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

Area of Science:

• Biochemistry
• Enzymology
• Yeast Metabolism

Background:

• 2,3-Butanediol (2,3-BDO) is a valuable platform chemical with diverse applications.
• Stereoselective synthesis of 2,3-BDO and its derivatives is crucial for various industries.
• Enzymatic approaches offer a sustainable and efficient alternative for chiral compound production.

Purpose of the Study:

• To isolate and characterize a novel NAD-dependent (R)-2,3-butanediol dehydrogenase from Saccharomyces cerevisiae.
• To investigate the enzyme's substrate specificity and catalytic activity in the reduction of diacetyl and acetoin.
• To determine the enzyme's biochemical properties, including molecular weight and subunit composition.

Main Methods:

• Enzyme isolation and purification using streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, affinity chromatography (Matrex Gel Blue A), and Superose 6 prep grade chromatography.
• Chiral product analysis via gas chromatography with pre-chromatographic derivatization.
• Enzyme characterization including molecular weight estimation (gel filtration, SDS-PAGE) and determination of optimal pH for substrate reduction.

Main Results:

• A 70-fold enriched (R)-2,3-butanediol dehydrogenase was purified with 44% yield.
• The enzyme selectively catalyzes the oxidation at the (R)-center of 2,3-butanediol.
• It reduces diacetyl to (R)-acetoin and subsequently to (R,R)-2,3-butanediol using NADH; accepts 1-hydroxy-2-ketones and C5-acyloins but not acetone or dihydroxyacetone.
• The enzyme has a relative molecular mass of 140,000 Da, composed of four identical subunits of 35,000 Da.
• Optimal pH for acetoin reduction is 6.7, and for the reverse reaction is 7.2.

Conclusions:

• Saccharomyces cerevisiae harbors a specific NAD-dependent (R)-2,3-butanediol dehydrogenase with potential for stereoselective synthesis.
• The enzyme's ability to convert diacetyl to (R)-acetoin and (R,R)-2,3-butanediol highlights its utility in producing chiral compounds.
• Further research into enzyme engineering could optimize its application in industrial biotransformations.