Efficient Production Of(S)-1-phenyl-1,2-ethanediol By(S)-carbonyl Reductase ? Using Xylan As Co-substrate

(S)-carbonyl reductase ?(SCR?)from Candida parapsilosis CCTCC M203011 cat alyzes 2-hydroxyacetophenone to(S)-1-phenyl-1,2-ethanediol.But nicotinamide coenzy me NADPH is required in the reaction.Since the cofactor is more expensive,unstable and difficult to be reused,resulting in the higher cost of chiral synthesis,its applicat ion in chiral catalysis is limited.In this work,we constructed co-expression and fusio n expression system containing SCR?,glucose dehydrogenase mutant Ala258Phe/GDH and xylanase XYN2 by overlapping extension PCR technique to achieve cofactors reg eneration.The recombinant system efficiently catalyzed the biotransformation of(S)-1-p henyl-1,2-ethanediol using 2-hydroxyacetophenone as substrate and xylan as co-substra te.Therefore,the xylan metabolism was successfully introduced into chiral synthesis.This work supplies research basis for the oxidoreductase-mediated chiral catalysis using xylan as co-substrate.The research contents are as follows:(1)The co-expression plasmids pET-S-SD-AS-G-SD-AS-2,pET-S-SD-AS-2-SD-ASG,pET-G-SD-AS-S-SD-AS-2,pET-G-SD-AS-2-SD-AS-S and the fusion expression plas mids pET-S-L-G-L-2,p ET-S-L-2-L-G,pET-G-L-S-L-2,pET-G-L-2-L-S carrying SCR?,A258F/GDH and XYN2 were constructed using overlap extension PCR technique with pET-SCR?,pET-A258F/GDH and pET-XYN2 as template.The SCR?,A258F/GDH a nd XYN2 were ligated with Shine-Dalgarno and Aligned Spacing sequence in the co-e xpression system and GGGGS sequences as linkers between them in the fusion expres sion system.The recombinant plasmids were transformed into Escherichia coli BL21.The results showed that three genes were successfully expressed in co-expression reco mbinant strain E.coli/p ET-G-SD-AS-S-SD-AS-2.The SDS-PAGE analysis revealed that three predominant bands corresponding to the theoretical sizes(33,30 and 22 kDa)of the target recombinant enzymes SCR?,A258F/GDH and XYN2,respectively.And t he fusion protein was expressed in recombinant strains E.coli/pET-S-L-G-L-2 and E.coli/pET-G-L-S-L-2 with corresponding to theoretical size 85 kDa by SDS-PAGE analy sis.The fusion protein was purified and a single protein band were obtained.(2)The enzyme activity of SCR?,A258F/GDH and XYN2 in the co-expression s train E.coli/p ET-G-SD-AS-S-SD-AS-2 were 0.85,1.36 and 55.33 U/mg,respectively,while the enzyme activity of SCR?,A258F/GDH and XYN2 were 4.58,3.71 and 188.49 U/mg in E.coli/p ET-S-L-G-L-2,and 2.43,6.54 and 231.78 U/mg in E.coli/p ETG-L-S-L-2.When the three enzymes SCR?,A258F/GDH and XYN2 were separately e xpressed,their activities were 8.56,14.58 and 695.63 U/mg,respectively,which were all higher than those in the co-expression and fusion expression stains.The optimum pH and temperature,pH stability and thermostability of three enzymes were determine d.The results indicated that SCR?,A258F/GDH and XYN2 showed the maximum cat alytic activity when the pH values were 7.0,6.5 and 5.0,respectively.SCR? and A258F/GDH performed a relative wide range of p H stability,when the p H values were in the range of 5.0–8.0 and 6.0–9.0,the enzymes activity remained above 60%.All three enzymes had good temperature stability,SCR?,A258F/GDH and XYN2 showed th e maximum catalytic activity at 35,50 and 50 ?,respectively.(3)The whole-biocatalyst biotransformation conditions of co-expression and fusion expression strains were optimized.The co-expression recombinant strain E.coli/pET-G-SD-AS-S-SD-AS-2 catalyzed 6 g/L 2-hydroxyacetophenone to(S)-1-phenyl-1,2-ethanedi ol with a highest optical purity of 100% and a highest yield of 98.3% under the opti mal conditions: 35 ? and,p H 6.5,the ratio between 2-hydroxyacetophenone and xylo se of 2:1,reaction duration of 28 h.Under the optimal conditions: 35 ?,pH 7.0,the ratio between 2-hydroxyacetophenone and xylose of 1:1 and 2:1,reaction duration of26 h,the recombinant strains E.coli/pET-S-L-G-L-2 and E.coli/pET-G-L-S-L-2 cataly zed 6 g/L 2-hydroxyacetophenone to(S)-1-phenyl-1,2-ethanediol with the highest yields of 98.8% and 95.6%.They both gave the highest optical purity of 100%.Compared to the recombinant strain E.coli/pET-SCR?,the co-expression and fusion expression st rains significantly improved the catalytic efficiency and shortened the reaction time ab out two-fold.