single band with an estimated molecular mass of approximately 68 kD upon SDS-PAGE under reduced denaturing conditions, cross-reacted with antisera against rat pancreatic ChE and showed 100% N-terminal sequence homology of the first 15 amino acids to that of rat pancreatic ChE. These results suggest that the purified protein has broad substrate specificity towards the conjugation of endogenous long chain fatty acids with substrates having hydroxyl and amino groups and is identical to ChE.     

Molecular cloning and characterization of CMCase gene (celC) from Salmonella typhimurium UR

The sequence coding for carboxymethylcellulase (CMCase, CelC) was isolated from the DNA of Salmonella typhimurium UR1. Comparison between the deduced amino acid sequence of CelC (368 amino acid residues, Molecular mass 41 kDa) and that of the previously published CMCase revealed that this enzyme belongs to the cellulase family 8 and D. The protein was overproduced in Escherichia coli using T7 expression system, and its activity was confirmed by CMC-SDS-PAGE. When the overexpressed CelC protein was tested on cellulose-type substrates, the recombinant protein is able to degrade cellulose-type substrates, such as CM-cellulose, xylan, avicel, lichenan, and laminarin. Optimal temperature and pH for enzyme activity were found to be 50 degrees C and pH 6.5, respectively.     

Substrate specificity of human carnitine acetyltransferase: Implications for fatty acid and branched-chain amino acid metabolism

Carnitine acyltransferases catalyze the reversible conversion of acyl-CoAs into acylcarnitine esters. This family includes the mitochondrial enzymes carnitine palmitoyltransferase 2 (CPT2) and carnitine acetyltransferase (CrAT). CPT2 is part of the carnitine shuttle that is necessary to import fatty acids into mitochondria and catalyzes the conversion of acylcarnitines into acyl-CoAs. In addition, when mitochondrial fatty acid β-oxidation is impaired, CPT2 is able to catalyze the reverse reaction and converts accumulating long- and medium-chain acyl-CoAs into acylcarnitines for export from the matrix to the cytosol. However, CPT2 is inactive with short-chain acyl-CoAs and intermediates of the branched-chain amino acid oxidation pathway (BCAAO). In order to explore the origin of short-chain and branched-chain acylcarnitines that may accumulate in various organic acidemias, we performed substrate specificity studies using purified recombinant human CrAT. Various saturated, unsaturated and branched-chain acyl-CoA esters were tested and the synthesized acylcarnitines were quantified by ESI-MS/MS. We show that CrAT converts short- and medium-chain acyl-CoAs (C2 to C10-CoA), whereas no activity was observed with long-chain species. Trans-2-enoyl-CoA intermediates were found to be poor substrates for this enzyme. Furthermore, CrAT turned out to be active towards some but not all the BCAAO intermediates tested and no activity was found with dicarboxylic acyl-CoA esters. This suggests the existence of another enzyme able to handle the acyl-CoAs that are not substrates for CrAT and CPT2, but for which the corresponding acylcarnitines are well recognized as diagnostic markers in inborn errors of metabolism.     

New thermophilic and thermostable esterase with sequence similarity to the hormone-sensitive lipase family, cloned from a metagenomic library

A gene coding for a thermostable esterase was isolated by functional screening of Escherichia coli cells that had been transformed with fosmid environmental DNA libraries constructed with metagenomes from thermal environmental samples. The gene conferring esterase activity on E. coli grown on tributyrin agar was composed of 936 bp, corresponding to 311 amino acid residues with a molecular mass of 34 kDa. The enzyme showed significant amino acid similarity (64%) to the enzyme from a hyperthermophilic archaeon, Pyrobaculum calidifontis. An amino acid sequence comparison with other esterases and lipases revealed that the enzyme should be classified as a new member of the hormone-sensitive lipase family. The recombinant esterase that was overexpressed and purified from E. coli was active above 30 degrees C up to 95 degrees C and had a high thermal stability. It displayed a high degree of activity in a pH range of 5.5 to 7.5, with an optimal pH of approximately 6.0. The best substrate for the enzyme among the p-nitrophenyl esters (C(4) to C(16)) examined was p-nitrophenyl caproate (C(6)), and no lipolytic activity was observed with esters containing an acyl chain length of longer than 10 carbon atoms, indicating that the enzyme is an esterase and not a lipase.     

