重组头孢菌素C酰化酶的固定化研究  

Immobilization of Recombinant Cephalosporin C Acylase

作　　者：朱琳琳[1] 常雁红[1] 姚舜[1] 罗晖[2] 于慧敏[3] 沈忠耀[3] 

机构地区：[1]北京科技大学环境工程系,北京100083 [2]北京科技大学生物科学与工程系,北京100083 [3]清华大学化学工程系,北京100084

出　　处：《化学与生物工程》2017年第2期19-24,共6页Chemistry & Bioengineering

基　　金：国家自然科学基金资助项目(21276023;21476025);北京市自然科学基金资助项目(2143041);中央高校基本科研业务费专项资金资助项目(FRF-SD-12-007A)

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50L发酵罐中发酵,发酵液OD600可达19.5,酶活达到11 542U·L^(-1);CPC酰化酶粗酶液经1%活性炭纯化后,比酶活由6.56U·mg^(-1)提高到10.77U·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 Lfermentor.When the OD600 value of the broth and the enzyme activity reached 19.5and 11 542U·L-1,respectively.Crude enzyme solution was purified by 1% activated carbons,and the specific enzyme activity increased from 6.56U·mg-1 to 10.77U·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-1000 HA immobilized enzyme was lower than that of LX-1000 EPC immobilized enzyme,while its thermal stability,pH value stability,and binding strength of LX-1000 HA immobilized enzyme and carrier were better than those of LX-1000 EPC immobilized enzyme.Catalytic performance of LX-1000 HA immobilized enzyme was better than that of LX-1000 EPC immobilized enzyme for the same enzyme dosage.The half-life of LX-1000 HA immobilized enzyme was about 39 cycles under such catalytic reaction conditions.

关 键 词：重组 头孢菌素C酰化酶 固定化酶 稳定性 生物催化 

分 类 号：TQ465.1[化学工程—制药化工] Q556[生物学—生物化学]