Characterization of a stereospecific acetoin(diacetyl) reductase from Rhodococcus erythropolis WZ010 and its application for the synthesis of (2S,3S)-2,3-butanediol

Appl Microbiol Biotechnol. 2014 Jan;98(2):641-50. doi: 10.1007/s00253-013-4870-5. Epub 2013 Apr 9.


Rhodococcus erythropolis WZ010 was capable of producing optically pure (2S,3S)-2,3-butanediol in alcoholic fermentation. The gene encoding an acetoin(diacetyl) reductase from R. erythropolis WZ010 (ReADR) was cloned, overexpressed in Escherichia coli, and subsequently purified by Ni-affinity chromatography. ReADR in the native form appeared to be a homodimer with a calculated subunit size of 26,864, belonging to the family of the short-chain dehydrogenase/reductases. The enzyme accepted a broad range of substrates including aliphatic and aryl alcohols, aldehydes, and ketones. It exhibited remarkable tolerance to dimethyl sulfoxide (DMSO) and retained 53.6 % of the initial activity after 4 h incubation with 30 % (v/v) DMSO. The enzyme displayed absolute stereospecificity in the reduction of diacetyl to (2S,3S)-2,3-butanediol via (S)-acetoin. The optimal pH and temperature for diacetyl reduction were pH 7.0 and 30 °C, whereas those for (2S,3S)-2,3-butanediol oxidation were pH 9.5 and 25 °C. Under the optimized conditions, the activity of diacetyl reduction was 11.9-fold higher than that of (2S,3S)-2,3-butanediol oxidation. Kinetic parameters of the enzyme showed lower K(m) values and higher catalytic efficiency for diacetyl and NADH in comparison to those for (2S,3S)-2,3-butanediol and NAD⁺, suggesting its physiological role in favor of (2S,3S)-2,3-butanediol formation. Interestingly, the enzyme showed higher catalytic efficiency for (S)-1-phenylethanol oxidation than that for acetophenone reduction. ReADR-catalyzed asymmetric reduction of diacetyl was coupled with stereoselective oxidation of 1-phenylethanol, which simultaneously formed both (2S,3S)-2,3-butanediol and (R)-1-phenylethanol in great conversions and enantiomeric excess values.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetoin Dehydrogenase / chemistry
  • Acetoin Dehydrogenase / genetics
  • Acetoin Dehydrogenase / isolation & purification
  • Acetoin Dehydrogenase / metabolism*
  • Butylene Glycols / metabolism*
  • Chromatography, Affinity
  • Cloning, Molecular
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • Enzyme Stability
  • Escherichia coli / genetics
  • Gene Expression
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Sequence Data
  • Molecular Weight
  • Protein Multimerization
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Rhodococcus / enzymology*
  • Rhodococcus / genetics
  • Sequence Analysis, DNA
  • Stereoisomerism
  • Substrate Specificity
  • Temperature


  • Butylene Glycols
  • DNA, Bacterial
  • Recombinant Proteins
  • 2,3-butylene glycol
  • Acetoin Dehydrogenase

Associated data

  • GENBANK/KC508606