We have investigated the performances 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, was previously reported to have a higher ratio between the rate of the antibiotic synthesis and the rate of the acylating agent hydrolysis than the wild type enzyme (vs/vh1 value). New immobilization studies were 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 to the commercial wild type PGA, the immobilized 3G3K acylase on glyoxyl agarose showed higher synthetic performances, in all the tested reactions, in terms of reaction rates and yields.

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

UBIALI, DANIELA;ALBERTINI, ALESSANDRA;TERRENI, MARCO
2009-01-01

Abstract

We have investigated the performances 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, was previously reported to have a higher ratio between the rate of the antibiotic synthesis and the rate of the acylating agent hydrolysis than the wild type enzyme (vs/vh1 value). New immobilization studies were 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 to the commercial wild type PGA, the immobilized 3G3K acylase on glyoxyl agarose showed higher synthetic performances, in all the tested reactions, in terms of reaction rates and yields.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11571/142011
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