GH42家族保守氨基酸位点累积突变对Geobacillus stearothermophilus来源β-半乳糖苷酶BgaB催化活性的影响

Cumulative effect of mutations at conserved sites of GH42 family on the catalytic activity ofβ-galactosidase BgaB from Geobacillus stearothermophilus

【背景】β-半乳糖苷酶转糖苷活性弱,产物低聚半乳糖(galactooligosaccharides,GOS)易被水解,致其催化得率普遍较低。【目的】以GH42家族Geobacillus stearothermophilus来源β-半乳糖苷酶BgaB为对象,探讨家族保守氨基酸位点突变对β-半乳糖苷酶BgaB催化活性的影响。【方法】在单点突变体功能研究基础上,采用定点突变与化学修饰相结合的方法,对保守氨基酸位点E303与F341进行累积突变。【结果】与野生型酶相比,所构建双点突变体Ox-E303C/F341S水解活性降低为30%;GOS最大得率由0.75%提高到19.50%。【结论】家族保守氨基酸位点累积突变能够使单点突变体功能得到共同进化,降低β-半乳糖苷酶水解活性和底物抑制作用,能够提高其转糖苷催化活性。

[Background]The weak transglycosidase activity of β-galactosidase and the hydrolyzable property of the product galactooligosaccharides(GOS)results in the low product yield.[Objective]To study the effect of mutations at highly conserved sites of glycoside hydrolase family 42(GH42)on the catalytic activity of the β-galactosidase BgaB from Geobacillus stearothermophilus.[Methods]On the basis of the functional analyses of single site mutations,the conserved sites E303 and F341 were cumulatively mutated by the combination of site-directed mutagenesis and chemical modification.[Results]The double-site mutant Ox-E303C/F341S was constructed.Compared with the wild-type enzyme,the double mutations reduced the hydrolysis activity to 30% and increased the GOS yield from 0.75% to 19.50%.[Conclusion]The cumulative mutations of conserved sites enable co-evolution of single-site mutant functions.Reducing the hydrolytic activity and substrate inhibition can improve the transglycosidase activity of β-galactosidases.This work provides a reference for the modification and regulation of the transglycosylating activity of GH42β-galactosidases.