1. A novel carbonyl reductase with anti-Prelog stereospecificity from Acetobacter sp. CCTCC M209061: purification and characterization.
- Author
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Chen XH, Wei P, Wang XT, Zong MH, and Lou WY
- Subjects
- 2-Propanol chemistry, 2-Propanol metabolism, Acetobacter genetics, Acetobacter growth & development, Alcohol Oxidoreductases chemistry, Alcohol Oxidoreductases genetics, Alcohols chemistry, Bacterial Proteins genetics, Bacterial Proteins isolation & purification, Base Sequence, Biocatalysis drug effects, Biomass, Electrophoresis, Polyacrylamide Gel, Enzyme Stability drug effects, Hydrogen-Ion Concentration, Ketones chemistry, Kinetics, Metals pharmacology, Molecular Sequence Data, NAD chemistry, NAD metabolism, NADP chemistry, NADP metabolism, Oxidation-Reduction, Sequence Homology, Nucleic Acid, Stereoisomerism, Substrate Specificity, Temperature, Acetobacter enzymology, Alcohol Oxidoreductases metabolism, Alcohols metabolism, Bacterial Proteins metabolism, Ketones metabolism
- Abstract
A novel carbonyl reductase (AcCR) catalyzing the asymmetric reduction of ketones to enantiopure alcohols with anti-Prelog stereoselectivity was found in Acetobacter sp. CCTCC M209061 and enriched 27.5-fold with an overall yield of 0.4% by purification. The enzyme showed a homotetrameric structure with an apparent molecular mass of 104 kDa and each subunit of 27 kDa. The gene sequence of AcCR was cloned and sequenced, and a 762 bp gene fragment was obtained. Either NAD(H) or NADP(H) can be used as coenzyme. For the reduction of 4'-chloroacetophenone, the Km value for NADH was around 25-fold greater than that for NADPH (0.66 mM vs 0.026 mM), showing that AcCR preferred NADPH over NADH. However, when NADH was used as cofactor, the response of AcCR activity to increasing concentration of 4'-chloroacetophenone was clearly sigmoidal with a Hill coefficient of 3.1, suggesting that the enzyme might possess four substrate-binding sites cooperating with each other The Vmax value for NADH-linked reduction was higher than that for NADPH-linked reduction (0.21 mM/min vs 0.17 mM/min). For the oxidation of isopropanol, the similar enzymological properties of AcCR were found using NAD+ or NADP+ as cofactor. Furthermore, a broad range of ketones such as aryl ketones, α-ketoesters and aliphatic ketones could be enantioselectively reduced into the corresponding chiral alcohols by this enzyme with high activity.
- Published
- 2014
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