Novel substrate specificity of a thermostable β-glucosidase from the hyperthermophilic archaeon, Thermococcus pacificus P-4

Based on the genomic analysis of Thermococcus pacificus P-4, we identified a putative GH1 β-glucosidase-encoding gene (Tpa-glu). The gene revealed a 1,464 bp encoding 487 amino acid residues, and the deduced amino acid residues exhibited 77% identity with Pyrococcus furiosus β-glucosidase (accession no. NP_577802). The gene was cloned and expressed in Escherichia coli system. The recombinant protein was purified by metal affinity chromatography and characterized. Tpa-Glu showed optimum activity at pH 7.5 and 75°C, and thermostability with a half life of 6 h at 90°C. Tpa-Glu exhibited hydrolyzing activity against various pNP-glycopyranosides, with k cat /K m values in the order of pNP-β-glucopyranoside, pNP-β-galactopyranoside, pNP-β-mannopyranoside, and pNP-β-xylopyranoside. In addition, the enzyme exhibited exo-hydrolyzing activity toward β-1,3-linked polysaccharide (laminarin) and β-1,3- and β-1,4-linked oligosaccharides. This is the first description of an enzyme from hyperthermophilic archaea that displays exo-hydrolyzing activity toward β-1,3-linked polysaccharides and could be applied in combination with β-1,3-endoglucanase for saccharification of laminarin.