| Transphosphatidylation reaction catalized by phospholipase D is the key technology used successfully in preparing high purity functional phospholipids. Immobilization as an enzyme modification method was used to improve the stability of free phospholipase D, reduce reaction costs, while making it more suitable for transphosphatidylation reaction. However, the resistance of mass transfer has been increased because of carrier and enzyme space conformation after immobilization. The competition between hydrolisis and transphosphatidylation reaction was found in the process, which influence the reaction velocity and yield. Therefore, it was imperative to evaluate the catalyzed performance and reaction mechanism, optimize the reaction conditions, and analysis the reaction kinetics. In this paper, the catalytic performances of Adsorbption-Aggregation-Cross-linking immobilized phospholipase D (enzyme activity:6.37 U/mg) and cross-linking phospholipase D aggregates (enzyme activity:28.62U/mg) were studied, which take the reaction of synthesis phosphatidylethanolamine (PE) as evaluation model. The optimum conditions were found catalized by these two kinds of immobilized PLD, and the kinetic model was been established to provide technical and theory support for the following industrialization application catalyzed by immobilized PLD.Through improving the traditional adsorption-cross-linking immobilization method, the Adsorbption-Aggregation-Cross-linking immobilization which based on the effect of precipitant has been proposed to prepare immobilized phospholipase D with higher activity and stability. The catalyzed performance of immobilized phospholipase D-AACL was evaluated and the optimum conditions were found:take ether as organic solvent, temperature is 30℃, pH is 6. The foundation dynamics factors were investigated too. It was found that the optimum concentration of substrate PC is 1mM, the concentration of EA is 50mM, the amount of immobilization enzyme is 0.5g, the rotational speed is 200rpm. Under these conditions, the optimum reaction velocity can achieve 8.37μmol/min·mg. It was discovered that under these conditions, the effect of hydrolitic reaction may be neglect. This will provide a broader platform for the application of immobilized phospholipase D.Moreover, the catalyzed performance of the cross-linking phospholipase D aggregates were evaluated, the optimum conditions of transphosphatidylation reaction catalyzed by immobilized PLD-CLEA were found:take ether as organic solvent, the reaction temperature is 32℃, pH is 7; The foundation dynamics factors were investigated too. It was found that the optimum concentration of substrate PC is 1mM, the concentration of EA is 50mM, the amount of immobilized enzyme is 0.2g, the rotational speed is 150rpm. Under this condition, the optimum reaction velocity can achieve 38.42μmol/min-mg. In addition, it was discovered that under the optimum conditions, the effect of hydrolitic reaction is very small. Obviously, immobilization PLD can elevate alternative specificity on the substrate.To compare the experimental results, because of the difference of preparative method and reaction mechanism, the kinetic parameters changed as well. Theoretical analysis on the enzymatic reaction kinetics model was made and the effect of exterior and interior diffusion was judged too. Under different substrates concentration, immobilized PLD (AACLs) and immobilized PLD (CLEAs) were used for transphosphatidylation reaction to synthesis phosphatidylethanolamine from phosphatidylcholine, the reaction rate was determined and the equilibrium constants of kinetic model were calculated too. Simulation of the kinetic equation was made using matlab software, it was founed that the model can directly reflect the process of enzymatic reaction dynamics, which provide a reliable basis for expansion of industrialization.
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