1. Trypsin crude production operation:
1. Extraction process:
1.1 For fresh pancreas, use forceps and scissors to remove the fatty connective tissue, etc., mince it in a mincer, and weigh 15Kg;
1.2 Put the minced material in a plastic bucket, soak it with 30L of 0.125mol/L sulfuric acid solution for 24 hours, and stir it once per hour during the soaking period;
1.3 Put the impregnated material into a filter bag for filtration, and then soak the filter residue with 15 L of 0.125mol/L sulfuric acid solution for 1 hour, and then discard the filter residue after filtration;
2. Salt dissolving and salting out process:
2.1 Combine the two dipping liquids with a volume of 44.9L, put them in a plastic bucket, add 242g of solid ammonium sulfate to every 1000ml of the solution to reach 40% saturation, add a total of 10.866Kg of solid ammonium sulfate, and let stand for 12 hours;
2.2 Filter, keep the filtrate, the filtrate volume is 44L, and the solid is discarded;
2.3 Add 205g of solid ammonium sulfate to every 1000ml of clear liquid to reach 70% saturation, share 9.020kg of ammonium sulfate, and let stand for 12 hours;
2.4 Filter, keep the filter cake, weigh 269.2g, and discard the filtrate;
2.5 Dissolve with 807.6ml of water, and then add solid ammonium sulfate to precipitate at nearly 40% and 70% saturation according to steps 2.1, 2.2, 2.3, and 2.4;
2.6 Take 70% saturation precipitate, weigh 178.5g, dissolve it in 267.8ml water, add 89.3ml saturated ammonium sulfate solution, and adjust the pH to 5.00 with 5mol/L sodium hydroxide solution;
2.7 The solution was left to stand in an incubator at 25°C for 48 hours, and a needle-shaped crude chymotrypsin product crystallized out;
2.8 Filtration, the mother liquor was adjusted to pH 3.0 with 0.25mol/L sulfuric acid, its volume was 328.4ml, 100.8g of ammonium sulfate was added, and it was allowed to stand for 12 hours;
3. Drying process:
The precipitate was taken and dried in a vacuum drying oven to obtain the crude trypsin product, weighing 100.0 g, that is, 6.67 g of trypsin per kilogram of pancreas.
2. Trypsin refining production operation:
1. Feeding
1.1 Weigh 100.0 g of crude trypsin, dissolve it with 300 ml of purified water, add 2.5 mol/L sulfuric acid solution to adjust the pH to 3.0 to help it dissolve, and stir for 12 hours;
1.2 Measure the volume of the solution to 298.4ml, add 140.9g of solid ammonium sulfate, and after it dissolves, let it stand for precipitation for 12 hours;
1.3 The solution was vacuum filtered, the filter cake weight was weighed 96.6g, 289.9ml of purified water was added, after stirring and dissolving, 2.5mol/L sulfuric acid solution was added to adjust pH to 3.0, 193.2ml of saturated ammonium sulfate solution was added, and static precipitation was performed for 12 hours;
1.4 Filter the solution, measure 479.3 ml of the filtrate volume, add 479.3 ml of saturated ammonium sulfate solution, and let it stand for precipitation for 12 hours;
1.5 Washing magnesium: discard the supernatant, filter the precipitate in vacuum, add saturated magnesium sulfate solution to the filter cake, let stand for 1 minute, and filter with suction. When the filtrate begins to flow out, pour the remaining magnesium sulfate solution on the funnel. Go, take out the filter cake and weigh 93.3g, to be activated;
2. Activation
Dissolve the filter cake in 373.2ml of 0.005mol/L hydrochloric acid solution, add 186.6ml of 1mol/L calcium chloride solution and 466.5ml of pH8.0 boric acid buffer, then add 746.4ml of purified water, adjust the pH to 7.4, and finally add 933mg Crystallized trypsin with high activity was activated, and the solution was left standing at 5-10°C for 72 hours for activation, and the pH value was measured at 7.4 the next day;
3. Calcium removal
The activated solution was adjusted to pH 3.1 with 2.5mol/L sulfuric acid solution, the solution volume was 1873ml, then 455.2g of solid ammonium sulfate was added, stirred to dissolve, stored at 0-10°C for 48 hours to precipitate calcium sulfate, filtered, and added to the filtrate. Solid ammonium sulfate 369.0g, left standing at 0～10 ℃ for 12 hours, filtered to obtain a filter cake weight of 45.6g;
4. Crystallization
Add 68.4 ml of boric acid buffer at pH 9.0 to the filter cake, adjust the pH to 8.0 with 2.5 mol/L sulfuric acid solution, stir evenly, then put the solution in a dialysis bag, immerse it in the external permeate at 5-10 °C, and crystallize for 5 days. Shake once every 2 hours;
5. Dialysis
Take out the crystal solution and filter to obtain 28.7g of crystals, add 43.1ml of purified water to dissolve, and adjust the pH to 3.0 with 2.5mol/L sulfuric acid; put the solution in a dialysis bag, and then hang it in a dialysis tank for 3 days to remove salt impurities. Change the water in the dialysis tank every 12 hours, and control the temperature at 5-10°C;
6. Lyophilization
Take out the dialysate, add a small amount of diatomaceous earth, and filter with a Buchner funnel to obtain the filtrate, adjust the pH to 6.0 with 5mol/L sodium hydroxide solution, and freeze-dry it in a freeze dryer to obtain 6.2g of crystalline trypsin finished product. Its active titer is 2600iu/mg;
