Common Expression Type Of Recombinant E. Coli Asymmetric Reduction Production D - Lactic Acid

D-lactic of high optical purity has wide perspective and practicability in biopolymer material, agriculture, chemical industry, etc. Bio-catalysis has become the most promising method in asymmetric synthesis because of the high conversion ratio, good stereo specificity and the mild reaction conditon. To solve the coenzyme regeneration problem, glucose dehydrogenase was used to construct co-expression plasmid with lactate dehydrogenase in E.Coli.The glucose dehydrogenase gene (gdh) was amplified from Bacillus subtilis through PCR technique, and inserted into the plasmid pETduet-1to construct the recombinant plasmid pETduet-GDH. The lactate dehydrogenase gene (ldh) was amplified from Serratia marcescens H30, and then inserted into pETduet-GDH to construct the recombinant plasmid pETduet-LG. The positive plasmid was transformed into E.Coli.BL21, and a recombinant strain E.Coli pETduet-LG was obtained. SDS analysis showed the glucose dehydrogenase and latate dehydrogenase were expressed simultaneously, and the molecular weight were31kD and37kD. The enzyme activity of lactate dehydrogenase was5.7u/mg and glucose dehydrogenase was9.2u/mg.The best condition for IPTG induction was:IPTG0.1mM; induce temperature:30℃induce time:7H; induce timing:not confined strictly, inducing result has no obvious difference when OD was in the range of0.3-1.5.To increase the substrate conversion, optimization of whole cell catalysis condition was studied. Adding NAD+helped in increasing the initial reaction velocity. Start of coenzyme regeneration system was blocked without initial coenzyme added, because the coenzyme needs to be cumulated through the tardy metabolism. The best catalysis condition was: temperature:37℃; buffer solution:0.2M phosphate buffer; pH:7.0; OD:40; substrate concentration:20g/L,15%enzyme activity was lost after each cell recycle use.