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

Abstract

A microreactor by immobilized trypsin on the activated glycidyl methacrylate-modified cellulose membrane packed column was constructed. Immobilized trypsin mirrored the properties of the free enzyme and showed high stability. A novel method to characterize the activity and reaction kinetics of the immobilized enzyme has been developed based on the frontal analysis of enzymatic reaction products, which was performed by the on-line monitoring of the absorption at 410 nm of p-nitroaniline from the hydrolysis of N-α-benzoyl- dl-arginine- p-nitroanilide (BAPNA). The hydrolytic activity of the immobilized enzyme was 55.6% of free trypsin. The apparent Michaelis–Menten kinetics constant ( K m) and V max values measured by the frontal analysis method were, respectively, 0.12 m M and 0.079 m M min −1 mg enzyme −1. The former is very close to that observed by the static and off-line detection methods, but the latter is about 15% higher than that of the static method. Inhibition of the immobilized trypsin by addition of benzamidine into substrate solution has been studied by the frontal analysis method. The apparent Michaelis–Menten constant of BAPNA ( K m), the inhibition constant of benzamidine ( K i) and V max were determined. It was indicated that the interaction of BAPNA and benzamidine with trypsin is competitive, the K m value was affected but the V max was unaffected by the benzamidine concentration.