| In this study, soybean as raw materials, with CaCl2, sodium acetate buffer extraction, centrifugal separation and preparation of semi-permeable dialysis membrane phospholipase D(PLD), and isolated by spectrophotometric method of determination of phospholipase D activity carried out. Of time,pH,temperature, extracted from soybean phospholipase D activity by the influence of single factor experiments and orthogonal test of the phospholipase D, the best extraction conditions for the extraction of phospholipase D to provide new technical methods. The results showed that the optimum enzyme extraction conditions were: soybean meal with 10 times the quality of extraction of sodium acetate buffer solution, Ca2+ concentration of 0.005mol/L, temperature 35℃, pH value of 4.5, time 20h. And the different varieties in different regions of the soybean as raw material to phospholipase D enzyme solution, determined by its activity, analysis showed that soybean phospholipase D activity in certain geographical differences. Phospholipase D will be extracted by vacuum freeze-drying, dry products after preparation synthesis of phosphatidylserine (PS) only.In the research process of synthesizing of PLD, this article has done two studies: water-organic solvent two-phase system research: In water - organic solvent two-phase system of the study, through the single factor and orthogonal experiment the basic reaction conditions: buffer solution: 0.02M acetic acid-sodium acetate,the concentration of substrate phospholipids: 1mg/mL,serine concentration: 0.1g/mL,the amount of enzyme: 1.5U/mL,adjust pH value: 4.5,liquid ratio: 1:4,reaction temperature: 35℃,PS generation rate: 39.47%,and established the basic reaction model;Single surfactant system of water-phase study: In the surface-active agent containing a single water phase of the study, mainly under the different active carrier system, the yield of PS synthesis were compared to arrive at with silica gel adsorption phospholipids as the substrate, by adding calcium sulfate as the active carrier system for the ultimate synthesis of PS, the highest yield achieved: compared to these two systems, the water - organic solvent two-phase system is very efficient PS synthesis system, in the short time, we can achieve equilibrium, but the organic solvents on the activity of PLD a great influence on the enzyme inactivation speed and is detrimental to the human body, compared with the PS maximum production rate is relatively low; containing surfactants single-phase system of water the reaction time is longer, but the water-phase system, inactivation of the enzyme is not appropriate and the production rate is relatively high. Adsorption with calcium sulfate as a carrier substrate, reaction time to take off at the end of adsorption concentration of 36mg/mL, 3.87M of L-serine, the concentration of 50mM, pH value of 5.5 sodium acetate buffer solution, 1.5u/mL phospholipids D enzyme reaction at 35℃when the 6 hours to get 53.2% of the PS.In this paper, column chromatography of the PS that have been synthesized product was purified separate effects of different mobile phase, flow of different speed and different load on the PS separation effect. The results showed that: The silica gel as the adsorption medium, chloroform / methanol (75% / 25%) as mobile phase purity of more than 65% of the PS product.
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