期刊
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
卷 98, 期 2, 页码 641-650出版社
SPRINGER
DOI: 10.1007/s00253-013-4870-5
关键词
Acetoin(diacetyl) reductase; Rhodococcus erythropolis WZ010; (2S,3S)-2,3-butanediol; Asymmetric reduction; Stereoselective oxidation
资金
- Natural Science Foundation of Zhejiang Province [LY12B06011, Y4110468]
- Zhejiang Provincial Top Academic Discipline of Biomedical Engineering [SWYX0905]
- Research Foundation of Education Bureau of Zhejiang Province [Y201122330]
- National High Technology Research and Development Program of China [2011AA02A210]
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 A degrees C, whereas those for (2S,3S)-2,3-butanediol oxidation were pH 9.5 and 25 A degrees 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.
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