Coupling of Site-Directed Mutagenesis and Immobilization for the Rational Design of More Efficient Biocatalysts: The Case of Immobilized 3G3K PGA from E-coli

摘要

We have investigated the synthetic performance of the immobilized 3G3K mutant of the Penicillin G acylase (PGA) from E. coli obtained by site-directed mutagenesis. The 3G3K mutant, characterized by a tag consisting of three lysines alternating with three glycines at the end of the beta-chain, has previously been reported to have a higher ratio for the rate of antibiotic synthesis and the rate of hydrolysis of the acylating agent (the vs/vh(1) value) than the wild-type enzyme. New immobilization studies have been carried out with the 3G3K mutant by using different glyoxyl supports (activated with aldehyde groups), The catalytic properties of the new immobilized preparations were tested in the synthesis of Cefamandole and Cefonicid by kinetically controlled N-acylation (kcNa). Compared with the commercial wild-type PGA, the 3G3K acylase immobilized on glyoxyl agarose showed higher synthetic performance (reaction rates and yields) in all the tested reactions. ((C) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)