On the specificity of porcine elastase.

The specificity of porcine elastase (EC 3.4.4.7) has been studied. Ethyl esters derived from benzoyl amino acids with straight side chains are better substrates than those with branched side chains; the best substrate is norvaline ester. In the series of benzoylalanine alkyl esters the alcohol moiety markedly affects the susceptibility. The benzyl ester was found to be the best nonactivated substrate derived from monomeric amino acid. With elastase acylation is rate limiting, in contrast to chymotrypsin and trypsin where deacylation is generally the rate determining step with specific ester substrates.     

Aromatase and testosterone fatty acid esters: the search for a cryptic biosynthetic pathway to estradiol esters.

The estradiol fatty acid esters (lipoidal derivatives, LE2) are extremely potent estrogens that accumulate in fat, including fat of menopausal women. These steroidal esters are protected from metabolism and are converted to the free, biologically active steroid through the action of esterases. Previous studies have shown that biosynthetic pathways in the adrenal gland exist in which steroid fatty acid esters are substrates. This led us to determine whether a cryptic aromatase pathway exists in which testosterone esters could be converted directly into LE2. We tested a representative fatty acid ester, testosterone stearate, both as an inhibitor and as a substrate for the aromatase enzyme from human placental microsomes. This ester had neither activity. In addition, we tested [1 beta-3H]testosterone acetate as a substrate for this enzyme complex, measuring the production of 3H2O as evidence of aromatization. Although the rate of reaction was considerably slower than that of testosterone, 3H2O was produced. However, when [2, 4, 6, 7-3H]testosterone acetate was incubated and the steroidal products isolated, we found that hydrolysis of the substrate had occurred. Both [3H]-labeled testosterone and estradiol were found, and very little if any [3H]estradiol acetate was formed. Thus, we conclude that an aromatase pathway involving testosterone esters does not exist and that the sole source of LE2 is through direct esterification of estradiol.     

Acyl coenzyme A:cholesterol acyltransferase in neonatal chick brain

An acyl coenzyme A:cholesterol acyltransferase activity which directly incorporates palmitoyl coenzyme A into cholesterol esters using endogenous cholesterol as substrate was demonstrated in microsomal preparations from neonatal chick brain. The enzyme showed, at pH 7.4, about 2-fold greater activity than that observed at pH 5.6. Nearly 10-times higher esterifying activity was found in brain microsomes using palmitoyl coenzyme A than that with palmitic acid. The acyltransferase activity was clearly different from the other cholesterol-esterifying enzymes previously found in brain, which incorporated free fatty acids into cholesterol esters and did not require ATP or coenzyme A as cofactors. Chick brain microsomes also incorporated palmitoyl coenzyme A into phospholipids and triacylglycerols. However, most of the radioactivity from this substrate was found in the fatty acid fraction, due to the presence of an acyl coenzyme A hydrolase activity in the enzyme preparations. Therefore, the formation of palmitate was tested during all the experiments. The brain acyltransferase assay conditions were optimized with respect to protein concentration, incubation time and palmitoyl coenzyme A concentration. Microsomal activity was independent of the presence of dithiothreitol in the incubation medium and microsomes can be stored at -40 degrees C for several weeks without losing activity. Addition of fatty acid-free bovine serum albumin to brain microsomal preparations produced a considerable increase in the acyltransferase activity, while acyl coenzyme A hydrolase was clearly inhibited. Results obtained show the existence in neonatal chick brain of an acyl coenzyme A:cholesterol acyltransferase activity similar to that found in a variety of tissues from different species but not previously reported in brain.     

