Affinity chromatography preparation method of high-activity trypsin

A trypsin, high-activity technology, applied in biochemical equipment and methods, enzymes, peptidases, etc., can solve the problems of weak purification ability, inconvenient large-scale production, low titer of finished products, etc., to avoid the zymogen precipitation step, The effect of improving market competitiveness and reducing production costs

Inactive Publication Date: 2017-05-31
宁波林叶生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The process of extracting trypsin in China has been using multiple salting out, crystallization and dialysis for a long time. The purification ability of this process is weak, the process is complicated, the dialysis process takes a long time, it is not convenient for large-scale production, and the finished product has a low potency , the activity of products produced by this traditional process is generally 2000-2500 units / mg, and the highest can only reach about 2700 units / mg
[0004] Due to the large process inertia of the trypsin production process in its manufacturer, once the process is stable, it is not easy to change, and is limited by the technical difficulty of the affinity chromatography technology itself, so that the development of the affinity chromatography process to prepare trypsin It has a high technical threshold and technical bottleneck, therefore, it is difficult to obtain a major breakthrough in the trypsin process
At present, in order to improve the activity of trypsin, many trypsin production companies have adopted arrowhead protease inhibitors as affinity chromatography column ligands, such as: patent CN102911926A discloses a "production of high-purity trypsin by affinity chromatography Method", the affinity chromatography medium used in this method is based on GE Health gel medium, and connected with arrowhead protease inhibitor as ligand, and the eluent used is 0.1mol / L formic acid-0.05mol / L KCl , pH2.2 buffer solution; however, the above-mentioned processes or similar processes require ammonium sulfate graded salting-out and zymogen precipitation steps without exception, the process is relatively complicated, and the space for improving trypsin activity is also very limited

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] (1) Grinding of raw materials, rough extraction of trypsin: take 150 kg of fresh frozen bovine pancreas, and directly crush the frozen raw materials with a pulverizer; add 450 L of pre-cooled 0.125M sulfuric acid solution, transfer it to a 1-ton stirring container, and place it in a 4-degree cold storage Stir and extract in medium for 6 hours, place overnight; filter with plate and frame filter equipment the next day, pack the separated residue into bags and treat according to environmental protection requirements, and the obtained supernatant is the crude extract of trypsin.

[0033] (2) Two-stage salting-out of ammonium sulfate: add reagent-grade ammonium sulfate to the above-mentioned trypsin crude extract to 25% saturation, and add a small amount of diatomaceous earth to aid in filtration, place it in a 4-degree cold storage overnight and filter, and take the supernatant Then add reagent-grade ammonium sulfate to the supernatant to 65% saturation, place it overnight ...

Embodiment 2

[0039](1) Raw material crushing, rough extraction of trypsin: take 450 kg of fresh frozen bovine pancreas, and use a pulverizer to directly crush the frozen raw material into a slurry; add 1350 L of pre-cooled 0.125M sulfuric acid solution, transfer it to a 2-ton stirring container, and Stir and extract in a cold storage for 6 hours, and place overnight; the next day, filter it with a plate-and-frame filter, bag the separated residue and treat it according to environmental protection requirements, and the obtained clear liquid is the trypsin crude extract.

[0040] (2) Two-stage salting-out of ammonium sulfate: Stir and add reagent-grade ammonium sulfate to the above-mentioned trypsin crude extract to 25% saturation, and add a small amount of diatomaceous earth to aid in filtering, place it in a 4-degree cold storage overnight, and then filter. Supernatant: Add reagent-grade ammonium sulfate to the supernatant to 65% saturation, place it overnight in a 4-degree cold storage, ab...

Embodiment 3

[0046] (1) Crushing of raw materials, rough extraction of trypsin: 200 kg of fresh frozen porcine pancreas was taken, and the frozen raw materials were directly pulverized into a slurry with a pulverizer. Add 600L of pre-cooled 0.125M sulfuric acid solution, transfer it to a 1-ton mixing container, stir and extract in a 4-degree cold storage for 6 hours, and place it overnight; filter it with a plate-and-frame filter the next day, and bag the separated residue according to environmental protection requirements After treatment, the obtained supernatant is the trypsin crude extract.

[0047] (2) Two-stage salting-out of ammonium sulfate: Stir and add reagent-grade ammonium sulfate to the above-mentioned trypsin crude extract to 25% saturation, and add a small amount of diatomaceous earth to aid in filtering, place it in a 4-degree cold storage overnight, and then filter. Supernatant: Add reagent-grade ammonium sulfate to the supernatant to 65% saturation, place it overnight in a...

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Abstract

The invention relates to an affinity chromatography preparation method of high-activity trypsin, which takes bovine or porcine pancreas as a raw material. The method comprises the following steps of crushing the raw material, performing rough extraction on tryptose, performing two-stage ammonium sulfate salting-out, performing enzymolysis and activation on a crude product, performing a reaction for 24h under an alkaline condition by taking a GE-Health gel medium Sepharose CL-4B as a framework and (2,6-dyhydroxyl)heptyl-(6-amino)acetyl-para-aminophenyl guanidine as a ligand to prepare an affinity chromatography packing for affinity chromatography, performing ultrafiltration, concentration and degerming, and then performing vacuum freeze drying to obtain a finished product. The method avoids the complicated zymogen precipitation step in the traditional technology, greatly simplifies a procedure, can obtain a high-purity and high-activity product at a higher yield, is stable in technology and controllable in quality, greatly lowers the production cost, and improves market competitiveness of the product.

Description

technical field [0001] The invention belongs to the technical field of biochemical separation and purification, in particular relates to an affinity chromatography preparation method for medicinal trypsin, more specifically relates to an affinity chromatography preparation method for high-activity trypsin. Background technique [0002] Trypsin is a proteolytic enzyme isolated and purified from bovine pancreas or porcine pancreas. It can hydrolyze proteins into polypeptides or amino acids, and has selective hydrolysis of arginine and lysine peptide chains. Dicer. The pharmacological and clinical effects of trypsin mainly include: decomposing liquefied blood clots, purulent secretions, necrotic tissue, etc., making pus thinner, easy to drain and promoting wound healing. [0003] The process of extracting trypsin in China has been using multiple salting out, crystallization and dialysis for a long time. The purification ability of this process is weak, the process is complicat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/76
CPCC12N9/6427C12Y304/21004
Inventor 林克叶昀
Owner 宁波林叶生物科技有限公司
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