S. Ezaki et al., Gene analysis and enzymatic properties of thermostable-beta-glycosidase from Pyrococcus kodakaraensis KOD1, J BIOSCI BI, 88(2), 1999, pp. 130-135
A beta-glycosidase with broad substrate specificity was identified from a h
yperthermophilic archaeon, Pyrococcus kodakaraensis KOD1. The gene encoding
beta-glycosidase (Pk-gly) consists of 1449 nucleotides corresponding to a
polypeptide of 483 amino acids. The protein showed similarity with other be
ta-glycosidases from family-1 glycosyl hydrolases, in particular, it showed
high identity to beta-mannosidase from P. furiosus (55.7%), beta-glycosida
se from Sulfolobus solfataricus (42.7%) and beta-glucosidase from P. furios
us (41.9%). The cloned gene was expressed in Escherichia coli and the recom
binant protein was purified. The beta-glycosidase showed optimal activity a
t pH 6.5 and at an extremely high temperature of 100 degrees C, and had a h
alf-life of 18 h at 90 degrees C. The beta-glycosidase hydrolyzed various p
Np-beta-glycopyranosides, with k(cat)/K-m values in the order of pNp-beta-g
lucopyranoside is approximately equal to pNp-beta-mannopyranoside > pNp-bet
a-galactopyranoside > pNp-beta-xylopyranoslde. pNp-beta-mannopyranoside was
the substrate exhibiting the lowest K-m value [0.254 mM] with a k(cat)/K-m
ratio comparable to that of pNp-beta-glucopyranoside. This substrate speci
ficity was distinct from previously reported beta-glycosidases. We observed
that the region in Pk-Gly corresponding to the fifth alpha-helix and beta-
strand region of beta-glycosidase from S. solfataricus, which constitutes a
large portion of the channel for substrate incorporation, displayed a chim
eric structure, with the N-terminal region similar to beta-glycosidases and
the C-terminal region similar to beta-mannosidases. An exo-type hydrolytic
activity and transglycosylation activity were also observed towards celloo
ligomers.