Study On Design And Preparation Of Novel Carriers Employed In Immobilization Of Porcine Pancreas Lipase

The thesis summarizes the progress of methods, carriers, application of immobilization enzyme, and the design and synthesis of novel reactive carrier materials, also includes study of the optimal conditions of immobilization enzyme and the properties of the immobilized enzyme.The experimental content which includes preparation, analysis of carriers and immobilization enzymes were all studied in this thesis. The main content for synthesis carriers is list below.1. Firstly, Hexagonal mesoporous silica (MCM-41) was synthesized via a silicate salt route in the presence of Cetyltrimethylammonium bromide (CTABr) as the structure directing agent at room temperature and normal pressure. Study the process of preparation and the influencing factors to the properties and structure of the materials detailedly. The material was functionalized by three kinds of method and employed as the carriers with reactive groups. The three functionalized supports since containt excellent reactivity groups will be covalent easily to amino groups on porcine pancreas lipase (PPL) in mild conditions.2. Secondly, through the design of molecule and structure, micron-sized magnetic poly(glycidyl methacrylate-divinylbenzene-methacrylic) (PGMA-DVB-MAA) micro-spheres with epoxy and carboxyl groups on the surface were prepared by a suspension polymerization method in the presence of oleic acid-coated magnetic Fe₃O₄ nan-oparticles. The relationship of properties and structure of the copolymer carriers were studied detailedly. The properties of the magnetic polymer microspheres were charactered by SEM, IR spectroscopy, TG, X-RD.The main content for study of immobilization enzyme is list below. 1. Three kinds of novel carriers with active reaction groups were firstly prepareded by functionalization of hexagonal mesoporous silica and secondly employed in the immobilization of porcine pancreas lipase. The maximal activity recovery (activity of immobilized yield) of the immobilized PPLs from functionalized carriers HMS-C, HMS-A, and HMS-G was 74.59%, 57.30%, and 44.04%, respectively, when the enzyme-adding amount was 1.10 ml[5mg/ml] with 60 mg of the carriers. The maximum value of relative activity was observed at pH 7.0 for free enzyme, where as the optimum pH was at7.5 for the immobilized PPLs. In other words, the resulting immobilized PPLs hold excellent adaptability in a wider and higher pH comparing with free enzyme. It was observed that the immobilized PPLs were still retained 35% of their original activities after the 10th reuse. This result further confirmed the three immobilized PPLs all have a good stability. It is worthwhile that the HMS-G (46% of the original activities at the 10th reuse) has the best reusability in them.2. Study the optimal conditions of immobilization PPL and the properties of the immobilized PPL after using magnetic polymer composite microsphere as carriers, and the results indicated that the structure and properties decide the activity and stability. As epoxy and carboxyl groups on the surface of the magnetic polymer composite microsphere, these carriers need not to be activised beforehand, in other hand, the magnetic microspheres show a superparamagnetic property, there are easy to be separate after reaction.3. Magnetic polymer composite microsphere immobilizaed PPL catalyze synthesis hexyl acetate by esterification between acetic acid and normal hexanol in organic solvents was studied, and the optimum condition for the esterification was investigated. The result indicated that using the immobilized PPL as catalyzer, the reaction is easy operated at mild condition, and the esterification conversion rate is high. The product is also easy to be separate. So this method has potential industry application value.The covalent immobilization enzymes are accomplished in mild condition after using hexagonal mesoporous silica (MCM-41) and magnetic polymer composite microsphere. The process of immobilization enzymes and the enzymatic reaction were all studied in this thesis. It is the novelty after study the synthesis hexyl acetate by esterification between acetic acid and normal hexanol in organic solvents by using the magnetic polymer composite microsphere immobilized lipase.