Purification and characterization of a (R)-2,3-butanediol dehydrogenase from Saccharomyces cerevisiae.
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 diastereomeric 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 C5-acyloins were also accepted as substrates, whereas the enzyme was inactive towards the reduction of acetone and dihydroxyacetone. The relative molecular mass (Mr) 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 Mr 35,000. Optimum pH was 6.7 for the reduction of acetoin to 2,3-butanediol and 7.2 for the reverse reaction.
Heidlas
J
J
Technische Universität Berlin, Fachbereich Lebensmitteltechnologie ünd Biotechnologie.
Tressl
R
R
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D002498:Centrifugation; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002849:Chromatography, Gas; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013237:Stereoisomerism; D013379:Substrate Specificity
D000429:Alcohol Oxidoreductases; C019801:butanediol dehydrogenase