Zinc oxide nanoparticles-impregnated chitosan surfaces for covalent immobilization of trypsin: Stability & kinetic studies

Trypsin (Try, EC. 3.4.21.4) was effectively immobilized on the surface of glutaraldehyde(GA)-activated ZnO/Chitosan nanocomposite through covalent attachment via Schiff-base linkages. Size, structure, surface morphology, & percentage elemental composition of the prepared ZnO nanoparticles and chitosan-coated ZnO nanocomposite were studied by UV-Visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), X-Ray diffraction analysis (XRD), transmission electron microscopy (TEM), Scanning electron microscopy (SEM), and Energy-Dispersive X-Ray Microanalysis (EDAX) techniques. Optimal immobilization conditions (incubation time (16 h), enzyme concentration (1.8 mg/ml), and pH (7.8)) were investigated to obtain the maximum expressed activity of the immobilized trypsin. Immobilized & solubilized trypsin exhibited the optimum catalytic activity at pH 8.5, 60 °C, and pH 7.8, 45 °C respectively. Kinetic parameters (Km, Vmax) of immobilized (27.12 μM, 8.82 μM/min) & free trypsin (25.76 μM, 4.16 μM/min) were determined, indicating that efficiency of trypsin improves after immobilization. Immobilized trypsin preserved 67% of initial activity at 50 °C during 2 h of incubation & sustained nearly 50% of catalytic activity until the 9th repeated cycle of utilization. Moreover, immobilized trypsin retained 50% of enzymatic activity after 90 days of storage at 4 °C. Hence, the current findings suggest that ZnO/Chitosan-GA-Trypsin would be a promising biocatalyst for large-scale biotechnological applications.