Gene analysis and enzymatic properties of thermostable-beta-glycosidase from Pyrococcus kodakaraensis KOD1

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.