title: Enhancement of the catalytic activity of a 27 kDa subtilisin-like enzyme from Bacillus amyloliquefaciens CH51 by in vitro mutagenesis.
abstract: 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 had a 2.5-fold higher k(cat) and a 2.3-fold lower K(m), which resulted in a 6-fold increase in catalytic efficiency (k(cat)/K(m)) relative to that of wild-type AprE51. In addition, AprE51-6 showed a relatively broader pH range and increased thermostability as compared to AprE51.
authors.affiliation: Department of Microbiology, College of Natural Sciences, Pusan National University, Busan, Korea.
authors.forename: Jieun
authors.initials: J
authors.lastname: Kim
authors.forename: Jong-Hyun
authors.initials: JH
authors.lastname: Kim
authors.forename: Kyoung-Hwa
authors.initials: KH
authors.lastname: Choi
authors.forename: Jeong Hwan
authors.initials: JH
authors.lastname: Kim
authors.forename: Young-Sun
authors.initials: YS
authors.lastname: Song
authors.forename: Jaeho
authors.initials: J
authors.lastname: Cha
mesh_terms: D000818:Animals; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002417:Cattle; D005342:Fibrinolysis; D006801:Humans; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D020860:Subtilisin / Q000235:genetics* / Q000378:metabolism*
chemical_list: D020860:Subtilisin