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

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

Key words:  tri-enzymatic system  D-amino acid oxidase  glutaryl-7-amino cephalosporin acid acylase  catalase  7-ACA

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400 mL的甘油混合液(15%甘油+7.5%鱼蛋白胨,W/V),发酵72 h后菌浓度达到32.79 g/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7 099.85 U/L和15 776.20 U/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。

Abstract:

[Objective] This work aimed to study the one-pot conversion of cephalosporin C(CPC) to 7-aminocephalosporanic acid(7-ACA) by different enzyme forms of tri-enzymatic system.[Methods] The recombinant E.coli was expressed in shake flask and fermenter to obtaine the highest enzyme activity.Then two different enzyme forms of tri-enzymatic system were converted CPC to 7-ACA in one-pot.[Results] Compared with shake flask fermentation, fermenter fermentation has higher enzyme activity.When fed-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 acylase (GA) and catalase (CAT) in the recombinant E.coli 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 immobilized 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 ability of GA to α-ketoadipyl-7-aminocephalosporanic acid(AKA-7-ACA) was poor, which restricted the application of the process.