Characterization of a Thermostable β-glucosidase from the Hyperthermophilic Archaeon Thermococcus pacificus

Abstract

Hyperthermophilc archaeon, Thermococcus pacificus (Topt of 80-88 °C) could grow on various substrates such as peptides, starch, and so on. Based on the genomic analysis of the strain, we identified a novel GH1 β-glucosidase encoding gene (PAC_orf01364), which was located in glycoside hydrolase gene clusters consisting of cellulose-, laminarin-, and agarose-degrading enzymes. The gene revealed an ORF of 1,464 bp encoding 487 amino acid residues, and the deduced amino acid sequence showed 77% identity with Pyrococcus furiosus β-glucosidase (accession no. NP_577802). The gene was cloned and expressed in Escherchia coli system. The recombinant protein was purified by metal affinity chromatography and characterized. The purified enzyme shows optimum activity at pH 5.5 and 75 °C, and thermostability with a half life of 6 h at 90 °C. Interestingly, the enzyme also exhibits laminarinase activity and β-xylosidase activity.(PAC_orf01364), which was located in glycoside hydrolase gene clusters consisting of cellulose-, laminarin-, and agarose-degrading enzymes. The gene revealed an ORF of 1,464 bp encoding 487 amino acid residues, and the deduced amino acid sequence showed 77% identity with Pyrococcus furiosus β-glucosidase (accession no. NP_577802). The gene was cloned and expressed in Escherchia coli system. The recombinant protein was purified by metal affinity chromatography and characterized. The purified enzyme shows optimum activity at pH 5.5 and 75 °C, and thermostability with a half life of 6 h at 90 °C. Interestingly, the enzyme also exhibits laminarinase activity and β-xylosidase activity.