Open AccessDirected evolution improves the fibrinolytic activity of nattokinase from B acillus natto
Author(s) -
Yongjun Cai,
Wei Bao,
Shujun Jiang,
Meizhi Weng,
Yan Jia,
Yan Yin,
Zhongliang Zheng,
Goulin Zou
Publication year - 2011
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2011.02423.x
Subject(s) - nattokinase , dna shuffling , subtilisin , bacillus amyloliquefaciens , mutant , bacillus licheniformis , directed evolution , biochemistry , linker , chemistry , enzyme , biology , gene , bacteria , bacillus subtilis , genetics , fermentation , computer science , operating system
Nattokinase (subtilisin NAT, NK ) is a relatively effective microbial fibrinolytic enzyme that has been identified and characterized from B acillus natto . In the current report, DNA family shuffling was used to improve the fibrinolytic activity of nattokinase. Three homologous genes from B . natto AS 1.107, B acillus amyloliquefaciens CICC 20164 and B acillus licheniformis CICC 10092 were shuffled to generate a mutant library. A plate‐based method was used to screen the mutant libraries for improved activity. After three rounds of DNA shuffling, one desirable mutant with 16 amino acid substitutions was obtained. The mutant enzyme was purified and characterized. The kinetic measurements showed that the catalytic efficiency of the mutant NK was approximately 2.3 times higher than that of the wild‐type nattokinase. In addition, the molecular modeling analysis suggested that the mutations affect the enzymatic function by changing the surface conformation of the substrate‐binding pocket. The current study shows that the evolution of nattokinase with improved fibrinolytic activity by DNA family shuffling is feasible and provides useful references to facilitate the application of nattokinase in thrombolytic therapy.