| With the development of biotechnology, multienzyme reaction system has been put on increasing emphasis on study. Being an important method for biotransformation, it has been widely used in medicine, foodstuff, and environment protection. In this paper, an enzymatic process for L-phenylalanine production from PPA and L-glutamic acid was established through coupled aspartate transaminase and glutamate dehydrogenase system, which released the production inhibition to transaminase process. By replacing the L-aspartic acid with less expensive L-glutamic acid, the cost of L-phenylalanine production would be reduced.In this paper, glutamate dehydrogenase was obtained from Corynebacterium Tianjinesel.58l, a glutamate-producing bacterium. Then by a series of fermentation experiments, the optimal fermentation medium was obtain: ureaO.8%, CSLO.6%, glucose 10%. By investigating the conditions of fermentation, the optimal conditions had been decided: pH7.2, temperature 35 , aeration l.Svvm, agitation 350rmp and time between 16 and 18 hours. Using the method of auto control feeding urea with constant pH, glutamate dehydrogenase with enzyme activity of 33.4U/g was obtained.The research on property of ammonium assimilation catalyzed by glutamate dehydrogenase demonstrated that the optimum condition was: NH4C1 used as amine donor, pH7.5, 35 癈, reaction time 12hr, enzyme concentration was 1.5g/L, the ratio of the substrates 1.2 : 1, and concentration of 2-oxoglutarate 6g/L. L-glutamic acid and PPA has inhibitory effect on the reaction, while L-phenylalanine has positive effect on it. Some bivalent ions such as Fe +, Mn2+could accelerate the reaction, while Cu2+ had inhibitive effects. 0.04% of CTAB or coenzyme NADPH could help the reaction moving forward, and Na had no obvious influence on the reaction.By researching on the reaction catalyzed by aspartate aminotransferase with L-glutamic acid as amine donor, the optimum reaction conditions were determined as followed: pH9.5, tetnperature37 , L-Glu : PPA = 1.2 '. 1 and concentration of PPA lOg/L. NFLiCl had inhibitory effect on the reaction, Zn2+, Fe2+, Mn2 could promotedthe reaction of aspartate transamination, Mg2 + had little effect, Cu2 + greatly restrained the reaction.The reaction property of the coupled enzymatic system of transaminase and glutamate dehydrogenase was studied, and optimum reaction condition was obtained: the ratio between glutamate dehydrogenase and transaminase 0.5 I 1 (ratio of cell quantity ), pH9.0, reaction temperature 35. When the ratio of substrates was 1 : 1.2 I 1.2 and concentration of PPA was 12g/L, the highest L-phenylalanine yield was obtained, 8.5% higher than single-enzyme system. In the processing of dual-enzymatic reaction system, it was found out that properly adding some CTAB, a kind of detergent, could promote the reaction , Zn2+, Fe2+, Mn2had active effect on the reaction of transamination, Mg2+had little effect, Cu2+greatly restrained the reaction.In this paper , the biosynthesis process was improved to release the inhibition of L-glutamic acid and PPA on glutamate dehydrogenase. On one aspect, by optimizing the adding time and interval of glutamate dehydrogenase and transaminase, the best result was obtained by adding glutamate dehydrogenase 1 hr after transaminase. On the other aspect, with reducing preliminary concentration of L-glutamic acid to 6g/L, the L-glutamic acid restrain on dual-enzyme reaction was released by feeding L-glutamic acid in concentration of 0.31g/ml, 0.5 ml per 2 hr. |
