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A method for separating pigment molecules in hirudin fermentation broth

A technology of hirudin and fermented liquid, which is applied in the field of separation and purification of biomedicine to achieve the effect of simple operation and short separation time

Active Publication Date: 2020-11-17
宁波博睿修存生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for rapidly separating the pigment molecules in the hirudin fermentation broth in view of the problems in the existing separation technology, using hydrophobic interaction chromatography to separate and purify the hirudin protein, and to improve the recovery rate of the hirudin protein

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  • A method for separating pigment molecules in hirudin fermentation broth

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] 100L tank high-density fermentation, centrifuged to obtain hirudin fermentation broth volume 35L, secreted hirudin expression: 1.2mg / ml, protein activity: 16800IU / ml.

[0029] Pretreatment of hydrophobic chromatographic packing: take 3000g of hydrophobic chromatographic packing, particle size of hydrophobic chromatographic packing is 20μm, pore size Put it in a 15000ml container, add 15000ml of 95% ethanol and shake it evenly, put it in an ultrasonic cleaner and vibrate for 15min, add it into a Buchner funnel and filter to remove ethanol, rinse with 15000ml of distilled water, and then rinse with 40mmol / L PB with a pH of 4.8 until The pH of the effluent was 4.8, drained and stored at 4°C for later use. Rinse with 6L of equilibrium solution before use, and then drain it and use it directly for subsequent adsorption.

[0030] Get 2800g of pretreated hydrophobic chromatographic filler (dry weight), put it in the centrifuged hirudin fermentation broth (add 4mol / L sodium c...

Embodiment 2

[0036] High-density fermentation in 100L tank, centrifuged to obtain hirudin fermentation broth volume 38L, secreted hirudin expression: 1.15mg / ml, activity: 16100IU / ml.

[0037] Pretreatment of hydrophobic chromatographic packing: take 3100g of hydrophobic chromatographic packing, particle size of hydrophobic chromatographic packing is 20μm, pore size Put it in a 15000ml container, add 15000ml of 95% ethanol and shake it evenly, place it in an ultrasonic cleaner and vibrate for 20min, add it into a Buchner funnel and filter to remove ethanol, rinse with 15000ml of distilled water, and then rinse with 60mmol / L PB with a pH of 5.2 until The pH of the effluent was 5.2, drained and stored at 6°C for later use. Rinse with 6L of equilibrium solution before use, and then drain it and use it directly for subsequent adsorption.

[0038] Get 3000g of pretreated hydrophobic chromatographic filler (dry weight), put it in the centrifuged hirudin fermentation broth (add 3mol / L sodium chl...

Embodiment 3

[0044] 100L tank high-density fermentation, centrifuged to obtain hirudin fermentation broth volume 40L, secreted hirudin expression: 1.08mg / ml, protein activity: 15800IU / ml.

[0045] Pretreatment of hydrophobic chromatographic packing: take 3200g of hydrophobic chromatographic packing, particle size of hydrophobic chromatographic packing is 20μm, pore size Put it in a 15000ml container, add 15000ml of 95% ethanol to shake well, put it in an ultrasonic cleaner and vibrate for 20min, add it into a Buchner funnel and filter to remove ethanol, rinse with 15000ml of distilled water, and then rinse with 50mmol / L PB with a pH of 5.0 to The pH of the effluent was 5.0, drained and stored at 4°C for later use. Rinse with 6L of equilibrium solution before use, and then drain it and use it directly for subsequent adsorption.

[0046] Get 3100g of pretreated hydrophobic chromatographic filler (dry weight), put it in the centrifuged hirudin fermentation broth (add 2mol / L sodium chloride ...

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Abstract

The invention relates to a method for separating pigment molecules from hirudin fermentation broth and belongs to separation and purification of biological medicine. The method comprises steps as follows: a hydrophobic chromatographic packing material is added to the hirudin fermentation broth for adsorption, and after adsorbed precipitates are eluted, hirudin protein of separated pigment molecules is obtained. The whole adsorption and elution process is simple to operate, a certain purification effect is achieved while pigment is separated, and the recovery rate of the hirudin protein can reach 70%.

Description

technical field [0001] The invention relates to a method for separating pigment molecules in hirudin fermentation liquid, which belongs to the separation and purification of biomedicine. Background technique [0002] In 1884, a strong anticoagulant substance was found in the medical leech (hirudo meduimalis), which was named hirudin Hir. After 1950, hirudin with biological activity was isolated from the salivary glands of medical leeches. It was determined in the early seventies that hirudin is a specific inhibitor of thrombin. In the 1980s, the amino acid sequence of hirudin was determined, and a series of studies were carried out on its structural relationship and its biological mechanism. Hirudin is a family composed of multiple isomers, and there are three common types: HV-1, HV-2 and HV-3 have a high degree of homology between isomers, and are all single-chain polypeptides composed of 65 amino acids, with a molecular weight of about 7000Da. Analysis of amino acid comp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/815C07K1/22C07K1/20
CPCC07K14/815
Inventor 朱文瑾陈平李浛民
Owner 宁波博睿修存生物科技有限公司
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