Kinetics of the hydrolysis of cellobiose and p-nitrophenyl-β-D-glucoside by cellobiase of Trichoderma viride

Abstract

Cellobiase was isolated from the crude cellulase mixture of enzymes of T. viride using column chromatographic and ion-exchange methods. The steady-state kinetics of the hydrolysis of cellobiose were investigated as a function of cellobiose and glucose concentrations, pH of the solution, temperature and dielectric constant, using isopropanol-buffer mixtures. The results show that there is a marked activation of the reaction by initial glucose concentrations of 4 .times. 10-3 M to 9 .times. 10-2 M and strong inhibition of the reaction at higher initial concentrations, the log rate-pH curve has a maximum at pH 5.2 and enzyme pK values of 3.5 and 6.8, the energy of activation at pH 5.1 is 10.2 kcal mol-1 over the temperature range 5-56.degree. C and the rate decreases from 0-20% (vol/vol) isopropanol. The hydrolysis by cellobiase (EC 3.2.1.21) of p-nitrophenyl-.beta.-D-glucoside was examined by pre-steady-state methods in which [enzyme]0 .mchgt. [substrate]0, and by steady-state methods as a function of pH and temperature. The results show a value for k2 of 21 s-1 at pH 7.0 (where k2 is the rate constant for the 2nd step in the assumed 2-intermediate mechanism .**GRAPHIC**. a log rate-pH curve, significantly different from that for hydrolysis of cellobiose, in which the rate increases with decreasing pH below pH 4.5, is constant in the region pH 4.5-6, and decreases above pH 6 (exhibiting an enzyme pK value of 7.3) and an activation energy of 12.5 kcal mol-1 at pH 5.7 over the temperature range 10.degree.-60.degree. C.