Novel phospholipid processing agent
A processing agent, phospholipid technology, applied in genetic engineering, medical preparations containing active ingredients, food preparation, etc., can solve the problem of low purity of phosphatidylinositol
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Embodiment 1
[0159] Embodiment 1 manufactures the method for PLB
[0160] A 30-liter fermenter with 20 liters of sterile medium was aseptically inoculated with 100 ml of the culture solution of Candida columnar strain ATCC14830 pre-incubated (4 days at 28° C.) in the same medium. The sterile medium contains 3% defatted cottonseed powder, 0.3% salt, 0.2% dipotassium hydrogen phosphate, 0.2% potassium phosphate, 0.1% magnesium sulfate and 0.3% defoamer. Incubation was performed at 28°C and aeration with sterile air (20 liters / min) while stirring at 300 rpm. After 50 hours it was confirmed that PLB had reached maximum activity (0.2 U / ml).
[0161] The resulting culture solution was centrifuged at 5,000 rpm for 10 minutes to obtain 16 liters of supernatant. The supernatant was concentrated by ultrafiltration. The precipitate formed by adding 9 liters of chilled acetone to 3 liters of the resulting concentrate was centrifuged at 5,000 rpm for 10 minutes to collect the precipitate, which was ...
Embodiment 2
[0163] Embodiment 2 measures the method for PLB activity
[0164] The enzymatic activity of PLB can be detected by performing an enzymatic method for measuring GPC produced from phosphatidylcholine. That is, 0.5 ml of the reaction solution prepared by dissolving in 0.5 ml of 1M MES-NaOH buffer solution containing 3% Triton X-100 (hereinafter referred to as MES-NaOH for short) was preheated at 37° C. for 2 minutes to 3 minutes. ) (pH 6) in the following composition: 0.05ml of 10mM egg yolk phosphatidylcholine, 0.025ml of 1M calcium chloride, 0.05ml of 0.2% TODB, 0.05ml of 0.2% 4-aminoantipyridine, 0.1 ml of 50 U / ml monoglyceride lipase, 0.1 ml of 300 U / m glycerol phosphate oxidase, 0.025 ml of 6 U / ml GPC phosphodiesterase and 0.05 ml of 100 U / ml peroxidase. Subsequently, the reaction solution was diluted with 10 mM Tris-HCl (pH 7.5) containing 0.05% BSA, and then 25 μl of PLB solution (0.03 U / ml to 0.15 U / ml) was added to the reaction solution, and the same dilution buffer was...
Embodiment 3
[0166] Embodiment 3 measures the method for lipase activity
[0167] The lipase activity was measured by converting monoglyceride produced using diglyceride as a substrate to glutelin by using monoglyceride lipase, and then measuring it by an enzymatic method. That is, 25 μl of an enzyme solution (0.03 U / ml to 0.15 U / ml) diluted with 10 mM Tris-HCl containing 0.05% BSA was added to 0.5 ml of the reaction solution, and then the reaction was carried out at 37° C. for 10 minutes. The solution consisted of the following dissolved in 0.1 ml of 1MMES-NaOH (pH 6.0) containing 3% Triton X-100: 0.05 ml of 10 mM 1,2 diglyceride, 0.025 ml of 0.5 M calcium chloride, 0.025 ml of 0.05M magnesium chloride, 0.05ml of 0.05MATP, 0.05ml of 10U / ml monoglyceride lipase, 0.05ml of 5U / ml glycerol kinase, 0.25ml of 400U / ml glycerol phosphate oxidase, 0.025ml of 100U / ml ml of peroxidase, 0.025 ml of 0.3% TOOS and 0.025 ml of 0.3% 4-aminoantipyridine. Thereafter, the reaction was stopped using 0.5% S...
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