Cloning and Expression ofPseudomonas putidaEsterase Gene inEscherichia coliand Its Use in Enzymatic Production of<scp>d</scp>-β-Acetylthioisobutyric Acid

Ozaki, Eiji; Sakimae, Akihiro; Numazawa, Ryozo

doi:10.1271/bbb.58.1745

Cited by 18 publications

(6 citation statements)

References 2 publications

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“…1996) and the synthesis of esters, as well as for the resolution of racemic mixtures (Margolin 1993). Enzymes with the specific esterase activity of hydrolysing (R)‐ or (S)‐carboxylic acid esters were identified from several Pseudomonas species, such as Pseudomonas fluorescens (Ozaki and Akihiro 1994), P. putida (Ozaki et al. 1994, 1995) and P. aeruginosa (Lee et al.…”

Section: Introductionmentioning

confidence: 99%

Cloning and expression of a novel esterase gene cpoA from Burkholderia cepacia

et al. 2004

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Aims: To screen and clone a novel enzyme with specific activity for the resolution of (R)-b-acetylmercaptoisobutyrate (RAM) from (R,S)-b-acetylmercaptoisobutyrate [(R,S)-ester]. Methods and Results: A micro-organism that produces a novel esterase was isolated and identified as the bacterium Burkholderia cepacia by using the analysis of cellular fatty acids, Biolog automated microbial identification/characterization system, and 16S rRNA gene sequence analysis. A novel esterase gene was cloned from the chromosomal DNA of B. cepacia and was designated as cpoA. The cpoA encodes a polypeptide of 273 amino acids which shows a strong sequence homology with many bacterial nonhaeme chloroperoxidases. In addition, a typical serine-hydrolase motif, Gly-X-Ser-X-Gly, and the highly conserved catalytic triad, Ser95, Asp224, and His253, were identified in the deduced amino acid sequence of cpoA by multiple sequence alignment. Conclusion: The cpoA cloned from B. cepacia encodes a novel esterase which is highly related to the nonhaeme chloroperoxidases. Significance and Impact of the Study: This is the first report that describes the isolation and cloning of a serine esterase gene from B. cepacia, which is useful in the chiral resolution of (R,S)-ester. The cloned gene will allow additional research on the bifunctionality of the enzyme with esterase and chloroperoxidase activity at the structural and functional levels.

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Section: Introductionmentioning

confidence: 99%

Cloning and expression of a novel esterase gene cpoA from Burkholderia cepacia

et al. 2004

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“…Examples are the esterases from Pseudomonas fluorescens (Choi, Jeohn, Rhee & Yoo, 1990;Hong, Jang, Choi & Yoo, 1991;Kim, Lee, Choi, Park & Yoo, 1994), P putida (Ozaki, Sakimae & Numazawa, 1994), and Pseudomonas sp. (McKay et al, 1992;Shimada et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

Crystallization and preliminary X-ray diffraction studies of thePseudomonas marginataesterase EstB

Wagner¹,

Sölkner²,

Petersen³

et al. 1997

Acta Cryst D

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“…A study of the hydrolytic reaction of methyl DL-␤-acetylthioisobutyrate (DL-MATI) with an esterase from Pseudomonas putida strain MR-2068 revealed that DAT had an optical purity of more than 98% when the reaction was done below pH 7.5 and below 50°C (34). Honda et al reported that 3,4-dihydrocoumarin hydrolase hydrolyzed DL-MATI to produce DAT with a high optical purity (Ͼ99.9% enantiomeric excess) (15).Esterases are widely distributed in Pseudomonas, and several have been identified, cloned, and sequenced, such as esterases of Pseudomonas fluorescens (6,7,16,20), Pseudomonas putida (27,28), and other Pseudomonas spp. (22,37,39,40).…”

mentioning

confidence: 99%

“…Esterases are widely distributed in Pseudomonas, and several have been identified, cloned, and sequenced, such as esterases of Pseudomonas fluorescens (6,7,16,20), Pseudomonas putida (27,28), and other Pseudomonas spp. (22,37,39,40).…”