On the specificity of lipid hydroperoxide fragmentation by fatty acid hydroperoxide lyase from Arabidopsis thaliana

Fatty acid hydroperoxide lyase (HPL) is a membrane associated P450 enzyme that cleaves fatty acid hydroperoxides into aldehydes and omega-oxo fatty acids. One of the major products of this reaction is (3Z)-hexenal. It is a constituent of many fresh smelling fruit aromas. For its biotechnological production and because of the lack of structural data on the HPL enzyme family, we investigated the mechanistic reasons for the substrate specificity of HPL by using various structural analogues of HPL substrates. To approach this 13-HPL from Arabidopsis thaliana was cloned and expressed in E. coli utilising a His-Tag expression vector. The fusion protein was purified by affinity chromatography from the E. coli membrane fractions and its pH optimum was detected to be pH 7.2. Then, HPL activity against the respective (9S)- and (13S)-hydroperoxides derived either from linoleic, alpha-linolenic or gamma-linolenic acid, respectively, as well as that against the corresponding methyl esters was analysed. Highest enzyme activity was observed with the (13S)-hydroperoxide of alpha-linolenic acid (13alpha-HPOT) followed by that with its methyl ester. Most interestingly, when the hydroperoxy isomers of gamma-linolenic acid were tested as substrates, 9gamma-HPOT and not 13gamma-HPOT was found to be a better substrate of the enzyme. Taken together from these studies on the substrate specificity it is concluded that At13HPL may not recognise the absolute position of the hydroperoxy group within the substrate, but shows highest activities against substrates with a (1Z4S,5E,7Z)-4-hydroperoxy-1,5,7-triene motif. Thus, At13HPL may not only be used for the production of C6-derived volatiles, but depending on the substrate may be further used for the production of Cg-derived volatiles as well.     

Substrate specificity of aminopeptidase from the mid-gut gland of the scallop (Patinopecten yessoensis)

An action for various peptides and a kinetic study for amino acid p-nitroanilides (pNAs) and 4-methylcoumaryl-7-amides (MCAs) were performed with purified aminopeptidase from the mid-gut of the scallop. The enzyme preferred dipeptides having Ala, Met, and Phe in the amino-terminal or the penultimate position from the amino-termini. The catalytic efficiencies, k(cat)/K(m) values for Ala-pNA and MCA were the highest in the tested substrates, and those for pNA and MCA substrates having Met or Phe were the next highest. The enzyme was found to be a new alanine-specific aminopeptidase.     

Over-expression and properties of a purified recombinant Bacillus licheniformis lipase: a comparative report on Bacillus lipases

The gene coding for an extracellular lipase of Bacillus licheniformis was cloned using PCR techniques. The sequence corresponding to the mature lipase was subcloned into the pET 20b(+) expression vector to construct a recombinant lipase protein containing 6 histidine residues at the C-terminal. High-level expression of the lipase by Escherichia coli cells harbouring the lipase gene-containing expression vector was observed upon induction with IPTG at 30 degrees C. A one step purification of the recombinant lipase was achieved with Ni-NTA resin. The specific activity of the purified enzyme was 130 units/mg with p-nitrophenyl-palmitate as substrate. The enzyme showed maximum activity at pH 10-11.5 and was remarkably stable at alkaline pH values up to 12. The enzyme was active toward p-nitrophenyl esters of short to long chains fatty acids but with a marked preference for esters with C(6) and C(8) acyl groups. The amino acid sequence of the lipase shows striking similarities to lipases from Bacillus subtilis and Bacillus pumilus. Based on the amino acid identity and biochemical characteristics, we propose that Bacillus lipases be classified into two distinct subfamilies of their own.     

A carboxylesterase from the thermoacidophilic archaeon Sulfolobus solfataricus P1; purification, characterization, and expression

The carboxylesterase, a 34 kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 85 degrees C and 8.0, respectively. The enzyme showed remarkable thermostability: 41% of its activity remained after 5 days of incubation at 80 degrees C. In addition, the purified enzyme exhibited stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity towards various PNP esters and short acyl chain triacylglycerols such as tributyrin (C4:0). Among the PNP esters tested, the best substrate was PNP-caprylate (C8) with Km and kcat values of 71 microM and 14,700 s(-1), respectively. The carboxylesterase gene consisted of 915 bp corresponding to 305 amino acid residues. We demonstrated that active recombinant S. solfataricus carboxylesterase could be expressed in Escherichia coli. The enzyme was identified as a serine esterase belonging to mammalian hormone-sensitive lipases (HSL) family and contained a catalytic triad composed of serine, histidine, and aspartic acid in the active site.     

