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

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.