Improvement of thermostability of beta-1,3-1,4-glucanase from Bacillus amyloliquefaciens BS5582 through in vitro evolution.
- Author:
Jiufu QIN
1
;
Weiwei GAO
;
Qi LI
;
Yongxian LI
;
Feiyun ZHENG
;
Chunfeng LIU
;
Guoxian GU
Author Information
1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
Bacillus;
enzymology;
Directed Molecular Evolution;
Endo-1,3(4)-beta-Glucanase;
biosynthesis;
chemistry;
genetics;
Enzyme Stability;
Hot Temperature;
Molecular Sequence Data;
Recombinant Fusion Proteins;
biosynthesis;
chemistry;
genetics
- From:
Chinese Journal of Biotechnology
2010;26(9):1293-1301
- CountryChina
- Language:Chinese
-
Abstract:
In vitro evolution methods are often used to modify protein with improved characteristics. We developed a directed evolution protocol to enhance the thermostability of the beta-1,3-1,4-glucanase. The thermostability of the enzyme was significantly improved after two rounds of directed evolution. Three variants with higher thermostability were obtained. The mutant enzymes were further analyzed by their melting temperature, halftime and kinetic parameters. Comparing to intact enzyme, the T50 of mutant enzymes 2-JF-01, 2-JF-02 and 2-JF-03 were increased by 2.2 degrees C, 5.5 degrees C and 3.5 degrees C, respectively, the halftime (t1/2, 60 degrees C) of mutant enzymes 2-JF-01, 2-JF-02 and 2-JF-03 were shortened by 4,13 and 17 min, respectively, the V(max) of mutant enzymes were decreased by 8.3%, 2.6% and 10.6%, respectively, while K(m) of mutant enzymes were nearly unchanged. Sequence analysis revealed seven single amino acid mutant happened among three mutant enzymes, such as 2-JF-01 (N36S, G213R), 2-JF-02 (C86R, S115I, N150G) and 2-JF-03 (E156V, K105R). Homology-modeling showed that five of seven substituted amino acids were located on the surface of or in hole of protein. 42.8% of substituted amino acids were arginine, which indicated that arginine may play a role in the improvement of the thermostability of the beta-1,3-1,4-glucanase.This study provide some intresting results of the structural basis of the thermostability of beta-1,3-1,4-glucanase,and provide some new point of view in modifying enzyme for future industrial use.
