Preparation Of SiO₂ Microspheres And Metal Chelate Microspheres And Application In Immobilized Papain

The immobilized enzymes have more stability, more use times and easier separation from substrate and product than enzymes in bulk solution. But current immobilization methods have many disadvantages, such as much enzyme activity lose after immobilization and high cost. So only a few immobilized enzymes have been used in industry widely though enzyme immobilization have been studied for about thirty years. In order to resolve these disadvantages, new enzyme carriers and immobilization methods need to be explored. In this paper we put forward some solutions and synthesized several kinds of new enzyme carriers.We choose papain as immobilized object, and investigate performance of these carriers used as papain carrier. Papain, a thiol protease, have great industrial and medicinal potential, and is well characterized kinetically and structurally being a suitable model to compare the efficiency of various immobilization procedures and methods.Among present enzyme carriers inorganic materials have lower cost and more stability than organic materials. But the present inorganic materials contain less active sites, and have to undergo some complicated processes before immobilizing enzyme. SiO2 particles containing amine groups were synthesized by synchronous hydrolysis of tetraethylorthosilicate(TEOS) and N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAPS) in W/O Microemulsion of TritonX-100/cyclohexane/ammonium hydroxide system. The obtained particles have a narrow diameter range, and its size can be controlled by adjusting water to surfactant molar ratio and water to TEOS and AEAPS molar ratio. Comparing with traditional porous silica beads, these panicles contain much more amine groups and its aminegroup content can be easily changed when synthesizing. After treated by glutaraldehyde, papain was immobilized on the particles by covalent method. The optimum immobilization conditions of enzyme were as follows: enzyme load was 15mg/g carrier, pH was 6.5.The pH and temperature optima were 6.5 and 70 C for immobilization enzyme. All above indicate this kind of particle can be a good enzyme immobilization carrier.The reverse phase suspension and embedment technique were adopted to prepare magnetic agar microsphere. The dispersion medium was mixture of some organic solvents. Span-80 was used as stabilizer. The magnetic agar microsphere was coupled with epichlorohydrin, then treated with iminodiacetic acid, and magnetic metal chelate carrier was prepared. After the carrier was treated with CuCl2 or NiCl2 papain was immobilized on this carrier. Papain can bind on this carrier tightly, and papain can be washed from the carrier when papain loses activity after being used many times. The carrier can be regenerate by treated with CuCl2 or NiCl2, and can absorb papain again. The enzyme immobilized on the carrier which was used three times have 90% activity of the first time. So this microsphere can be as a good regenerable carrier. The result indicates metal chelate carrier will be a promising enzyme carrier.