A novel β-glucosidase from higher termite Microcerotermes annandalei (MaBG) was obtained via a screening method targeting β-glucosidases with increased activities in the presence of glucose. The purified natural MaBG showed a subunit molecular weight of 55 kDa and existed in a native form as a dimer without any glycosylation. Gene-specific primers designed from its partial amino acid sequences were used to amplify the corresponding 1,419-bp coding sequence of MaBG which encodes a 472-amino acid glycoside hydrolase family 1 (GH1) β-glucosidase. When expressed in Komagataella pastoris, the recombinant MaBG appeared as a ~ 55-kDa protein without glycosylation modifications. Kinetic parameters as well as the lack of secretion signal suggested that MaBG is an intracellular enzyme and not involved in cellulolysis. The hydrolytic activities of MaBG were enhanced in the presence of up to 3.5-4.5 M glucose, partly due to its strong transglucosylation activity, which suggests its applicability in biosynthetic processes. The potential synthetic activities of the recombinant MaBG were demonstrated in the synthesis of para-nitrophenyl-β-D-gentiobioside via transglucosylation and octyl glucoside via reverse hydrolysis. The information obtained from this study has broadened our insight into the functional characteristics of this variant of termite GH1 β-glucosidase and its applications in bioconversion and biotechnology.

Abstractβ-Glucosidases play an important role in biomass degradation as they hydrolyze cellobiose to glucose in a final step of cellulolysis. In particular, ruminant animals rely onβ-glucosidases from rumen microorganisms for conversion of plant cellulosic materials into glucose. In this study, we are interested in characterization of a novelβ-glucosidase from rumen microorganisms. However, most rumen microorganisms are obligate anaerobes, which require special cultivation conditions. Presently, the metagenomic techniques, which enable isolation and characterization of microbial genes directly from environmental samples, have been applied to overcome these problems. In this study, the sequence-based screening approach was successfully applied to identify a novelβ-glucosidase gene,Br2, from a bovine rumen metagenomic sample. A 1338-bp complete coding sequence ofBr2encodes a 51-kDa GH1β-glucosidase of 445 amino acid residues with 59% sequence identity to aβ-glucosidase fromCellulosilyticum ruminicolaJCM 14822. The recombinantly expressed Br2 exhibited an optimal activity at pH 6.5 and 40°C, reflecting its rumen bacterial origin, and relatively higher catalytic efficiencies toward glucoside and fucoside substrates than other glycosides, similar to many previously reported bacterialβ-glucosidases. Our sequence-based screening approach can be applied to identify other genes of interest from environmental samples.

No abstract