Synthesis, characterization and kinetic analysis of chitosan coated magnetic nanobiocatalyst and its application on glucose oleate ester synthesis

Abstract

In this work, we report the covalent immobilization of lipase enzyme on the surface of chitosan coated magnetic nanoparticles (MNPs) using N-(3-dimethylaminoproyl)-N-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) as coupling agents. Surface functional groups of chitosan coated MNPs, lipase enzyme, and immobilized lipase enzyme were characterized by Fourier transform infrared spectroscopic (FT-IR) analysis. The structural characteristics of the chitosan coated magnetic nanoparticles were analyzed by X-ray Diffraction (XRD) studies. The statistical methodology with central composite design (CCD) was applied to evaluate the effects of immobilization, including the magnetic nanoparticles, lipase enzyme, pH and immobilization time on the enzyme activity. Based on the statistical analysis, the optimum immobilization conditions of magnetic nanoparticles (40mg), lipase enzyme (40mg), pH (9), and immobilization time (6h), the maximum enzyme activity obtained was 16.94U/mL. The optimum pH and temperature for maximum enzyme activity of immobilized lipase are found to be pH 9 and 40°C respectively. The immobilized lipase exhibited excellent catalytic activity over eight successive cycles and retain 64% original stability. The Michaelis–Menten enzyme kinetic studies of immobilized lipase on chitosan coated MNPs showed maximum activity (Vmax) and Michaelis–Menten constant (Km) of 33.7 (μmol/mLmin) and 0.89mM respectively. The thermal stability of lipase was significantly improved after immobilization. The thermal deactivation rate of immobilized lipase was studied to follow the Arrhenius law with deactivation energy of 73kJ/mol. Glucose ester yield of 80.8% was achieved in this study revealed that immobilized lipase on MNPs have promising application in industrial scale for sugar ester production.