【摘要】 目的:单因素试验研究大豆磷脂酶D(phospholipase D,PLD)的固定化工艺以及固定化PLD催化合成大豆磷脂酰丝氨酸(phosphatidylserine, PS)的最佳工艺。方法:首先通过非极性溶剂辅助St9ber法制备纳米级介孔二氧化硅作为固定化酶的载体，通过吸附-沉淀-交联方式将PLD固定，单因素试验优化固定化条件，并对固定化后的PLD进行结构表征和热稳定性及贮存稳定性研究；然后利用固定化PLD以大豆卵磷脂和L-丝氨酸为底物催化合成PS,并通过单因素试验确定最佳合成工艺；最后对固定化PLD的循环使用性能进行研究。结果:非极性溶剂辅助St9ber法制备的介孔二氧化硅BET比表面积为948 m~2/g,气孔体积为1.35 cm~3/g,孔径为8.20 nm,平均粒径为335.10 nm,扫描电镜确定载体的形貌为近球形结构；当加入5.00 mL乙醇、1.00 mL酶液(温度为35℃、pH为6.50)、0.40 mL戊二醛时，固定化PLD的相对酶活达到最大值(88.39%±1.00%),蛋白固定化率为80.76%±1.30%;70℃时游离PLD的相对酶活剩余15.30%,而固定化PLD的相对酶活仍然有66.40%,且游离PLD被固定化后半衰期由20 d延长到50 d;当乙酸乙酯与乙酸钠/醋酸缓冲溶液的比为6∶1,卵磷脂与L-丝氨酸的质量比为1∶8,反应温度为40℃,pH为6.50,固定化PLD的加入量为40%,反应时间为12 h时，PS的产率达86.60%±1.40%,纯度为90.30%,且固定化PLD循环使用7次PS的产率仍有61.70%±1.60%,而游离PLD循环使用4次PS的产率只剩余21.60%±1.80%。结论:介孔二氧化硅固定化PLD的稳定性、催化性能均显著提高，实现了PLD的回收重复使用，降低了生产成本；同时为PS的固定化酶法工业化生产提供技术支持。更多还原

【Abstract】 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 St9ber 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 St9ber method was 948 m~2/g, the stomatal volume was 1.35 cm~3/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.更多还原