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A kind of method utilizing immobilized enzyme to extract heparin sodium

A technology of immobilized enzyme and heparin sodium, which is applied in the field of extracting heparin sodium, can solve the problems of uncontrollable reaction, insufficient enzyme activity, and decreased catalytic efficiency, and achieve the effects of improving purity, increasing enzyme activity, and reducing content

Active Publication Date: 2016-01-06
PUJIANG CAREX BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, enzymatic hydrolysis is mostly carried out in the form of free enzymes. In this way, the enzyme activity is insufficient, and self-degradation occurs in the solution, which makes the reaction difficult to control, and the catalytic efficiency decreases, which is not conducive to the purification of the product and affects the extraction of heparin sodium.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Take 0.2 g of trypsin, 5 g of silica with a particle size of 300 nanometers, add phosphate buffer solution with a pH of 7.5 (the configuration uses: take 1.36 g of potassium dihydrogen phosphate, add 79 ml of 0.1 mol / L sodium hydroxide solution, and dilute with water to 200ml) 100ml, stirred at room temperature (magnetic stirrer, stirring speed 500 rpm) for adsorption for 4 hours, repeated contact with trypsin and adsorbent, and then centrifuged at room temperature and 5000 rpm for 8 minutes, Discard the supernatant and take 20 g of the precipitate (i.e. immobilized enzyme) for later use.

[0035] Take 72 grams of porcine small intestinal mucosa, homogenate (at room temperature, put the washed small intestinal mucosa in 100ml distilled water, 10000 rpm homogenate for 3 minutes, centrifuge, take the precipitate), in 100ml, pH8.7 boric acid buffer Medium (the configuration adopts: take 0.05mol / L (19.07g / L) borax solution 60ml, 0.2mol / L (12.37g / L) boric acid solution 40ml,...

Embodiment 2

[0039] Take 0.2g of trypsin, 4g of silicon dioxide with a particle size of 300nm, add phosphate buffer solution with a pH of 7.5 (the configuration uses: take 1.36g of potassium dihydrogen phosphate, add 79ml of 0.1mol / L sodium hydroxide solution, and dilute with water to 200ml) 120ml, stirred at room temperature (stirring speed 300 rpm) for adsorption for 6 hours, then centrifuged at room temperature and 8000 rpm for 10 minutes, discarded the supernatant, and took 20g of the precipitate (i.e. immobilized enzyme) spare.

[0040] Take 90 grams of porcine small intestinal mucosa, homogenate (at room temperature, put the washed small intestinal mucosa in 200ml distilled water, homogenate for 5 minutes at 6000 rpm, centrifuge, and take the precipitate), in 150ml, pH8.7 boric acid buffer solution (Its configuration adopts: get 0.05mol / L (19.07g / L) borax solution 60ml, 0.2mol / L (12.37g / L) boric acid solution 40ml mix and shake, to obtain final product.

[0041] Add 9 grams of the i...

Embodiment 3

[0044] Take 0.15 g of heparin sodium enzyme, 10 g of silicon dioxide with a particle size of 200 nanometers, add a phosphate buffer solution with a pH of 7.5 (the configuration uses: take 1.36 g of potassium dihydrogen phosphate, add 79 ml of 0.1 mol / L sodium hydroxide solution, and add water Dilute to 200ml) 80ml, stir at room temperature (stirring speed 500 rpm) for adsorption for 2 hours, then centrifuge at room temperature and 4000 rpm for 15 minutes, discard the supernatant, and take the precipitate (i.e. immobilized enzyme) About 15g for spare.

[0045]Take 50 grams of porcine small intestinal mucosa, homogenate (at room temperature, put the washed small intestinal mucosa in 150ml distilled water, homogenate for 2 minutes at 12000 rpm, centrifuge, and take the precipitate), and put it in pH8.7 boric acid buffer ( Its configuration adopts: take 60ml of 0.05mol / L (19.07g / L) borax solution, 40ml of 0.2mol / L (12.37g / L) boric acid solution, mix and shake well to obtain).

[...

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Abstract

The invention provides a method of extracting heparin sodium by using immobilized enzyme. The method comprises the following steps: first, adding a silicon dioxide adsorbent into protease under the presence of a phosphate buffer with the pH of 7.5-8.5 to adsorb, and centrifugalizing to obtain an immobilized enzyme precipitate; carrying out enzymolysis on the mucous membrane of small intestine after homogenization under the presence of a phosphate buffer with the pH of 8-9 by adopting the immobilized enzyme, filtering, taking the filtrate which is centrifugalized, and separating to obtain an enzymatic hydrolysate and a precipitate; finally, purifying the enzymatic hydrolysate. According to the method provided by the invention, as protease does not take part in the chemical reaction in the treatment of the immobilized enzyme, the integral structure of the protease is remained unchanged, and the catalytic activity of the enzyme is better remained. By adopting the enzymolysis treatment provided by the invention, the protease is not removed as an impurity, so that the content of proteins in heparin sodium is effectively reduced, thereby improving the purity of heparin sodium.

Description

technical field [0001] The invention relates to a method for extracting heparin sodium by enzymolysis of small intestine, in particular to a method for extracting heparin sodium by using immobilized enzyme. Background technique [0002] At present, heparin sodium is the most effective and clinically used anticoagulant drug in the world. It is mainly used in the treatment of cardiovascular and cerebrovascular diseases and hemodialysis. Among them, it is the only effective specific drug in hemodialysis treatment. Clinical application and research have shown that in addition to its anticoagulant effect, heparin sodium also has a variety of other biological activities and clinical uses, including lowering blood lipids, anti-proliferation of smooth muscle cells (SMC) in the media, and promoting fibrinolysis. In addition, low-molecular-weight heparin sodium is a large class of antithrombotic drugs that are further processed from heparin sodium raw materials. The drug of choice fo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/26
Inventor 吴飞虹
Owner PUJIANG CAREX BIOTECH
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