Investigation For Functional Regions And Catalytic Reaction Systems Of Phospholipase D To Produce Phosphatidylserine

Phospholipase D(PLD)-catalyzed transphosphatidylation was widely used to convert phosphatidylcholine(PC)into phosphatidylserine(PS).Basing on the biomolecular simulation technology,the recognition mechanism of functional sites of PLD was investigated systematically to guide the construction of reaction system and the design of PLD structure,which were used to improve the reaction efficiency of transphosphatidylation.The molecular docking and molecular dynamics were combined to study the catalytic mechanism of PLD in the water-ethyl ether system.The binding order of two substrates and the inhibition mechanism of byproduct choline were illustrated.The kinetic model was built.The reaction process of transphosphatidylation can be predicted.The accumulation of byproduct choline was monitored.The use of the initial amount of PC and the reaction time were controlled to avoid the byproduct inhibition.Moreover,the optimal times of the recycling of the enzyme solution were predicted.γ-(2,3-epoxypropoxy)propytrimethoxysilane was covalently bond on the surface of silica and,then,esterified to construct an aqueous-solid system for transphosphatidylation,which could adsorb PC in the aqueous solution by hydrophobic interactions.Modified silica-adsorbed PC would interact with free PLD.The side reaction hydrolysis was suppressed and the reaction efficiency was improved Similarly,PLD contains a large number of hydrophobic secondary domains(such as α-helix,β-fold,etc.)in the outer surface,which can rapidly adsorb PC through hydrophobic interactions in the water solution.The adsorbed PC entered into the active center of PLD through three diffusion modes(adsorption desorption equilibrium,parallel movement of protein surface and combined diffusion).Therefore,in this paper,PLD was covalently immobilized on the surface of the carrier at a specific concentration,thus the water-solid immobilized PLD system was constructed.In this system,the loading rate of PC and the yield of PS reached 94% and 95% respectively.The biomolecular simulation was used to explore the catalytic mechanism of transphosphatidylation in different liquid-liquid reaction systems.The effect of the solvent on the internal migration mechanism of substrates on PLD was clarified.The structure function relationship of the enzyme protein was analyzed.The coconut and olive oil were used as solvent to build a biphasic system for transphosphatidylation.The side reaction hydrolysis is effectively suppressed,and the maximum PS yield can reach more than 95%.After the reaction,the water phase and all the water-soluble impurities were removed by the vacuum distillation.The high-purity PS edible oil solution was obtained and manufactured into microcapsules.Due to the poor regioselectivity of the active center of PLD for L-serine,a large substrate molar ratio is always required during the transphosphatidylation.PLD with primary sequence number BAA75216 was used as the target enzyme protein.The selectivity of the second substrate L-serine and the adjustment of the active pocket size were mutation strategies.After the key residues(L205 and Q418)were confirmed,the homology modeling and molecular dynamics simulation were used to build and optimize the virtual mutant library.Through molecular docking,molecular dynamics simulation and binding free energy calculation,three types of simulation technology were used to predict and judge the positive and negative of mutations.