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Extraction method of trypsin-chymotrypsin

A technology of chymotrypsin and extraction methods, which is applied in the direction of biochemical equipment and methods, enzymes, hydrolytic enzymes, etc., can solve the problems of high cost, difficulty in widespread use, and high technical requirements, and achieve simple and feasible extraction and separation methods, saving costs, The effect of shortening the time

Inactive Publication Date: 2010-02-10
马忠仁 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

They are first prepared into crude products of chymotrypsin, then purified by CM-cellulose column chromatography and affinity chromatography, and freeze-dried to obtain chymotrypsin with high purity. However, due to its complicated preparation process, technical requirements High, expensive, not easy to be widely used

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] The present invention has the following sequential steps:

[0016] a. Take 1 kg of fresh bovine pancreas as raw material, remove fat and connective tissue, wash it with water, and grind it with a meat grinder.

[0017] b. Move the minced beef pancreas into a stainless steel bucket, add 3000 ml of tap water W / V, and use 15% H 2 SO 4 Solution V / V, adjusted to pH 3.0, stirred continuously at room temperature for 24 hours, and then centrifuged at 4000 rpm for 30 minutes to obtain supernatant I and pancreatic residue precipitate I.

[0018] c. Pour the obtained 3000 ml supernatant I into the supernatant bucket for later use, stir and wash the pancreatic residue sediment I with 1000 ml W / V of tap water with a pH value of 3.0 for 20 minutes, and then use 4000 rpm Centrifuge at a speed of 30 minutes for 30 minutes to obtain supernatant II and pancreatic residue pellet II.

[0019] d. Pour the supernatant II into the supernatant bucket, wash the pancreatic residue sediment II...

Embodiment 2

[0025] The present invention has the following sequential steps:

[0026] a. Take 2 kg of frozen bovine pancreas as raw material, remove fat and connective tissue, wash it with clean water, and grind it with a meat grinder.

[0027] b. Move the minced cow pancreas into a stainless steel bucket, add 6000 ml of tap water W / V, and use 15% H 2 SO 4 Solution V / V, adjusted to pH 3.0, stirred continuously at room temperature for 24 hours, and then centrifuged at 4000 rpm for 30 minutes to obtain supernatant I and bovine pancreas residue precipitate I.

[0028] c. Pour the obtained 6000 ml supernatant I into the supernatant bucket for later use, and stir and wash the bovine pancreas residue sediment I with 2000 ml W / V of tap water with a pH value of 3.0 for 20 minutes, and then use 4000 rpm Centrifuge at a speed of 1 / min for 30 minutes to obtain supernatant II and bovine pancreas residue precipitate II.

[0029]d. Pour the supernatant II into the supernatant bucket, and stir and wa...

Embodiment 3

[0035] The present invention has the following sequential steps:

[0036] a. Take 5 kg of fresh bovine pancreas as raw material, remove the fat and connective tissue, wash it with water, and grind it with a meat grinder.

[0037] b. Move the minced beef pancreas into a stainless steel bucket, add 15,000 ml of tap water W / V, and use 15% H 2 SO 4 Solution V / V, adjusted to pH 3.0, stirred continuously at room temperature for 24 hours, and then centrifuged at 4000 rpm for 30 minutes to obtain supernatant I and pancreatic residue precipitate I.

[0038] c. Pour the obtained 15,000 ml of supernatant I into the supernatant bucket for later use, stir and wash the pancreatic residue precipitate I with 3000 ml of tap water with a pH value of 3.0 W / V for 20 minutes, and then use 4000 rpm Centrifuge at a speed of 30 minutes for 30 minutes to obtain supernatant II and pancreatic residue pellet II.

[0039] d. Pour the supernatant II into the supernatant bucket, stir and wash the pancrea...

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PUM

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Abstract

The invention relates to the field of biological engineering, in particular to an extraction method of trypsin-chymotrypsin. In the invention, CaCl2 is adopted to activate chymotrypsinogen into the trypsin-chymotrypsin, and simultaneously (NH4)2SO4 is added to generate CaSO4 precipitation and adsorb the trypsin-chymotrypsin; the white freeze-dried powder can be obtained directly through the stepsof elution, salting out, ultrafiltration and freeze-drying, with no need of preparing by the following steps: firstly salting out multiple crystallization joint dialysis and an organic solvent methodof alcohol and acetone and the like is adopted to prepare the trypsin-chymotrypsin crude product, and then the complex steps of CM-cellulose column chromatography and affinity chromatography and the like are carried out for purifying preparation. The extraction method in the invention is convenient and feasible, and is applicable to industrial production, thus saving cost and shortening time whichis shortened to 3 to 4 days from the original required 7 to 8 days. 4.0-4.5 grams of freeze-dried trypsin-chymotrypsin powder can be obtained from per kilogram of pancreas, wherein, the specific activity of chymotrypsin is 355 U. / mg protein (nitrogen) and the specific activity of trypsin is 1361 U. / mg protein (nitrogen).

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a method for extracting chymotrypsin. Background technique [0002] Both chymotrypsin and trypsin are typical serine proteases. Their properties are very similar and difficult to separate, but they have different specificities for proteolysis. Trypsin acts only on the C-terminal peptide bond formed by the carboxyl group of the basic amino acid L-arginine or lysine, while chymotrypsin selectively hydrolyzes aromatic or aliphatic amino acids (such as phenylalanine , tyrosine) peptide bond. Therefore, the two of them have the effect of synergistically hydrolyzing protein peptide bonds, which is better than the effect of using one enzyme alone, and has a wider application range. At present, a lot of research has been done on them at home and abroad, and they have been used clinically as a kind of anti-inflammatory and detumescence drugs. Other uses such as: leather industry to remove...

Claims

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Application Information

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IPC IPC(8): C12N9/76C12N9/64
Inventor 马忠仁印明善冯玉萍李明生蔡翠萍乔自林冯若飞阮小华
Owner 马忠仁
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