Cloning, Characteration Of S-specific Short Chain Dehydrogenase/Reductase And Catalyzation To Produce S-CHBE

Biocatalysis,also called Biotransformation,is an engineering using cells or enzymes as catalyst to realize the chemical transformation. Recently, with the develop of the biocatalysis, industrial application of biocatalysis is more and more. As the biocatalysis has so many advatages like safe.. short cycle-high return、less pulution, it can be seen as a long-term project for having basis in our country. So that we get the gene from Candida parapsilosis by cloning, test enzyme properties; mutate active sites by analysising the Amino acid sequence,get the new mutation; By Co-expression of keto reductase and glucose dehydrogenase, coenzyme recycling is realized. In this way,we can develop the yield by using less money.Ketoreductase gene from Candidaparapsilosis was obtained by cloning and then expressed in E coli.. After adding0.1mmol/L inducer IPTG,getting production of the recombinant E. coli.. The substrate4-ethyl chloride (COBE) was convertedto4-chloro-3-hydroxybutyrate (CHBE) with NADPH coenzyme system. The optical purity ofthe product was over99%enantiomeric excess for the (S)-isomer, and the molar conversion yieldof the product was91.1%. The apparent Km value for COBE was0.19±0.01mM, which is anorder of magnitude lower than that of other enzymes in the literature.Because more similar motifs and residue patterns between extended SDRs and CPE are identified,it is appropriate to define CPE as member of the extended SDR family. Change the G195and S126using twice PCR,It said that the key amino acid G195can meta Substrate spectrum, the coenzyme binding site as well as the tolerance of the substrate, enzyme activity, chiral. Compare with CPE, the Conversion addition rate ofG195A and G195is10%,as well as they can using NADP instead of NADPH,which can cost less money,that is very important in the reality production.By successfμlly building the Co-expression of keto reductase and glucose dehydrogenase that is called CPE-GDH2and CPE-GDH104,NADPH coenzyme recycling is realized,which can develop the production and use less money. It can emhance the reaction and cost lower.however, during the course of protein expression, due to the interaction of the two proteins, their configuration slight change occurs, chiral has undergone a change from the beginning ofthe (S) form becomes now the (R)-type in the same e.e.