Application of Fe3O4−chitosan nanoparticles for Mucor racemosus NRRL 3631 lipase immobilization

Abstract

Introduction and purpose Magnetic Fe 3 O 4 -chitosan (CS) nanoparticles are prepared by the coagulation of an aqueous solution of CS with Fe 3 O 4 nanoparticles. Various factors such as time, elevated pH and temperatures affecting magnetic Fe 3 O 4 -CS nanoparticles were studied using the immobilization technology. To improve the efficient use of lipase and reduce the cost of production, enzyme immobilization technology is applied. Materials and methods Fe 3 O 4 nanoparticles were prepared by the coprecipitation method. The CS solution was prepared by dissolving 0.5 g of CS in 50 ml of 1% (v/v) acetic acid, followed by the addition of 12.5 ml of 1 mg/ml Tripolyphosphate (Tpp) solution as a cross-linker to enhance colloidal stability. The solution was used to resuspend the magnetic Fe 3 O 4 nanoparticles. The resulting Fe 3 O 4 -CS was stored at 4°C until use. Uncoated and coated magnetite nanoparticles for immobilizing the lipase were characterized according to the particle size, as measured by atomic force microscopy or scanning force microscopy using contact mode by means of Agilent 5500. The infrared spectra were detected by a Fourier-transform infrared spectrophotometer. Results and conclusion To solve leaching problems of the adsorbed lipase and improve the conventional method of lipase immobilization, different concentrations of CS were used. A concentration of CS nanoparticles of 0.3 mg was proved to be more suitable, with an immobilization efficiency of 95.6%. On the basis of atomic force microscopy three-dimensional images, the diameters of the uncoated magnetite particles were determined to be 12 nm. The immobilized lipase shows better operational stability, including wider pH and thermal ranges. The pH optimum of the immobilized enzyme exhibited an acidic shift (pH 5-6). The immobilized lipase was stable in the pH range from 3 to 5 as compared with free lipase. Lipase immobilized by Fe 3 O 4 -CS nanoparticles remained fully active up to 40°C. At a temperature of 60°C, free lipase preserved about 40% of its residual activity for 1 h; however, the immobilized enzyme preserved about 72.9% of its residual activity at the same time. The kinetic constants V max and K m were determined to be 250 U/mg protein and 20 mmol/l, respectively, for immobilized lipase. The resulting immobilized lipase had better resistance to pH and temperature inactivation compared with free lipase and exhibited good reusability; after eight repeated uses, the immobilized lipase retained over 63.5% of its original activity.