Cloning，expression，directed evolution in vitro and structural simulation of β-glycosidase from Bacillus subtilis

Abstract:

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 BSGLY_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 ℃ and optimum pH at 7.0．The specific activity of BS-GLY_M1 was 17.1U/mg，with optimum temperature at 55 ℃ and optimum pH at 7.0．Moreover，the halflife time of the mutant enzyme at 55 ℃ was 3.5 h，2 h longer than that of wild-type enzyme． Furthermore，the catalytic efficiency (Km/Kcat) 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.［Conclusion］β-glycosidase from Bacillus subtilis could be expressed in E.coli BL21 (DE3)，meanwhile its hydrolysis efficiency could be further improved by directed evolution.