| Lipases are a kind of ester bond hydrolase.They have been widely used in food industry,biosensor and preparation of chiral chemical compounds because of their specific characteristic.They can catalyze the hydrolysis of triglyceride to diglyceride,glycerol and fatty acids at oil-water interface.In most cases,enzymes are preferably in their immobilized states owning to a lot of advantages,such as improved stability,ready reutilization of the catalyst and possibility of continuous operation.Therefore,enzyme immobilization techniques have always been one of the hot research issues.In this thesis,chitosan microspheres were prepared and modified.Lipase was immobilized onto modified chitosan microspheres.The conditions for the immobilization of lipase were optimized by single factor experiments.Furthermore,the application of immobilized lipase in the enrichment of docosahexenoic acid and eicosapentaenoic acid was also investigated.The main results obtained in this work are as follows:1.Neutralization sedimentation method was employed to prepare chitosan microspheres,which then underwent the acylation with succinic anhydride,providing N-Succinyl-Chitosan microspheres.The results showed that the concentration of chitosan has a great effect on the formation of chitosan microspheres,and the concentration of NaOH has less effect.Fourier Transform Infrared spectra confirmed the conjugation of chitosan with succinic anhydride,generating N-succinylchitosan.When the reaction temperature was 60 0C,the reaction time was 12h and the addition amount of succinic anhydride was 2.5 g/g chitosan,the substitution degree of chitosan was determined to be 48.2%.2.Lipase was immobilized onto the modified chitosan microspheres using EDC/NHS as the activating agents.The conditions for the immobilization of enzyme were explored.The properties of the immobilized enzyme were also investigated as compared with the free enzyme.The results indicated that the optimal immobilized conditions as follows:lipase loading 60 mg per gram carrier,reaction time for 3h,temperature 40 0C,pH 7.5.The immobilized enzyme obtained under these conditions possessed a enzyme loading of 28 mg/g carrier,enzyme activity of 1480 u/g carrier;and the enzyme activity recovery is 74.5%.The stabilities of immobilized lipase against temperature,acid and base have been greatly improved as compared with those of free lipase.The immobilized lipase was found to remain 76.6%activity after it was reused for 5 times,indicating a good operational stability.The immobilized enzyme was also found to possess a high storage stability,remaining more than 80%of the initial enzyme activity at low temperature after five days,whereas the free enzyme lost more than half activity under the same conditions.3.The immobilized lipase was used to hydrolyze the crude fish oil to enrich polyunsaturated fatty acids including docosahexenoic acid and eicosapentaenoic acid.The hydrolysis conditions were investigated.The components,the physical and chemical properties of fish oil before and after hydrolysis were analyzed.The results showed that the physical and chemical indicators of the fish oil before and after hydrolysis have reached the primary standard of the aquaculture industry standard in China.The fatty acids in the fish oil were mainly composed of polyunsaturated fat acid.The hydrolysis of fish oil was carried out in nitrogen atmosphere under the following conditions:oil-water ratio 4:1,immobilized lipase dosage 60mg per gram fish oil,reaction time 12 hours.In the hydrolyzed fish oil,the contents of saturated fatty acids and monounsaturated fatty acid decreased obviously,while the polyunsaturated fatty acid content increased significantly,the content of docosahexenoic acid and eicosapentaenoic increasing from 16.25%to 27.04%. |
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