Cloning, expression and mutation of a novel thermophilic GH1 β-galactosidase from Vulcanisaeta thermophila with transglycosylation activity for tyrosol galactoside synthesis

Abstract

β-Galactosidase is a crucial class of biocatalysts, renowned for its ability to catalyze the hydrolysis of non-reducing terminal β-d-galactoside bonds, while also demonstrating significant potential in galactosylation synthesis reactions. In this study, a novel GH1 β-galactosidase gene VthBga was screened and cloned from Vulcanisaeta thermophila. Soluble expression of the VthBga was induced at 47 °C in Escherichia coli BL21. VthBga exhibited optimal activity at 95 °C and pH 4.5 with p-nitrophenyl-β-d-galactoside (pNPGal) as a substrate and displayed excellent thermotolerance below 90 °C. In addition, VthBga possessed not only a diverse array of substrate hydrolytic activities but also inherent galactosylation activity, rendering it a promising candidate for the galactosylation of the natural compound. To enhance its transglycosylation activity, a superior mutant Vth-A266G/F441T was obtained by adjusting the transglycosylation/hydrolysis ratio through a semi-rational design. Under optimal conditions, this mutant produced 44.5 % tyrosol galactoside, representing a significant 2.3-fold increase compared to the wild-type strain, highlighting its practical value in related biosynthesis applications.