MUTANT ESCHERICHIA-COLI PENICILLIN ACYLASE WITH ENHANCED STABILITY ATALKALINE PH

Increased stability at alkaline pH should be a valuable attribute for the utilization of penicillin acylase in bioreactors employed to conve rt penicillins into 6-amino-penicillanic acid, a precursor of semisynt hetic penicillins. In these systems, base is added for pH control, whi ch results in local alkaline conditions that promote enzyme inactivati on. Hydrolysis and synthesis reactions are also pH dependent. Here, we report work in which the gene coding for Escherichia coli penicillin acylase was subjected to oligonucleotide-directed random mutagenesis a t regions coding for amino acids predicted to be at the surface of the enzyme. The resulting mutant library, cloned in E. coli, was screened by a filter paper assay of the colonies for the presence of penicilli n acylase activity with enhanced stability at alkaline pH. Characteriz ation of one of the selected clones revealed the presence of a mutatio n, Trp431-Arg, which would presumably alter the surface charge of the protein. In vitro experiments demonstrated a near twofold increase in the half-life of the mutant enzyme when stored at pH 8.5 as compared w ith the wild-type enzyme, with a comparable specific activity at sever al pH values. In general, the mutant displayed increased stability tow ard the basic side in the pH-stability profile. (C) 1995 John Wiley & Sons, Inc.