Metagenomic exploration of novel β-galactosidases for glycosylation engineering

β-Galactosidases are important enzymatic tools for glycosylation, but their properties vary greatly with the source. Here, ten putative β-galactosidase genes, designated as bga 1 to bga 10, encoding proteins Bga1 to Bga10, were mined from an environmental metagenomic dataset comprising 119,152 sequences. Five of the encoded enzyme proteins exhibited less than 80% sequence similarity to known enzymes, but displayed conserved catalytic sites in their predicted three-dimensional models. After heterologous expression and characterization, two recombinant enzymes showed specific hydrolysis activity toward o -nitrophenyl-β- d -galactopyranoside. One of them, Bga4R, exhibited remarkable activity at pH 7.4 and 50℃, with excellent alkaline stability. Notably, Bga4R tolerated a wide range of acceptors for transglycosylation. It catalyzed galactosyl transfer to various monosaccharides and sugar alcohols, and enabling the synthesis of diverse glycosylated derivatives. This study identifies a novel GH 1 β-galactosidase as a powerful tool for glycosylation engineering, with promising potential for synthesizing galactosides valuable to food and pharmaceutical industries.