Biological Catalysis Prepared By Acetophenone Keto Acids

Phenyl gyoxilic acid (PGA), also called benzoylformic acid (BFA) is applied widely as an organic synthetic intermediate. Mandelic acid racemase gene (mdlA) and dehydrogenase gene (mdlB) have cloned from Pseudomonas aeruginosa, then we constructed recombinant strains E.coli BL21 (DE3)/pEt30a (mdlA+mdlB), in which one MA can be converted into PGA, and with a higher expression in IPTG induced. Based on the work above, in this paper, medium and culture conditions are optimizated, to achieve high cell density while a high protein expression.Another important aspect of the biocatalysis is the optimization of conversion conditions, only in the appropriate conditions, cell can be fully demonstrated the best activity. Concrete works are as followed:The qualities and induction conditions of the recombinant strain strain have been stuied. we chosed lactose instead of IPTG as inducer; and concentration of lactose was 0.5% inducer added time was 4h, culture time was 8h, cultrued with 37℃at frist, and induced temperature was 30℃.we used statistic method to optimize the medium composition and culture conditions of recombinant strain. First, Plackett-Burman experimental design was used to selecte three main factors, namely, the concentration of C-source and N-source, and pH. Based on this, the steepest ascent has been used to approach to the the region of maximum response; with CCD experimental design and response surface analysis, the optimum conditions are determinded, namely, the C-source concentration is 6.34g/L, complex N-source is 13.56 g/L, pH is 7.7. With this condition, the highest predictive value of the response is 18.5276 U/g. With the five batches of training validation experiments, the average prediction and validation tests close to the optimal conditions.In order to further improve the the converse ability of the bacteria and improve the acetophenone keto acid production, with single-factor test, we determined the modested condition was:substrate concentration with 20g/L, cell concentration with lOg/L, pH 6.0,37℃,200rpm. At this conditions, the transformation ability was improved to 20.5273U/g, and the end time of conversion was 8h.After the optimization experiments, when the substrate concentration was 2%, the conversion reaction just need 8h, compared to original conditions, the substrate concentration was upgraded 1 times while the time was shorted 40h.