Purification of α-Hydroxysteroid Dehydrogenase from Lactobacillus xylosus
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概要
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α-Hydroxysteroid dehydrogenase was purified to an electrophoretically homogeneous state by DEAE-Sephadex column chromatography, dehydrocholic acid-AH-Sepharose affinity chromatography and preparative polyacrylamide gel electrophoresis from the cell-free extract of Lactobacillus xylosus DC1115,and its specific activity was 102 units/mg.The molecular weight of the enzyme was found to be 84,000 by Sephadex G-200 column chromatography and to be 83,000 by sodium dodecylsulfate polyacrylamide gel electrophoresis.The enzyme had an optimum pH of 4.8 in the system of NADH and dehydrocholic acid and 9.0 in the system of NAD and chenodeoxycholic acid. It was stable in a pH range of 6.8 to 8.0. The optimum temperature for the activity was around 35℃ and the enzyme was stable up to 45℃ for 30 min.The Michaelis-Menten constant for NADH was 83 μM, and that for dehydrocholic acid was 1.8 mM. The maximum reaction rate was 406 μmol/min/mg.In the oxidation of NADH, the enzyme acted on the 3-carbonyl group of bile acids preferentially, and on the 7-carbonyl group, but did not act on the 12-carbonyl group. In the reduction of NAD, the enzyme acted on the 3α-hydroxyl group preferentially, and the rate was lowered by the presence of a substitution group(s) at the 7α- and /or 12α-position. α-Hydroxysteroid dehydrogenase was classified as a new oxidoreductase (EC. 1.1.1.-). The purified enzyme could produce 12-ketochenodeoxycholic acid via 7,12-diketolithocholic acid from dehydrocholic acid and NADH.
- 公益社団法人日本生物工学会の論文
- 1983-02-25
著者
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Kinoshita Shinichi
Department Of Fermentation Technology Faculty Of Engineering Osaka University
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Kinoshita S
Hokkaido Univ. Hokkaido Jpn
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Nishimura Akira
Department Of Forensic Medicene Health Bioscience Institute Tokushima University Graduate School
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Sawada Haruji
Department Of Fermentation Technology Faculty Of Engineering Osaka University
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Taguchi Hisaharu
Department Of Fermentation Technology Faculty Of Engineering
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KADOTA KEIICHI
Department of Fermentation Technology, Faculty of Engineering, Osaka University
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Kadota Keiichi
Department Of Fermentation Technology Faculty Of Engineering Osaka University
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Nishimura Akira
Department Of Applied Chemistry Faculty Of Engineering Osaka University
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Kinoshita Shinichi
Department Of Chemical Process Engineering Faculty Of Engineering Hokkaido University
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Nishimura Akira
Department Of Fermentation Technology Faculty Of Engineering Osaka University
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NISHIMURA AKIRA
Department of Fermentation Technology, Faculty of Engineering, Osaka University
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