The Fermentation Process Of Penicillin Acylase And Preparation Of Catalytic By Enzymatic Method

Penicillin acylase is an important industrial and medical enzyme which is used in semisynthetic β-lactam antibiotics and production of pharmaceutical intermediates, peptide synthesis and separation of racemic chiral compounds. Penicillin acylase has a broad market demand and well prospect. With the developing of genetic engineering and enzyme engineering, the researchers had build high yield and good quality genetically engineered bacteria which were used to produce penicillin acylase, and penicillin acylase had been used in synthetic penicillins and cephalosporins. Compared with conventional chemical synthesis, the advantages of enzymatic synthesis method are the absolute specificity of the reaction, avoiding environmental pollution, providing mild reaction condition and reducing production cost more than10%. Therefore, to gain the process perameters of producing penicillin acylase by fermentation, immobilization and transformation of penicillin acylase, would promote application in medicine, food and other industries.Using recombinant Escherichia coli as the test strain, the conditions and kinetic model of batch fermentation, the optimizing reaction conditions of immobilizing penicillin acylase and catalytic synthesis of cefaclor with immobilized penicillin acylase enzyme were studied. The main results were listed as follows:Optimization of fermentation conditions:To optimize the fermentation conditions, monofactorial experiment and reponse surface methodology were combined. After the monofactorial experiment, the optimal glycerol concentration、peptone concentration、 IPTG concentration、pH and temperature could be determined as followed:15-20g/L,15～20g/L,6.5～7.5,50μg/mL,28-36℃. Box-Behnken and response surface method-ology were designed for the optimization:glycerol concentration20g/L, peptone con-centration21g/L, pH7.1, and in this case, enzyme activity of PGA was reached to8425.31U/L,11%higer than before.The fermentation kinetics modeling:The fermentation was magnified in fermenter of5L. According to Logistic equation and Luedeking-piret equation, the nonlinear fitting was applied to analyze fermentation experiment data of penicillin acylase produced by Escherichia coli by Origin8.0software. The kinetic models of cell growth penicillin acylase synthesis, and substrate consumption were build. The result was as followed: (1) The kinetic model of cell growth(2) The kinetic model of synthesis of PGA(3) The kinetic model of substrate consumptionThis model described the dynamic process of batch fermentation of recombinant Echerichia coli well and could provide references for latter scale experiment.The process conditions of immobilization penicillin acylase were explored. Monofactorial experiment and the Box-Behnken experimental design were designed for the optimization:pH7.8, temperature25℃,carrier usage0.11g/mL, time24h, and in this case, the enzyme activity was229.78U/L and the enzyme recovery rate was61.66%, which showed it was feasible to use LX-1000HA to immobilize penicillin acylase.The reaction conditions of immobilized penicillin acylase catalytic synthesis of cefaclor:temperature15～25℃, pH5.5-6.5, side chain to substrate feed ratio:1.4, the dosage of immobilized enzyme:5.5U/mL. Under this optimized condition, experiments were repeated with the recovery of self-made immobilized penicillin acylase for125batch. The results showed that:the average response time is138min, the conversion rate of96.23%. It showed that the effect of self-made immobilized penicillin acylase was good, and its dynamic expression was stable under this condition. Immobilized penicillin acylase would be fully applicable to large-scale industrial production.