Thermal stable and oxidation-resistant variant of subtilisin E

A remarkable thermal stable and oxidation-resistant mutant was obtained usi ng the random mutagenesis PCR technique on the mutant M222A gene of subtili sin E. Sequencing analysis revealed an A was replaced by G at nucleotide 67 1 of the subtilisin E gene, converting the asparagine codon (AAT) to serine codon (AGT) at position 118. The half-life of M222A/N118S enzyme activity, when heated at 65 degrees C, was approximately 80 min while the half-life of M222A and wild-type subtilisin E were 13 min and 15 min, respectively. T his suggested the stability of the M222A/N118S mutant was five times greate r than that of the wild-type enzyme. The mutant was also as oxidation resis tant as the mutant M222A of subtilisin E. These results indicated the M222A /N118S mutant is both an oxidation-resistant and a heat-stable variant of s ubtilisin E. (C) 2000 Elsevier Science B.V. All rights reserved.