Unstable character of β-glucosidase in B. stearothermophilus

Abstract

β-Glucosidase of $\underline{Bacillus} \underline{stearothermophilus}$ was studied. This was to establish an enzymatical foundation on which cloning of β-glucosidase gene will be initiated in the future. $\underline{B}$. $\underline{stearothermophilus}$ was found to grow well on cellobiose as a sole source of carbon suggesting this organism as a potential gene donor for the cloning experiments. The β-glucosidase in $\underline{B}$. $\underline{stearothermophilus}$ has pH optimum at 8.0 and temperature optimum at 37℃. The enzyme was strongly inhibited by the presence of glucose or sucrose. The β-glucosidase of $\underline{B}$. $\underline{stearothermophilus}$, however, was found to be extremely unstable and easily lost its activity by rupturing the cells. Even in the intact cells suspended in 0.05M sodium phosphate buffer the enzyme lost its activity completely within six hours of storage at ice-cold temperature. The activity was also lost simply by changing from phosphate buffer to SMM buffer which contains sucrose to suspend the intact cells. The loss of activity in the ruptured cells was not due to the loss of some cofactors, because addition of various ions, ATP, and DTT to the ruptured cells could not restore the β-glucosidase activity. The negative data in the ruptured cell fractions was assumed to be due mainly to the unstable characteristics of the enzyme. Therefore, an establishment of standard assay conditions which yields accurate and reproducible determinations of β-glucosidase seemed to be greatly needed.