Immobilization Of Phospholipase A₁ And C And Its Application In Combined Degumming

Phospholipids have function of emulsification and dispersion,also promote fat metabolism,muscle growth,development of nervous system and antioxidant damage in the body.However,it remains in the vegetable oil,which will make the oil become not stable,accelerated rancidity,deepens the color,increases the difficulty of filtration and operation,causes coking of equipment and increases the burden of decoloration and deacidification.Enzymatic degumming can reduce water consumption and increase oil yield and harmless.Immobilization of phospholipase to a suitable carrier not only increase enzyme stability,but also can realize reutilization of phospholipase.In this paper,phospholipase C was immobilized on macroporous resin,phospholipase A₁ was immobilized on magnetite nanoparticles.immobilized phospholipase A₁ and immobilized phospholipase C were subjected to single and combined degumming.Different degumming processes were compared and analyzed by determine residual phosphorus content and oil yield,and the results are as follows.?1?Take the enzyme activity and protein adsorption rate of immobilized phospholipase as indicators,the weakly basic ion-exchange resin D345 was selected from several macroporous resins such as 001×10,001×7 and D345 as the preferred immobilized carrier.The optimal preparation conditions of immobilized phospholipase A₁ conditions were determined with the single factor experiment and the oahogonal experiment:enzyme dosage 6.0 was mL,pH was 5.5,time was 6.0 h,glutaraldehyde concentration was 1.0%,temperature was 30?.The immobilized enzyme activity reached 1832 U/g,the enzyme protein adsorption rate was 78.7%;the optimum reaction temperature of immobilized phospholipase C was 50?,pH was 6.5,thermal stability and pH stability were a certain improved,in the 4?refrigerator save 40 days remaining 67.0%enzyme activity.?2?The Fe₃O₄-SiO₂ magnetic carrier was prepared by reacting Fe₃O₄ nanoparticles with TEOS?tetraethyl silicate?,and then the carrier was reacted with 3-APTES?3-aminopropyltriethoxysilane?to prepare aminated Fe₃O₄-SiO₂,finally,the Fe₃O₄-SiO₂magnetic composite carrier was crosslinked with glutaraldehyde,eventually obtain modified immobilized PLA1 carrier preparation.By measuring the enzyme activity recovery rate and protein immobilization rate were determined,obtain the best condition of immobilization,the amount of enzyme 8mL/50mg carrier,temperature 30?,pH 6.0,time 8.0 h,glutaraldehyde concentration 8.0%,under this condition,the enzyme activity recovery rate of magnetic immobilized PLA1 enzyme was 63.3%,and the protein immobilization rate was 68.0%.?3?Scanning electron microscopy analysis shows that the basic morphology of Fe₃O₄ magnetic nanoparticles is spherical,with good dispersibility and less agglomeration,and its average particle size is about 15 nm.The Fe₃O₄-SiO₂ magnetic composite carrier has a uniform particle size,a smooth surface and a spherical shape,and the dispersion between the carriers is good.The average particle size of the carrier is about 200 nm.By analyzing the transmission electron microscopy analysis,it can be seen that the immobilized carrier of Fe₃O₄ nanoparticles has a core-shell shape,the solid black in the middle is Fe₃O₄ nanoparticles,the solid black in the middle is Fe₃O₄nanoparticles,and the uniform gray thin layer around the carrier is SiO₂ layer.By Fourier transform infrared spectroscopy,it was shown that the phospholipase was successfully immobilized on the magnetic carrier.It can be seen by X-ray diffraction that the inverse spinel crystal structure in the immobilized support material is the crystal plane of magnetic Fe₃O₄.?4?Phosphorus content was used as indicators in optimization of immobilized phospholipase C and immobilized phospholipase A₁ for degumming oil.For immobilized phospholipase C,the enzyme addition amount was 1.2 g/kg,time 4.0 h,The temperature was 55?and the pH was 6.5.For the immobilized phospholipase A₁,the enzyme addition amount was 0.6 g/kg,the time was 2.0 h,the temperature was 60?,and the pH was 5.5.Compared with different degumming processes,immobilized phospholipase A₁ degumming can effectively reduce the phosphorus content in the hair oil.Immobilized phospholipase C can improve the oil yield of all oil samples,but the phosphorus content has not yet reached the physical refining requirements?<10 mg/kg?,combined with immobilized phospholipase C and immobilized phospholipase A₁degumming can combine the advantages of both.It can not only combine the advantages of both,but also effectively reduce the phosphorus content in the oil sample,while also improving the oil production rate and improving economic benefits.