[Site-directed mutagenesis at Ser177 of penicillin G acylase gene]

Site-directed mutagenesis was performed at Ser177 of Penicillin G Acylase (PGA) gene of E. coli by gap duplex method. Screened by 2-nitro-5-phenylacetaminobenzoic acid (NIPAB) test paper assay and confirmed by DNA sequencing, the mutants Cys177, Gly177, Arg177 and Asn177 were obtained. They have no activity of PGA. The mutant enzyme was expressed and detected by PAGE. It is suggested that Ser177 is at the substrate binding center of PGA and can not be replaced.