Immobilization and Kinetic Properties of ß-N-Acetylhexosaminidase from Penicillium oxalicum

The application of immobilized enzymes often plays a key role in successfully implementing an economically feasible biocatalytic process at an industrial scale. Designing an immobilized biocatalyst involves solving several tasks, from the selection of the carrier and immobilization method to the characterization of the kinetic properties of the immobilized enzyme. In this study, we focused on the kinetic properties of free and immobilized ß-N-acetylhexosaminidase (Hex), a promising enzyme for application in the field of biotechnology, especially for the synthesis of bioactive carbohydrates. Hex was immobilized via covalent binding in methacrylate particles. The effect of immobilizing Hex from Penicillium oxalicum into porous particles on kinetic properties was investigated, and mathematical and experimental modeling showed that the kinetic behavior of the enzyme was significantly influenced by diffusion in the particles. Along with the study on kinetics, a simple method was developed to investigate the reversible inhibition of the immobilized enzyme in a continuous-flow system. The method is suitable for application in cases where a chromogenic substrate is used, and here it was applied to demonstrate the inhibitory effects of N-acetyl-glucosaminyl thiazoline (NAG-thiazoline) and O-(2-Acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenyl carbamate ((Z)-PugNAc) on Hex.