Development And Research On The Syntheses Of Phosphatidylglycerol By Immobilized Phosphlipase D Catalyzed Transphosphatidylation

As one special kind of rare phospholipids, Phosphatidylglycerol (PG) plays a certain role for the diagnosis and treatment of neonatal respiratory distress syndrome and other lung diseases. More than that, phosphatidylglycerol is the carotene lipid biosynthesis intermediates. Although the phosphatidylglycerol distributes widely in nature, it is generally less in the content of the in-vivo. The PG of several single aliphatic chain is important accessories of the liposomal formulations of drugs, and thus the synthesis of phosphatidylglycerol is very necessary. The enzyme modified synthesis of PG is an ideal way to produce a large number of phosphatidylglycerol with low production costs and mild conditions. In this paper, the enzyme modified method is the use of phospholipase D transfer characteristics which can catalyze synthesis of phosphatidylglycerol from soybean lecithin.Phospholipase D plays a very important role for the reaction of the syntheses of ghosphatidylglycerol by phosphlipase D catalyzed transphosphatidylation. A PLD-high-producing strain was selected by ultraviolet mutagenesis from wild Streptomyces and its PLD activity was increased by27.8%compared with the original strain. By means of response surface methodology analysis to further optimize fermentation medium, the optimum fermentation medium was determined in shake flask fermentation. The optimized components (g/L) of fermentation medium were13.0of glucose,6.0of beef extract,6.0of peptone,1.5of MgSO4·7H2O,3.0of CaCl2,2.0of NaCl, and0.6of Tween80. Under the above conditions, the enzyme transphosphatidylation activity reached2.034U/ml.Taking the single factor experiment method to the syntheses of ghosphatidylglycerol by free phosphlipase D catalyzed transphosphatidylation, the results showed that the reatction temperature28℃, pH5.5, molar ratio (glycerol:soybean PC)165, organic solvent-water ratio1.5:1, reaction time4hours. Under the above conditions, the yield of phosphatidylglycerol was77.3%. By means of three factors and five levels of test design through the center combination in Design-expert8.0, the optimum reaction conditions was determined, which included that the reatction temperature28.2℃, pH6.2, molar ratio (glycerol:soybean PC)165, organic solvent-water ratio2.0:1, reaction time4hours. Under this above conditions, the yield of phosphatidylglycerol was81.5%.The inactivation of the free phospholipase D is contacted by organic phase, and it has high price, so it is necessary for the immobilized enzyme which is easily recycled and has a repetition frequency. Taking the single factor experiment method to the syntheses of ghosphatidylglycerol by Immobilized phosphlipase D catalyzed transphosphatidylation, the results showed that the reatction temperature30℃, pH6.5, molar ratio (glycerol:soybean PC)110, organic solvent-water ratio1:1.5, reaction time6hours. Under the above conditions, the yield of phosphatidylglycerol was64.1%. By means of three factors and five levels of test design through the center combination in Design-expert8.0, the optimum reaction conditions was determined, which included that the reatction temperature30.6℃, pH6.9, molar ratio (glycerol:soybean PC)110, organic solvent-water ratio1:1.8, reaction time6hours. Under this above conditions, the yield of phosphatidylglycerol was66.7%.On the basis of the experiments, it enlarged budget for the process of the annual output of6.34tons of phosphatidylglycerol. The equipments were selected with the material balance and energy balance. Finally, it can carry out the economic evaluation. The results showed that the proceeds of the project the annual earnings was2.1438million RMB and has a better economic efficiency.