Objective:A one-way test was performed to investigate the immobilization process of soybean phospholipase D(PLD)and the optimal process for immobilized PLD to catalyze the synthesis of soybean phosphatidylserine(PS).Methods:Firstly,nanoscale mesoporous silica was prepared as a carrier for immobilized enzyme by non-polar solvent-assisted Stöber method,and PLD was immobilized by adsorption-precipitation-cross-linking,and the one-way test was carried out to optimize the immobilization conditions and to study the structural characterization and thermal and storage stability of immobilized PLD.Then,PS was catalytically synthesized from immobilized PLD by using soybean lecithin and L-serine as substrates,and the optimal synthesis process was determined by a one-way test.Finally,the recycling performance of immobilized PLD was investigated.Results:The specific surface area of the mesoporous silica BET prepared by the non-polar solvent-assisted Stöber method was 948 m2/g,the stomatal volume was 1.35 cm3/g,the pore size was 8.20 nm,the average particle size was 335.10 nm,and the morphology of the carriers was determined to be a near-spherical structure by scanning electron microscopy.When ethanol was added in a volume of 5.00 mL,temperature was 35℃,pH was 6.50,volume of enzyme was 1.00 mL,and volume of glutaraldehyde was 0.40 mL,the relative enzyme activity of the immobilized PLD reached a maximum of 88.39%±1.00%,and the protein immobilization rate was 80.76%±1.30%.The relative enzyme activity of free PLD at 70℃ was 15.30%remaining,while the relative enzyme activity of immobilized PLD was still 66.40%,and the half-life of free PLD was prolonged from 20 to 50 days after it was immobilized.When the ratio of ethyl acetate to sodium acetate/acetic acid buffer solution was 6∶1,the mass ratio of lecithin to L-serine was 1∶8,the reaction temperature was 40℃,the pH was 6.50,the amount of immobilized PLD added was 40%,and the reaction time was 12 h,the yield of PS reached 86.60%±1.40%,the purity was 90.30%,and the yield of PS remained 61.70%±1.60%after 7 cycles of immobilized PLD,while only 21.60%±1.80%of PS yield remained after 4 cycles of free PLD.Conclusion:The stability and catalytic performance of mesoporous silica-immobilized PLD were significantly improved,and the recycling and reuse of PLD was realized to reduce the production cost.At the same time,technical support was provided for the industrialized production of immobilized enzyme method for PS.
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