Stabilization of cellobiase by covalent coupling to soluble polysaccharide.

Cellobiase from Aspergillus niger was glycosylated by covalent coupling to cyanogen bromide activated dextran. The conjugated enzyme retained 62% of the original specific activity exhibited by the native cellobiase. The optimum pH as well as the pH stability of the conjugated form remain almost the same as for the native enzyme. Compared to the native enzyme, the conjugated form exhibited a higher optimal reaction temperature and energy of activation, a higher K(m) (Michaelis constant) and lower Vmax (maximal reaction rate), and improved thermal stability. The thermal deactivation of the native and conjugated cellobiase obeyed the first-order kinetics. The calculated half-life values of heat inactivation at 60, 70 and 80 degrees C was 10.7, 6.25, and 4.05 h, respectively, whereas at these temperatures the native enzyme was less stable (half-life of 3.5, 1.69, and 0.83 h, respectively). The deactivation rate constant at 80 degrees C for the conjugated cellobiase is about 7.9 x 10(-2) h-1, which is lower than that of the native enzyme (36.0 x 10(-2) h-1). The activation energy for denaturation of the native enzyme is about 10.58 kcal/mol, which is 7.25 kcal/mol lower than that of the conjugated enzyme. The effect of different surfactants and some metal ions on the activity of the conjugated cellobiase has been investigated.