IMPROVEMENT OF A USEFUL ENZYME (SUBTILISIN BPN') BY AN EXPERIMENTAL EVOLUTION SYSTEM

In order to improve a natural enzyme so as to fit industrial purposes, we have applied experimental evolution techniques comprised of succes sive in vitro random mutagenesis and efficient screening systems. Subt ilisin BPN', a useful alkaline serine protease, was used as the model enzyme, and the gene was cloned to an Escherichia coli host-vector sys tem. Primary mutants with reduced activities of below 80% of that of t he wild type were first derived by hydroxylamine mutagenesis directly applied to subtilisin gene DNA, followed by screening of clear-zone no n-forming transformant colonies cultured at room temperature on plates containing skim-milk. Then, secondary mutants were derived from each primary mutant by the same mutagenic procedure, but screened by detect ing transformant colonies incubated at 10 degrees C with clear zones t hat were greater in size than that of the wild type. One such secondar y mutant, 12-12, derived from a primary mutant with 80% activity, was found to gain 150% activity (k(cat)/K-m value) of the wild-type when t he mutant subtilisin gene was subcloned to a Bacillus subtilis host-ve ctor system, expressed to form secretory mutant enzyme in the medium, and the activity measured using N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-n itroanilide as the substrate. When N-succinyl-L-Ala-L-Ala-L-Pro-L-Leu- p-nitroanilide was used, 180% activity was gained. Genetic analysis re vealed that the primary and secondary mutations corresponded to D197N and G131D, respectively. The activity variations found in these mutant subtilisins were discussed in terms of Ca2+-binding ability. The ther mostability was also found to be related to the activity.