Study On The Modification Of Acetyl Cellulose And Lipase Immobilization

Enzyme as a biological catalyst is extremely sensitive to the environment and vulnerable to be inactivated for physical, chemical and biological factors.What's more,it mixes with the product after the reaction.For this reason,it is difficult to purify and operate continuously.All these problems astrict the enzyme catalytic reaction in the industrial application. In such conditions, the enzyme immobilization techniques proposed and developed.Lipase is a term for a class of enzymes which are catalyzed to hydrolyze grease. According to classification principle of the International Enzyme Nomenclature Committee, the system ID: EC3.1.1.3 and the System Name: Three lipid triacylglycerol acyl hydrolase.Lipase whose basic function is to catalyze natural substrate hydrolysis to glycerol and fatty acids is a special ester hydrolase.It can catalyze many organic synthesis reactions in oil-water interface such as, ester hydrolysis or alcoholysis, ester synthesis, ester exchange, lactone synthesis, high Oligomer Synthesis and so on.It is an very important enzyme catalyst being studyed now.In this study, the raw material CA was modified by amino-silane coupling agent KH-792.In this way the surface of CA grafted amino group. And then the lipase was cross-linked by glutaraldehyde. This method is simple and fresh. The main content as follows:First,we study on the free basic properties of lipase initially, including the free enzyme optimum pH, optimum reaction temperature, storage stability, and Michaelis constant(K_m) value. In this part,we established the method to measure protein content with Coomassie brilliant blue and titrated with NaOH to analyse fatty acids with phenolphthalein as indicator.We determined the best reaction conditions of free lipase-catalyzed hydrolysis of olive by experiment.The optimum temperature is 40℃.The optimum pH value: 7.0. The Michaelis of free lipase constant is 7.68mol·L^-1.Secondly,in order to make it a good enzyme immobilization carrier materials the modification of acetyl cellulose (CA) with amino-silane coupling agent KH-792 was studied in the article. Secondly. We mainly studyed the modification time, coupling agent concentration on the lipase immobilization and the activity yield.Infrared spectroscopy proves that the silane agent was grafted on the CA surface by chemical bonds and the elemental analysis gave the amount of amino on the CA surface. At the same time, the study of immobilizing enzyme on the modified CA was done. The most anount of immobilized Lipase enzyme and the relative activity are 25.8mg·g^-1 and 43.3 percent when the modified time is 24h and silane concentration is 6%.Thirdly,we use the modified CA as a carrier material to cross-link lipase.Glutaraldehyde concentration, activation time, crosslinking time, enzyme volume were studyed to optimize the immobilization conditions.Then experiment test the enzymatic properties of immobilized enzyme. The results show the optimal conditions are as follows:0.1g modified CA,5%glutaraldehyde,activation 8h,4mL concentration of 1.64 mg·mL^-1 of lipase solution (dissolved in pH = 7.0 phosphate buffer solution),at 4℃crosslink 10h.In this way immobilization of enzyme efficiency is the best. the greatest amount of enzyme immobilization is 34.5 mg·g^-1.Immobilized lipase on modified CA has better performance then the free lipase in the follow aspects: reaction temperature range and pH range, thermal stability and storage stability and so on. The immobilized lipase still retain 52.05% activity after after 8 successive batch reactions.The enzyme activity has not declined obviously for 12day(stored at 4℃).Finally,we study the application of immobilized lipase in membrane bioreactor. A simple membrane bioreactor which mainly includes the membrane and process selection has been designed.And it is used to hydrolyze olive oil.