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.
Highlights
Enantiopure alcohols are widely used as building blocks in pharmaceutical, agrochemical, flavor, and functional material industries
Carbonyl reductases belong to the oxidoreductase family that often need cofactors such as NAD(H) or NADP(H) to be functionally active, which could be divided into three subtypes based on the coenzyme requirements: (1) NAD(H)-specific, like carbonyl reductase originated from Candida viswanathii [7]; (2) NADP(H)-specific, such as carbonyl reductase from Neurospora crassa [5]; (3) dual coenzyme specific, like carbonyl reductase from Candida parapsilosis [8]
The apparent Km value (S0.5) for 49-chloroacetophenone was 4.13 mM, which was higher than 49-chloroacetophenone Km (2.96 mM) using NADPH as cofactor. These results showed that the enzyme preferred NADPH when catalyzing reduction of 49-chloroacetophenone
Summary
Enantiopure alcohols are widely used as building blocks in pharmaceutical, agrochemical, flavor, and functional material industries. The ‘‘green’’ synthesis catalyzed by enzymes, microorganisms and plant cells for the production of various enantiopure alcohols has attracted considerable attention due to its high efficiency, high enantioselectivity, mild reaction condition, and low environmental pollution [1,2]. Carbonyl reductase is one of the attractive biocatalysts for synthesis of enantiopure alcohols [3], and widely expressed in various organisms from microorganisms to mammals such as Bacillus [4], Neurospora crassa [5] and human brain [6]. The carbonyl reductase from Neurospora crassa could catalyze asymmetric reduction of ketones, diketones, a-keto esters, and b-keto esters [5] and the carbonyl reductase from Streptomyces coelicolor could be used for synthesis of chiral alcohols from aryl ketones, a-keto esters, and b-keto esters with high enantioselectivity [9]
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