重组头孢菌素C酰化酶的固定化研究

摘要

将含头孢菌素C(CPC)酰化酶的重组大肠杆菌E.coli BL21 (DE3)/pET28-CPCAcy在50 L发酵罐中发酵,发酵液OD600可达19.5,酶活达到11 542 U·L-1;CPC酰化酶粗酶液经1％活性炭纯化后,比酶活由6.56 U· mg-1提高到10.77 U· mg-1;然后将纯化的CPC酰化酶共价结合固定在氨基载体LX-1000HA和环氧基载体LX-1000EPC上,并对固定化酶的热稳定性及pH值稳定性进行考察.结果表明,LX-1000HA固定化酶的初始酶活较LX-1000EPC固定化酶低,但其热稳定性、pH值稳定性以及酶与载体的结合强度均高于LX-1000EPC固定化酶;并且在固定化酶投加量相同时,LX-1000HA固定化酶的催化性能优于LX-1000EPC固定化酶.对LX-1000HA固定化酶进行催化批次实验,在催化39批次时达到半衰期.%In this paper,the recombinant E.coli BL21 (DE3)/pET28-CPCAcy which contains cephalosporin C(CPC) acylase was fermented in a 50 L fermentor.When the OD600 value of the broth and the enzyme activity reached 19.5 and 11 542 U · L-1,respectively.Crude enzyme solution was purified by 1％ activated carbons,and the specific enzyme activity increased from 6.56 U · mg-1 to 10.77 U · mg-1.Then the purified CPC acylase was immobilized on LX-1000HA(amino carrier) and LX-1000EPC(epoxy carrier) by covalent binding,and the thermal stability and pH value stability of the immobilized enzyme were investigated.The results showed that the initial enzyme activity of LX-1000HA immobilized enzyme was lower than that of LX-1000EPC immobilized enzyme,while its thermal stability,pH value stability,and binding strength of LX-1000HA immobilized enzyme and carrier were better than those of LX-1000EPC immobilized enzyme.Catalytic performance of LX-1000HA immobilized enzyme was better than that of LX-1000EPC immobilized enzyme for the same enzyme dosage.The half-life of LX-1000HA immobilized enzyme was about 39 cycles under such catalytic reaction conditions.