CN101503682A - Fungal fibrinolytic enzyme and cultivating method thereof - Google Patents
Fungal fibrinolytic enzyme and cultivating method thereof Download PDFInfo
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- CN101503682A CN101503682A CNA2008101375644A CN200810137564A CN101503682A CN 101503682 A CN101503682 A CN 101503682A CN A2008101375644 A CNA2008101375644 A CN A2008101375644A CN 200810137564 A CN200810137564 A CN 200810137564A CN 101503682 A CN101503682 A CN 101503682A
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- plasmin
- cordyceps militaris
- enzyme
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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
The present invention relates to a kind of fungi plasmin, the invention still further relates to a kind of training system method of this fungi plasmin.
Background technology
As everyone knows, thrombus disease is harm humans health for many years always, causes one of main cause of disease of death.The most frequently used means of modern medicine treatment thrombus rely on plasmin or its activator to carry out thrombolysis exactly, and employed thrombolytics medicine has now also carried out three generations's renewal.At present, its source of third generation thrombolysis medicament that is in the middle of developing mainly contains two kinds: the one, utilize protein engineering, and as the molecule rite-directed mutagenesis, means such as the deletion of structural domain, increase, splicing or fusion are carried out molecular modification to existing thrombolytics.The 2nd, exploitation derives from the thrombolysis material of various biologies, and people have successfully extracted multiple product with fibrinolytic from snake venom, earthworm, microorganism and hematophagous bug and marine organisms at present.The applicant has successfully cultivated a kind of number of patent application in 2006 be 200610163497.6 biological plasmin, it is to separate, screen acquisition in March, 1999 by the applicant in China's northern sauce class fermentation intermediate that this plasmin is cultivated employed bacterial classification, and providing preservation to China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on October 12nd, 2006, preserving number is the bacterial strain of CGMCC No.1836.Confirm that by multinomial conscientious research trial this plasmin is solution fibrin and the indirect solution fibrin of plasminogen activation directly, and shows good and stable enzyme activity, has very big technology and market development potential.Certainly, because being a kind of being difficult to, bacterial classification that this project is used repeats to obtain kind from occurring in nature, therefore its resource is relatively limited, and in order to make it can keep original good characteristic for a long time, in use also need it is constantly tamed and manages, this applies all in large area to it can cause certain restriction.Therefore the applicant is striving to find some similar bacterial classifications in the easier acquisition of occurring in nature always in recent years, and the present invention is exactly the partial content of the research progress.
Summary of the invention
Goal of the invention of the present invention is to provide a kind of fungi plasmin, and this plasmin has good dissolving fibrinogen performance.Another goal of the invention of the present invention also is to provide a kind of training system method of this plasmin.
Product Cordyceps militaris (L.) Link. plasmin of the present invention employed bacterial classification in culturing process is a Cordyceps militaris (L.) Link., wherein preferred bacterial classification is the C.LSG-1 bacterial strain, this bacterial strain provides culture presevation on July 4th, 2008 to China Committee for Culture Collection of Microorganisms common micro-organisms center by the applicant, and preserving number is CGMCC N
O.2577.Its taxonomy called after Cordyceps militaris.
Cordyceps militaris (L.) Link. is China be otherwise known as Cordyceps militaris (L.) Link. or Wu Shi Chinese caterpillar fungus, similar with Cordyceps sinensis, be a kind of very precious Chinese medicine and high tonic, have the raising immunologic function, protect liver clearing lung-heat, relieving cough and reducing sputum, antifatigue, anti-hypoxia, effect such as antiviral, have important medical value.Because the relative Cordyceps sinensis of its resource of Cordyceps militaris (L.) Link. is abundant, add the artificial culture of part, be widely used as the surrogate of traditional rare Chinese medicine Cordyceps sinensis.Cordyceps militaris (L.) Link. has caused quite a lot of people's very big concern as a kind of medicine, food dual-purpose bacterium of preciousness in recent years, and relevant exploitation report is also quite a lot of to be seen.The major progress in this field at present has three aspects, and the one, there is a large amount of application products continually developing, wherein see so that nutrient health more, also comprise a spot of therapeutic pharmaceuticals.The 2nd, cultivate raising technology research constantly, its core purpose is for abundant and expansion resource.The 3rd, important component is extracted the starting of research, is mainly used in scientific research and medical fundamental research.From present related technique means, though it is enormously potential to utilize this raw material to adopt microbiology fermentation process to obtain newtype drug aspect, the progress that has obtained is relatively very little.
Product of the present invention is to be bacterial classification with the cordyceps militaris link bacterial strain, and through slant culture, dull and stereotyped cultivation, liquid fermentation and culture, separation and purification, plasmin product of the present invention is made in training at last.This Cordyceps militaris (L.) Link. plasmin enzyme molecule can directly dissolve fibrinogen, and relative molecular mass is about 21000, and pH can preserve vigor preferably when being neutral.
