Method for producing chymotrypsin

Production method: using bovine pancreas as raw material, after extraction and fractionation, the chymotrypsinogen is prepared first, and then activated by trypsin, and then recrystallized with ammonium sulfate and ethanol.

The pancreas was taken out within 1 h after the cattle were slaughtered and leached with sulfuric acid at 0 °C. The leachate is fractionated and precipitated with ammonium sulfate. The salt is dissolved in cold distilled water (pH is adjusted with sulfuric acid), and the pH is adjusted with ammonium sulfate and sodium hydroxide to complete the crystallization, repeated dissolution, and recrystallization to obtain chymotrypsin crystals. . The chymotrypsin element crystals are activated, salted out, dialyzed, and dried to obtain chymotrypsin.

Details: After mincing and extracting cattle, the bovine pancreas (usually within 0.5 to 1 h) is quickly removed, washed, and the fat, connective tissue and other impurities are removed, and immediately immersed in a pre-frozen sulfuric acid solution (0.125 mol/L). Suddenly cooled, stored at around 0C, and filled after 100kg. The pancreas was taken into a meat grinder and smashed into pancreatic juice (should be twisted 3 times if necessary), and twice the amount of ice-cold sulfuric acid (0.125 mol/L) was added to the pancreas. In the cold room, stir once every 1 to 2 hours, and dip and extract for 24 hours. The impregnated material is filtered with a coarse filter bag (or two layers of gauze), and after filtration, the filter residue is further treated with 1 times of cold sulfuric acid solution (0.125 mol/L, and then filtered, and the filter residue is discarded, and the filtrate is combined twice per liter of filtrate. Adding solid ammonium sulfate to a concentration of 242g, at which time the ammonium sulfate concentration reaches 40% saturation, placed in a cold room, overnight, siphon supernatant, the bottom layer precipitate is added with appropriate amount of diatomaceous earth as a filter aid, filtered under reduced pressure, supernatant The liquid and the filtrate were combined to obtain an extract.

Bovine pancreas ice-cold H2SO4→extract extract

Grade salting out, crystallization of solid ammonium sulfate 205g per liter of extract, so that the ammonium sulfate concentration reaches 70% saturation, the cold room is placed overnight, the next day the supernatant is discarded, the bottom layer precipitate is filtered under reduced pressure to dry, added 3 times Dissolve in the ice water of the filter cake, repeat the precipitation with ammonium sulfate 40% and 70% saturation, take the second 70% saturation precipitation cake, add 1.5 times the weight of the filter cake to dissolve, and Add 0.5 times the weight of the filter cake to the saturated ammonium sulfate solution, adjust the Ph5 with sodium hydroxide (5mol/L), let stand in a 250C incubator, and incubated for 48 hours for crystallization (take a drop of crystal solution on a glass slide and observe with a microscope of 100 times , there should be obvious needle crystals), and then the crystals are vacuum filtered to dryness to obtain crude chymotrypsin products, and the yield is 5% to 6% according to the mass of bovine pancreas. Add crude trypsinogen to 7 times the amount of ice-cold distilled water to dissolve it, and add sulfuric acid (2.5mol/L) to adjust Ph to about 2, and place the solution on the Buchner funnel with acid-washed talcum powder. The filtrate should be clarified. Add 2 times the amount of saturated ammonium sulfate solution, add sodium hydroxide (5mol / L) to neutralize the acidity to make Ph about 5, in the condition of 20 ~ 250C, keep warm for more than 4h, that is, there is white precipitate precipitation, under the microscope It was observed as a rod crystal, and the filtrate was discarded by filtration, and the precipitate was further crystallized three times according to this method to obtain chymotrypsinogen crystals.

Extract (NH4)2SO4→graded salting-out precipitation (NH4)2SO4; NaOH; Ph4^→ crystalline chymotrypsinogen

Activation, salting out, crystallization, weigh the original chymotrypsin crystals, add 3 times the amount of ice-cold distilled water and add sulfuric acid solution to dissolve, then add the same amount of Ph7.6 phosphate buffer solution (0.5mol / L) And a certain amount of sodium hydroxide [corresponding to the mass of 2.5 mol/L sulfuric acid in (2)], keeping Ph at 7.6, and adding 150 times more than 150 mg of trypsin per 100 g of chymotrypsinogen. Place in a 50C refrigerator and activate it for 48 hours. Adjust the Ph to about 4 with sulfuric acid (0.5mol/L), add 0.5kg of solid ammonium sulfate per liter of the activation solution, salt out and let it stand for 2h. The solution is filtered under a Buchner funnel and the filtrate is discarded. The precipitate is used. 3/4 times the amount of sulfuric acid (0.005 mol/L), the acid-washed talc powder is clarified, a small amount of seed crystal is added, and it is allowed to stand at 20 to 250 C for 24 hours. When a large amount of crystals are formed, it is filtered to obtain chymotrypsin crystals.

Chymotrypsinogen ice cold; trypsin; 50→ activation activation H2SO4; Ph4→ salting out; crystallization of chymotrypsin

Dialysis, sterilization, drying, weigh chymotrypsin crystals, add 3 times the amount of distilled water, and add sulfuric acid solution (0.005mol / L), dissolve it, put a dialysis bag every 350ml, so that the dialysis bag dipped In a water bath of 50, the inner and outer solutions are placed on the same plane, and dialysis is continued for 2 to 3 days with water, and the dialysis should be complete.

Chymotrypsin crystal 50→dialysis dialysate freezing; decompression→sterilization; drying finished product