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A method for concentrating hirudin in fermented liquid

A technology of hirudin and fermentation broth, applied in the field of biomedicine, can solve the problems of high concentration cost, large consumption of ultrafiltration membrane, low protein recovery rate and the like, and achieve the effects of short concentration time, simple operation and cost reduction.

Active Publication Date: 2020-10-09
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

Traditionally concentrated hirudin mostly adopts ultrafiltration membranes such as ceramic membranes and cellulose membranes to concentrate. This kind of concentration technology has the following difficulties: 1. After ultrafiltration and concentration, the hirudin active protein recovery rate is about 40%, and the protein recovery rate is relatively low. 2. Because protein degrading enzymes will be produced in the fermentation process, and the degrading enzymes will hydrolyze the hirudin activity, the ultrafiltration concentration time is too long, which is not conducive to the stability of the hirudin activity, and the consumption of the ultrafiltration membrane is large, and the concentration cost high

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] 100L tank high-density fermentation, centrifuged to obtain hirudin fermentation broth volume 35L, secreted hirudin expression: 1.2mg / ml, protein activity: 16800IU / ml.

[0027] Pretreatment of reversed-phase chromatographic packing: take 2500 g of reversed-phase chromatographic packing, particle size 20 μm, pore size Put it in a 15000m1 container, add 7500ml of 95% ethanol and shake well, put it in an ultrasonic cleaner and vibrate for 15min, put it into a Buchner funnel and filter it to remove ethanol, rinse it with 10000ml of distilled water and 10000ml of distilled water with a pH of 2.8, drain it and store it at 4°C for later use.

[0028] Take 2100 g of the pretreated reversed-phase chromatographic filler (dry weight), put it in the centrifuged hirudin fermentation broth, and stir it magnetically for 20 min. After standing still for 1.5 hours, the reversed-phase chromatography packing completely settled, and the supernatant was removed by siphoning, and the precipi...

Embodiment 2

[0033] High-density fermentation in 100L tank, centrifuged to obtain hirudin fermentation broth volume 38L, secreted hirudin expression: 1.18mg / ml, activity: 16100IU / ml.

[0034] Pretreatment of reversed-phase chromatographic packing: take 2500 g of reversed-phase chromatographic packing, particle size 20 μm, pore size Put it in a 15000m1 container, add 7500ml of 93% ethanol, 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 10000ml of distilled water and 10000ml of distilled water with a pH of 3.2, and then drain and store at 5°C for later use.

[0035]Take 2300g of the pretreated reversed-phase chromatographic filler (dry weight), put it in the centrifuged hirudin fermentation broth, and stir it magnetically for 15 minutes. After standing still for 2 hours, the reversed-phase chromatography packing completely settled, and the supernatant was removed by siphoning, and the precipitate was s...

Embodiment 3

[0040] 100L tank high-density fermentation, centrifuged to obtain hirudin fermentation broth volume 40L, secreted hirudin expression: 1.08mg / ml, protein activity: 15800IU / ml.

[0041] Pretreatment of reversed-phase chromatographic packing: take 2500 g of reversed-phase chromatographic packing, particle size 20 μm, pore size Put it in a 15000m1 container, add 7500ml of 96% ethanol to shake well, put it in an ultrasonic cleaner and vibrate for 20min, put it into a Buchner funnel and filter to remove ethanol, rinse with 10000ml of distilled water and 10000ml of distilled water with a pH of 3.0, and then drain and store at 6°C for later use.

[0042] Take 2400g of the pretreated reversed-phase chromatographic filler (dry weight), put it in the centrifuged hirudin fermentation broth, and stir it magnetically for 20 minutes. After standing still for 2 hours, the reversed-phase chromatography packing completely settled, and the supernatant was removed by siphoning, and the precipita...

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Abstract

The invention relates to a method for concentrating hirudin in fermentation broth and belongs to the technical field of biological medicines. The method comprises the steps of adding reversed-phase chromatography filler in hirudin fermentation broth for adsorption, performing eluting adsorption precipitation, and collecting effluent, namely hirudin concentrated solution, wherein the reversed-phase chromatography filler is formed by bonding hydrophobic groups to the surface of silica gel substrate. The whole process of adsorption and elution is simple to operate, a purification effect is also achieved while concentration is performed, and the hirudin egg white recovery rate is 70% or above.

Description

technical field [0001] The invention relates to a method for concentrating hirudin in fermented liquid, belonging to the technical field 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 composition shows that hirudin molecul...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/815C07K1/20
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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