Aqueous-solid System For Phospholipase D-catalyzed Transphosphatidylation To Produce Phosphatidylglycerol

Phospholipase D?PLD?,as a biocatalyst for transphosphatidylation,which is usually used to catalyze the relatively abundant natural lecithin or phosphatidylcholine?PC?to synthesize various functional and high purity single phospholipids,rare phospholipids and new phospholipid derivatives,such as phosphatidylglycerol?PG?.Clinical studies have indicated that PG can reduce pulmonary surface tension and maintain the structure and function of alveolar stability;PG has a good emulsification effect,it is often combined with other emulsifiers to emulsifie food;PG can also be used as the drug carrier to achieve targeted drug delivery.In this paper,PG was synthesized by PLD-mediated transphosphatidylation of PC with glycerol.We was investigated that two kinds of liquid-solid systems effects on the transphosphatidylation.First,the bio-imprinting technique was introduced to over-activate the enzyme by imprinting pretreatment,but the imprint-induced conformational changes could be completely erased in the presence of water.Therefore,compared with the traditional immobilization method,the PLD was adsorbed and precipitated on the surface of the nanosilica,then,these attached enzyme molecules form a“enzyme net”covering the surface of nanosilica through intra-and intermolecular cross-linking.The combination of bio-imprinting with traditional immobilization methods was introduced to achieve increased PLD activity and selectivity.After that,the modified bio-imprinting immobilized enzyme is used as a solid phase,PC is dissolved in the organic phase,and the substrate and the imprinted immobilized enzyme are dissolved in the aqueous phase,in order to establish a liquid-liquid-solid system catalyzed PC by transphosphatidylation reaction to synthesize PG.Second:Considering that phospholipid products are used in the fields of medicinal and food,the residual toxic organic solvents can cause harm to users and operators,increasing the environmental pollution and post-production processing costs.Water is generally recognized as the most ideal reaction medium.At the same time,it has been reported that surfactants can form mixed micelles with amphiphilic substrate phospholipids to achieve the dispersion of phospholipids in aqueous solution.A highly efficient and environmentally friendly liquid-solid phase PLD catalytic reaction system was constructed.Triton X-100 was covalently bound to the surface of nanosilica,and obtained Triton X-100-modified nanosilica was used for the adsorption of PC in the purely aqueous solution.PG was successfully synthesized using the carrier-adsorbed PC under PLD-catalyzed transphosphatidylation.The main experimental results was as follows:?1?Phospholipase D,fermented by streptomycete,which has been screened and conserved by the laboratory.The bio-imprinting immobilized enzyme,good selectivity and high stability,which was prepared by four main processes of imprinting,adsorption,precipitation and cross-linking.We were investigated the optimal process conditions for imprinted immobilized enzyme.The results were as follows:the best candidate of precipitant was isopropanol,precipitant content was 4 mL,pH 5.5,the substrate molar ratio of glycerol to PC was 65:1,the content of glutaraldehyde was 320?L.Under these conditions,the bio-immobilized enzyme activity reached 1658.8U/g_protein.In addition,the performance of the prepared bio-imprinting immobilized enzyme was evaluated.Compared with the free enzyme,the thermal,storage,and operational stability of the bio-imprinting immobilized enzyme were significantly improved.?2?Usingthepreparedbio-imprintingimmobilizedenzymetocatalyzePC transphosphatidylation for the preparation of PG,and the appropriate operational conditions were systematically obtained,including the main effect factors of organic solvents,pH,temperature etc.the results was as follows:the best candidate of organic solvents was ethyl ether,the reaction temperature was 30?,pH was 6.0,under this conditions,the yield of PG reached 73%.The reaction process was simulated by the enzymatic reaction kinetic equation.The fitting results were basically consistent with the experimental data,indicating that the enzyme had good affinity with the substrate.?3?In order to circumvent the drawback of the traditional liquid phase,it is proposed to use acetic acid buffer as the aqueous phase,Triton X-100 was covalently bound to the surface of nanosilica,and obtained Triton X-100-modified nanosilica was used for the adsorption of phosphatidylcholine?PC?in the purely aqueous solution.PG was successfully synthesized usingthecarrier-adsorbedPCunderPLD-catalyzedtransphosphatidylation.We systematically investigated the effect of the content of Triton X-100 on the surface of nanosilica on transphosphatidylation.The optimal value of Triton X-100 content on the surface of nanosilica was 8.5×10^-8mol/g.Results indicated that the highest PC loading and yield of PG reached 98%and 90.5%,respectively.The reaction kinetics conforms to the PC-limited Michaelis-Menten equation.In addition,We were explored the optimal process conditions:the reaction temperature was 30?,pH 6.0,the recyclability and stability of free PLD solution were studied,the relative yield of PG was still up to 80%in the seventh batch.The carrier was recycled for 16 times,the adsorption capacity for PC and the yield of PG did not obviously decrease.