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Process method for extracting three enzymes from animal pancreas

A process method, pancreas technology, applied in the field of bioengineering, can solve the problems of high price, low product purity, and other enzyme structure damage, and achieve the effects of reducing resource waste, high product purity, and easy operation

Active Publication Date: 2020-05-08
江西浩然生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Pancreatic kininogenase, chymotrypsin, and elastase are widely used clinically and are expensive
All three enzymes can be obtained from animal pancreas. In the past, when one enzyme was extracted, the other enzymes were treated as impurities and the structure of the other enzymes was so bad that it was greatly destroyed, making it difficult to form multi-enzyme co-production.
Because the isoelectric point of chymotrypsin is 8.1, the isoelectric point of pancreatic kininogenase is 4.05, and diacetylamino agarose gel chromatography is an anion exchange column, the purity of purified chymotrypsin is not high; the isoelectric point of trypsin is 10.1 , the isoelectric point of elastase is 9.5, and the bisacrylamide dextran gel is also an anionic column, so this process has the disadvantages of low purity of the obtained product and complicated operation.

Method used

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  • Process method for extracting three enzymes from animal pancreas
  • Process method for extracting three enzymes from animal pancreas
  • Process method for extracting three enzymes from animal pancreas

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Such as figure 1 As shown, the invention provides a process for extracting three enzymes from animal pancreas.

[0045] The concrete implementation process of this method is as follows:

[0046] Step 101: Mincing the tissue: Mincing the pancreas of the animal to obtain a tissue pulp.

[0047] Wherein, in combination with specific implementation scenarios, the animal pancreas can be fresh porcine pancreas or bovine pancreas, and the fat and connective tissue are removed, washed and minced with a meat grinder.

[0048] Step 102: Activation and soaking: adding calcium chloride solution to the tissue pulp, adjusting the pH to the first pH range, and incubating at 35°C-45°C.

[0049]Wherein, in combination with specific implementation scenarios, the calcium chloride solution can be 1-5% calcium chloride solution, and calcium ions in calcium chloride can activate pancreatic kininogenase during soaking, and calcium ions can act on chymotrypsin Protective effect, inhibiting ...

Embodiment 2

[0062] Step 201: Grinding the tissue: take 1000 grams of fresh frozen pig, remove the fat and connective tissue, wash it with water, and grind it with a meat grinder.

[0063] Step 202: Activation and soaking: Add 5 liters of 3% calcium chloride solution, adjust the pH to 7.0, stir, and put it in a 40°C water bath for 4 hours.

[0064] Step 203: Filtration and Concentration: After the heat preservation is completed, add 5% diatomaceous earth for filtration, and the obtained filtrate 1 is ultrafiltered with a 10KD ultrafiltration membrane to obtain a 300 mL solution.

[0065] Step 204: Q Sepharose FF anion exchange chromatography: After the ion exchange chromatography column is equilibrated with a balance solution (0.1M HAc-NaAc, pH 5), adjust the pH of the concentrated solution after the ultrafiltration to 5, and put it on at a flow rate of 5ml / min Column, collect the column liquid 1, elute with the eluent (0.1M HAc-NaAc+0.5M NaCl, pH5), and collect the eluate 1.

[0066] Ste...

Embodiment 3

[0070] Step 301: Grinding the tissue: take 10 kg of fresh frozen pigs, remove the fat and connective tissue, wash them with clean water, and grind them with a meat grinder.

[0071] Step 302: Activation and soaking: Add 50 liters of 3% calcium chloride solution, adjust the pH to 7.0, stir, and put it in a 40°C water bath for 4 hours.

[0072] Step 303: Filtration and Concentration: After the heat preservation is completed, add 5% diatomaceous earth for filtration, and the obtained filtrate 1 is ultrafiltered with a 10KD ultrafiltration membrane to obtain 1000 ml of a solution.

[0073] Step 304: Q Sepharose FF anion exchange chromatography: After the ion exchange chromatography column is equilibrated with an equilibrium solution (0.1M HAc-NaAc, pH 5), adjust the pH of the concentrated solution after ultrafiltration to 5, and put it on at a flow rate of 5ml / min Column, collect the column liquid 1, elute with the eluent (0.1M HAc-NaAc+0.5M NaCl, pH5), and collect the eluate 1. ...

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Abstract

The invention discloses a process method for extracting three enzymes from animal pancreas. The process method comprises the following steps: by taking animal pancreas as a raw material, performing tissue crushing, activation, soaking, filtration, ultrafiltration, anion exchange chromatography and hydrophobic chromatography so as to obtain high-purity pancreatic kallidinogenase; performing cation-exchange chromatography on an anion exchange chromatography column-through fluid so as to obtain elastase; activating Sepharose, arming aminocaproic acid, coupling o-aminoanisole so as to obtain an O-Anisidin-ACA-Sepharose affinity adsorbent, and adsorbing a cation-exchange chromatography column-through fluid, so as to obtain high-purity chymotrypsin. By adopting the method, co-production of threeenzymes, namely pancreatic kallidinogenase, chymotrypsin and elastase, can be achieved, the operation is simple and convenient, the product purity is high, and resource waste can be reduced. Due to synthesis and use of an O-Anisidin-ACA-Sepharose medium, the production cost is reduced, and the product quality is improved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a process method for extracting three kinds of enzymes from animal pancreas. Background technique [0002] Pancreatic Kallidinogenase (Pancreatic Kallidinogenase), also known as pancreatic kallikrein and kallikrein, is a kind of endo-type proteolytic enzyme, which exists in various tissues in mammals, especially in the pancreas. Rich. Pancreatic kininogenase is a glycoprotein composed of 18 amino acids and 4 sugars, with a molecular weight of about 26800 and an isoelectric point of 4.05. [0003] Chymotrypsin, also known as chymotrypsin, is a kind of protease extracted from animal pancreas, which belongs to serine peptide chain endonuclease, and its isoelectric point is 8.1. [0004] Elastase is a broad-spectrum proteolytic enzyme characterized by decomposing insoluble elastin, with an isoelectric point of 9.5, mainly found in animal pancreas, skin, aorta, platelets and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64C12N9/76
CPCC12N9/6427C12N9/6445C12N9/6448C12Y304/21004C12Y304/21118
Inventor 万偲徐富勇陈洁玉耿卫锋李国龙
Owner 江西浩然生物制药有限公司
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