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A chymotrypsin and process technology, which is applied in the field of chymotrypsin to achieve the effects of improving drug safety, high purity and good purification effect
Active Publication Date: 2012-08-01
SHANDONG TOPSCI BIO TECH
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[0004] In order to solve the above problems of low chymotrypsin activity and low purity, the present invention provides a chymotrypsin with high chymotrypsin activity and high purity Purification process
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Embodiment 1
[0031] (1) Take 10 kg of fresh bovine pancreas, remove fat, connective tissue, etc., and grind it into pancreas pulp with a meat grinder;
[0032] (2) Add 20 L (twice the volume of pancreas pulp) 0.25 N sulfuric acid solution, stir and extract in a cold room for 24 hours, filter, filter cake with 10 L of cold 0.25 N sulfuric acid, stir and extract for 1 hour in the same way, filter, and combine twice the filtrate;
[0033] (3) Stir the filtrate and gradually add ammonium sulfate to 0.4 saturation, and place it in a cold room at low temperature overnight;
[0034] (4) Filtration, the filtrate is ultrafiltered with an ultrafiltration membrane with a molecular weight cut-off of 50kDa, and the filtrate is collected.
[0035] (5) Perform ultrafiltration with an ultrafiltration membrane with a molecular weight cut-off of 10kDa;
[0036] (6) Add ammonium sulfate to the filtrate to a saturation of 0.7, and place it overnight at low temperature;
[0037] (7) Collect 390g of filter c...
Embodiment 2
[0044] (1) Take 5kg of frozen bovine pancreas, remove fat, connective tissue, etc., and mince it into pancreas pulp;
[0045] (2) Add 15 L (3 times the volume of pancreatic pulp) 0.25 N sulfuric acid solution, stir and extract in a cold room for 24 hours, filter, and then use cold 0.25 N sulfuric acid solution with 1 times the volume of pancreatic pulp as the filter cake Stir and extract for 1 h, filter, and combine the two filtrates;
[0046] (3) Stir the filtrate and gradually add ammonium sulfate to 0.4 saturation, add diatomaceous earth to aid in filtering, and place it in a cold room at low temperature overnight;
[0047] (4) Filtration, the filtrate is ultrafiltered with an ultrafiltration membrane with a molecular weight cut-off of 50kDa;
[0048] (5) The filtrate is ultrafiltered with an ultrafiltration membrane with a molecular weight cut-off of 10kDa;
[0049] (6) Add ammonium sulfate to the filtrate to a saturation of 0.7, place it overnight at low temperature, an...
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Abstract
The invention relates to a purifying process for chymotrypsin, which belongs to the field of chymotrypsin in a bio-pharmaceutical technology. The purifying process comprises the following steps of: pulping; salting out, and filtering; adding ammonium sulfate till the saturation degree is 0.4, and standing in a cold chamber over night; filtering and performing ultrafiltration by using an ultrafiltration film of 50kDa; performing ultrafiltration by using an ultrafiltration film of 50kDa; adding ammonium sulfate into a filtrate till the saturation degree is 0.7, and standing at a low temperature over night; filtering and crystallizing; activating; precipitating; performing ultrafiltration by using an ultrafiltration film of 10kDa; purifying with an SP ion exchange column; and performing ultrafiltration by using an ultrafiltration film of 10-30kDa, and sterilizing to obtain the chymotrypsin. In the process, the chymotrypsin is purified by combining the ultrafiltration film with the ion exchange column, so that a good purifying effect is achieved, and an obtained product has high purity; and the chymotrypsin obtained through the process has high activity which is more than 1500 U / mg, the dosage can be reduced, the medical expense of a patient is reduced, the administration safety of the chymotrypsin is improved.
Description
technical field [0001] The invention relates to the field of chymotrypsin in biopharmaceutical technology, in particular to a purification process of chymotrypsin. Background technique [0002] Chymotrypsin is a kind of protease extracted and purified from bovine pancreas or porcine pancreas. It can cut off the carboxy-terminal peptide chain of tyrosine and phenylalanine in the protein peptide chain, so it can effectively enzymatically hydrolyze protein, so it has the functions of debridement and pus removal, digestion of pus and removal of necrotic tissue, and promote the growth of new granulation. Clinically, it is mainly used for anti-inflammation and detumescence, and it has been reported that it also has a good effect in assisting anti-cancer. In addition, in the leather industry, chymotrypsin is often used to remove hair from animal skins. [0003] At present, a lot of research on this enzyme has been done in China, and the extraction and purification process mostl...
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