Construction And Application Of Gigaporous Polymeric Microspheres Immobilized Enzyme

Immobilized enzyme is an important part of research and application of enzyme. Porous microspheres are fine carriers for enzyme immobilization because of their advantage of high specific surface area, controllable hydrophilicity/hydrophobicity and functional groups. In the meantime, frequently-used mesoporous microspheres might limit the catalytic performance of enzyme due to the high mass transfer resistance in the small pores. In the previous studies of our laboratory, the gigaporous polystyrene (PST) microspheres were used to immobilize lipase and the activity, reusability, stability and kinetic properties of immobilized lipase were superior to those of mesoporous ones. In this study, the reasons of advantages of gigaporous microspheres as immobilization carriers were analyzed as well as the mechanism of pore size influencing enzyme immobilization. The gigaporous microspheres with different surface groups as lipase immobilization carriers were researched. Lipase immobilized in gigaporous microspheres was applied in synthesis of esters. The matching relations between pore sizes of carriers and molecular sizes of enzymes were initially investigated.There are mainly four sections in this dissertation.(1) The differences of mesoporous particles, gigaporous particles and a flat surface for enzyme immobilization were further investigated. The deep reasons of the advantages of gigaporous microspheres were analyzed. By regulating the adsorption time and the enzyme concentration, it can be concluded that enzyme adsorbed in gigaporous microspheres can attain higher activity and specific activity due to the low pore curvature, large pore space, the multilayer adsorption of enzyme and better mass transfer effects. When lipase adsorbed on a flat surface, the activity continued to grow and the specific activity had little change during the whole process without the limitation of pore structrue. In addition, due to the accumulation of the substrate, the activity of enzyme will decrease with substrate of high concentration.(2) The gigaporous PGMA, P(ST-co-GMA), PST microspheres were used to be the carriers of lipase, which were compared with commercial immobilized lipase Novozym 435. Lipase immobilized in PGMA carriers showed the highest activity, the best reusability and stability. The influences of covalent binding and the hydrophobicity of carriers on the performance of immobilized lipase were investigated. Through the characterization of adsorption curve, the determination of density of epoxy groups, the contact angles measurement and the simple physical adsorption, it can be concluded that the advantages of PGMA microspheres were mainly due to the covalent binding between lipase and the carriers other than the relatively weaker hydrophobicity.(3) The synthesis of isooctyl palmitate catalyzed by lipase immobilized in gigaporous microspheres was investigated. Under the optimal reaction conditions, lipase in PGMA carriers can attain the highest esterification rate, which was 91.3%. And after ten-time reuse, the esterification rate was still 87.1%. The inactive immobilized lipase by reusing can be partially renatured by adding PBS. The activity of inactive PGMA-lipase can be renewed to over 80% of the initial activity.(4) Several kinds of enzymes with different molecular sizes were immobilized on meso-and giga-porous microspheres which were hydrophilic or hydrophobic. The selection of immobilized enzyme carrier is complicated. The pore size, hydrophobic and hydrophilic performance, immobilization method and mass transfer of substrate should be comprehensively considered. Carriers with large pore size are beneficial to the enzyme immobilization. The hydrophilic or hydrophobic carriers are selected according to the properties of enzyme and substrate. Covalent binding can improve the activity and the stability of certain enzymes. The relationship between molecular size of enzyme and pore size of carriers can be attained, which is not positive correlation. In addition, the influence of protein loading and the mass transfer request of substratre and product also should be considered.In a word, gigaporous microspheres as enzyme immobilization carriers have great advantages due to the structure, the good mass transfer performance and the multilayer adsorption of enzyme. Lipase immobilization in gigaporous microspheres combined with covalent binding can improve its activity and stability. High catalytic efficiency, good stability and reusability can be attained as lipase immobilized in gigaporous carriers.They were appropriate to immobilize various kinds of enzyme with different molecular sizes by regulating the microspheres, which can be ideal enzyme immobilization carriers.