Preparation Of Novel Magnetic Polymer Carrier,lipase Immobilization And Their Application On Chiral Separation Of Ketoprofen

This dissertation reviews the studies of enzyme immobilization technologies, novel immobilization materials of today and application of lipase and immobilized lipase on chiral separation of 2-Arylpropionic Acid. Novel magnetic polymer composites were synthesized via soap-free seeded emulsion polymerization and DPE controlled radical polymerization, respectively. After that, these novel magnetic polymer composites were used as carriers for lipase immobilization. Then free lipase and the synthesized immobilized lipases were used as catalyst to catalyze the asymmetric hydrolysis of ketoprofen ester.1,Fe3O4 nanoparticles were synthesized by the chemical co-precipitation method, and the magnetic nanoparticles were chemically modified by oleic acid and y-aminopropyltriethoxysilane (APTS).2,Superparamagnetic composites based on polymer colloids were prepared by seeded emulsion. Firstly, styrene (St), glycidyl methacrylate (GMA) and 2-hydroxyethyl methacrylate (HEMA) were copolymerized by soap-free seeded emulsion polymerization, and then APTS-Fe3O4 nanoparticles were introduced into above copolymer emulsion by covalent bonding or physically absorption. Secondly, butyl methacrylate (BMA), vinyl acetate (VAc) and ethylne glycol dimethacrylate (EGDMA) were added drop wise to the the superparamagnetic seed latexes without addition of any emulsifier. The results showed that the magnetic composites of polymer colloids were successfully synthesized and had a structure with a thinner shell (around 100 nm) and a bigger cavity (around 200 nm) and also possessed a certain level of magnetic response (4.16emu/g carrier). Candida rugosa lipases (CRL) were covalently immobilized or physically absorbed on the magnetic microspheres. The immobilized lipase could be separated, recovered and reused easily and rapidly. The immobilization conditions were optimized; the properties of immobilized lipase (such as themal stability, reusability and kinetic properties) prepared by the two methods were compared. The results indicated that immobilized lipase prepared by covalent bonding held better themal stability and reusability than ICRL prepared by physically absorption.3,OA-Fe3O4 nanoparticles were introduced into 1,1-Diphenylethylene (DPE) controlled radical polymerization system to prepare superparamagnetic microspheres for enzyme immobilization by two steps of polymerization. In the presence of DPE, glycidyl methacrylate (GMA) and 2-hydroxyethyl methacrylate (HEMA), methacryloxyethyl trimethyl ammonium chloride (MATAC) with charge were selected as copolymering monomers based on their reactive functional group and excellent biocompatibility which were suitable for immobilization of Candida rugosa lipase (CRL). SDS-PAGE analysis was also conducted to demonstrate whether CRL is covalently immobilized or only physically adsorbed. The results indicated that the polymerization was successfully carried out, and lipase was immobilized on the magnetic microspheres through ionically adsorption and covalent binding under mild conditions. The immobilized lipase exhibited high activity recovery (69.7%), better resistance to pH and temperature inactivation in aqueous phase, as well as superior reusability in nonaqueous phase. The data showed that the resulting carrier could hold an amphiphilic property.4,The methyl ester, ethyl ester, propyl ester, isopropyl ester and butyl ester of ketoprofen were synthesized. Candida rugosa lipase was used as catalyst to catalyze the asymmetric hydrolysis reaction of ketoprofen esters. The reaction conditions (such as structure of substrate, pH, addition of DMSO and reaction time) were optimized. The obtained products were analyzed by HPLC. The results showed that the conversion% of substrate and eep value of products will increase with the addition of DMSO.5,Candida rugosa lipase was immobilized on magnetic polymer colloids and P(GMA-b-HEMA). Then the immobilized CRL were used to catalyze asymmetric hydrolysis reaction of ketoprofen esters. The effects of the two immobilized CRL on the conversion% of substrate and eep value of products were compared. The reaction conditions (such as structure of substrate, pH, addition of DMSO and reaction time) were optimized. The reusability of immobilized CRL was also investigated.