Chitosan membrane-immobilized urease. Kinetic behavior in phosphate bufferin the pH range 5.76-8.19

The effect of phosphate buffer on the kinetic behavior of jack bean urease covalently immobilized on chitosan membrane was studied in the pH range 5.7 6-8.19, and compared with that of the free enzyme in an attempt to elucidat e the effects of heterogeneity of the system on its kinetics. The chemical inhibition by the buffer, occurring between pH 5.76 and 7.50, was found to consist of two antagonistic effects: a decrease in the intrinsic enzyme act ivity and a reduction in the degree of environment-related inhibition. The apparent kinetic constants of the immobilized urease: upsilon(max)(x), K-M( x) and K-i,K-buffer exhibited both pH-constants and buffer concentration-de pendence, anomalous as compared to the free enzyme: the optimum pH and the PKi,buffer values were displaced toward more acidic pH values, and the pK(M )(x) values were leveled off. The anomalies were gradually suppressed by in creasing the buffer concentration. The anomalous behavior of chitosan membr ane-immobilized urease was accounted for by a combined effect of: a) the in crease in local pH on the membrane produced by both the enzymatic reaction and the electric charge of the support, and b) diffusional limitations impo sed on substrate and product in the external solution.