Nanjing Forestry University
以磁性Fe3O4为载体，采用吸附-聚集-交联的方法固定来源于Streptomyces chromofuscus的磷脂酶D （scPLD）。该方法制备的磁性磷脂酶D交联酶聚集体（MCLEA）酶活回收率可达72.89%，酶活为（437±6.60）U/g。与游离酶相比，MCLEA在不同温度和pH下的稳定性都得到了一定程度的提升，但由于与底物的亲和力降低，需要更高浓度的磷脂酰胆碱（PC）和Ca2+作为反应底物与MCLEA结合。另外，还研究了MCLEA的有机溶剂耐受性，发现乙醇、四氢呋喃、叔丁醇、乙酸乙酯、乙醚和甲苯等有机溶剂对MCLEA的酶促反应具有促进作用，此发现为scPLD催化转磷脂酰作用生成磷脂酰丝氨酸（PS）过程中构建双相体系时有机溶剂的选择提供了参考。MCLEA在进行连续13次酶促反应后，酶活仍可保留在76%以上，其半衰期为331.67 min，说明所选择的固定化方案可以提高酶的稳定性，从而提高其在催化反应过程中的重复利用性。
The magnetic Fe3O4 was used as the carrier, and the magnetic cross-linked scPLD aggregates (MCLEA) were prepared by adsorption-aggregation-crosslinking. The enzyme activity recovery of the MCLEA was 72.89%, and the enzyme activity was 437±6.60 U/g. Compared to free scPLD, the MCLEA showed better tolerance at different temperatures and pH levels. However, due to the reduced affinity of the MCLLEA with the substrate, it requires higher concentrations of phosphatidylcholine (PC) and Ca2+ as the reaction substrate to bind to MCLEA. Furthermore, the organic solvent tolerance of the MCLEA was also investigated, and organic solvents such as ethanol, tetrahydrofuran, tert-butanol, ethyl acetate, ether, and toluene were found to improve the enzymatic reactions of MCLEA. These findings provide a reference for the selection of organic solvents in the construction of a biphasic system in the production of phosphatidylserine (PS) catalyzed by scPLD. After 13 cycles of continuous reaction, the activity of the MCLEA remained above 76%, giving a half-life of 331.67 min, which indicated that the immobilizing method developed in this work could improve the stability of the PLD and its reusability in the catalytic reaction.