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.
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Acknowledgments
This work was financially supported by Qianjiang Talent Program of Zhejiang Province (No. 2009R10048), Scientific Research Foundation of Zhejiang University of Technology (No. 20090172), Zhejiang Provincial Top Academic Discipline of Applied Chemistry and Eco-Dyeing and Finishing Engineering (No. ZYG2010016 and YR2010011) and Key Scientific and Technological Program of Zhejiang Province, China (No. 2009C13033-3). The authors thank Feng Cheng of Lehrstuhl für Biotechnologie, RWTH Aachen University for reading the manuscript.
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Chi Yang and Xiangxian Ying contributed equally to this study.
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Yang, C., Ying, X., Yu, M. et al. 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. J Ind Microbiol Biotechnol 39, 1431–1443 (2012). https://doi.org/10.1007/s10295-012-1160-7
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DOI: https://doi.org/10.1007/s10295-012-1160-7