Biochemical characterization of two novel β-glucosidase genes by metagenome expression cloning

Two novel β-glucosidase genes designated as bgl1D and bgl1E, which encode 172- and 151-aa peptides, respectively, were cloned by function-based screening of a metagenomic library from uncultured soil microorganisms. Sequence analyses indicated that Bgl1D and Bgl1E exhibited lower similarities with some putative β-glucosidases. Functional characterization through high-performance liquid chromatography demonstrated that purified recombinant Bgl1D and Bgl1E proteins hydrolyzed d-glucosyl-β-(1–4)-d-glucose to glucose. Using p-nitrophenyl-β-d-glucoside as substrate, Km was 0.54 and 2.11 mM, and kcat/Km was 1489 and 787 mM−1 min−1 for Bgl1D and Bgl1E, respectively. The optimum pH and temperature for Bgl1D was pH 10.0 and 30 °C, while the optimum values for Bgl1E were pH 10.0 and 25 °C. Bgl1D exhibited habitat-specific characteristics, including higher activity in lower temperature and at high concentrations of AlCl3 and LiCl. Bgl1D also displayed remarkable activity across a broad pH range (5.5–10.5), making it a potential candidate for industrial applications.