Preparation Of Amino Group Coated Magnetic Microspheres And Its Application Study On Immobilizing Papain

Magnetic microsphres are one of the most promising complex materials, which are often composed of Fe₃O₄ core and polymers shell. These kinds of particles are not magnetic, but also with properties like small particle size, high couple capacity, fast movement and long-time suspension etc, which make it an important tool in both biology research and other fields. In this paper, amino-group modified magnetic microsphres were prepared and well characterized. The as-synthesized particles were used for immobilization of papain and then the immobilized papain was used for quality improvement study of beer.1. Preparation of oleic oil coated magnetic nano Fe₃O₄The superparamagnetic nano Fe₃O₄ with mean diameter of 5-10nm was prepared by chemical co-precipitation method. The magnetic Fe₃O₄ has cubic syngony structure and saturation magnetization of 55emu/g. The remanence and coercivity approximate to 0. This method was simple, fast and the particle size of product was comparatively uniform. The oleic oil coated Fe₃O₄ could be used as the core of magnetic microspheres.2. Synthesis of amino group modified magnetic microspheresEpoxy group coated Magnetic microspheres were synthesized by dispersion polymerization. The particle diameter is approximately 40nm and saturation magnetization was 50emu/g. Amino group coated magnetic microspheres are attained by means of ammonification treatment, the functional amino groups were quantitatively determined by linear potentiometric titration method. The result showed that the the concentration of amino groups was about 486μmol/g.3. Immobilization of papain on amino-group functionalized magnetic microspheres and its applicationThe amino group coated magnetic microspheres were used to immobilize papain. The reaction condition for the immobilization of enzyme was optimized and the similarities and differences of both immobilized enzyme and free enzyme were investigated, either. The result showed that, the optimum condition for immobilization of papain were enzyme concentration 1.2mg/ml, glutaraldehyde concentration 5%, reaction time 4 hours, respectively; the optimal reaction condition for immobilized enzyme and free enzyme was pH 6.8 and 7.2, reaction temperature 77℃and 87℃, respectively. The Substrate binding capability of immobilized enzyme is 7 times higher than that of free enzyme. Compared with incipient enzyme activity, the enzyme activity of immobilized and free enzyme maintained 98% and 91% respectively after storage in refrigerator at 4℃for 30 days. The surplus enzyme activity of immobilized enzyme was 97% of incipient enzyme activity after resused for 5 times.In order to elevate the quality of beer such as color and clarity, immobilized papain was applied to hydrolyze proteins in beer. It turned out that, the turbidity of beer decreased by 0.013 light absorption units, and the amount of free amino acids raised in different degree. The results demonstrated the feasibility of magnetic microspheres based papain on hydrolyzation of proteins in beer. This method was not only fast and efficient, but facilitates the separation of enzyme from beer.