Kinetic study of a thermostable beta-glycosidase of Thermus thermophilus. Effects of temperature and glucose on hydrolysis and transglycosylation reactions.

Abstract

A beta-glycosidase of a thermophile, Thermus thermophilus, belonging to the glycoside hydrolase family 1, was cloned and overexpressed in Escherichia coli. The purified enzyme (Ttbetagly) has a broad substrate specificity towards beta-D-glucoside, beta-D-galactoside and beta-D-fucoside derivatives. The thermostability of Ttbetagly was exploited to study its kinetic properties within the range 25-80 degreesC. Whatever the temperature, except around 60 degreesC, the enzyme displayed non-Michaelian kinetic behavior. Ttbetagly was inhibited by high concentrations of substrate below 60 degreesC and was activated by high concentrations of substrate above 60 degreesC. The apparent kinetic parameters (kcat and Km) were calculated at different temperatures. Both kcat and Km increased with an increase in temperature, but up to 75 degreesC the values of kcat increased much more rapidly than the values of Km. The observed kinetics might be due to a combination of factors including inhibition by excess substrate and stimulation due to transglycosylation reactions. Our results show that the substrate could act not only as a glycosyl donor but also as a glycosyl acceptor. In addition, when the glucose was added to reaction mixtures, inhibition or activation was observed depending on both substrate concentration and temperature. A reaction model is proposed to explain the kinetic behavior of Ttbetagly. The scheme integrates the inhibition observed at high concentrations of substrate and the activation due to transglycosylation reactions implicating the existence of a transfer subsite.