Immobilization Of Lipase And Its Application In Catalytic Synthesis

Lipase is one of the most widely used enzymes in the chemical, pharmaceutical, food, energy and many other fields that catalyzes the hydrolysis of the ester, ester synthesis, transesterification, peptide synthesis reactions, to become the research focus of industrial enzymes. As for the free enzyme in organic solvents easily gathering into a group, the catalytic efficiency being low, and the poor stability, in this study, with catalytic synthesis of citronellyl acetate as an object, preparing high catalytic activity and high stability of the immobilized lipase as the goal, the influence of the catalytic properties of the enzyme was investigated from two aspects of immobilization conditions including materials and methods, and immobilized enzyme at different process conditions on the preparation of the impact citronellyl acetate, as well as the influence on the catalytic synthesis of citronellyl acetate at different process conditions with immobilized enzyme.(1) This thesis choosed lipase as the research object, using the entrapment of lipase in sol-gels. Experimental results show that the specific activity of immobilized lipase has been significantly improved than free enzyme, and ultimately determine the immobilized enzyme conditions: With ratio of1:1r-methacryloxypropyl trimethoxysilane (MAPTMS) and tetramethoxysilane (TMOS) as the silicon source, the amount of enzyme is3mg/mL, the molar ratio R of water and the silicon source is20, the pH of50mM of phosphate buffer solution is7.0, the hydrolysis time is30min; At this point, the immobilized enzyme has the highest specific activity,9.1times for the free enzyme. The study for esterification with citronellol as a model reaction investigated the stability of the enzyme. After stored at room temperature after56days, immobilized enzyme retained92%of original, while free enzyme activity only31%; Reused seven times, the activity of immobilized enzyme remained only31%.(2) Use o-phenylenediamine as monomers to prepare poly-o-phenylenediamine of hollow and solid particles, which were used as a novel support for lipase immobilization by adsorption and covalent attachment. It was shown that its specific activity had been significantly improved4.9fold than free enzyme with hollow microsphere-based poly-o-phenylenediamine as immobilization support, and the enzyme ratio of2:5to the support, with phosphate buffer in pH being7.0, and concentration being50mM, the adsorption,time of4h. In addition, this study also immobilized enzyme with covalent attachment to support modified through glutaraldehyde. When the hollow microsphere-based poly-o-phenylenediamine was modified with2%glutaraldehyde in2h,40℃with phosphate buffer in pH7.0,50mM, it was shown that its specific activity has been improved5.3fold. The study for esterification with citronellol as a model reaction investigated the stability of the enzyme. Stored at room temperature after56days, immobilized enzyme retained60%of original; After reused seven times, immobilized enzyme by covalent attachment method residues was95%.(3) With immobilized lipase for the study, research the impact of immobilized enzyme at different process conditions on the catalytic synthesis of citronellyl acetate. The results showed that the best optimizing conditions with immobilized enzyme produced by entrapment: the amount of immobilized enzyme was O.lg/mL, reaction temperature was40℃, the shaker speed was200rpm. While the best optimizing conditions with immobilized enzyme produced by covalent attachment: the amount of immobilized enzyme was0.1g/mL, reaction temperature was50℃, the shaker speed was200rpm. Meanwhile, the results showed that when the immobilized enzyme with both the covalent attachment and sol-gel entrapment catalyzed synthesis of citronellyl acetate, changing the substrate concentration and product concentrations, the reaction did not appear significant inhibition.