Excavation Of Novel Alcohol Dehydrogenases From Lactobacillus Curieae S1L19 And Asymmetric Reduction For Preparation Of Ethyl (R)-4-Chloro-3-Hydroxybutanoate

Chiral alcohols are the most important chiral building blocks for the synthesis of chiral drugs,agricultural chemicals and fine chemicals.Compared with conventional chemical synthesis,biocatalytic asymmetric reduction of prochiral ketones is an effective method in the synthesis of chirality drugs due to its mild reaction conditions,high enantioselectivity and high conversion(R)-ethyl-4-chloro-3-hydroxybutanoate[(R)-CHBE]is a very valuable intermediate in the synthesis of L-carnitine,afatinib and macrolide A.In this study,we focused on excavation of five novel ketoredutases from Lactobacillus curieae S1L19 and establish an enzynatic preparation technology of(R)-CHBE.The results are as follows:1.Five open reading frames(ORF)encoding putative alcohol dehydrogenases(LCRI?LCR??LCR??LCRIV and LCRV)from Lactobacillus curieae S1L19 were successfully cloned and expressed.Multiple sequence alignment revealed that LCR? and LCR? are the members of short-chain alcohol dehydrogenase(SDR).LCRI,LCRIV and LCRV showed high sequence identity with the quinine oxidoreductase(QOR)family,which are the members of the medium-chain dehydrogenase(MDR)family.Five alcohol dehydrogenases exhibit activity in the reduction of COBE.LCR? exhibits the highest activity and excellent enantioselectivity(R-type,ee>99%)in the reduction of COBE.LCR? was choosed for further study.2.Characterized LCRIII indicated that the optimal pH for LCRIII were pH 6.0,the LCRIII was more stable in acidic environment.The optimum temperature was found to be 35? and more stable at 30 and 40?.The addition of Li+ resulted in the highest relative activity.The addition of 7 mM Li+ resulted in 218%relative activity compared with the control.LCRIII displayed a relatively broad spectrum of carbonyl substrate.LCRIII showed reductive activities toward a variety of ketones,including ?-,?-keto esters and aromatic ketones.LCRIII showed the highest activity toward methyl pyruvate of 284.2 U/mg.Kinetic analysis of experiment data reveral that LCRIII exhibited a high affinity for COBE.The Km value was 4.28 mM.The Vmax was 10.8 U/mg.3.A cofactor regeneration system was constructed by co-expressing.The high cost of cofactors is a restrictive factor for industrial biosynthesis of optically active alcohols.For the lower cost in the industrial production,LCRIII and glucose dehydrogenase(GDH)genes were cloned into plasmid pET-28a(+),and then transformed into expressioin host E.coli BL21(DE3)cells to achieve the heterologous expression of two enzymes.Analysis found that LCRIII and BsGDH are both realized the efficient expression LCRIII and BsGDH demonstrated activity of 113.46 and 454.2 U per gram of dried cells,respectively.Compared with cell-coupled system,the co-expression system has the higher transfer rate.4.An asymmetric reduction conditions was constructed.To further develop an economical process and efficient method for synthesis of(R)-CHBE.Various parameters,such as the concentration of substrate(0.5-1.5 M),dosage of coenzymes(0-0.5 mM),biocatalysts(30-50 g/L)and metal ion(0-7 mM)were evaluated.The results demonstrate that 1.5 M(246.8 g/L)COBE could be completely reduced to(R)-CHBE within 6 h under optimal conditions(0 mM cofactor,40 g/L of dry cells and 7 mMLi+ addition)in a monophasic aqueous system.The space-time yield of the process was 980 g/L/d.Moreover,the process could be performed even without external addition of cofactors,which results in reduced costs and demonstrate the great potential of this process in industrial applications.