Enhancement of the catalytic activity of a 27 kDa subtilisin-like enzyme from Bacillus amyloliquefaciens CH51 by in vitro mutagenesis.

AprE51 from Bacillus amyloliquefaciens CH51 is a 27 kDa subtilisin-like protease with fibrinolytic activity. To enhance the catalytic activity of AprE51, two residues, Gly-169 and Ser-101, which, according to the three-dimensional structural model of subtilisin, are located in the P1 substrate-binding site and S3 subsite, respectively, were mutated by site-directed mutagenesis. Results of the mutational analysis showed that substitution of alanine for Gly-169 increased the fibrinolytic activity 1.4-fold. All four Ser-101 mutations, that is, replacements with arginine, leucine, lysine, and tryptophan, also increased the fibrinolytic activity up to 3.9-fold. The S101W mutant with a bulky side chain was more active than mutants with a positively charged or nonpolar small side chains. The fibrinolytic activity of the S101W mutant was further increased by error-prone polymerase chain reaction. The AprE51–6 mutant (S101W/G169A/V192A) had stronger fibrinolytic activity than the S101W mutant. Purified AprE51–6 ha...