Purification and characterization of an extremely thermostable β‐glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus

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Abstract
Cell-free extracts of cellobiose-grown cells of the hypermophile Pyrococcus furiosus contain very high activities (19.8 U/mg) of a β-glucosidase. The cytoplasmic enzyme was purified 22-fold to apparent homogeneity, indicating that the enzyme comprises nearly 5% of the total cell protein. The native β-glucosidase has a molecular mass of 230 ± kDa, composed of 58 ± 2-kDa subunits. The enzyme has a pI of 4.40. Thiol groups are not essential for activity, nor is the enzyme dependent on divalent cations or a high ionic strength. The enzyme shows optimum activity at pH 5.0 and 102–105°C. From Lineweaver-Burk plots, Vmax values of 470 U/mg and 700 U/mg were found for cellobiose (Km= 20 mM) and p-nitrophenyl-β-d-glucopyranoside (Km= 0.15 mM), respectively. The purified enzyme also exhibits high βgactosidase activity and β-xylosidase activity, but shows no activity towards α-linked disaccharides or β-linked polymers, like cellulose. The purified β-glucosidase shows a remarkable thermostability with a half life of 85 h at 100°C and 13 h at 110°C.
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