Enhancement of the Thermostability of a Fibrinolytic Enzyme from Bacillus amyloliquefaciens CH51

Abstract

AprE51 from Bacillus amyloliquefaciens CH51 is a 27 kDa subtilisin-like protease with fibrinolytic activity. AprE51-6 showing increased catalytic activity was produced previously. To enhance the thermostability of AprE51-6, 2 residues, Gly-166 and Asn-218 based on B. subtilis subtilisin E were mutated by site-directed mutagenesis. The results of the mutational analysis showed that substitution of arginine for Gly-166 (AprE51-7) increased the fibrinolytic activity 1.8-fold. An N218S mutant (AprE51-8) also increased the fibrinolytic activity up to 4.5-fold in a fibrin plate assay. Purified AprE51-7 and AprE51-8 mutants had a 1.9-and a 2.5-fold higher kcat, respectively, and a 2.1-1.9-fold lower Km, respectively. This resulted in a 3.8-and a 4.7-fold increase in catalytic efficiency (kcat/Km), respectively, relative to that of wild-type AprE51. AprE51-8 had a broader pH range than AprE51-6 and nattokinase, especially at an alkaline pH value. In addition, AprE51-8 showed higher thermostability than AprE51-6 at 60 o C. The half-lives of AprE51-7 and AprE51-8 at 50 o C were 21.5 and 27.3 min, respectively, which are 2.0 and 2.6 times longer, respectively, than that of the wild-type AprE51.