The concrete steps of product preparation of the present invention:
1, with the cordyceps militaris link bacterial strain is bacterial classification, produces the Cordyceps militaris (L.) Link. plasmin through slant culture, dull and stereotyped cultivation, liquid fermentation and culture;
2, cultural method: slant culture → flat board cultivation → liquid fermentation and culture;
Cordyceps militaris link bacterial strain is inserted slant medium: glucose 1%, agar 2.5%, potato juice 20%, peptone 0.5%, KH
2PO
40.1%, MgSO
40.05%, pH nature, 23 ℃ of constant temperature culture 10d.Culture is transferred into 23 ℃ of constant temperature culture 10d of plate culture medium (the same slant medium of medium component).
Fermention medium contains sucrose 1.25%, zein 5%, KH
2PO
40.01%, KCl 0.005%, MgSO
40.001%, CuSO
40.001%, liquid amount is the 40mL/250mL triangular flask, the initial pH value nature of fermention medium.Liquid fermentation condition: connect bacterium amount and be 1 of the lawn disk of diameter 1cm; 21～28 ℃, the 180rpm/min fermentation got the liquid fermentation and culture thing in 5 days.
3, the Cordyceps militaris (L.) Link. plasmin of slightly purifying: with liquid fermentation and culture thing 10000rpm/min, 4 ℃, centrifugal 10min, centrifuged supernatant is crude enzyme liquid behind the ammonium sulfate precipitation of 20% saturation ratio.
4, smart purification Cordyceps militaris (L.) Link. plasmin: adopt modern chromatographic technique that crude enzyme liquid is carried out essence and purify, mainly adopt following method to obtain chromatographically pure Cordyceps militaris (L.) Link. plasmin:
A, Phenyl Sepharose HP hydrophobic interaction chromatography: adopt the gradient elution mode: sample salt loading degree is transferred to 40%, and with 40%～0 ammoniumsulphate soln gradient elution, collecting has the fibrinolytic component behind the last sample;
B, Superdex75 prepacked column gel permeation chromatography, collecting has the fibrinolytic component.
Advantage of the present invention: this Cordyceps militaris (L.) Link. plasmin has good thrombolytic performance, and does not have tangible acute toxicity, thereby has the prospect of carrying out test, Application and Development clinically, has also increased a new member for thin plasmin family.Bacterial strain preferred for this invention is the high yield plasmin cordyceps militaris link bacterial strain C.LSG-1 that the strict screening of process obtains, and growth conditions is more extensive, produces enzyme cycle weak point, can gather in the crops a large amount of Cordyceps militaris (L.) Link. fungus filaments simultaneously at the product enzyme, is the good bacterial classification of a strain production performance.Cordyceps militaris (L.) Link. plasmin of the present invention is a kind of extracellular enzyme, is particularly conducive to follow-up separation and purifying in preparation.Because product of the present invention is that zein is a main medium to originate widely, not only raw materials cost is cheap, and provides new approach for the development and use of these original resources.
Description of drawings
Fig. 1 utilizes fibrinogen albumen flat band method to confirm the picture of the molten fine effect of product plasmin of the present invention
Product Cordyceps militaris fibrinolysin of the present invention employed preferred bacterial classification C.LSG-1 bacterial strain in incubation provides culture presevation on July 4th, 2008 to China Committee for Culture Collection of Microorganisms common micro-organisms center by the applicant, and preserving number is CGMCC No.2577. Its taxology called after Cordyceps militaris.
Specific embodiment
Embodiment 1:
1, bacterial classification: cordyceps militaris link bacterial strain C.LSG-1, preserving number are CGMCC N
0.2577
2, substratum and culture condition:
(1) slant medium: glucose 1%, agar 2.5%, potato juice 20%, peptone 0.5%, KH
2PO
40.1%, MgSO
40.05%, the pH nature.Culture condition: 23 ℃ of constant temperature culture 10d.(preparation of 20% potato juice: claim peeling potato 200g, be cut into fragment, adding distil water 1000ml boils 30min, and it is standby to cross leaching juice with double gauze)
(2) plate culture medium: glucose 1%, agar 2.5%, potato juice 20%, peptone 0.5%, KH
2PO
40.1%, MgSO
40.05%, the pH nature.Culture condition: 23 ℃ of constant temperature culture 10d.
(3) liquid fermentation medium: sucrose 1.25%, zein 5%, KH
2PO
40.01%, KCl0.005%, MgSO
40.001%, CuSO
40.001%, liquid amount is the 40mL/250mL triangular flask, the initial pH value nature of fermention medium.Liquid fermentation condition: connect bacterium amount and be 1 of the lawn disk of diameter 1cm; 21～28 ℃, the 180rpm/min fermentation got the liquid fermentation and culture thing in 5 days.
Embodiment 2
1, slant culture is with embodiment 1
2, liquid fermentation and culture is with embodiment 1
3, the separation and purification of Cordyceps militaris (L.) Link. plasmin: liquid fermentation production is through 20% saturation ratio ammonium sulfate precipitation, Phenyl Sepharose HP hydrophobic interaction chromatography, Superdex75 gel permeation chromatography, and collecting has the fibrinolytic component.
Embodiment 3
Cordyceps militaris (L.) Link. plasmin fibrinolytic proves:
Adopt fibrin plate method to check the solvability of this enzyme to fibrinogen.
