Construction Of Recombinant Escherichia Coli For Asymmetric Carbonyl Reduction Of Prochiral Aromatic Ketone To L-Ephedrine

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. In this thesis, the model of ephedrine producing from 1-phenyl-2-methylamine-acetone (MAK) was built, a recombinant of E. coli expressing single carbonyl reductase was constructed, and then the reaction process of l-ephedrine producing from MAK was investigated.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. After transformation, it was found that the expression product could produce l-ephedrine from 1-phenyl-2-methylamine-acetone (MAK). Compared with the recombinant strain E.coli BL21 (pET28a-mldh), the enzyme specific activity increased by three times. After the inducing conditions optimization, the enzyme specific activity of the recombinant E.coli JM109 (pKK223-mldh) reached 0.56 U/mg protein by 0.4 mmol/L IPTG induction, and 200 r/min shaking flask culture at 37℃for 6 h.In order to solve the problem of co-enzyme regeneration, the glucose dehydrgenase gene gdh from Bacillus subtili was cloned, and then the expression vectors pET28a-gdh was constructed and transformed into E.coli BL21. Then the recombinant E.coli BL21 (pET28a-gdh) was constructed to express glucose dehydrogenase capable of regenerating NAD+ to NADH. Then the recombinant plasmids pKK223-mldh and pET28a-gdh were transformed into E.coli. Investigation on the stability of the recombinant plasmids indicated that coexistence could last for 14 h with the initial ampicillin and kanamycin concentrations at 100 mg/L and 30 mg/L. After IPTG inducion, the co-expression of the carbonyl reductase gene mldh and glucose dehydrogenase gene gdh was achieved. Further more, it showed that the glucose dehydrogenase from the recombinant E.coli could provide sufficient reducing power for the asymmetric reduction reaction with no extra glucose dehydrogenase and co-enzyem addition.