| Compared with traditional acid degumming and hydration degumming,vegetable oils enzymatic degumming process has the advantages of little pollution,energy saving,better degumming effect,ect.If the free phospholipase is used directly in the degumming process of vegetable oil,it is difficult to reuse the enzyme,so it will increase the production cost of the oil and fat enterprise.By immobilized phospholipase on a suitable carrier,not only can it increase the stability of the enzyme,but also convenient for the recovery and reuse of phospholipase.Prepared immobilized phospholipase with high enzyme activity and stability is very important for the large-scale application of enzymatic degumming in industrial production.In this paper,macroporous resin and self-made magnetic nanoparticles were selected as the immobilized carrier for phospholipase Ai immobilization.The effect of the change of immobilization conditions on the enzyme activity of the immobilized enzyme was investigated and the immobilized enzyme was used in the degumming experiment of rapeseed oil.The process conditions of degumming reaction were optimized by response surface method,and the effect of degumming was very good.The specific contents and results were as follows:(1)Using macroporous resin as a carrier to immobilize phospholipase has the advantages of simple operation and mild reaction conditions.And the reutilization of the macroporous resin can be achieved through the treatment of the regenerated liquid.The biggest disadvantage of using macroporous resin as a carrier is that the enzyme is connected mainly by non-covalent bonds with carriers,and the enzyme is easier to fall off from the resin.In this experiment,two kinds of macroporous adsorption resins(D1400 and DA201)and six kinds of ion exchange resins(D001,D113,D201,D301,D314 and SA-2)were selected as carriers for immobilized phospholipase A1.By comparing the enzyme activity of the immobilized enzyme,it is found that the cationic exchange resin D001 has the best immobilization effect.The cation exchange resin D001 immobilized phospholipase A1 on its surface and aperture mainly through ion bond.Through the optimization experiment,it is found that the immobilization effect of D001 resin is the best when the immobilization condition as follows:adding amount of phospholipase A1 is 1.5mL/g resin,the reaction time is 4h and the reaction pH is 5.0.Under this condition the enzyme activity of the immobilized enzyme was 665.8U/g,and the adsorption rate of the enzyme protein was 62.3%.After the immobilization reaction,the stability of the enzyme was improved.Compared with the free enzyme,the immobilized enzyme can maintain higher relative enzyme activity in the wider temperature and pH range.The immobilized enzyme has good storage stability,and it still contains 74.6%initial enzyme activity after 10 weeks of storage at 4℃.(2)The cation exchange resin D001 immobilized phospholipase A1 was used in the enzymatic degumming experiment of rapeseed oil.At the same time,the response surface method was used to optimize the conditions of the degumming reaction.The optimum degumming conditions are as follows:the dosage of immobilized enzyme is 1.8 g/kg,the reaction time is 3.6 h,the reaction temperature is 51 ℃,and the reaction pH is 5.5.Under this condition,the content of phosphorus in the oil of rapeseed oil can be reduced from 92.6mg/kg to 5,82mg/kg.Because the main force between the resin D001 and phospholipase is the ionic bond,the interaction force is weak,in the process of degumming,the enzyme is easier to fall off from the carrier.This causes the reutilization of the immobilized enzyme can not to be too much.After reusing the immobilized enzyme for 5 times,the enzyme activity of the immobilized enzyme was only less than 50%,and the phosphorus content in the degumbed oil increased to 9.78mg/kg.(3)In order to conveniently separate the immobilized enzyme from the reaction system,phospholipase Ai was immobilized on the modified magnetic Fe3O4 nanoparticles carrier in this experiment.Because magnetic Fe3O4 nanoparticles have superparamagnetic,that means at the presence of an external magnetic field,it can be easily separated from the reaction system,when the magnetic field was removed,the magnetic will disappear,so it won’t gather together.Magnetic Fe3O4 nanoparticles were prepared by chemical coprecipitation method under the condition of nitrogen protection.Because magnetic Fe3O4 nanoparticles wew easily oxidized and lack of groups that can react with the enzyme under mild conditions.So the SiO2 was wrapped on its surface to increase its stability.And the chitosan(CS)was wrapped on the surface of Fe3O4-SiO2 nanoparticles.The magnetic Fe3O4-SiO2-CS nanoparticles with a large number of amino groups on the surface are prepared.The results of X ray diffractometer and transmission electron microscopy showed that the anti spinel type Fe3O4 was obtained by co precipitation,and its size was about 13.43nm.The results of Fourier transform infrared spectroscopy showed that both Si0O2 and chitosan were successfully wrapped on magnetic Fe3O4 nanoparticles.The two aldehyde groups of glutaraldehyde can react with amino group on phospholipase and the amino group on the prepared carriers respectively.Through the bridging action of glutaraldehyde,the phospholipase A1 can be immobilized on the magnetic Fe3O4-SiO2-CS nanoparticles carrier.The optimum conditions for immobilization are as follows:the addition amount of phospholipase A1 is 2mL/g carrier,the immobilized reaction time is 3h,and the immobilization reaction pH is 5.Under this condition,the enzyme activity of the immobilized enzyme was 2760U/g,and the immobilization rate was 61.4%.The immobilization reaction enhanced the stability of the enzyme and reduced the effect of temperature and pH on the enzyme activity.The immobilized enzyme has good storage stability.Storage the immobilized enzyme at 4 ℃ after 10 weeks,it still remains 86.8%enzyme activity.(4)The immobilized phospholipase A1 on magnetic Fe3O4-SiO2-CS nanoparticles was used in the degumming experiment of rapeseed oil.The conditions of the degumming reaction were optimized by the response surface method.The optimum conditions for the degumming reaction are as follows:the reaction time is 3.2 h,the reaction temperature is 51℃,and the reaction pH is 5.Under the optimum degumming condition,the phosphorus content of the degumbed oil is 5.41 mg/kg.Because the phospholipase and the carrier are linked by covalent bonds,the enzyme is not easy to fall off the carrier.When immobilized enzyme was reused for 8 times,it still had 58.4%of the initial enzyme activity.The phosphorus content of the obtained degumming oil was 9.67mg/kg,which still could meet the requirements of industrial production.It can be seen that the immobilized enzyme has good operating stability,and it can be used in degumming process of rapeseed oil. |
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