Isolation of a galactose-free 20-kDa fragment exhibiting butyrylcholine esterase and aryl acylamidase activity from human serum butyrylcholine esterase by limited alpha-chymotrypsin digestion

Purified human serum butyrylcholine esterase (approximately 90-kDa subunit), which also exhibits aryl acylamidase activity, was subjected to limited alpha-chymotrypsin digestion. Three major protein fragments of approximately 50 kDa, approximately 21 kDa and approximately 20 kDa were found to be produced, as observed by SDS-gel electrophoresis of the chymotryptic digest. The purified butyrylcholine esterase could fully bind to a Ricinus-communis-agglutinin-Sepharose column but after chymotryptic digestion about 15-20% of the enzyme activity remained unbound and was recovered in the run-through fractions. Sephadex G-75 chromatography of the chymotryptic digest showed an enzymatically active fragment eluted at an approximate molecular mass of 20 kDa, apart from the undigested butyrylcholine esterase eluted at the void volume. The butyrylcholine esterase fragment that did not bind to Ricinus communis agglutinin also was eluted at an approximate molecular mass of 20 kDa from a Sephadex G-75 column. This enzymatically active low-molecular-mass fragment from Sephadex G-75 chromatography showed a single protein band of approximately 20 kDa on SDS-gel electrophoresis. Neutral sugar analysis of the approximately 20 kDa fragment showed the presence of mannose only, whereas the undigested butyrylcholine esterase showed the presence of both mannose and galactose. Amino-terminal-sequence analysis of the approximately 20 kDa fragment showed the sequence Arg-Val-Gly-Ala-Leu, which agrees with amino acid residues 147-151 reported for human serum butyrylcholine esterase [Lockridge et al. (1987) J. Biol. Chem. 262, 549-557]. Both cholinesterase and aryl acylamidase activities were co-eluted in all chromatographic procedures. The results suggested that limited alpha-chymotrypsin digestion of human serum butyrylcholine esterase resulted in the formation of a approximately 20-kDa enzymatically active fragment with Arg147 as its N-terminal residue and which was devoid of galactose.     

Extracellular esterase activity from Bacillus stearothermophilus

Bacillus stearothermophilus has been reported to produce an extracellular esterase with molecular weight of 42–47 kDa. Extracellular esterase activity in Bacillus stearothermophilus (NCIB 13335) was found to reside in protein with a molecular weight less than 10 kDa. This small esterase was responsible for all the esterase activity observed in this strain under the conditions studied.     

Isolation of two forms of carboxylester lipase (cholesterol esterase) from porcine pancreas

Carboxylester lipase (cholesterol esterase, EC 3.1.1.13) has been purified to homogeneity from porcine pancreas. The enzyme is isolated in two molecular mass forms, a monomer of 74 kDa and a dimer of 167 kDa. The dimer consists of two catalytically-active subunits which have molecular masses approximately 9 kDa greater than the monomers. The difference in size was not attributable to carbohydrate or lipid content. The catalytic properties of the two forms are comparable on a weight basis, the amino acid compositions are quite similar, and the N-terminal sequences are nearly identical for 24 residues. These similarities suggest a possible precursor-product relationship between the two carboxylester lipase forms.     

Cloning and characterization of thermostable esterase from <Emphasis Type="Italic">Archaeoglobus fulgidus</Emphasis>

Thermostable esterase gene was cloned (Est-AF) from extremophilic microorganisms, Archaeoglobus fulgidus DSM 4304. The protein analysis result showed that Est-AF is monomer with total 247 amino acids and molecular weight of estimated 27.5 kDa. It also showed repeating units G-X-S-X-G (GHSLG) (residues 86 approximately 90) which is reported as active site of known esterases, and the putative catalytic triad composed of Ser88, Asp198 and His226. The esterase activity test with various acyl chain length of rho-nitrophenol resulted that Est-AF showed highest specific activity with rho-nitrophenylbutyrate (pNPC4) and rapidly decrease with rho-nitrophenyl ester contain more than 8 carbon chain. These results represent that cloned enzyme is verified as a carboxylesterase but not a lipase because esterase activity is decreased with rho-nitrophenyl ester contains more than 8 carbon chains but lipase activity does not affected with carbon chain length. Optimum temperature of esterase reaction with rho-nitrophenylbutyrate (pNPC4) was 80 degrees C. When ketoprofen ethyl ester was used as a substrate, activity of Est-AF showed the highest value at 70 degrees C, and 10% of activity still remains after 3 h of incubation at 90 degrees C. This result represents Est-AF has high thermostability with comparison of other esterases that have been reported. However, Est-AF showed low enantioselectivity with ketoprofen ethyl ester. Optimum pH of Est-AF is between pH 7.0 and pH 8.0. Km value of ketoprofen ethyl ester is 1.6 mM and, Vmax is 1.7 micromole/mg protein/min. Est-AF showed similar substrate affinity but slower reaction with ketoprofen ethyl ester compare with esterase from mesophilic strain P. fluorescens.     

