Alcohol Dehydrogenases:Novel Screening Procedure,Application On The Preparation Of Chiral Alcohols And Engineering Of Coenzyme Specificity

Alcohol dehydrogenases (ADHs) are a class of ubiquitous NAD(P)+-dependent oxidoreductases catalyzing the interconversion between alcohols and aldehydes and ketones. They have also attracted much biotechnological interest for their potentials in the production of industrially valuable chiral alcohols via asymmetric ketone reduction. In this study, a simple and rapid screening procedure for the discovery of alcohol dehydrogenases was developed. Furthermore, a versatile biocatalyst was isolated and identified as Rhodococcus erythropolis WZ010and then thoroughtly characterized in terms of stereoselective oxidation and asymmetrical reduction. In addition, engineering of a NADP(H)-specific alcohol dehydrogenase from Thermococcus guaymasensis was carried out to alter its coenzyme specificity.1、A simple and efficient 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 appearance of NAD(P)H originated from the oxidation of alcohols, which was also suitable for identifying substrate specificity of any NAD(P)+-dependent alcohol dehydrogenase.2、The primary and secondary screenings from soil samples yielded a versatile bacterial biocatalyst R. erythropolis WZ010, which demonstrated potentials for the preparation of chiral aryl secondary alcohols via stereoselective oxidation and asymmetric reduction. In terms of activity and stereoselectivity, the reaction conditions in the stereoselective oxidation was optimized to be30℃, pH10.5and250rpm on a rotary shaker, wheras bioreduction using glucose as co-substrate was the most favorable at35℃and pH7.5in the statical reaction mixture. Under the optimized conditions, fresh cells of the strain stereoselectively oxidized (S)-enantiomer of racemic1-phenylethanol (120mM) to aceto-phenone and afforded the unoxidized (R)-1-phenylethanol in49.4%yield and>99.9%enantiomeric excess (e.e.). In the reduction of10mM acetophenone,100mM glucose addition significatly increased the conver-sion rate from3.1%to97.4%.80mM acetophenone was enantioselectivery reduced to corresponding (S)-1-phenylethanol with64.5%yield and97.8%e.e. In addition, other aromatic ketones (80mM) were enantioselectively reduced to corresponding (S)-alcohols, giving excellent e.e. values. Both stereoselective oxidation and asymmetrical reduction required no external cofactor regeneration system.3、Alcohol dehydrogenase from T. guaymasensis(TGADH)was highly active, thermostabe and NADP+-specific. The loop region between β-sheet9and α-helix6in the dinucleotide-binding fold was predicted as a principal determinant of coenzyme specieity. Structure analyse revealed five residues that could account for the observed coenzyme specicity. Based on the strategy of site-directed mutagenesis, five single-site mutations (Gly208Asp, Ser209Asp, Arg210Gln, Arg228Phe and Tyr278Phe) and two double-site mutations (Gly208Asp/Arg228Phe, Ser209Asp/Arg228Phe) were successfully constructed. Among the mutants, seven of them exhibited activity using NAD+as coenzyme, whereas the mutants Ser209Asp and Tyr278demonstrated higher activity than the control using NADP+as coenzyme. Particularly, the specific activity of the mutant Ser209Asp using NAD+and NADP+were HU/mg (4.9%of the control) and264U/mg (117.9%of the control), respectively.