Determination of kinetic parameters of pectolytic enzymes at low pectin concentrations by a simple method

A simple and sensitive method for determination of the pectolytic enzyme activity was improved for soluble and immobilized forms of the enzyme. During enzymatic hydrolyzation of pectin, samples were collected from substrate solution at certain time intervals. Pectin in the samples was precipitated by alcohol and then pectin concentration was determined by measuring the refractive index of a prepared aqueous solution of these precipitates. The validity of this technique was evaluated using the kinetic behavior of soluble and immobilized enzymes. The kinetics of free and Duolite A568-immobilized pectinase was investigated. Michaelis-Menten constants and maximal reaction rates were found as K-m=20.71 g l(-1) and V-max=81.30 g l(-1) s(-1) for free enzyme and K-m=1.02 g l(-1) and V-max=0.035 g s(-1) g(-1) particles for immobilized pectinase at 20 degreesC.