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Producing new fibrinolysin from rhizopchin

A production method and technology of plasminase, which is applied in the field of production of new-type plasminase by Rhizopus sinensis, can solve the problems of high price, bleeding complications, non-antigenicity, etc.

Inactive Publication Date: 2005-03-02
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it can only recanalize 55% of patients with coronary artery blockage. The thrombolysis time is longer, the dosage is large, and the price is expensive: APSAC causes less bleeding adverse reactions, but because it contains SK, it is antigenic and can cause allergic reactions; scu-PA has a strong thrombolytic effect, has no antigenicity, and has no allergic reaction, but it can cause bleeding complications

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1. Strain: Rhizopus sinica TK317

[0037] 2. Culture medium and culture method: Glucose soybean juice medium is used for slant preservation, and the slant bacteria are cultured at 28-30°C for 5 days;

[0038] The medium used for body fermentation contains 5% bran water, 6% soybean meal, 0.1% ammonium chloride, 0.2% sodium nitrate and 1% to 2.5% trypsin

[0039] Peptone, pH 4.5, inoculum size 10%-20%, shake culture at 28-30°C for 60-72h.

[0040] 3. Rough purification of enzyme: the obtained fibrinolytic enzyme fermentation liquid is micro-filtered to remove bacteria and solids; use D301 anion exchange resin to

[0041] The liquid containing plasmin is decolorized; ammonium sulfate with 30% to 70% saturation is used for salting out step by step, and the precipitate is collected.

[0042] 4. Fine purification of enzymes: sequential ultrafiltration to remove salt and some impurity proteins; use sulfonic acid propyl as the ligand for ion exchange chromatography, ...

Embodiment 2

[0045] Culture medium and culture method: Glucose soybean juice medium is used for slant preservation, and the slant bacteria are cultured at 28-30°C for 5 days; liquid fermentation medium contains 5.6% bran water and 5.3% soybean meal hydrolyzate (hydrolyzed with 0.1N HCl in advance) , 1% to 2.5% tryptone, inoculum size 10% to 20%, cultured with shaking at 28 to 30°C for 60 to 72 hours. D296 anion-exchange resin was used for decolorization of plasmin-containing solution, and plasmin was precipitated with 65% ethanol. Other steps are the same as in Example 1.

Embodiment 3

[0047] The medium and culture method steps are the same as in Example 1. The purified enzyme was used to determine the N-terminal amino acid residue sequence of the protein, and the following results were obtained:

[0048] NH 2 -Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly was determined to have a molecular weight of 30.5 kDa; the isoelectric point was 8.5. Low concentration of Cu' 2+ 、Co 2+ 、K + , Fe 3+ , Fe 2+ , Ca 2+ ,, Mg 2+ ,, Mn2+ 、Na + , Zn 2+ Does not affect the activity of thrombolytic enzymes.

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PUM

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Abstract

The present invention relates to the extraction of fibrinolysin as new type of thromboliytic medicine from rhizopchin. The fibrinolysin has molecular weight 30.5 KD, isoelectric point 8.5, and double effects of directly dissolving thrombus and activating human plasminogen. The production process includes three stages, of culture of rhizopchin including slant culture, seed culture and fermentation culture; and separating and purifying fermented liquid to obtain fibrinolysin. The product is safe, non-toxic and cheap and the production process uses cheap agricultural side product as main material and is a liquid fermentation process. The purification process includes microfiltering to eliminate thallus, decolorizing with ion exchange resin, salting out, ultrafiltering to eliminate salt and hybrid protein and ion exchanging to purify. The present invention has short technological process, low cost and high production efficiency.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceutical production, and in particular relates to a novel plasminase produced by rhizopus sinensis and a method thereof. Background technique [0002] Cardiovascular and cerebrovascular diseases, such as acute myocardial infarction and cerebral thrombosis, are one of the three major diseases that cause human death, and their incidence rates are increasing. The main component of thrombus is fibrin. Thrombolytic therapy is to dissolve the main matrix fibrin in thrombus by drugs, or activate the inactive plasma plasminogen in the blood to form active plasmin to catalyze the formation of fibrin. The hydrolysis of the thrombus dissolves and recanalizes blood vessels. Thrombolytic therapy is known as one of the top ten discoveries in cardiovascular and cerebrovascular sciences in the 20th century, and has been highly valued by various countries. The development of high...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N9/68C12P21/02
Inventor 路福平杜连祥蔡兴旺张清忠刘晓兰迟文鹤
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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