Novel microorganism, a novel esterase and method for preparing the same
AssigneeSumitomo Chemical Company, Limited
InventorsChiaki Sugiki, Kanji Nishizawa, Masako Sugimoto, Kazumi Sonoda, Fumitaka Kishimoto
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Abstract
A novel esterase (molecular weight 54,000.+-.2,000) derived from a microorganism belonging to genus Bacilus is extensively utilizable for organic synthetic reaction, especially for asymmetric hydrolysis of ethyl 2,2-dimethyl-3-(2,2-dichlorovinyl)cyclopropane carboxylate (ethyl permethrate), in the same manner as pig liver esterase. The microorganism suitable for production of the above novel esterase is Bacillus sp. DC-1 (FERM BP-1254).
Claims2
A process for preparing a novel esterase having the following physicochemical properties, which process comprises cultivating the microorganism Bacillus sp. DC-1 (FERM BP-1254) which is capable of producing an esterase having the following physicochemical properties, and recovering said esterase from the cells;
- (1) Activity;
- hydrolyzes the ester bond of organic carboxylic acid ester;
- (2) Substrate specificity:
- active mainly to an alcohol ester moiety of organic carboxylic acid and highly active to water-soluble short chain fatty acid ester;
- active to short chain mono- or tri-acyl glycerides but not to long chain mono-, di- or tri-acyl glycerides;
- active to palmitoyl-CoA but not to acetyl-CoA, choline ester or cholesterol ester;
- (3) Optimum pH and pH stability:
- optimum pH for hydrolysis in the case of using p-nitrophenyl ester of 2,2-dimethyl-3-(2,2-dichlorovinyl)-cyclopropane carboxylic acid of the formula (I) as a substrate is 8.5-9.0;
- ##STR3## when 10 mM Tris-HCl buffer solution (pH 7.0-9.0) is used for hydrolysis of the above substrate at 4.degree. C., remaining activity after 48 hours is about 100%;
- when 10 mM of phosphate buffer solution (pH 6.0) is used, the remaining activity is about 45%;
- and when 10 mM of phosphate buffer solution (pH 5.0) is used, the remaining activity is about 20%;
- (4) Optimum temperature and heat stability:
- optimum temperature for hydrolysis in the case of using p-nitrophenyl ester of 2,2-dimethyl-3-(2,2-dichlorovinyl)-cyclopropane carboxylic acid as a substrate in 100 mM Tris-HCl buffer (pH 9.0) is 50.degree.-55.degree. C.;
- remaining activities after treating in 100 mM Tris-HCl buffer solution (pH 9.0) for 10 minutes at 35.degree. C., is about 100%, at 40.degree. C. about 80%, and at 50.degree. C. about 15%;
- (5) Molecular weight:
- 54. 000.+-.2,000 (by gel filtration and SDS-polyacrylamide gel electrophoresis)
- (6) Isoelectric point:
- 4.8.+-.0.1
- (7) Inactivating conditions:
- (a) completely inactivated by treatment with 100 .mu.M phenylmethylsulfonyl fluoride or 80 .mu.M diisopropyl fluorophosphate at 37.degree. C. for 100 minutes;
- (b) activity decreased about 25% and about 70% by treatment with 100 mM Tris-HCl buffer solution (pH 9.0) containing 1 mM sodium deoxycholate and 5 mM sodium lauryl sulfate, respectively, at 37.degree. C. for 10 minutes.
A biologically pure culture of Bacillus sp. DC-1 (FERM BP-1254) capable of producing recoverable amounts of an enzyme having the following physiochemical properties:
- (1) Activity:
- hydrolyzes the ester bond of organic carboxylic acid ester;
- (2) Substrate specificity:
- active mainly to an alcohol ester moiety of organic carboxylic acid and highly active to water-soluble short chain fatty acid ester;
- active to short chain mono- or tri-acyl glycerides but not to long chain mono-, di-, or tri-acyl glycerides;
- active to palmitoyl-CoA but not to acetyl-CoA, choline ester or cholesterol ester;
- (3) Optimum pH and pH stability:
- optimum pH for hydrolysis in the case of using p-nitrophenyl ester of 2,2-dimethyl-3-(2,2-dichlorovinyl)cyclopropane carboxylic acid of the formula (I) as a substrate is 8.5-9.0;
- ##STR4## when 10 mM Tris-HCl buffer solution (pH 7.0-9.0) is used for hydrolysis of the above substrate at 4.degree. C., remaining activity after 48 hours is about 100%;
- when 10 mM of phosphate buffer solution (pH 6.0) is used, the remaining activity is about 45%;
- and when 10 mM of phosphate buffer solution (pH 5.0) is used, the remaining activity is about 20%;
- (4) Optimum temperature and heat stability:
- optimum temperature for hydrolysis in the case of using p-nitrophenyl ester of 2,2-dimethyl-3-(2,2-dichlorovinyl)-cyclopropane carboxylic acid as a substrate in 100 mM Tris-HCl buffer (pH 9.0) is 50.degree.-55.degree. C.;
- remaining activities after treating in 100 mM Tris-HCl buffer solution (pH 9.0) for 10 minutes at 35.degree. C., is about 100%, at 40.degree. C. about 80%, and at 50.degree. C. about 15%;
- (5) Molecular weight:
- 54. 00.+-.2,000 (by gel filtration and SDS polyacrylamide gel electrophoresis)
- (6) Isoelectric point:
- 4.8.+-.0.1
- (7) Inactivating conditions:
- (a) completely inactivated by treatment with 100 .mu.M phenylmethylsulfonyl fluoride or 80 .mu.M diisopropyl fluorophosphate at 37.degree. C. for 10 minutes;
- (b) activity decreased about 25% and about 70% by treatment with 100 mM Tris-HCl buffer solution (pH 9.0) containing 1 mM sodium deoxycholate and 5 mM sodium lauryl sulfate, respectively, at 37.degree. C. for 10 minutes.
Dependencies are inferred from the claim text.