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

被引:26
作者
Fourage, L [1 ]
Dion, M [1 ]
Colas, B [1 ]
机构
[1] Fac Sci & Tech, CNRS, FRE 2230, Unite Rech Biocatalyse, F-44322 Nantes 3, France
关键词
beta-glycosidase; temperature dependence; kinetics; glucose; transglycosylation; (Thermus thermophilus);
D O I
10.1023/A:1007104030314
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 (Tt beta gly) has a broad substrate specificity towards beta -D-glucoside, beta -D-galactoside and beta -D-fucoside derivatives. The thermostability of Tt beta gly 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. Tt beta gly 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 (k(cat) and K-m) were calculated at different temperatures. Both k(c)at and K-m increased with an increase in temperature, but up to 75 degreesC the values of k(cat) increased much more rapidly than the values of K-m. 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 Tt beta gly. 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.
引用
收藏
页码:377 / 383
页数:7
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