Construction Of Recombinant E Coli.of ?-Phenylalanine Aminomutase And Optimization Of Producing Conditions

?-phenylalanine aminomutase belongs to MIO-dependentamonomutase,itable to catalytic?-phenylalanine becomes ?-phenylalanine,which plays a vital part in medicine.As a significant prerequisite matter of taxol which is most effective anti-cancer medicine,?-phenylalanine has been drawing extensive attention.Currently,chemical resolution and Mannich reaction are major approaches of acquiring a large quantity of ?-phenylalanine.But these approaches are complex,and products are difficult to purify,while production cost is high and positions and harmful items generate.In contrast with the above approaches,enzymatic catalysis has great strength including high effectiveness,quickness,and safety.As one of biological catalysts which can catalyze and produce ?-phenylalanine highly effectively,?-phenylalanine aminomutase has been drawing more and more attention.Based on PAM gene sequence of ?-phenylalanine aminomutase,the experiment designs and compounds primer up and primer down.Expand target gene by utilizing PCR technology,link the target gene with expression vector pET-28a and thus expression plasmid pET-28a-pam is formulated.Transfer expression plasmid into E.coli expressing strain pET-28a,and induce its expression.Screen positive bacterial strain.Acquire one genetic recombination strain which can generate p-phenylalanine aminomutase.Test the optimum reaction temperature and optimum catalytic pH of IPTG.Induced expression of recombinant bacteria,The enzymatic properties of pH,temperature and thermal stability were studied in three aspects.The optimal pH was determined to be 9.0 and the optimum temperature was 50 ?.The recombinant bacteria had good thermal stability.Fermentation conditions are optimized by using single factor method and response surface method,respectively.The optimal fermentation condition is 12.8 h of inoculum,6.7 of initial pH value,25.2 h of fermentation time,by which recombined highest enzyme activity can reach 11.15 U/ml,128.9%higher than without optimization.Expanded culture of the recombinant bacteria was observed in 3L fermntor.Single factor test was used to determine the optimum solvent oxygen content of 30%.The different fermentation methods,fermentation without adding feeding 30 mL medium at 20 h and feeding 40 mL medium at 30 h,were compared.The results showed that the best fermentation method was feeding 30 mL medium at 20 h.The highest enzyme activity was 16.63 U/mL,increased by 49.1%and 241.5%,compared with shake flask fermentation and non-optimized fermentation conditions.