On-line characterization of the activity and reaction kinetics of immobilized enzyme by high-performance frontal analysis

A microreactor by immobilized trypsin on the activated glycidyl methacrylat e-modified cellulose membrane packed column was constructed, Immobilized tr ypsin mirrored the properties of the free enzyme and showed high stability. A novel method to characterize the activity and reaction kinetics of the i mmobilized enzyme has been developed based on the frontal analysis of enzym atic reaction products, which was performed by the on-line monitoring of th e absorption at 410 nm of p-nitroaniline from the hydrolysis of N-alpha -be nzoyl-DL-arginine-p-nitroanilide (BAPNA). The hydrolytic activity of the im mobilized enzyme was 55.6% of free trypsin. The apparent Michaelis-Menten k inetics constant (K-m) and V-max values measured by the frontal analysis me thod were, respectively, 0.12 mM and 0.079 mM min(-1) mg enzyme(-1). The fo rmer is very close to that observed by the static and off-line detection me thods, but the latter is about 15% higher than that of the static method. I nhibition of the immobilized trypsin by addition of benzamidine into substr ate solution has been studied by the frontal analysis method. The apparent Michaelis-Menten constant of BAPNA (K-m), the inhibition constant of benzam idine (K-m) and V-max were determined. It was indicated that the interactio n of BAPNA and benzamidine with trypsin is competitive, the K-m value was a ffected but the V-max was unaffected by the benzamidine concentration. (C) 2000 Elsevier Science B.V. All rights reserved.