固定化青霉素G酰化酶合成头孢氨苄过程中酶活稳定性研究

Study on the enzymatic activity stability of penicillin G acylase during the process of enzymatic synthesis of cephalexin

目的改善固定化青霉素G酰化酶(PGA)的酶活稳定性,从而提高其重复使用次数。方法采用模拟法,研究了酶法合成头孢氨苄过程中影响PGA酶活稳定性的一些主要因素。结果 7-氨基-3-脱乙酰氧基头孢烷酸(7-ADCA)、苯甘氨酸甲酯(PGM)对固定化PGA表现出了很强的底物抑制作用,而且它们的抑制作用与其浓度几乎无关。固定化PGA加入到7-ADCA、PGM溶液后,其酶活迅速降低,但反应时间超过12h后,PGA酶活几乎保持恒定不变。头孢氨苄对固定化PGA表现出了产物抑制作用,这种抑制作用主要是由其水解产物造成的。随着与头孢氨苄反应时间的延长,固定化PGA的酶活一直持续降低。当控制反应温度≤30℃时,固定化PGA表现出良好的酶活稳定性。当酶反应液p H值为6.5时,固定化PGA酶活稳定性最好。结论合理控制头孢氨苄合成反应条件能够有效改善固定化PGA酶活稳定性。

Objective The reuse number of immobilized penicillin G acylase（PGA） is directly related with the production cost of semi-synthetic -lactam antibiotics. The higher the enzymatic activity stability of PGA, the more its reuse number. The aim of this study is to improve the enzymatic activity stability of PGA. Methods Some influencing factors on the enzymatic activity stability of PGA were investigated during the enzymatic synthesis of cephalexin using a simulation method in this study. Results 7-Amino-3-desacetoxy-cephalosporanic acid （7-ADCA） and D-phenylglycine methyl ester （PGM） had very strong substrate inhibitions on immobilized PGA, and their inhibitions were hardly related to their concentrations. PGM could be hydrolyzed to produce phenylglycine under the action of PGA and the hydrolysis of itself. Phenylglycine would also precipitate on the surface of immobilized PGA as it was insoluble in water, which might be another reason why PGM showed inhibition on immobilized PGA. The enzymatic activity of immobilized PGA decreased rapidly and greatly when it was added into the 7-ADCA or PGM solution. After 12h reaction time with 7-ADCA or PGM, the enzymatic activity almost kept constant. Cephalexin showed a product inhibition, and its inhibition was caused mainly by its hydrolysates. As the reaction time with cephalexin increased the enzymatic activity of immobilized PGA continuously decreased. When the reaction temperature was lower than 30℃, immobilized PGA presented perfect enzymatic activity stability. Immobilized PGA had the best enzymatic activity stability when the pH value of reaction solution was 6.5. Conclusion The enzymatic activity stability of PGA can be improved by reasonably controlling the reaction conditions of cephalexin synthesis.