Study On Asymmetric Carbonyl Reduction Of Prochiral Aromatic Ketone To D-pseudoephedrine By Co-expression Recombinant Escherichia Coli

Recently, chiral technology has highly attracted attentions from industry and academia, the asymmetrical synthesis of chiral compounds and their chiral intermediates have become an active field in research and development. Due to the mild reaction condition, high conversion ratio and outstanding stereo chemical specificity, biocatalysis has become the most promising approach in asymmetric synthesis. This thesis focuses on the problem of coenzyme regeneration on asymmetric reduction MAK to d-pseudoephedrine. Glucose dehydrogenase and formate dehydrogenase are used to constructe co-expression plasmid with carbonyl reductase respectively for the coenzyme regeneration enzyme.With the recombinant plasmid pET28a-mldh as the plate, the gene mldh was obtained by PCR amplification. The recombinant plasmid pKK223-mldh was constructed and expressed in E.coli JM109. The glucose dehydrogenase gene gdh was amplified from Bacillus subtilis by PCR technique. Then the purified PCR products were inserted into plasmid pKK223-3-mldh which had been ever constructed previously to construct plasmid pKK223-3-gdh-mldh. The positive plasmid was transformed into E. coli JM109, and a recombinant strain E. coli pKK223-3-gdh-mldh was obtained. It was shown by SDS-PAGE analysis that two enzymes were expressed simultaneously and the molecular weights were 43 kD and 31 kD respectively. It was illustrated by HPLC assay that the recombinant strain could be used to catalyze MAK to d-ψ-ephedrine without adding glucose dehydrogenase. In further whole-cell transformation test, 0.1 g thallus incubated with 0.15 mg MAK and 6 mg glucose could produce 0.058 mg d-ψ-ephedrine at 30°C for 10 h.The formate dehydrogenase gene fdh was amplified from Candida boidinii by PCR technique. Then the purified PCR products were inserted into plasmid pKK223-3-mldh which had been ever constructed previously to construct plasmid pKK223-3-pro-fdh-mldh. It was illustrated by HPLC assay that the recombinant strain could be used to catalyze MAK to d-ψ-ephedrine.In whole-cell transformation test, 0.1 g thallus incubated with 0.09 mg MAK and 3 mg sodium formate could produce 0.037.1 mg d-ψ-ephedrine at 30°C for 10 h.By comparing the two co-experssion recombinant bacteria,the glucose dehydrogenase recombinant bacteria is better than the formate dehydrogenase recombinant bacteria on d-ψ-ephedrine yield.