Enzyme catalysts for a biotechnology-based chemical industry. Quarterly progress report, September 29--December 28, 1997

The goal of this research is to engineer enzymes to be efficient and economically attractive catalysts for the chemical industry. The author is attempting to demonstrate generally-applicable approaches to enzyme improvement as well as develop specific catalysts for potential industrial application. In this report attention is focused on random mutagenesis of pNB esterase -- improved activity and stability. The most thermostable esterases obtained by sequential random mutagenesis (6H7) and random mutagenesis plus recombination (6sF9) each contain 9 amino acid mutations and a number of silent mutations, relative to the wild-type sequence. Eight of the mutations are present in both genes, for a total of ten potentially adaptive mutations. Because several of these mutations occurred in the same generation, it is difficult to identify the mutations responsible for the increases in activity and stability. In order to aid in this identification, the thermostable genes were recombined with the wild-type gene, in hopes of removing neutral mutations. The gene from the first-generation variant, with five amino acid substitutions was also recombined with wild-type