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Volume 271, Number 39, Issue of September 27, 1996 pp. 23749-23755
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Comparison of a -Glucosidase and a -Mannosidase from the Hyperthermophilic Archaeon Pyrococcus furiosus
PURIFICATION, CHARACTERIZATION, GENE CLONING, AND SEQUENCE ANALYSIS

(Received for publication, November 27, 1995, and in revised form, June 12, 1996)

Michael W. Bauer , Edward J. Bylina ^§ , Ronald V. Swanson ^§ and Robert M. Kelly

From the  Department of Chemical Engineering, North Carolina State University, Raleigh, North Carolina 27695-7905 and ^§ Recombinant BioCatalysis, Inc., Sharon Hill, Pennsylvania 19079-1005

Two distinct exo-acting, -specific glycosyl hydrolases were purified to homogeneity from crude cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus: a -glucosidase, corresponding to the one previously purified by Kengen et al. (Kengen, S. W. M., Luesink, E. J., Stams, A. J. M., and Zehnder, A. J. B. (1993) Eur. J. Biochem. 213, 305-312), and a -mannosidase. The -mannosidase and -glucosidase genes were isolated from a genomic library by expression screening. The nucleotide sequences predicted polypeptides with 510 and 472 amino acids corresponding to calculated molecular masses of 59.0 and 54.6 kDa for the -mannosidase and the -glucosidase, respectively. The -glucosidase gene was identical to that reported by Voorhorst et al. (Voorhorst, W. G. B., Eggen, R. I. L., Luesink, E. J., and deVos, W. M. (1995) J. Bacteriol. 177, 7105-7111; GenBank accession no. U37557[GenBank]). The deduced amino acid sequences showed homology both with each other (46.5% identical) and with several other glycosyl hydrolases, including the -glycosidases from Sulfolobus solfataricus, Thermotoga maritima, and Caldocellum saccharolyticum. Based on these sequence similarities, the -mannosidase and the -glucosidase can both be classified as family 1 glycosyl hydrolases. In addition, the -mannosidase and -glucosidase from P. furiosus both contained the conserved active site residues found in all family 1 enzymes. The -mannosidase showed optimal activity at pH 7.4 and 105 °C. Although the enzyme had a half-life of greater than 60 h at 90 °C, it is much less thermostable than the -glucosidase, which had a reported half-life of 85 h at 100 °C. K_m and V_max values for the -mannosidase were determined to be 0.79 mM and 31.1 µmol para-nitrophenol released/min/mg with p-nitrophenyl--D-mannopyranoside as substrate. The catalytic efficiency of the -mannosidase was significantly lower than that reported for the P. furiosus -glucosidase (5.3 versus 4, 500 s¹ mM¹ with p-nitrophenyl--D-glucopyranoside as substrate). The kinetic differences between the two enzymes suggest that, unlike the -glucosidase, the primary role of the -mannosidase may not be disaccharide hydrolysis. Other possible roles for this enzyme are discussed.

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