title: Directed evolution of a β-1,3-1,4-glucanase from Bacillus subtilis MA139 for improving thermal stability and other characteristics.
abstract: In order to improve some characteristics of a β-1,3-1,4-glucanase from Bacillus subtilis MA139, directed evolution was conducted in this study. After error-prone PCR, the β-1,3-1,4-glucanase gene, glu-opt, was cloned into the vector pBGP1 and transformed into Pichia pastoris X-33 to construct a mutant library. Three variants named as 7-32, 7-87, and 7-115 were screened from 8000 colonies. Amino-acid sequence analysis showed that these mutants had one or two amino-acid substitutions (7-32: T113S, 7-87: M44V/N53H, and 7-115: N157D). The variants were over-expressed in P. pastoris by methanol induction. After purification of the enzyme proteins, the characteristics of the variants were analyzed in detail. It indicated that these mutant enzymes had broader ranges of pH value and better pH stability than the wild-type enzyme. The mutant enzyme 7-87 had the best ability to tolerate an acid environment (pH 2.0), while the wild-type enzyme had no activity under this condition. Moreover, all these mutants demonstrated improved thermal stability. In particular, the mutant enzyme 7-32 had residual enzymatic activity of 60% and 40% after being incubated at 80 °C and 90 °C for 10 min. While, the wild-type enzyme had no residual enzymatic activity after being incubated at 80 °C for 4 min. In addition, the mutant enzymes had better tolerance to some chemicals than the wild-type enzyme. The improved stability could enhance the prospects for this enzyme to have use in the feed industry to reduce the effects of the anti-nutritional factor β-glucan.
authors: National Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, PR China.
authors: Honglei
authors: H
authors: Pei
authors: National Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, PR China.
authors: Xiaojing
authors: X
authors: Guo
authors: National Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, PR China.
authors: Wenhan
authors: W
authors: Yang
authors: National Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, PR China.
authors: Junnan
authors: J
authors: Lv
authors: National Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, PR China.
authors: Yiqun
authors: Y
authors: Chen
authors: National Key Laboratory of Animal Nutrition, China Agricultural University, Beijing, PR China.
authors: Yunhe
authors: Y
authors: Cao
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution; D044644:Endo-1,3(4)-beta-Glucanase / Q000235:genetics* / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D010843:Pichia / Q000235:genetics; D016133:Polymerase Chain Reaction
chemical_list: D044644:Endo-1,3(4)-beta-Glucanase
keywords: Directed evolution; Improved stability; Pichia pastoris; β-1,3-1,4-Glucanase