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confidence: 99%

Stereoselective Esterase from Pseudomonas putida IFO12996 Reveals α/β Hydrolase Folds for d -β-Acetylthioisobutyric Acid Synthesis

Elmi

Lee²,

Huang

et al. 2005

J Bacteriol

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Esterase (EST) from Pseudomonas putida IFO12996 catalyzes the stereoselective hydrolysis of methyl DL-␤-acetylthioisobutyrate (DL-MATI) to produce D-␤-acetylthioisobutyric acid (DAT), serving as a key intermediate for the synthesis of angiotensin-converting enzyme inhibitors. The EST gene was cloned and expressed in Escherichia coli; the recombinant protein is a non-disulfide-linked homotrimer with a monomer molecular weight of 33,000 in both solution and crystalline states, indicating that these ESTs function as trimers. EST hydrolyzed DL-MATI to produce DAT with a degree of conversion of 49.5% and an enantiomeric excess value of 97.2% at an optimum pH of about 8 to 10 and an optimum temperature of about 57 to 67°C. The crystal structure of EST has been determined by X-ray diffraction to a resolution of 1.6 Å, confirming that EST is a member of the ␣/␤ hydrolase fold superfamily of enzymes and includes a catalytic triad of Ser97, Asp227, and His256. The active site is located approximately in the middle of the molecule at the end of a pocket ϳ12 Å deep. EST can hydrolyze the methyl ester group without affecting the acetylthiol ester moiety in DL-MATI. The examination of substrate specificity of EST toward other linear esters revealed that the enzyme showed specific activity toward methyl esters and that it recognized the configuration at C-2.Esterases belong to the hydrolase family and are ubiquitous enzymes identified in various species (24). These enzymes exist as either monomers or oligomers with subunit molecular weights (MW) that range from 25,000 to 60,000 (13), with a conserved pentapeptide (Gly-X 1 -Ser-X 2 -Gly) sequence around the catalytic serine residue (3), despite sharing a low sequence homology. Unlike lipases, esterases preferentially hydrolyze water-soluble esters or short-chain fatty acid triglycerides. The proposed mechanism of catalysis for esterases is similar to the serine protease mechanism, which involves nucleophilic attack by the catalytic serine hydroxyl on the carbonyl carbon of the scissile bond. This hydroxyl group becomes more nucleophilic and the reaction is stabilized through hydrogen bonding to the imidazole group of the catalytic histidine that is stabilized by the carboxyl group of the acidic member of the catalytic triad (9, 12). The importance of esterases for application in pharmaceutical and agrochemical industries is increasing because their properties as stereo-selective catalysts in the synthesis of optically pure molecules are diverse. On the basis of their protein structures, the ␣/␤ hydrolase architecture of lipases possesses an additional movable helical lid that controls access to the active site, providing a molecular explanation for lipase-enhanced activity upon contact with a lipase-water interface (41).D-␤-acetylthioisobutyric acid (DAT), also known as S-␤-acetylthio-2-methylpropionic acid, is a key intermediate for the synthesis of angiotensin-converting enzyme inhibitors, such as captopril and alacepril, which are used to treat hypertension and congestive he...

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Cloning and Expression ofPseudomonas putidaEsterase Gene inEscherichia coliand Its Use in Enzymatic Production ofd-β-Acetylthioisobutyric Acid

Cited by 18 publications

References 2 publications

Cloning and expression of a novel esterase gene cpoA from Burkholderia cepacia

Cloning and expression of a novel esterase gene cpoA from Burkholderia cepacia

Crystallization and preliminary X-ray diffraction studies of thePseudomonas marginataesterase EstB

Stereoselective Esterase from Pseudomonas putida IFO12996 Reveals α/β Hydrolase Folds for d -β-Acetylthioisobutyric Acid Synthesis

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