一种具有立体选择性新酯酶的菌种筛选和基因克隆

从土样分离到一株产生具有立体选择性酯水解酶的恶臭假单孢菌(Pseudomonas putida NH33)。构建P.putida NH33的基因组文库,并在E.coli中进行酯酶活性筛选,得到一个含有4.7kb插入片段的阳性克隆。对这个克隆的DNA片段进行序列分析,表明它含有一个1142个碱基的开放阅读框,为编码381个氨基酸的酯酶。推测的酯酶氨基酸序列与其它丝氨酸酯水解酶具有共同的保守基序GXSXG。把该蛋白在E.coli BL21(DE3)中进行表达,并用金属亲和层析纯化至单一条带。利用纯化的酶水解2-芳基丙酸乙酯制备2-芳基丙酸的S型异构体,产物的光学纯度ee_p>99%,说明此酶可8用于手性药物的合成。该酯酶是一个新酶,其基因序列已递交GenBank,登记号为AY896293。     

Purification and characterization of esterases D1 and D2 from human erythrocytes. Evidence that they are monomers

Esterase D1 and esterase D2, two common esterase D (EC 3.1.1.1) isozymes, were isolated and purified from human erythrocytes. Their substrate specificity, pH profile and Km values were essentially identical. Their molecular mass was the same at 34 kDa on sodium dodecyl sulfate/polyacrylamide electrophoresis and at 27 kDa on Sephadex G-100 gel filtration. Antisera to each of the esterase D1 and esterase D2 isozymes were successfully raised in chickens; each antiserum reacted identically with both isozymes. These findings indicate that the isozymes are close to each other in structure. The fact that the molecular mass of the esterase D1 and esterase D2 isozymes computed on sodium dodecyl sulfate/polyacrylamide electrophoresis was close to that obtained on Sephadex G-100 gel filtration in non-dissociating buffer indicates that the isozymes are not dimers bound by disulfide bonds or a noncovalent force. These facts together indicate that the esterase D isozymes are monomers, contrary to the prevailing view that they are dimers.     

Plant biochemistry of xenobiotics. Purification and properties of a wheat esterase hydrolyzing the plasticizer chemical, bis(2-ethylhexyl)phthalate

A soluble wheat esterase, catalyzing a cleavage of the mass-produced plasticizer chemical, bis(2-ethylhexyl)phthalate (DEHP), has been discovered. Although wheat plants and seeds as well as cultured wheat cells contained more than 12 non-specific esterase activities, only a single protein with a marked preference for a substrate chain-length of 6-8 carbon atoms was active with DEHP. This enzyme is shown to differ from all previously characterized plant lipases and esterases. The enzyme was purified 10-fold from wheat plants and 280-fold, to electrophoretic homogeneity, from cultured wheat cells. An apparent functional molecular mass of 38 000 Da and an apparent subunit molecular mass of 22 000 Da were determined. Inhibitor experiments pointed to the catalytic involvement of a serine residue. Cleavage of DEHP by the purified enzyme was about 10(4) times slower than cleavage of 4-nitrophenyl octanoate. This was consistent with previous evidence for a rate-limiting role of the esterase reaction in DEHP metabolism by intact wheat cells.     

