On the use of chitosan immobilized b-glucosidase in wine-making: kinetics and enzyme inhibition

The kinetics of chitosan-immobilized beta-glucosidase and enzyme inhibition by several components of wine and must (glucose, fructose and terpenols) were studied. Optimum immobilization conditions were: temperature 25 degrees C, pH between 5.5 and 6.0, polymeric support dimension in the range 38-75 mu m, cross-linking time 30 min, glutaraldehyde concentration 0.5-1.0% w/v, 1 g of chitosan per 1000 units of beta-glucosidase. The immobilized enzyme retained 29% of the wet biocatalyst activity when freeze-dried and showed good stability (half-life roughly 2 years) when stored at 4 degrees C. Kinetics were tested at 25 degrees C following the hydrolysis of beta-nitrophenyl beta-D glucopyranoside and obey the Michaelis-Menten rate equation. K-m = 1.3 mM and the activation energy, 62.84 kJ mol(-1), are close to those of the free enzyme. The operational half-life was roughly 500 h. Glucose only depressed the enzyme activity according to a reversible non-competitive inhibition mechanism with K-i = 11.2 mM.