PURIFICATION AND CHARACTERIZATION OF AN ALPHA-GLUCOSIDASE FROM A HYPERTHERMOPHILIC ARCHAEBACTERIUM, PYROCOCCUS-FURIOSUS, EXHIBITING A TEMPERATURE OPTIMUM OF 105-DEGREES-C TO 115-DEGREES-C

Pyrococcus furiosus is a strictly anaerobic hyperthermophilic archaebacterium with an optimal growth temperature of about 100°C. When this organism was grown in the presence of certain complex carbohydrates, the production of several amylolytic enzymes was noted. These enzymes included an α-glycosidase that was located in the cell cytoplasm. This α-glucosidase has been purified 310-fold and corresponded to a protein band of 125 kilodaltons as resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited optimum activity at pH 5.0 to 6.0 and over a temperature range of 105 to 115°C. Kinetic analysis conducted at 108°C revealed hydrolysis of the substrate p-nitrophenyl-α-D-glucopyranoside (PNPG), methyl-α-D-glucopyranoside, maltose, and isomaltose. Trace activity was detected towards p-nitrophenyl-β-D-glucopyranoside, and no activity could be detected towards starch or sucrose. Inhibition studies conducted at 108°C with PNPG as the substrate and maltose as the inhibitor yielded a K(i) for maltose of 14.3 mM. Preincubation for 30 min at 98°C in 100 mM dithiothreitol and 1.0 M urea had little effect on enzyme activity, whereas preincubation in 1.0% sodium dodecyl sulfate and 1.0 M guanidine hydrochloride resulted in significant loss of enzyme activity. Purified α-glycosidase from P. furiosus exhibited remarkable thermostability; incubation of the enzyme at 98°C resulted in a half life of nearly 48 h.