Biosynthesis pathway of gp90MEL-14, the mouse lymph node-specific homing receptor

The mouse lymph node specific homing receptor gp90MEL-14 is a 95-kDa molecular mass ubiquitinated cell surface molecule involved in the binding of lymphocytes to high endothelial venules in peripheral lymph nodes. The molecule is thought to consist of a core protein to which ubiquitin side chains are covalently bound. Recently we cloned the cDNA encoding the core protein; this cDNA clone encodes for a polypeptide with an estimated molecular mass of 37 kDa. We have studied the biosynthesis of gp90MEL-14 in an effort to explain the difference in molecular mass between the core protein and the 95-kDa mature molecule. Pulse labeling experiments show a rapid synthesis of a 70-kDa precursor form that contains high-mannose N-linked oligosaccharides. On processing of the high-mannose oligosaccharides into complex N-linked oligosaccharides, the precursor matures in a single step into the 95-kDa form. Experiments using deglycosylating enzymes and inhibitors of N-linked glycosylation demonstrate that the molecular mass of deglycosylated gp90MEL-14 is 45 kDa; extensive N-linked glycosylation is responsible for the difference in molecular mass with the mature 95-kDa form. The core protein molecular weight of in vitro transcribed and translated gp90MEL-14 cDNA is consistent with the estimated molecular mass of 37 kDa, calculated from the cDNA sequence of the core protein, and 8 to 10 kDa less than the protein molecular mass of gp90MEL-14 translated in vivo in the presence of tunicamycin (45 kDa). Inasmuch as we have ruled out glycosylation as accounting for this discrepancy, this is consistent with the addition of one ubiquitin moiety to the core protein during biosynthesis. Limited proteolysis confirms the similarity between in vitro transcribed gp90MEL-14 cDNA and the tunicamycin form of gp90MEL-14.     

A new esterase, belonging to hormone-sensitive lipase family, cloned from Rheinheimera sp. isolated from industrial effluent

The gene for esterase (rEst1) was isolated from a new species of genus Rheinheimera by functional screening of E. coli cells transformed with the pSMART/HaeIII genomic library. E. coli cells harboring the esterase gene insert could grow and produce clear halo zones on tributyrin agar. The rEst1 ORF consisted of 1,029 bp, corresponding to 342 amino acid residues with a molecular mass of 37 kDa. The signal P program 3.0 revealed the presence of a signal peptide of 25 amino acids. Esterase activity, however, was associated with a homotrimeric form of molecular mass 95 kDa and not with the monomeric form. The deduced amino acid sequence showed only 54% sequence identity with the closest lipase from Cellvibrio japonicus strain Ueda 107. Conserved domain search and multiple sequence alignment revealed the presence of an esterase/ lipase conserved domain consisting of a GXSXG motif, HGGG motif (oxyanion hole) and HGF motif, typical of the class IV hormone sensitive lipase family. On the basis of the sequence comparison with known esterases/ lipases, REst1 represents a new esterase belonging to class IV family. The purified enzyme worked optimally at 50 degrees C and pH 8, utilized pNP esters of short chain lengths, and showed best catalytic activity with p-nitrophenyl butyrate (C?), indicating that it was an esterase. The enzyme was completely inhibited by PMSF and DEPC and showed moderate organotolerance.     

Purification and properties of an esterase from the yeast Saccharomyces cerevisiae and identification of the encoding gene.

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

Tissue kallikrein of human seminal plasma is secreted by the prostate gland

Samples of human seminal plasma were subjected to gel filtration, and the eluted fractions were analysed for their contents of tissue kallikrein-like antigen, arginine esterase activity and kininogenase activity. Two peaks of tissue kallikrein-like antigen were detected with apparent molecular masses of about 72 and 48 kDa. As judged by the criteria of molecular mass, immunoreactivity, kininogenase activity, identification of the released kinin as kallidin and inhibition studies, a genuine tissue kallikrein has been identified in the 48-kDa peak. In addition, this peak contains one or more species of immunoreactive tissue kallikrein which differ in molecular mass and enzymatic activities. The 72-kDa peak probably represents the complex of tissue kallikrein with alpha 1-proteinase inhibitor rather than a true high molecular mass tissue kallikrein. The prostate gland was identified as the site of origin of the tissue kallikrein in the seminal fluid by indirect methods and by demonstrating immunoreactive tissue kallikrein in prostatic tissue and secretion.     

Purification and properties of an acetylxylan esterase from Fibrobacter succinogenes S85.

