Immobilization of trypsin onto Fe3O4@SiO2 -NH2 and study of its activity and stability

Abstract

The preparation of biocatalysts based on immobilized trypsin is of great importance for proteomic research, industrial applications and organic synthesis. Here in, we have developed a facile method to immobilize trypsin on magnetic nanoparticles. Fe₃O₄ nanoparticles were synthesized by co-precipitating Fe²⁺and Fe³⁺in an ammonia solution and then coated by silicon dioxides were developed by sol-gel method. The silica-coated Fe₃O₄ nanoparticles were further modified with 3-aminopropyltriethoxysilane, resulting in attaching of primary amine groups on the surface of the particles. Trypsin from porcine pancrease was then immobilized on the magnetic core-shell particles by using glutaraldehyde as a cross-linker. The synthesis steps and characterizations of immobilized trypsin were examined by FT-IR, XRD, TGA, EDX and SEM. The results showed that the enzyme immobilization increased the enzyme activity in different pHs and temperatures, without any changes in the optimum pH and temperature for enzyme activity. The Kinetic results showed that the enzyme immobilization decreased and increased V_max and K_m values, respectively. The stability results showed that the enzyme immobilization improved trypsin thermostability in the absence and presence of 10% (v/v) of the used solvents (DMF, THF, DMSO, ACN and 1, 4-Dioxane). The reusability results indicated that the immobilized enzyme maintained 85% of its activity after 6 periods of activity.

Keywords: Activity; Immobilization; Magnetic nanoparticles; Stability; Trypsin.