Discovery and characterization of a new β-glucosidase from Wolfiporia cocos through RNA sequencing and heterologous expression in Escherichia coli.

De novo RNA-sequencing of Wolfiporia cocos mycelia cultured with filter paper composed of cellulose as the sole carbon source revealed a total of five expressed β-glucosidase genes. Among these, the β-glucosidase named Wcbg1B-1, which is composed of 539 amino acid residues and belongs to the GH1 family, had the highest mRNA abundance, accounting for 65 % of the total mRNA of the five expressed β-glucosidases. The recombinant Wcbg1B-1 was successfully expressed in Escherichia coli, with an optimal pH of 6.0 and an optimal temperature of 40°C using p-nitrophenyl-β-d-glucopyranoside (pNPG) as the substrate. Recombinant Wcbg1B-1 has a K value of 0.32 mmol/L, a V of 416 μmol/min/mg and specific activity of 461.5 U/mg. Recombinant Wcbg1B-1 has strict β-glycosidic bond specificity, the highest hydrolysis activity to cellobiose, followed by lactose, and the lowest hydrolysis activity to gentian. Recombinant Wcbg1B-1 exhibits hydrolytic activity towards macromolecular cellulose such as sodium carboxymethyl cellulose, microcrystalline cellulose and filter paper. Additionally, its high tolerance to high concentrations of glucose and salt makes it more practical for cellulose hydrolysis. Recombinant Wcbg1B-1 can hydrolyze lactose at low temperatures, indicating that it could also be a potential biocatalyst for lactose-reduced dairy industry.