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

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 p I 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.