In-situ Cross-linking Of Phospholipase D On Nanocomposite Support And Base Exchange Catalytic Reaction

The base exchange reaction catalyzed by phospholipase D（PLD）[EC 3.1.4.4]is of great significance in the preparation of high-purity natural rare phospholipids or the synthesis of some important phospholipids.Therefore,modification of the free enzyme is necessary since it is easily inactivated and difficult to reuse.At present,modification of free PLD by immobilized enzyme means is a commonly used method.In this paper,ZnO nanowire/macroporous SiO₂（ZnO NWs/SiO2）composite was used as support,and anionic diepoxides was used as cross-linker to immobilize PLD through in-situ Adsorption-Cross-linking and in-situ Co-coss-linking.And the obtained nano-biocatalysts were used to catalyze base exchange reaction.1.PLD was effectively immobilized on a ZnO NWs/SiO₂ composite support through an in-situ Adsorption-Cross-linking method.An anionic and long-chained bisepoxide cross-linker was used by adsorbing on the surface of ZnO nanowires through static interaction before cross-linking.The results showed that under the fine control of in-situ cross-linking,the loading amount of immobilized PLD reached as high as 113.7 mg/g_support,possessing high specific activity from13,987 to 16,142 U/g_protein in all the range of loading amount.The immobilized PLD showed high activity and stability in catalyzing the conversion of phosphatidylcholine（PC）to phosphatidylserine（PS）.In addition,reaction conditions such as loading amount of PLD,substrate molar ratio,temperature,solution pH,and reaction time were optimized for the finding of best synthetic process.Under optimized conditions and the PS yield reached 94.8%within 40 min at50°C.The immobilized PLD exhibited not only better thermostability and resistance to pH inactivation than free PLD but also the greatly improved storage stability and reusability.It was found that 81.5%of initial activity retained after incubation at 4°C for 60 days and that 80.4%of PS yield retained after 13 cycling reuses.The enzyme activity of immobilized PLD is hoped to further boost.2.In order to further improve the enzyme activity,this paper attempts to immobilize PLD on a nanocomposite support composed of ZnO nanowires and SiO₂ through in-situ Co-cross-linking with poly-L-lysine（PLL）.Under the optimal pH,6.5,the loading amount of PLD reached an average of 114 mg/g_supportupport for PLL concentrations in the range of 0-0.6 mg/mL.Co-cross-linking of PLD with PLL enhanced the activity of PLD remarkably.The highest specific activity of co-cross-linked PLD reached 25.8 U/mg_protein,which was 1.62 times as high as that of the cross-linked PLD(15.9 U/mg_protein).In the base exchange reaction of co-cross-linked PLD catalyzed conversion of PC and glycerol to phosphatidylglycerol（PG）,the yield of PG reached98.6%under the optimized conditions（45°C,pH 6.5,80 min）.Compared to cross-linked PLD,the co-cross-linked PLD not only exhibited good resistance to pH and heat inactivation but also improved storage stability and reusability.The yield of PG remained≥85.4%after 13 repeat uses.