title: [Cloning, expression, directed evolution in vitro and structural simulation of β-glycosidase from Bacillus subtilis].
abstract: OBJECTIVE
  To further study physiological functions and structure of β-glycosidase, we cloned the bglC gene of Bacillus subtilis and expressed it in E. coli BL21 (DE3), followed by the characterization and structural simulation of the enzyme.
  
  
  METHODS
  We amplified the bglC gene and transferred it into E. coli BL21 (DE3), then we obtained a mutant with higher hydrolytic activity by directed evolution. After purifying the enzymes through a nickel-nitrilotriacetic acid agarose column, we characterized the wild-type and mutant enzymes. By means of CD spectrum, Native-PAGE and protein 3-D structure modeling, we analyzed the higher structure of the β-glycosidase.
  
  
  RESULTS
  We got one mutant enzyme BS-GLY_M1 (A242T/T385A/S425L) with improved hydrolytic activity by directed evolution and screening. The specific activity of wild-type enzyme was 9.7 U/mg, with optimum temperature at 60 degrees C and optimum pH at 7.0. The specific activity of BS-GLY_M1 was 17. 1U/mg, with optimum temperature at 55 degrees C and optimum pH at 7.0. Moreover, the half-life time of the mutant enzyme at 55 degrees C was 3.5 h, 2 h longer than that of wild-type enzyme. Furthermore, the catalytic efficiency (K(m)/K(cat)) of BS-GLY_M1 on the substrates 4-nitrophenyl-β-galactoside, lactose, and arbutin improved obviously. The polymer forms of the enzyme under the native conditions were of dimer and tetramer, but the dimer was the most probable functional unit. Result of structural simulation also showed slight changes occurred in the tertiary structure of the mutant enzyme, which may be the main reason for the enhanced thermal stability and catalytic efficiency of BS-GLY _M1. [
  
  
  CONCLUSIONS
  β-glycosidase from Bacillus subtilis could be expressed in E. coli BL21 (DE3), meanwhile its hydrolysis efficiency could be further improved by directed evolution.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liu
authors.forename: Qian
authors.initials: Q
authors.lastname: Chen
authors.forename: Yinnan
authors.initials: Y
authors.lastname: Chen
authors.forename: Zuxin
authors.initials: Z
authors.lastname: Wang
authors.forename: Qiuxiang
authors.initials: Q
authors.lastname: Zhu
authors.forename: Xian'ai
authors.initials: X
authors.lastname: Shi
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D013696:Temperature; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001616:beta-Galactosidase