ИСТИНА

Penicillin acylase (penicillin amidohydrolase, PA; EC 3.5.1.11) catalyzes hydrolysis of amide bond in different amides including penicillins. In our laboratory the gene of PA from Alcaligenes faecalis VKM B 1518 (AfPA) was cloned and expressed in E. coli cell as active and soluble enzyme. AfPA is superior to well-known PA from bacteria E. coli (EcPA) in many aspects (thermal stability, operational pH-optimum, catalytic parameters, stereoselectivity and synthetic performance in chiral synthesis). The main problem of PA production is complex posttranslational multistep processing of the enzyme in bacterial cell. Point mutation introduced frequently influences on the efficiency and rate of active enzyme formation. Optimization of expression conditions for each mutant is time-consuming and expensive procedure, therefore development of standardized expression conditions is of great importance. The solution of the problem is to develop new genetic construction, encoding the permuted form of the enzyme. We created the gene of permuted AfPA (pAfPA), found optimal conditions for expression and obtained high-purity enzyme preparation. Investigation of its properties revealed that pAfPA has similar kinetic parameters as compared to wild-type enzyme, but surpasses it manifold in thermal stability. This work was supported by Russian Foundation for Basic Research (grant 13 04 01907).