title: [Improvement of thermostability of beta-1,3-1,4-glucanase from Bacillus amyloliquefaciens BS5582 through in vitro evolution].
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.
authors.affiliation: The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
authors.forename: Jiufu
authors.initials: J
authors.lastname: Qin
authors.forename: Weiwei
authors.initials: W
authors.lastname: Gao
authors.forename: Qi
authors.initials: Q
authors.lastname: Li
authors.forename: Yongxian
authors.initials: Y
authors.lastname: Li
authors.forename: Feiyun
authors.initials: F
authors.lastname: Zheng
authors.forename: Chunfeng
authors.initials: C
authors.lastname: Liu
authors.forename: Guoxian
authors.initials: G
authors.lastname: Gu
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D019020:Directed Molecular Evolution; D044644:Endo-1,3(4)-beta-Glucanase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D004795:Enzyme Stability; D006358:Hot Temperature; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D044644:Endo-1,3(4)-beta-Glucanase