Chymotrypsin

Lyophilized powder, dialyzed

A serine endopeptidase secreted by the pancreas as its zymogen, CHYMOTRYPSINOGEN and carried in the pancreatic juice to the duodenum where it is activated by TRYPSIN. It selectively cleaves aromatic amino acids on the carboxyl side.

After mincing and extracting the slaughtered cattle, quickly remove the bovine pancreas (usually within 0.5 ~ 1h), wash it away, remove fat, connective tissue and other debris, and immediately immerse it in the pre-frozen sulfuric acid solution (0.125mol/L). Cool, store at about 0C, and feed after 100kg is accumulated. Take the pancreas into the meat grinder and grind it into a pancreatic slurry (shred three times if necessary), add twice the amount of ice cold sulfuric acid (0.125mol/L) of the pancreatic slurry. Place in the cold room, stir once every 1-2 hours, immerse and extract for 24 hours. The impregnation is filtered with a coarse filter bag (or two layers of gauze). After filtering, the filter residue is then repeated with 1 times the amount of cold sulfuric acid solution (0.125mol/L) and the process is repeated. The filter residue is discarded and the filtrate is combined twice. Add solid ammonium sulfate to the concentration of 242g. At this time, the concentration of ammonium sulfate reaches 40% saturation. Place in a cold room overnight, siphon the supernatant, add an appropriate amount of diatomaceous earth as a filter aid to the bottom sediment, filter under reduced pressure, and supernatant The liquid and the filtrate were combined to obtain an extract. The bovine pancreas ice-cold H2SO4→Extraction extract was graded and salted out, and crystallization. 205 g of solid ammonium sulfate was added to each liter of extract to bring the concentration of ammonium sulfate to 70% saturation, and placed in a cold room overnight, the next day The supernatant was discarded, and the bottom precipitate was filtered under reduced pressure to dryness. Add 3 times the weight of the filter cake to make it dissolve. Repeat the stage precipitation with ammonium sulfate 40% and 70% saturation. Take the second 70% saturation Precipitate the filter cake, add 1.5 times the weight of the filter cake in ice water to dissolve, and add 0.5 times the weight of the filter cake in saturated ammonium sulfate solution, adjust the Ph5 with sodium hydroxide (5mol/L), stand in a 250C incubator, keep warm Crystallize at 48h (take a drop of crystallization solution on a glass slide and observe with a 100x microscope, there should be obvious needle-like crystals), and then filter the crystals under reduced pressure to dry to obtain the crude crude chymotrypsin. The rate is 5% ~ 6%. Take the crude trypsinogen and add 7 times the amount of ice-cold distilled water to dissolve it, and add sulfuric acid (2.5mol/L) dropwise to adjust Ph to about 2, the solution is placed on the Büchner funnel and washed with talc Filter the powder, the filtrate should be clarified, then add twice the amount of saturated ammonium sulfate solution, dropwise add sodium hydroxide (5mol/L) to neutralize the acidity so that Ph is about 5, under 20 ~ 250C, keep it warm for more than 4h, namely A white precipitate precipitated, which was observed as rod-shaped crystals under a microscope. The filtrate was filtered and discarded. The precipitate was recrystallized three times in this way to obtain the original chymotrypsin crystal. Extract (NH4) 2SO4 → graded salting-out precipitate (NH4) 2SO4; NaOH; Ph4^→ Crystalline chymotrypsinogen activation, salting out, crystallization Weigh chymotrypsinogen crystals, add 3 times the amount of ice-cold distilled water and dropwise add sulfuric acid solution to dissolve it, then add an equal amount of Ph7.6 phosphate buffer Solution (0.5mol/L) and a certain amount of sodium hydroxide [equivalent to the mass of 2.5mol/L sulfuric acid in (2)], keep Ph at 7.6, then add more than 150 times per 100g of chymotrypsinogen 5mg of trypsin. Place it in a refrigerator at 50C and activate it for 48h. Adjust Ph to about 4 with sulfuric acid (0.5mol/L), add 0.5kg of solid ammonium sulfate per liter of activation solution, salt out and place for 2h The solution was filtered under reduced pressure in a Buchner funnel, the filtrate was discarded, the precipitate was clarified with 3/4 times the amount of sulfuric acid (0.005mol/L), the acid washed talc was clarified, a small amount of seed crystal was added, and it was allowed to stand at 20-250C for 24h. When a large amount of crystals are formed, filter to obtain chymotrypsin crystals. The chymotrypsin is ice-cold; trypsin; 50→activate H2SO4; Ph4→salting out ; Crystallization chymotrypsin dialysis, sterilization, drying Weigh chymotrypsin crystals, add 3 times the amount of distilled water, and dropwise add sulfuric acid solution (0.005mol/L) to dissolve it, put it into a dialysis bag every 350ml , Immerse the dialysis bag in a water bath of 50, and keep the internal and external solutions on the same plane. Continuous dialysis with water for 2 to 3 days. Dialysis should be complete. Chymotrypsin crystallizing 50→dialysis dialysate freezing; decompression→sterilization; drying finished products

α-Chymotrypsin from bovine has been used in a study to inform proteasome inhibition in order to advance anticancer research. α-Chymotrypsin from bovine has also been used in a study that functionalized surface anchored poly(methylhydrosiloxane) thin films on oxidized silicon wafers.

(1977) No Data|(1979) No Data

Molecular Weight:499.5
XLogP3:0.4
Hydrogen Bond Donor Count:5
Hydrogen Bond Acceptor Count:8
Rotatable Bond Count:12
Exact Mass:499.17031277
Monoisotopic Mass:499.17031277
Topological Polar Surface Area:200
Heavy Atom Count:36
Complexity:801
Undefined Atom Stereocenter Count:1
Covalently-Bonded Unit Count:1
Compound Is Canonicalized:Yes