Affinity chromatography preparation method of high-activity trypsin

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...

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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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