An acetylxylan esterase (EC 3.1.1.6) was purified to apparent homogeneity from the nonsedimentable extracellular culture fluid of Fibrobacter succinogenes S85 grown on cellulose. This enzyme had an apparent molecular mass of 55 kDa and an isoelectric point of 4.0. The temperature and pH optima were 45 degrees C and 7.0, respectively. The apparent Km and Vmax were 2.7 mM and 9,100 U/mg, respectively, for the hydrolysis of alpha-naphthyl acetate. The enzyme cleaved acetyl residues from birchwood acetylxylan but did not hydrolyze carboxymethylcellulose, larchwood xylan, ferulic acid-arabinose-xylose polymer, p-nitrophenyl-alpha-L-arab-inofuranoside, or longer-chain naphthyl fatty acid esters. The esterase enzyme may play a role in enhancing hemicellulose degradation by F. succinogenes, thereby allowing it greater access to cellulose present in forage cell walls.     

Purification and properties of an acetylxylan esterase from Fibrobacter succinogenes S85

An acetylxylan esterase (EC 3.1.1.6) was purified to apparent homogeneity from the nonsedimentable extracellular culture fluid of Fibrobacter succinogenes S85 grown on cellulose. This enzyme had an apparent molecular mass of 55 kDa and an isoelectric point of 4.0. The temperature and pH optima were 45 degrees C and 7.0, respectively. The apparent Km and Vmax were 2.7 mM and 9,100 U/mg, respectively, for the hydrolysis of alpha-naphthyl acetate. The enzyme cleaved acetyl residues from birchwood acetylxylan but did not hydrolyze carboxymethylcellulose, larchwood xylan, ferulic acid-arabinose-xylose polymer, p-nitrophenyl-alpha-L-arab-inofuranoside, or longer-chain naphthyl fatty acid esters. The esterase enzyme may play a role in enhancing hemicellulose degradation by F. succinogenes, thereby allowing it greater access to cellulose present in forage cell walls.     

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

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 α/β hydrolases (G × S × G) was detected from position 104 to 108 together with a putative catalytic triad consisting of Ser¹⁰⁶, Asp¹⁹⁶, and His²²⁵. 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°C with more than 50% retained activity at the freezing point of water. The thermostability of EstO (50% activity after 5 h at 40°C) dramatically increased in the truncated variant (50% activity after 2.5 h at 90°C). Furthermore, the esterase displays broad substrate specificity for esters of short-chain fatty acids (C₂–C₈).     

Isolation and characterization of p-coumaroyl esterase from the anaerobic fungus Neocallimastix strain MC-2.

An extracellular p-coumaroyl esterase produced by the anaerobic fungus Neocallimastix strain MC-2 released p-coumaroyl groups from 0-[5-0-((E)-p-coumaroyl)-alpha-L-arabinofuranosyl]-(1----3)-0-beta -D-xylopyranosyl-(1----4)-D-xylopyranose (PAXX). The esterase was purified 121-fold from culture medium in successive steps involving ultrafiltration column chromatography on S-sepharose and hydroxylapatite, isoelectric focusing, and gel filtration. The native enzyme had an apparent mass of 11 kDa under nondenaturing conditions and a mass of 5.8 kDa under denaturing conditions, suggesting that the enzyme may exist as a dimer. The isoelectric point was 4.7, and the pH optimum was 7.2. The purified esterase had 100 times more activity towards PAXX than towards the analogous feruloyl ester (FAXX). The apparent Km and Vmax of the purified p-coumaroyl esterase for PAXX at pH 7.2 and 40 degrees C were 19.4 microM and 5.1 microM min(-1), respectively. p-Coumaroyl tetrasaccharides isolated from plant cell walls were hydrolyzed at rates similar to that for PAXX, whereas a dimer of PAXX was hydrolyzed at a rate 20-fold lower, yielding 4,4'-dihydroxy-alpha-truxillic acid as an end product. Ethyl and methyl p-coumarates were hydrolyzed at very slow rates, if at all. The purified esterase released p-coumaroyl groups from finely, but not coarsely, ground plant cell walls, and this activity was enhanced by the addition of xylanase and other cell wall-degrading enzymes.     

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.     

