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

Abstract

<jats:p>Cell‐free extracts of cellobiose‐grown cells of the hypermophile <jats:italic>Pyrococcus furiosus</jats:italic> 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<jats:italic>I</jats:italic> 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, <jats:italic>V</jats:italic><jats:sub>max</jats:sub> values of 470 U/mg and 700 U/mg were found for cellobiose (<jats:italic>K</jats:italic><jats:sub>m</jats:sub>= 20 mM) and <jats:italic>p</jats:italic>‐nitrophenyl‐β‐<jats:sc>d</jats:sc>‐glucopyranoside (<jats:italic>K</jats:italic><jats:sub>m</jats:sub>= 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.</jats:p>

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