Chromatography of air-conditioner in restructuring the modification application of phospholipase C

by:CBFI     2020-10-07
Chromatography freezers in restructuring the modification application of phospholipase C, whose character is: includes the following steps:

1, modifier SS ‑ mPEG preparation: to add 2 g flask molecular weight is 5000 polyethylene glycol monomethyl oxygen radicals mPEG, add 4 MLN, N ‑ dimethyl formamide, fully dissolved; Then add 0. 12 g succinic anhydride, sealed blending, at 100 ℃, stirring and heating 3 h; By drops in the reactant chromatography freezer after cooling, add 10 ml ether, proceeds to precipitate filter dry; After drying the precipitate with methylene chloride compound solution, reoccupy ether precipitation, repeat four times. The precipitation product vacuum drying, kept in a dryer spare; Will the end product soluble in 4 MLN, N ‑ dimethyl formamide, adding 0. 46 designed. the gn ‑ hydroxy succinimide and 0. 825 g dicyclohexyl carbon 2 imide, 30 ℃ dissolve overnight, the reaction product put in chromatography of air-conditioner cooling to 0 ℃ and 40 ml ether by drops to join, to filter the precipitation dry; After drying the precipitate with methylene chloride compound solution, reoccupy ether precipitation, repeat four times. Is the activation of the polyethylene glycol precipitation products, it is dry, alternate;

2, modification enzyme preparation: the SS ‑ mPEG and phospholipase C in the molar ratio of 0 ~ 30 dissolved for 10 ml volume, pH of 7. 2, the concentration of 25 mm Tris ‑ HCl buffer, shock response time at room temperature, with the dialysis bag 4 ℃ dialysis for the night, the modification enzyme surface dry cold backup.
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