Construction and characterization of a recombinant esterase with high activity and enantioselectivity to (S)-ketoprofen ethyl ester

The ester-hydrolyzing enzyme families, including lipase and esterase, mediated a broad range of reactions and, thus, were able to act on a variety of ester compounds that are found naturally or exploited industrially. With the increasing demand for pharmacological use, attempts to produce an enantiomer (S)-ketoprofen from the corresponding ethyl ester have recently been proliferating, but information about the structure and function of related enzymes has not been reported to date in detail. Here, we reported the construction, expression, and one-step purification of a potential esterase in Escherichia coli with a hexahistidine tag at its N-terminus. The expression level of the enzyme was more than 20% of the total protein in E. coli, resulting in approximately 1.2mg of the purified proteins by an affinity resin, Ni-NTA, from a 0.2L of bacterial culture in a single step. As typical properties, its innate traits that revealed favorable reactions at alkaline pH and high activity to the triglycerides composed of short chain fatty acids (99% ee(p)). The small-scale conversion using the recombinant enzyme strongly suggested the enzyme to be useful for enzyme-mediated chiral resolution of (S)-ketoprofen.     

Molecular analysis of an esterase-encoding gene from a lipolytic psychrotrophic pseudomonad.

An esterase gene (estA) from a lipolytic psychotroph (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.     

Partial purification and characterization of sialate O-acetylesterase from bovine brain

From bovine brain an esterase was purified 2,600-fold in an overall yield of 5.6%. For the isolation ion-exchange chromatographies, gel filtration, and preparative isoelectric focusing were used. The molecular mass is 56 kDa after gel chromatography on Sephacryl S-200 and 51 kDa after HPLC, the pH-optimum at 7.4, and the isoelectric point in the range of pH 5.8-6.1, as estimated from preparative isoelectric focusing. The substrate specificity of this enzyme was tested with various naturally occurring O-acylated sialic acids, synthetic carbohydrate acetates, and other esters. Besides aromatic acetyl esters such as e.g. alpha-naphthyl acetate, the highest preference was for N-acetyl-9-O-acetylneuraminic acid, followed by N-acetyl-4-O-acetylneuraminic acid. Other primary acetyl esters such as 6-O-acetylated D-glucose and 2-acetamido-2-deoxy-D-mannose were not hydrolyzed. The 9-O-acetyl derivative of the naturally occurring unsaturated sialic acid 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, however, is a substrate for this esterase. Whereas N-acetyl-9-O-acetylneuraminic acid as a component of sialyllactose is nearly as well hydrolyzed as the corresponding free sialic acid, O-acetylated sialoglycoconjugates with high molecular weights (mucins, serum glycoproteins, gangliosides) are not hydrolyzed by this esterase. N-Acetylated sialic acids are better substrates than the analogous N-glycoloyl derivatives. Esterification of the carboxyl function of sialic acids prevents the action of the esterase on the O-acetyl groups. The enzyme has no carboxyl esterase or amidase activity, and does not act on acetylcholine. It hydrolyzes almost exclusively acetyl esters. Inhibition studies suggest that it has a catalytically active serine residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and some properties of an acryloyl-CoA reductase of Clostridium kluyveri

Acryloyl-CoA reductase, a presumably previously unknown soluble enzyme, is present in Clostridium kluyveri. It catalyses the reduction of the carbon-carbon double bond of acryloyl-CoA or ethyl vinyl ketone and other alpha, beta-unsaturated carbonyl compounds at the expense of reduced methylviologen. On the basis of a Vmax/Km ratio, which is at least 18 times higher than that for the next best substrate (E)-2-butenoyl-CoA, the enzyme is called acryloyl-CoA reductase. A purity of over 90% was achieved. The apparent molecular mass, as determined by gel chromatography, is 28.4 kDa. Dodecyl sulfate gel electrophoresis shows subunits with a molecular mass of 14.2 kDa. Based on a molecular mass of 28.4 kDa about 1.5 mol FMN have been observed. Less than 0.2 g-atom iron per mol protein were determined. Ferredoxin or flavodoxin seem to be able to carry electrons from hydrogenase to the acryloyl-CoA reductase. The addition of hydrogen to the alpha-carbon of ethyl vinyl ketone occurs from the re-side.