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Antiplasmin of Verticillium lecanii, preparation method and usage

A technology of Verticillium and plasmin, applied in the directions of enzymes, bacteria, biochemical equipment and methods, etc., can solve problems such as no reports, and achieve the effects of convenient industrial production, good fibrinolytic activity, and wide pH stability.

Inactive Publication Date: 2005-01-12
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there are some reports on the isolation of plasmin from microorganisms, including Bacillus sp., Streptomyces, Bacillus amyloliquefaciens, Plerotus otreatus and Flammulina velutipes, etc. However, , using Verticillium, especially plant endophytes to isolate plasmin, so far there is no report

Method used

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  • Antiplasmin of Verticillium lecanii, preparation method and usage
  • Antiplasmin of Verticillium lecanii, preparation method and usage
  • Antiplasmin of Verticillium lecanii, preparation method and usage

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1. The preparation of verticillium plasmin:

[0024] (1) fermentation

[0025] The endophytic fungus Verticillium sp. from Trachelospermum jasminoides was cultured on potato-dextrose (PDA) medium at 25-29°C for about 3 days, and then inoculated in Chase medium (sucrose 1- 3%, NaNO 3 0.1-0.3%, KCl 0.01-0.05%, MgSO 4 0.01-0.05%, FeSO 4 0.0005-0.001%, K 2 HPO 4 0.01-0.03%, yeast extract 0.1-0.5%), cultured for 4 days, then replanted in the same medium, cultured at 25-29°C for 7 days, centrifuged or filtered the culture to obtain the fermentation broth.

[0026] (2) Salting out

[0027] Add 600g (NH 4 ) 2 SO 4 Stir to dissolve to 90% saturation. Place in the refrigerator at 4°C overnight, then centrifuge at 7500rpm at 4°C for 15min to collect the precipitate.

[0028] (3) Desalination

[0029] After dissolving the precipitate obtained in step (2), centrifuge at 10,000 rpm at 4° C. for 15 min, take the supernatant, and dialyze with Tris-HCl buffer ...

Embodiment 2

[0035] Embodiment 2. The preparation of verticillium plasmin:

[0036] Except that step (5) is different from embodiment 1, other steps are the same as embodiment 1. Step (5) uses the following method instead:

[0037] Dialyze 10 mL of the crude enzyme solution against phosphate buffer, then use CM-cellulose ion exchange chromatography, perform gradient elution with phosphate buffer containing 0-0.6M NaCl, detect with 280 nm ultraviolet light, and collect the active peak.

Embodiment 3

[0038] Embodiment 3. The preparation of verticillium plasmin:

[0039] Steps (1) and (2) are the same as in Example 1.

[0040] (3) After dissolving the precipitate obtained in step (2) with Tris-HCl buffer solution, centrifuge at 10000rpm at 4°C for 15min, take the supernatant, and conduct preliminary separation with SephadexG-100 column (2.6×100cm) chromatography, and use ultraviolet spectrophotometry Measure the absorbance at 280nm, collect the active peak, and concentrate by ultrafiltration.

[0041](4) The eluate containing the active part obtained in step (3) is chromatographed on DEAE-cellulose, eluted with Tris-HCl buffer, detected by 280nm ultraviolet light, and the active peak is collected.

[0042] (5) ultrafiltering and concentrating (concentrating to 1 / 10 of the original volume) the eluent containing the active part obtained in step (4) to obtain the crude enzyme of Verticillium plasmin.

[0043] (6) Dialyze 10 mL of the crude enzyme solution with phosphate buff...

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Abstract

A verticillium plasminogen is a fermentation liquid to be salted out and deposited to be desalinated by dialysis then to be chromatographed by DEAE and active peaks are collected to get rough enzymes after frozen and dried to be prepared with electrophoresis to be washed and dewateres to and get pare verticillium plasminogen with the molecular weight of 33KD, isoelectric point of 8.5 which has the fibrinolysis activation directly degradating fibrin and fine thrombus solving action.

Description

1. Technical field [0001] The invention relates to a thrombolytic fibrinolytic enzyme capable of degrading fibrin, which can be used for treating thromboembolic diseases, prepared from fermented liquor of endophytic fungus Verticillium verticillium of stromatolites——Verticillium fibrinolytic enzyme. 2. Background technology [0002] Thromboembolic disease is currently one of the diseases that endanger human health and has the highest morbidity and mortality. At present, clinical treatment mostly uses thrombolytic drugs such as urokinase and streptokinase. However, streptokinase has a strong antigenicity. Although urokinase has a small antigenicity, its half-life in the body is short, and long-term large-scale administration may cause systemic hemorrhage. There are also antithrombotic enzymes from Agkistrodon halys in my country, oral lumbrokinase and hirudin, etc., but the application effects are not very good, accompanied by adverse immune reactions such as fever, headache...

Claims

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Application Information

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IPC IPC(8): A61K38/49A61P7/02C12N1/20C12N9/68C12P21/02
Inventor 谭仁祥李英双惊雷黄午阳袁伟伟
Owner NANJING UNIV
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