Biosynthesis Of α,β-Unsaturated Alcohols Using A Newly Isolated Yokenella Sp.Wzy002 And Characterization Of Its Purified Alcohol Dehydrogenase

α,β-unsaturated alcohols are valuable intermediates in the synthesis of spices, flavors, and drugs. Up to date,α,β-unsaturated alcohols are usually produced through selective hydrogenation of α,β-unsaturated aldehydes and ketones using chemical catalysts. Chemical approaches require precious metal catalysts and demonstrated lower selectivity in terms of α,β-unsaturated alcohols as product, whereas biological approaches have drawn more and more attention because of their high region-selectivity in the reduction of α,β-unsaturated aldehydes and ketones. Here, an excellent biocatalyst-Yokenella sp.WZY002 was screened from the soil samples, which demonstrated high regioselectivity and activity in the reduction of α,β-unsaturated aldehydes and ketones to unsaturated alcohols. The alcohol dehydrogenase was sucessfully purified from Yokenella sp.WZY002 and then characterized in detail. Moreover, the gene encoding the alcohol dehydrogenase was cloned and functionally over-expressed in the Escherichia coli BL21(DE3).1. Based on morphological, physiological and 16S rDNA characteristics, the isolated strain with the potentials for the synthesis of a,p-unsaturated alcohols was identified and named as Yokenella sp.WZY002. In the reduction of α,β-unsaturated aldehydes and ketones, the optimal values of temperature and pH were 30 ℃ and pH8.0 respectively. When glucose was supplemented as co-substrate, both the yield and conversion of the reduction reaction were significantly improved and the addition of coenzymes were not necessary. Under the optimized conditions, the reduction of 50mM trans-2-hexenal (crotonaldehyde) to crotyl alcohol led to>98% conversion and 85% yield. Yokenella sp. WZY002 could also reduce 2-hydroxyacetophenone to (S)-1,2-phenylethanediol, giving>99% e.e. value. In addtion, the strain remained active when the substrate concentration reached up to 400mM.2. An alcohol dehydrogenase (ADH) was purified to homogeneity from Yokenella sp.WZY002 using a three-step procedure:ion-exchange chromatography, hydrophobic chromatography and size-exclusion chromatography. The specific activity of the purified enzyme was 25.9U/mg and its subunit size was 37+1 kDa determined by SDS-PAGE. The optimal values of temperature and pH were 30 ℃ and 7.5, respectively. The ions Zn2+, Mn2+ and Al3+ inhibited the enzyme activity, while other ions such as Na+, Ca2+, Mg2+ and K+ had no significant effect on the enzyme activity. The enzyme demonstrated high solvent tolerance. The presence of 20%(v/v) solvents including acetonitrile, ethanol, methanol and aceton improved the enzyme activity. Particularly, the enzyme retained >85% of the initial activity when the solvent concentration was increased up to 40%(v/v). The enzyme was NADP(H) specific. When 0.4mM NADPH was used as coenzyme, apparent Km and Vmax values of the enzyme for benzaldehyde were 0.12mM and 35.3U/mg, respectively.3. The gene encoding the purified alcohol dehydrogenase was cloned, which gene product consisted of 339 amino acid residues and belonged to the family of medium chain alcohol dehydrogenases. The motifs of putative active site and NADP+binding were identified to be G62H63E64X2G67X5G73 and G175XG177XXG120, respectively. The three-dimensional structure of the ADH monomer was composed of two domains:a dinucleotide binding domain containing a classic Rossmann fold and a substrate binding domain. The structure information also indicated that it had the binding motifs for both structural and catalytic zinc ions, which might be critical for enzyme activity and stability. The gene was functionally over-expressed in E. coli, in which the specific activity of crude extracts was 2.1U/mg.