title: Towards the discovery of alcohol dehydrogenases: NAD(P)H fluorescence-based screening and characterization of the newly isolated Rhodococcus erythropolis WZ010 in the preparation of chiral aryl secondary alcohols.
abstract: A simple and reliable procedure was developed to screen biocatalysts with high alcohol dehydrogenase activity, efficient internal coenzyme regeneration, and high stereoselectivity. The strategy of activity screening in a microtitre plate format was based on the detection of fluorescence of NAD(P)H originating from the oxidation of alcohols. The primary and secondary screenings from soil samples yielded a versatile bacterial biocatalyst Rhodococcus erythropolis WZ010 demonstrating potential for the preparation of chiral aryl secondary alcohols. In terms of activity and stereoselectivity, the optimized reaction conditions in the stereoselective oxidation were 30 °C, pH 10.5, and 250 rpm, whereas bioreduction using glucose as co-substrate was the most favorable at 35 °C and pH 7.5 in the static reaction mixture. Under the optimized conditions, fresh cells of the strain stereoselectively oxidized the (S)-enantiomer of racemic 1-phenylethanol (120 mM) to acetophenone and afforded the unoxidized (R)-1-phenylethanol in 49.4 % yield and >99.9 % enantiomeric excess (e.e.). In the reduction of 10 mM acetophenone, the addition of 100 mM glucose significantly increased the conversion rate from 3.1 to 97.4 %. In the presence of 800 mM glucose, acetophenone and other aromatic ketones (80 mM) were enantioselectively reduced to corresponding (S)-alcohols with excellent e.e. values. Both stereoselective oxidation and asymmetric reduction required no external cofactor regeneration system.
authors: College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, 310014 Zhejiang, China.
authors: Chi
authors: C
authors: Yang
authors: Xiangxian
authors: X
authors: Ying
authors: Meilan
authors: M
authors: Yu
authors: Yinjun
authors: Y
authors: Zhang
authors: Bin
authors: B
authors: Xiong
authors: Qingqing
authors: Q
authors: Song
authors: Zhao
authors: Z
authors: Wang
mesh_terms: D000426:Alcohol Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism*; D000438:Alcohols / Q000737:chemistry* / Q000378:metabolism*; D055162:Biocatalysis; D005453:Fluorescence; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007659:Ketones / Q000378:metabolism; D009249:NADP / Q000032:analysis / Q000378:metabolism*; D010084:Oxidation-Reduction; D012240:Rhodococcus / Q000145:classification / Q000201:enzymology* / Q000302:isolation & purification* / Q000378:metabolism; D013237:Stereoisomerism; D013696:Temperature
chemical_list: D000438:Alcohols; D007659:Ketones; D009249:NADP; D000426:Alcohol Dehydrogenase; D005947:Glucose