Proteolytic anaerobic bacteria from lake sediments of Antarctica

Amongst twenty five proteolytic bacteria isolated from lake sediment samples of Antarctica, six isolates were selected based on SDS PAGE protein profile and zone of hydrolysis on casein agar at 10 degrees C. Most of the cultures were rod shaped and motile with two showing terminal bulging spores. Isolates grew between 5 degrees C to 37 degrees C and protease was induced in the late log, stationary or death phase. Isolate SPA-3 grew maximally at 10 degrees C and SPA-6 at 37 degrees C while others preferred 20 degrees C-30 degrees C for growth. The growth and protease production on casein, skimmed milk, bovine serum albumin and gelatin varied with the isolates. Acetate was the dominant volatile fatty acid (24-66% of total VFA) produced during hydrolysis of protein substrate.     

Isolation and characterization of proteinases from the sarcocarp of snake-gourd fruit

Seven proteinases were isolated from the fruit of snake-gourd, Trichosanthes cucumeroides Maxim. Their isozymes are all serine proteinases, and homologous in their respective molecular weights, amino acid compositions, and enzymatic properties. Their molecular weight was estimated to be about 50,000. Using casein as a substrate, the maximum activity was found in the alkaline pH region. The optimum temperature using casein was 70 degrees C at pH 7.3. The enzymes were strongly inhibited by diisopropyl fluorophosphate and not inhibited by inhibitors of sulfhydryl or metalloproteases. The reduced and S-carboxymethylated insulin B-chain was used as a substrate in an investigation of the specificity. The enzyme was found to have a wide specificity for this substrate but preferentially hydrolyzed the peptide bonds involving the carboxyl groups of charged amino acid such as S-cm-cysteine, glutamic acid, histidine, arginine, and lysine. Experimental evidence indicated that the snake-gourd proteinases are similar in their properties to cucumisin, which is isolated from the sarcocarp of melon fruit.     

Hydrolysis of penicillins and related compounds by the cell-bound penicillin acylase of Escherichia coli

1. A method is given for the preparation of penicillin acylase by using Escherichia coli N.C.I.B. 8743 and a strain selected for higher yield. The enzyme is associated with the bacterial cells and removes the side chains of penicillins to give 6-amino-penicillanic acid and a carboxylic acid. 2. The rates of penicillin deacylation indicated that p-hydroxybenzylpenicillin was the best substrate, followed in diminishing order by benzyl-, dl-alpha-hydroxybenzyl-, 2-furylmethyl-, 2-thienylmethyl-, d-alpha-aminobenzyl-, n-propoxymethyl- and isobutoxymethyl-penicillin. Phenylpenicillin and dl-alpha-carboxybenzylpenicillin were not substrates and phenoxymethyl-penicillin was very poor. 3. Amides and esters of the above penicillins were also substrates for the deacylation reaction, as were cephalosporins with a thienylmethyl side chain. 4. For the deacylation of 2-furylmethylpenicillin at 21 degrees the optimum pH was 8.2. The optimum temperature was 60 degrees at pH7. 5. By using selection A of N.C.I.B. 8743 and determining reaction velocities by assaying yields of 6-amino-penicillanic acid in a 10min. reaction at 50 degrees and pH8.2, the K(m) for benzylpenicillin was found to be about 30mm and the K(m) for 2-furylmethylpenicillin, about 10mm. The V(max.) values were 0.6 and 0.24mumole/min./mg. of bacterial cells respectively.     

