PURIFICATION AND PROPERTIES OF RECOMBINANT BETA-GLUCOSIDASE OF THE HYPERTHERMOPHILIC BACTERIUM THERMOTOGA-MARITIMA

A beta-glucosidase of the hyperthermophilic bacterium Thermotoga marit ima has been purified from a recombinant Escherichia coli clone expres sing the corresponding gene. The enzyme was found to be a dimer with a n apparent molecular mass of approximately 95 kDa as determined by siz e exclusion chromatography. It was composed of two apparently identica l subunits of about 47 kDa (determined by sodium dodecyl sulphate-poly acrylamide gel electrophoresis). The enzyme had a broad substrate spec ificity and attacked beta-glucoside, beta-galactoside, beta-fucoside, and, to a very small extent, also beta-xyloside substrates. Alpha-glyc osidic bonds were not hydrolysed. Kinetic measurement of the hydrolysi s of o-nitrophenyl-beta-D-glucopyranoside (oNPGlc) and o-nitrophenyl-b eta-D-galactopyranoside (oNPGal) in the concentration ranges 0.05-20 m M and 0.1-10 mM, respectively, at 75-degrees-C resulted in non-linear Lineweaver-Burk and Eadie-Hofstee plots whereas cellobiose and lactose did not induce this type of effect. Lactose caused substrate inhibiti on above 350 mM. The enzyme was optimally active at about pH 6.1. The T. maritima beta-glucosidase represents the most thermostable beta-glu cosidase described to date. In 50 mM sodium phosphate buffer, pH 6.2, at an enzyme concentration of 50 mug/ml, the pure enzyme without addit ives retained more than 60% of its initial activity after a 6-h incuba tion at 95-degrees-C.