Screening novel β-galactosidases from a sequence-based metagenome and characterization of an alkaline β-galactosidase for the enzymatic synthesis of galactooligosaccharides.

βgalactosidases have wide industrial applications in lactose hydrolysis and transglycosylation reactions. Therefore, there is a need to mine novel and high-quality β-galactosidases with good tolerance and novel features from harsh environments and genomic databases. In this study, an Escherichia coli β-galactosidase-deficient host, ΔlacZ(DE3)pRARE, was constructed by the CRISPR-Cas9 system for screening active β-galactosidases. Of thirty selected β-galactosidases, twelve novel enzymes showed β-galactosidase activity, four of which were purified for further study. BGal_375 exhibited maximal activity at pH 8 and 50 °C. The concentrations of two types of galactooligosaccharides, tri- and tetra-saccharides, produced by BGal_375, reached 64.53 g/l and 8.32 g/l, respectively. BGal_375 displayed a Km value of 1.65 mM and kcat value of 53 s-1 for p-nitrophenyl-β-d-galactopyranoside (pNPG). BGal_137, BGal_144-3, and BGal_145-2 showed promising hydrolytic activity for pNPG. BGal_137 is a homodimer while BGal_144-3, BGal_145-2, and BGal_375 were all monomeric. This study provided an efficient solution for the identification of new β-galactosidases from metagenomic data, and an alkaline β-galactosidase efficient for the synthesis of galactooligosaccharides was obtained, which is important for potential industrial applications.