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 of β-galactosidase BgaB from Geobacillus stearothermophilus

摘要:

【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。

关键词:β-半乳糖苷酶;累积突变;低聚半乳糖;GH42糖苷水解酶家族;转糖苷催化活性

Abstract:

[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 GH42 β-galactosidases.

Key words:β-galactosidase;cumulative mutation;galactooligosaccharides (GOS);glycoside hydrolase family 42 (GH42);transglycosylating activity