酶形式差异的三酶系统一步法转化头孢菌素C制备7-氨基头孢烷酸  

Study on the One?Pot Conversion of Cephalosporin C to 7-Aminocephalosporanic Acid by Different Enzyme Forms of Tri-Enzymatic System

作　　者：邱杰 刘玉婷 陈钰泉 廖威[2] 袁晶 谭强 QIU Jie;LIU Yuting;CHEN Yuquan;LIAO Wei;YUAN Jing;TAN Qiang(Institute of Pharmacy,Guangxi University of Chinese Medicine,Nanning,Guangxi,530001,China;The Food and Biotechnology,Guangxi Vocational and Technical College,Nanning,Guangxi,530226,China)

机构地区：[1]广西中医药大学药学院,广西南宁530001 [2]广西职业技术学院食品与生物技术系,广西南宁530226

出　　处：《广西科学》2018年第4期438-443,共6页Guangxi Sciences

基　　金：国家自然科学基金项目(31360213);广西高校科研项目(2013ZD034)资助

摘　　要：【目的】对酶形式差异的三酶系统一步法裂解头孢菌素C(CPC)制备7-氨基头孢烷酸(7-ACA)进行研究。【方法】对重组大肠杆菌分别进行摇瓶和发酵罐高密度表达,获得最高酶活后使用两个形式差异的三酶系统一步法转化CPC制备7-ACA。【结果】相较于摇瓶发酵,发酵罐发酵获得的酶活更高,发酵罐上对重组大肠杆菌的高密度表达发现,补料流加总量为400mL的甘油混合液(15%甘油+7.5%鱼蛋白胨,W/V),发酵72h后菌浓度达到32.79g/L、最高的戊二酰-7-氨基头孢烷酸酰化酶(GA)和过氧化氢酶(CAT)活力分别为7 099.85U/L和15 776.20U/L。利用一个三酶系统包括固定化D-氨基酸氧化酶(DAAO)、游离GA和CAT,一步法催化CPC获得的7-ACA生成率为87.28%;而另一个三酶系统包括固定化DAAO、冻融细胞GA和CAT一步法催化CPC获得的7-ACA生成率为87.10%。【结论】两种酶形式差异的三酶系统一步法制备7-ACA的得率大致相等。GA对α?酮己二酰-7-氨基头孢烷酸(AKA-7-ACA)的特异性和水解能力较差,限制了该工艺运用。[Objective]This work aimed to study the one pot conversion of cephalosporin C（CPC） to 7 aminocephalosporanic acid （7 ACA） bydifferent enzyme forms of tri enzymatic system. [Methods] The recombinant E. coli was ex pressed in shake flask and fermenter to obtaine the highest enzyme activity. Then two different enzyme forms of tri enzymatic system were con verted CPC to 7 ACA in one pot. [Results]Corn pared with shake flask fermentation, fermenter fermentation has higher enzyme activity. Whenfed batch fermentation by adding with 400 mL feed （15% glycerin and 7.5% fish peptone） ,the biomass was 32.79 g/L and the highest activities of glutaryl 7 amino cephalosporanic acid acy lase （GA） and catalase （CAT） in the recombinant E. coZi reached up to 7 099.85 U/L and 15 776.20 U/L in 72 h of fermentation, respectively. Using a three enzyme system including im mobilized D amino acid oxidase （DAAO）,free GA and CAT,the yield of 7 ACA obtained by one pot conversion of CPC was 87.28%. Another three enzyme system included immobilized DAAO, freeze thaw cells GA and CAT. The yield of 7 ACA obtained by one pot conversion of CPC was 87.10%. [Conclusion]The yield of 7 ACA prepared by two different enzyme forms of tri enzymatic system in one pot was approximately equal. And the specificity and hydrolysis a bility of GA to u ketoadipyl 7 aminocephalosporanic acid（AKA 7 ACA ） was poor,which re stricted the application of the process.

关 键 词：三酶系统 D-氨基酸氧化酶 戊二酰-7-氨基头孢烷酸酰化酶 过氧化氢酶 7-氨基头孢烷酸 

分 类 号：Q81[生物学—生物工程]