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Fungal fibrinolytic enzyme and cultivating method thereof

A technology of fibrinolytic enzyme and fungus, applied in the field of cultivation of the fungal fibrinolytic enzyme, can solve the problems of limited popularization and application, limited resources, etc., and achieve the effects of good thrombolytic performance, short enzyme production cycle, and low raw material cost

Active Publication Date: 2009-08-12
QIQIHAR UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Of course, because the strain used in this project is a species that is difficult to obtain repeatedly from nature, its resources are relatively limited, and in order to keep its original excellent characteristics for a long time, it needs to be constantly improved during use. Domestication and management, which will cause certain restrictions on its large-scale promotion and application

Method used

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  • Fungal fibrinolytic enzyme and cultivating method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] 1. Strain: Cordyceps militaris strain C.LSG-1, the preservation number is CGMCC N 0 .2577

[0021] 2. Medium and culture conditions:

[0022] (1) Incline medium: glucose 1%, agar 2.5%, potato juice 20%, peptone 0.5%, KH 2 PO 4 0.1%, MgSO 4 0.05%, pH natural. Culture conditions: culture at a constant temperature of 23°C for 10 days. (Preparation of 20% potato juice: Weigh 200g of peeled potatoes, cut into pieces, add 1000ml of distilled water, boil for 30min, filter with double gauze to get the juice for later use)

[0023] (2) Plate medium: glucose 1%, agar 2.5%, potato juice 20%, peptone 0.5%, KH 2 PO 4 0.1%, MgSO 4 0.05%, pH natural. Culture conditions: culture at a constant temperature of 23°C for 10 days.

[0024] (3) Liquid fermentation medium: 1.25% sucrose, 5% zein, KH 2 PO 4 0.01%, KCl0.005%, MgSO 4 0.001%, CuSO 4 0.001%, the filling volume is 40mL / 250mL Erlenmeyer flask, and the initial pH value of the fermentation medium is natural. Liquid fer...

Embodiment 2

[0026] 1. Incline cultivation is the same as in Example 1

[0027] 2, liquid fermentation culture is the same as embodiment 1

[0028] 3. Separation and purification of cordyceps militaris fibrinolytic enzyme: the liquid fermentation product was subjected to 20% saturation ammonium sulfate salting out, Phenyl Sepharose HP hydrophobic interaction chromatography, and Superdex75 gel filtration chromatography to collect fibrinolytic active components.

Embodiment 3

[0030] Proof of fibrinolytic activity of Cordyceps militaris fibrinolytic enzyme:

[0031] The solubility of the enzyme to fibrin was tested by the fibrin plate method.

[0032] fibrin plate method

[0033] The fibrin plate contains fibrinogen (commercially available fibrinogen may contain fibrin) and thrombin. The soluble fibrinogen forms fibrin monomers under the action of thrombin, and the fibrin monomers spontaneously associate. Polymerization forms a visible fibrin gel, and the plasminase solution is added to the surface of the slab gel. After a short period of incubation, the enzyme dissolves the fibrin, forming a transparent circle visible to the naked eye on the surface of the slab gel. See figure 1 .

[0034] Bacterial strains used in the present invention can use existing commercially available products, and the high-yield fibrinolytic enzyme Cordyceps militaris bacterial strain C.LSG-1 that preferably uses through strict screening obtains, and its growth conditio...

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Abstract

The invention discloses a fungal fibrinolytic enzyme and a preparation method thereof. The fungal fibrinolytic enzyme is prepared by taking a Cordyceps militaris strain as a strain and performing liquid fermentation culture, separation and purification. The relative molecular weight of the fibrinolytic enzyme is about 21,000. The Cordyceps militaris fibrinolytic enzyme has good thrombolytic performance, is free from obvious acute toxicity, has prospects for clinical trial, development and application, and adds a new member to a rare fibrinolytic enzyme family. Particularly, a preferable fibrinolytic-enzyme Cordyceps militaris strain C.LSG-1 obtained through severe screening is rough in growth conditions, short in enzyme production cycle and capable of harvesting a large amount of Cordyceps militaris mycelium during enzyme production, and is the strain excellent in production performance. The Cordyceps militaris fibrinolytic enzyme is an extracellular enzyme which is particularly beneficial to subsequent separation and purification during preparation. The fungal fibrinolytic enzyme takes corn protein with extensive sources as a main culture medium, thereby having low cost for raw materials and providing a novel way for developing and utilizing the prior resources.

Description

technical field [0001] The invention relates to a fungal plasmin, and also relates to a method for preparing the fungal plasmin. Background technique [0002] As we all know, thrombotic diseases have been one of the most important causes of endangering human health and causing death of patients for many years. The most commonly used means of treating thrombosis in modern medicine is to rely on plasmin or its activator for thrombolysis, and the thrombolytic drugs used have been updated for three generations. At present, there are two main sources of the third-generation thrombolytic agents under development: one is to use protein engineering, such as molecular site-directed mutagenesis, deletion, addition, splicing or fusion of structural domains, to modify existing thrombolytic agents. carry out molecular modification. The second is to develop thrombus-dissolving substances derived from various organisms. At present, people have successfully extracted a variety of products...

Claims

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Application Information

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IPC IPC(8): C12N9/68C12R1/645
Inventor 刘晓兰郑喜群邓永平李巍时晰李瑶
Owner QIQIHAR UNIVERSITY
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