|
KMID : 0613820130230010015
|
|
Journal of Life Science
2013 Volume.23 No. 1 p.15 ~ p.23
|
|
|
Enhancement of the Thermostability of a Fibrinolytic Enzyme from Bacillus amyloliquefaciens CH51
|
|
Kim Ji-Eun
Choi Kyoung-Hwa
Kim Jeong-Hwan
Song Young-Sun
Cha Jae-Ho
|
|
|
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 K_cat, 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 (K_cat/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℃. The half-lives of AprE51-7 and AprE51-8 at 50℃ 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.
|
|
|
KEYWORD
|
|
|
Bacillus amyloliquefaciens, Cheonggukjang, fibrinolytic enzyme, in vitro mutagenesis, thermostability
|
|
|
FullTexts / Linksout information
|
|
|
|
|
|
Listed journal information
|
|
|
|