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Bacillus licheniformis fibrinolytic enzyme and production method thereof

A Bacillus licheniformis and production method technology, applied in the field of bioengineering, can solve the problems of low enzyme production level, high price, and defects in enzymatic properties, and achieve the effects of short process flow, high production efficiency, and high enzyme activity

Inactive Publication Date: 2011-06-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with the first-generation thrombolytic agents, tissue plasminogen activator (tPA) and prourokinase (proUK) have improved fibrin affinity, but the half-life is short, the dosage is large, Expensive and can still cause side effects of internal bleeding when taken orally
[0006] At present, microorganisms are mainly used to produce plasmin, but the existing plasmin-producing bacteria have problems such as generally low enzyme production levels, enzymatic property defects and safety.

Method used

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  • Bacillus licheniformis fibrinolytic enzyme and production method thereof
  • Bacillus licheniformis fibrinolytic enzyme and production method thereof
  • Bacillus licheniformis fibrinolytic enzyme and production method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Medium and culture method:

[0047] The strains were stored on a slant in LB medium. Firstly, the strains were cultured at 37°C for 24 hours, and then inoculated into the liquid fermentation medium.

[0048] The liquid fermentation medium contains 2% glucose, 2% yeast extract, 10% soybean juice (preparation method: soak 10% soybeans for 24 hours, simmer for 30 minutes, and extract the juice), KH 2 PO 4 1%, K 2 HPO 4 0.5%, MgSO 4 0.45%, pH natural.

[0049] The inoculum size is 1%, the fermentation temperature is 37°C, the shaker speed is 140r / min, and the maximum enzyme activity obtained is 320IU / ml after culturing for 38 hours.

[0050] Rough purification of enzymes: The fermentation broth was subjected to high-speed refrigerated centrifugation at a speed of 12,000r / min and a temperature of 4°C to remove the bacteria to obtain a supernatant, which was decolorized by a gel column Sephadex G-50 and then used in mass percent concentration Carry out segmental salti...

Embodiment 2

[0054] The strains were stored on a slant in LB medium. Firstly, the strains were cultured at 37°C for 24 hours, and then inoculated into the liquid fermentation medium.

[0055] The liquid fermentation medium contains 2% bran, 2% yeast extract, 10% soybean juice (preparation method: soak 10% soybeans for 24 hours, simmer for 30 minutes, filter to obtain juice), KH 2 PO 4 1%, K 2 HPO 4 0.5%, MgSO 4 0.45%, pH natural. The inoculation amount is 1%, the fermentation temperature is 37° C., the shaking table rotation speed is 120 r / min, and the maximum enzyme activity obtained is 510 IU / ml after culturing for 72 hours.

[0056] According to the rough purification of enzyme in embodiment 1 and the fine purification method of enzyme, obtain the higher bacillus licheniformis plasmin of enzymatic activity, through SDS-PAGE analysis, its molecular size is 30KD; The specific activity of this enzyme is 52000IU / mg protein.

Embodiment 3

[0058] The strains were stored on a slant in LB medium. Firstly, the strains were cultured at 37°C for 24 hours, and then inoculated into the liquid fermentation medium.

[0059] The liquid fermentation medium contains 4% bran, 2% yeast extract, 10% soybean juice (preparation method: soak 10% soybeans for 24 hours, simmer for 30 minutes, filter to obtain juice), CaCl 2 0.1%, KH 2 PO 4 1%, K 2 HPO 4 0.5%, MgSO 4 0.45%, pH natural. The inoculum size is 1%, the fermentation temperature is 37°C, the shaking table rotation speed is 140r / min, and the maximum enzyme activity obtained is 590IU / ml after culturing for 72 hours.

[0060] According to the rough purification of the enzyme in Example 1 and the refined purification method of the enzyme, the higher Bacillus licheniformis plasmin of enzyme activity is obtained, and through SDS-PAGE analysis, its molecular size is 30KD; Finally freeze-drying obtains the higher enzyme activity eluent samples. The specific activity of ...

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Abstract

The invention discloses a bacillus licheniformis fibrinolytic enzyme produced by the method: bacillus licheniformis ZJUEL31410 CGMCC 1397 is fermented and cultured to get fermented liquid, then separation and purification are carried out to the fermented liquid to get the bacillus licheniformis fibrinolytic enzyme. The invention firstly discovers that the bacillus licheniformis ZJUEL31410 CGMCC 1397 can be used for producing fibrinolytic enzyme. The bacillus licheniformis fibrinolytic enzyme products produced by the invention take cheap agricultural by-products of soybean pulp, expanded soybeans and bran as raw materials, thereby being nontoxic. Besides, a liquid fermentation method is adopted in the production, resulting in short technique process, low cost, high industrial production efficiency and high activity of enzyme, thereby being with development value.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a bacillus licheniformis fibrinolytic enzyme and a production method thereof. Background technique [0002] Thromboembolic disease is a type of cardiovascular disease that seriously endangers human health, and its incidence is on the rise. Currently, the most effective and reliable means of treating thromboembolic diseases is thrombolytic therapy using fibrinolytic enzymes or their activators. Thrombolytic drugs may be converted into fibrinolytic enzymes by activating plasminogen, which catalyzes the hydrolysis of fibrin, the main matrix of thrombus, so as to dissolve thrombus and recanalize blood vessels to achieve the purpose of thrombolysis; or directly dissolve fibrin in thrombus , to dissolve the thrombus. Thrombolytic enzymes can be derived from human body [such as urokinase (UK), tissue plasminogen activator (t-PA), etc.], animals (such as snake antithrombotic enzyme, month...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/68C12R1/10C12P21/06
Inventor 陈启和朱建良何国庆章海锋阮晖
Owner ZHEJIANG UNIV
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