Biocatalytic properties of a recombinant aldo-keto reductase with broad substrate spectrum and excellent stereoselectivity.
In the screening of 11 E. coli strains overexpressing recombinant oxidoreductases from Bacillus sp. ECU0013, an NADPH-dependent aldo-keto reductase (YtbE) was identified with capability of producing chiral alcohols. The protein (YtbE) was overexpressed, purified to homogeneity, and characterized of biocatalytic properties. The purified enzyme exhibited the highest activity at 50°C and optimal pH at 6.5. YtbE served as a versatile reductase showing a broad substrate spectrum towards different aromatic ketones and keto esters. Furthermore, a variety of carbonyl substrates were asymmetrically reduced by the purified enzyme with an additionally coupled NADPH regeneration system. The reduction system exhibited excellent enantioselectivity (>99% ee) in the reduction of all the aromatic ketones and high to moderate enantioselectivity in the reduction of α- and β-keto esters. Among the ketones tested, ethyl 4,4,4-trifluoroacetoacetate was found to be reduced to ethyl (R)-4,4,4-trifluoro-3-hydroxy butanoate, an important pharmaceutical intermediate, in excellent optical purity. To the best of our knowledge, this is the first report of ytbE gene-encoding recombinant aldo-keto reductase from Bacillus sp. used as biocatalyst for stereoselective reduction of carbonyl compounds. This study provides a useful guidance for further application of this enzyme in the asymmetric synthesis of chiral alcohol enantiomers.
Ni
Yan
Y
Laboratory of Biocatalysis and Bioprocessing, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, People's Republic of China.
Li
Chun-Xiu
CX
Ma
Hong-Min
HM
Zhang
Jie
J
Xu
Jian-He
JH
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase