Luo Wei,Du Hui-Jun,Bonku Emmanuel Mintah,Hou Ya-Li,Li Ling-Ling,Wang Xiao-Qiang,Yang Zhong-Hua
Abstract Enantiopure alcohols have received much attention due to their widespread use as pharmaceutical intermediates. In the asymmetric biosynthesis of enantiopure alcohols, the excellent performance of carbonyl reductase makes it be the best choice as the biocatalysts. In this work, an alkali-tolerant carbonyl reductase ( Bs CR, encoded by yueD ) from Bacillus subtilis (strain 168) was obtained through gene mining, and successfully heterologously expressed in Escherichia coli with pET-32a. Bs CR showed excellent alkali resistance and even can keep more than 70% of its peak activity after incubation in Tris–HCl buffer at pH 9.0 for 40 h. The Michaelis constants and maximal velocity of the Bs CR to NADPH (A) and ethyl 4-chloroacetoacetate (B) are \(K_{m}^{A}\) = 5.390 × 10 −2 mmol/L, \(K_{m}^{B}\) = 1.855 mmol/L, and \(V_{max}\) = 147.3 μmol·min −1 ·mg −1 , respectively. Applying the E. coli BL21(DE3)/pET-32a-yueD to catalyze asymmetric reduction of ethyl 4-chloroacetoacetate and acetophenone, the yield of S -CHBE reached 89.9% and S -1-phenyl ethanol reached 66.7%, and e.e. of both products reached more than 99%. This work provides a novel CR for asymmetric reduction. Graphic Abstract A carbonyl reductase ( Bs CR) and its gene were identified through gene mining, and overexpressed in Escherichia coli BL21(DE3) for whole-cell biocatalytic asymmetric reduction
In this study, sugarcane bagasse (SB) was pretreated with combination pretreatment (e.g., sequential KOH extraction and ionic liquid soaking, sequential KOH extraction and Fenton soaking, or sequential KOH extraction and glycerol soaking). After the enzymatic hydrolysis of pretreated SBs, it was found that all these three concentrated hydrolyzates could be used for the asymmetric bioreduction of ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl ( S )-4-chloro-3-hydroxybutanoate [( S )-CHBE]. Compared with glucose, arabinose and cellobiose couldn’t promote the initial reaction rate, and xylose could increase the intracellular NADH content. Moreover, it was the first report that hydrolyzates could be used for the effective biosynthesis of ( S )-CHBE (∼500 g/L; 98.0% yield) from 3000 COBE by whole cells of Escherichia coli CCZU-K14 in the presence of β-CD (0.4 mol β-CD/mol COBE), l -glutamine (200 mM) and glycine (500 mM). In conclusion, it is a new alternative to utilize bioresource for the synthesis of key chiral intermediate ( S )-CHBE.
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