枯草芽孢杆菌β-糖苷酶的克隆表达、体外定向进化及结构模拟

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

摘要:

摘要:【目的】从枯草芽孢杆菌基因组DNA中扩增出bglC基因并在大肠杆菌中表达，分析表达产物的酶学性质并进行结构模拟，为进一步研究其生理功能及结构解析奠定基础。【方法】将bglC基因克隆到大肠杆菌(Escherichia coli)BL21(DE3)中表达，通过定向进化获取水解效率提高的突变株，经Ni-NTA镍离子层析柱纯化后，测定野生枯草芽孢杆菌β-糖苷酶与突变酶的性质。利用CD光谱，非变性聚丙烯酰胺凝胶电泳及三维结构建模，分析野生酶与突变酶的高级结构。【结果】野生酶的比活力为9.7 U/mg，最适催化温度为60℃，最适pH值是7.0。经过突变和筛选，我们得到一个突变体BS-GLY_M1(A242T/T385A/S425L)，其比活力达到17.1 U/mg，最适温度为55 ℃，最适pH值是7.0，在55 ℃下的半衰期为3.5 h，比野生酶增加2 h。突变酶对4-硝基苯基-β-半乳糖苷、乳糖和熊果苷的催化效率(Km/Kcat)有所提高。酶在天然条件下以二聚体、四聚体状态存在，推测它以二聚体为基本功能单位。结构模拟结果表明突变后酶的三维结构有轻微地变化，这可能是酶热稳定性和催化效率提高的原因。【结论】枯草芽孢杆菌β-糖苷酶可以在大肠杆菌中高效表达并可以通过定向进化提高其水解效率。

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.