Comparison of a ß-glucosidase and a ß-mannosidase from the
Hyperthermophilic Archaeon Pyrococcus furiosus: Purification, Characterization,
Gene cloning, and Sequence Analysis
Bauer M.W., Bylina E.J., Swanson R.V., Kelly R.M.
Department of Chemical Engineering, North Carolina State University, Raleigh, North
Carolina 27695-7905, USA.
Two distinct exo-acting, ß-specific glycosyl hydrolases were purified to homogeneity from
crude cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus: a
ß-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 ß-mannosidase. The ß-mannosidase and ß-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 ß-mannosidase and the ß-glucosidase, respectively. The
ß-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. U37557U37557). The deduced amino acid sequences showed homology both
with each other (46.5% identical) and with several other glycosyl hydrolases, including
the ß-glycosidases from Sulfolobus solfataricus, Thermotoga maritima,
and Caldocellum saccharolyticum. Based on these sequence similarities, the
ß-mannosidase and the ß-glucosidase can both be classified as family 1 glycosyl
hydrolases. In addition, the ß-mannosidase and ß-glucosidase from P. furiosus
both contained the conserved active site residues found in all family 1 enzymes. The
ß-mannosidase showed optimal activity at pH 7.4 and 105 degrees C. Although the enzyme
had a half-life of greater than 60 h at 90 degrees C, it is much less thermostable than
the ß-glucosidase, which had a reported half-life of 85 h at 100 degrees C. Km
and Vmax values for the ß-mannosidase were determined to be 0.79 mM and 31.1
micromol para-nitrophenol released/min/mg with p-nitrophenyl-ß-D-mannopyranoside as
substrate. The catalytic efficiency of the ß-mannosidase was significantly lower than
that reported for the P. furiosus ß-glucosidase (5.3 versus 4, 500 s-1 mM-1 with
p-nitrophenyl-ß-D-glucopyranoside as substrate). The kinetic differences between the two
enzymes suggest that, unlike the ß-glucosidase, the primary role of the ß-mannosidase
may not be disaccharide hydrolysis. Other possible roles for this enzyme are discussed.