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Preparation method of actinomycetes fibrinolytic enzyme

A technology of fibrinolytic enzymes and actinomycetes, applied in the biological field, can solve the problems of reduced fibrinolytic enzyme activity, reduced fibrinolytic effect, and many separation and purification steps, so as to protect catalytic activity and stability, reduce losses, and process The effect of simple process

Inactive Publication Date: 2016-01-06
QIQIHAR UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The applicant has been exploring the application potential of microorganisms in the preparation of modern biothrombolytic agents. In 2006 and 2010, two microbial fibrinolytic enzymes with patent application numbers 200610163497.6 and 200810137564.4 were successfully cultivated. The strains used in the cultivation of plasmin are all fungi, and there are more fungal fermentation metabolites, which lead to more subsequent separation and purification steps of plasmin. Too many separation and purification steps will reduce the activity of plasmin and lead to fibrinolytic effect Therefore, for many years, the applicant has been trying to find an excellent enzyme-producing strain with fewer types of fermentation metabolites and easy product separation and purification.

Method used

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  • Preparation method of actinomycetes fibrinolytic enzyme
  • Preparation method of actinomycetes fibrinolytic enzyme
  • Preparation method of actinomycetes fibrinolytic enzyme

Examples

Experimental program
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Effect test

Embodiment 1

[0022] 1. Landscape medium: 0.4-0.5%glucose, 0.3-0.6%yeast paste, 0.5-1.5%malt extract, 0.1-0.2%of calcium carbonate, 1.5%agar, pH6.7.

[0023] Crowding conditions: 4-7D cultivation at 28 ℃.

[0024] Seed medium: 0.4-0.5%glucose, 0.3-0.6%yeast paste, 0.5-1.5%malt extract, 0.1-0.2%of calcium carbonate, adjustment pH to 6.7, sterilization after sterilization.

[0025] Cultivation conditions: The speed is 170-190r / min, and 28 ° C is erected 28-32h as liquid seeds.

[0026] Fermentation medium: 6-8%of millet powder, 2-4%glucose, 0.1-0.3%calcium carbonate, 0.4-0.6%sodium chloride, 0.6-0.7%protein, pH6.7, 5%inoculation amount.

[0027] Cultivation conditions: 150-170r / min, 22 ° C fermentation 90-100h.

[0028] The pine bacteria YY21 liquid fermentation product is 10000R / min 20min under 4 ° C, obtained the upper liquid, and the ostelase ratio is 12.01mg / ml.

[0029] Preparation of picked bacteria fibrobine liquid: Add ammonium sulfate powder to the fermented liquid containing moltencease...

Embodiment 2

[0031] 1. Landscape training with the same embodiment 1

[0032] 2. Seed training and embodiment 1

[0033] 3. Ferment training and embodiment 1

[0034] 4. Preparation and embodiments of the compound enzyme liquid of the lines of the lines 1

[0035] 5. Octyl-sephaaroseff interaction method of hydrophobic interaction

[0036] Add ammonium sulfate powder to the crude enzyme solution to fully dissolved, so that the ammonium sulfate saturation can reach 30 %.Collect fibrous activity components and obtain a compounding ratio of 112.07mg / ml.

Embodiment 3

[0038] 1. Landscape training with the same embodiment 1

[0039] 2. Seed training and embodiment 1

[0040] 3. Ferment training and embodiment 1

[0041] 4. Preparation and embodiments of the compound enzyme liquid of the lines of the lines 1

[0042] 5. Octyl-sephaaroseff interaction layer analysis method is the same as embodiment 2

[0043] 6. Phenyl-sephaarosehp hydrophobic interaction layer analysis method

[0044] Adjust the OCTYL-sephaaroseff interaction layer to collect the ammonium sulfate of the active component to 15 %, and perform Phenyl-SEPHAROSEHP hydrophobic interaction layer.The collection of fibrous activity components is collected, and the enzyme ratio is 173.16U / mg, and the vitality recovery rate is 19.35%.

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Abstract

The invention relates to a preparation method of an actinomycetes fibrinolytic enzyme. Specifically, the method comprises the following steps: (1) adding ammonium sulfate powder to a fermentation supernatant containing actinomycetes fibrinolytic enzyme until a saturation degree is 25-35%W / V, standing by for a whole night at 4 DEG C and centrifuging so as to obtain a supernatant, adding ammonium sulfate powder to the supernatant once again until a saturation degree is 55-65%W / V, standing by for a whole night at 4 DEG C, centrifuging so as to obtain a precipitate, and dissolving the precipitate by virtue of a PB buffer solution so as to obtain crude enzyme liquid; and (2) centrifuging the crude enzyme liquid obtained from the step (1) at low temperature so as to remove impurities; purifying an obtained supernatant subsequently through Octyl-Sepharose FF hydrophobic interaction chromatography and Phenyl-Sepharose HP hydrophobic interaction chromatography so as to obtain a chromatographically pure enzyme; dialyzing and desalting the enzyme, and freeze-drying the enzyme so as to obtain fibrinolytic enzyme freeze-dried powder which can be preserved for a long time at minus 20 DEG C. The separation and purification method of the actinomycetes fibrinolytic enzyme disclosed by the invention has the advantages of simple operation steps and high recovery rate.

Description

Technical field [0001] The present invention involves a preparation method for plane in the lines of lines, which belongs to the field of biotechnology. Background technique [0002] Thromacbolic diseases are seriously harmful to human life and health. The thrombolysis is currently the main means to treat and prevent thrombosis diseases. New thrombolysis drugs that develop high -efficiency, fast, prevent re -embolism and reduce bleeding are modern medicine.urgent need. [0003] Alasses have the function of dissolving blood fiber protein, and blood fiber protein is the main component of thrombosis.The use of microbial fermentation to produce fibrobine iconic has a short production cycle, small area, and high product content.Therefore, the development of high -efficiency and specific microbioplasics is the main way to produce new thrombolytic drugs. [0004] This applicant has been exploring the application potential of microorganisms in the preparation of modern biological thrombo...

Claims

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Application Information

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IPC IPC(8): C12N9/68C12R1/01
CPCC12N9/52C12Y304/21007
Inventor 邓永平刘晓兰郑喜群艾瑞波
Owner QIQIHAR UNIVERSITY
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