产GL-7-ACA酰化酶重组菌的构建及高表达研究

Construction and Overexpression Accomplishment of GL-7-ACAacylasein a Recombinant Escherichia coli

戊二酰基-7-氨基头孢烷酸(GL-7-ACA)酰化酶是7-氨基头孢烷酸(7-ACA)两步酶法生产中的关键酶。成功构建组成型表达的产GL-7-ACA酰化酶重组大肠杆菌JM105/pMKC-ACY,并对其高表达条件进行了研究,得到了组成简单、廉价的国产培养基配方及操作简便、易于实现工业化的发酵工艺。在优化条件下,上罐补料高密度发酵的酶活高达6668.9U/L,是优化前的12.4倍,产率最高可达275.5U/(L.h),达到了工业生产的要求。

Glutaryl-7-aminocephalosporanic acid（GL-7-ACA） acylase is one of the key enzymes received considerable recognition as a biocatalyst for two-step enzymatic production of 7-ACA. A new recombinant Escherichia coli, E. coli JM105/pMKC-ACY, was constructed and used for the overexpression studies of the GL-7-ACAacylase. The superior culture conditions were figured out, Fed-batch culture of the recombinant strain was further accomplished under the optimized conditions in a 5L fermentor. The GL-7-ACAacylase activity increased to as high as 6668.9 U/L with the highest productivity of 275. 5U/（ L·h）, which was highly promising for the scale-up enzymatic production of 7-ACA in industry.