Manganese-phospholipid-stimulated protein kinase activity of human leukemic cells

A protein kinase system with unusual characteristics was noted in extracts of HL-60 cells using endogenous proteins as substrates. This system exhibited a cation preference for manganese at an optimal concentration of 0.5 mM. Moderate activity was detectable with magnesium at an optimal concentration of 5.0 mM, but calcium was inactive. Activity was markedly stimulated by phospholipid, with phosphatidylglycerol and phosphatidylinositol exhibiting greater activity than phosphatidylserine. In isolated plasma membranes, the major substrate of this system was a 73-kDa protein, while cytoplasmic extracts exhibited larger amounts of a 42-kDa substrate. 73-kDa phosphorylating activity of membranes was comparably active at 0 and 31 degrees C, although in cytosol activity was greater at 31 degrees C. No 73-kDa protein phosphorylation was observed in the presence of Ca2+, Mg2+, and phosphatidylserine. Phosphoamino acid analysis of the 73-kD band revealed phosphothreonine and phosphoserine. The 42-kDa substrate was distinguishable from actin by two-dimensional gel electrophoresis, which disclosed that both major substrates were highly basic in the isoelectric focusing dimension. Protamine and histones (H2B greater than H1 greater than H3) exhibited phospholipid-stimulated phosphorylation in the presence of Mn2+, but phosvitin, casein, and vinculin were not substrates. High levels of phosphorylative activity involving the 73-kDa substrate were noted in nuclear extracts. Complex patterns of increase of this activity were noted in both cytosol and nuclear extracts following induction of differentiation with dimethyl sulfoxide, retinoic acid, or phorbol 12-myristate 13-acetate. This study thus demonstrated the presence of a previously undescribed type of protein kinase activity which exhibited alterations during leukemic cellular differentiation.     

p-Nitroanilides of amino acids and peptides and fluorescence peptide with inner fluorescence quenching as substrates for cathepsins H, B, D and high molecular weight aspartic peptidase in the brain

p-Nitroanilides of amino acids and peptides were used to study the specificity of cathepsins H and B from human and bovine brain, respectively. The specific activity of cathepsin H decreased in the following order: Arg-pNa greater than or equal to Leu-pNa greater than Ala-pNa greater than or equal to Phe-pNa greater than Pro-pNa greater than Glu-pNa; Arg-pNa was split by the enzyme 12 times as fast as Bz-Arg-pNa. Among other oligopeptide p-nitroanilides tested (Ala-Ala, Ala-Leu, Ala-Ala-Ala, Ala-Ala-Leu, Gly-Gly-Leu, Gly-Gly-Phe, Gly-Leu-Phe, pGlu-Phe-Leu, pGlu-Phe-Ala, pGlu-Phe), PGlu-Phe-Leu and pGlu-Phe-Ala appeared to be the best substrates for cathepsin B; Km for hydrolysis were 0.1 mM and 0.165 mM, respectively, kcat were 5.1 and 8.3 s-1, respectively. A comparative study of substrate specificity of cathepsin D and high molecular weight aspartic peptidase with the use of fluorescent substrate with inner fluorescence quenching, Abz-Ala-Ala-Phe-Phe-pNa, revealed that both peptidases hydrolyzed the single bond between two phenylalanine residues, resulting in the increase of fluorescence (4.5-5-fold) of anthraniloyl tripeptide. The Km values for the substrate hydrolysis by cathepsin D and high molecular weight aspartic peptidase were 6.2 microM and 11.2 microM; kcat were 7.2 s-1 and 1.3 s-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and Characterization of a Bacterial Cell-Bound Type B Carboxylesterase from Bacillus sp. BP-7

A clone producing halos on tributyrin plates was isolated from a genomic library of Bacillus sp. BP-7. The insert contained an open reading frame that coded for a protein of 487 amino acids with homology to carboxylesterases. The cloned enzyme showed clear preference for esters of short-chain fatty acids, being classified as an esterase. Maximum activity was found at 45 degrees C and pH 7.5. The enzyme displayed stability in the pH range from 6 to 9.5, and at temperatures from 4 degrees to 45 degrees C. Zymogram analysis of the protein revealed a molecular mass of 53 kDa and a pI of 5.1. The enzyme showed homology to members of the bacterial subclass of type B carboxylesterases, a set of proteins potentially useful for biotechnological applications.