Purification and characterization of a (R)-2,3-butanediol dehydrogenase from Saccharomyces cerevisiae.

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摘要：

A NAD-dependent ( R )-2,3-butanediol dehydrogenase (EC 1.1.1.4), selectively catalyzing the oxidation at the ( R )-center of 2,3-butanediol irrespective of the absolute configuration of the other carbinol center, was isolated from cell extracts of the yeast Saccharomyces cerevisiae . Purification was achieved by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, affinity chromatography on Matrex Gel Blue A and Superose 6 prep grade chromatography leading to a 70-fold enrichment of the specific activity with 44% yield. Analysis of chiral products was carried out by gas chromatographic methods via pre-chromatographic derivatization and resolution of corresponding diasteromeric derivatives. The enzyme was capable to reduce irreversibly diacetyl (2,3-butanediol) to ( R )-acetoin (3-hydroxy-2-butanone) and in a subsequent reaction reversibly to ( R,R )-2,3-butanediol using NADH as coenzyme. 1-Hydroxy-2-ketones and C 5 -acyloins were also accepted as substrates, whereas the enzyme was inactive towards the reduction of acetone and dihydroxyacetone. The relative molecular mass ( M r ) of the enzyme was estimated as 140 000 by means of gel filtration. On SDS-polyacrylamide gel the protein decomposed into 4 (identical) subunits of M r 35 000. Optimum pH was 6.7 for the reduction of acetoin to 2,3-butanediol and 7.2 for the reverse reaction.

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关键词：

Yeast Saccharomyces cerevisiae R)-2,3-Butanediol dehydrogenase Stereospecificity Gas chromatographic analysis of enantiomers

DOI：

10.1007/BF00248966

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年份：

1990