COMPARISON OF A BETA-GLUCOSIDASE AND A BETA-MANNOSIDASE FROM THE HYPERTHERMOPHILIC ARCHAEON PYROCOCCUS-FURIOSUS - PURIFICATION, CHARACTERIZATION, GENE CLONING, AND SEQUENCE-ANALYSIS

Two distinct exo-acting, beta-specific glycosyl hydrolases were purifi ed to homogeneity from crude cell extracts of the hyperthermophilic ar chaeon Pyrococcus furiosus: a beta-glucosidase, corresponding to the o ne previously purified by Kengen et al. (Kengen, S. W. M., Luesink, E. J., Stams, A. J. M., and Zehnder, A. J. B. (1993) fur. J. Biochem. 21 3, 305-312), and a beta-mannosidase. The beta-mannosidase and beta-glu cosidase genes were isolated from a genomic library by expression scre ening. The nucleotide sequences predicted polypeptides with 510 and 47 2 amino acids corresponding to calculated molecular masses of 59.0 and 54.6 kDa for the beta-mannosidase and the beta-glucosidase, respectiv ely. The beta-glucosidase gene was identical to that reported by Voorh orst ct al. (Voorhorst, W. G. B., Eggen, R. I. L., Luesink, E. J., and deVos, W. M. (1995) J. Bacteriol. 177, 7105-7111; GenBank accession n o. U37557). The deduced amino acid sequences showed homology both with each other (46.5% identical) and with several other glycosyl hydrolas es, including the beta-glycosidases from Sulfolobus solfataricus, Ther mo-toga maritima, and Caldocellum saccharolyticum. Based on these sequ ence similarities, the beta-mannosidase and the beta-glucosidase can b oth be classified as family 1 glycosyl hydrolases. In addition, the be ta-mannosidase and beta-glucosidase from P. furiosus both contained th e conserved active site residues found in all family 1 enzymes. The be ta-mannosidase showed optimal activity at pH 7.4 and 105 degrees C. Al though the enzyme had a half-life of greater than 60 h at 90 degrees C , it is much less thermostable than the beta-glucosidase, which had a reported half-life of 85 h at 100 degrees C. K-m and V-max values for the beta-mannosidase were determined to be 0.79 mM and 31.1 mu mol par a-nitrophenol released/min/mg with p-nitrophenyl-beta-D-mannopyranosid e as substrate. The catalytic efficiency of the beta-mannosidase was s ignificantly lower than that reported for the P. furiosus beta-glucosi dase (5.3 versus 4, 500 s(-1) mM(-1) with p-nitrophenyl-beta-D-glucopy ranoside as substrate). The kinetic differences between the two enzyme s suggest that, unlike the beta-glucosidase, the primary role of the b eta-mannosidase may not be disaccharide hydrolysis. Other possible rol es for this enzyme are discussed.