Fibrin plate method
Fibrin plate contains Parenogen (may contain scleroproein in the commercially available Parenogen) and zymoplasm, soluble fibrin is former to form fibrin monomer in thrombin action, fibrin monomer is spontaneous to be concluded, multimerization forms the visible fibrin gel, plasmin liquid is added to this slab gel surface, be about to fibrinolysis through this enzyme after the short period of time cultivation, form macroscopic transparent circle on the slab gel surface.See Fig. 1.
Bacterial strain uses therefor of the present invention can use existing commercially available prod, the high yield plasmin cordyceps militaris link bacterial strain C.LSG-1 that the strict screening of the preferred process of using obtains, its growth conditions is more extensive, produce enzyme cycle weak point, simultaneously can gather in the crops a large amount of Cordyceps militaris (L.) Link. fungus filaments at the product enzyme, be the good bacterial classification of a strain production performance.
Claims (6)
1, a kind of fungi plasmin, it is characterized in that: this plasmin is to cultivate bacterial classification with the cordyceps militaris link bacterial strain, makes through fermentation, separation and purification, and this Cordyceps militaris (L.) Link. plasmin enzyme molecule can directly dissolve fibrinogen, relative molecular mass is about 21000, and pH can preserve vigor preferably when being neutral.
2, a kind of fungi plasmin according to claim 1 is characterized in that: employed bacterial classification is that preserving number is CGMCC N
0.2577, the cordyceps militaris link bacterial strain C.LSG-1 of taxonomy called after Cordyceps militaris.
3, a kind of training system method of the plasmin of fungi as claimed in claim 1 or 2, it is characterized in that: this training system method may further comprise the steps: be bacterial classification with the cordyceps militaris link bacterial strain, prepare plasmin through slant culture, dull and stereotyped cultivation, liquid fermentation and culture, separation, purifying plasmin from the tunning crude enzyme liquid.
4, a kind of training system method of fungi plasmin as claimed in claim 3: it is characterized in that: described slant culture adopts following improvement glucose agar medium: glucose 1%, agar 2.5%, potato juice 20%, peptone 0.5%, KH
2PO
40.1%, MgSO
40.05%, the pH nature, 23 ℃ of constant temperature culture 10d, the same slant culture of dull and stereotyped cultivation, fermention medium contains sucrose 1.25%, zein 5%, KH
2PO
40.01%, KCl0.005%, MgSO
40.001%, CuSO
40.001%, liquid amount is the 40mL/250mL triangular flask, the initial pH value nature of fermention medium; Liquid fermentation condition: connect bacterium amount and be 1 of the lawn disk of diameter 1cm; 21～28 ℃, the 180rpm/min fermentation got the liquid fermentation and culture thing in 5 days.
5, a kind of training system method of fungi plasmin as claimed in claim 3: it is characterized in that: described crude enzyme liquid sepn process is through 20% saturation ratio ammonium sulfate precipitation with resulting filtered liquid, the centrifugal impurity of removing, obtain crude enzyme liquid, adopt modern chromatographic technique purifying plasmin.
6, a kind of training system method as fungi plasmin as described in the claim 3: it is characterized in that: described crude enzyme liquid purification process is as follows:
A, Phenyl Sepharose HP hydrophobic interaction chromatography: sample salt loading degree is transferred to 40%, and with 40～0% ammoniumsulphate soln gradient elutions, collecting has the fibrinolytic component behind the last sample;
B, Sephadex 75 gel permeation chromatographies: with pH7.4 phosphate buffered saline buffer wash-out, elutriant contains 0.3mol/LNaCl, and collecting has the fibrinolytic component.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101962637A (en) * | 2009-12-04 | 2011-02-02 | 齐齐哈尔大学 | Cordyceps militaris plasmin and culturing method thereof |
| CN102604919A (en) * | 2012-04-16 | 2012-07-25 | 山东省农业科学院农业资源与环境研究所 | Method for fermenting and producing plasmin by use of cordyceps militaris liquid |
| CN103333874A (en) * | 2013-05-02 | 2013-10-02 | 齐齐哈尔大学 | Actinomycete fibrinolytic enzyme and preparation method thereof |
| CN104911169A (en) * | 2015-04-07 | 2015-09-16 | 齐齐哈尔大学 | Method for culturing cordyceps militaris mycelium and plasmin |
| CN105219754A (en) * | 2015-09-30 | 2016-01-06 | 齐齐哈尔大学 | A kind of preparation method of Actinomycete fibrinolytic enzyme |
| CN106148308A (en) * | 2016-08-31 | 2016-11-23 | 武汉真福医药股份有限公司 | A kind of isolation and purification method of recombination bacillus subtilis fibrinolysin |
| CN106222151A (en) * | 2016-08-31 | 2016-12-14 | 武汉真福医药股份有限公司 | A kind of recombination bacillus subtilis fibrinolysin and the preparation method of enteric coated capsule thereof |
| CN106978411A (en) * | 2017-05-09 | 2017-07-25 | 江西师范大学 | Fermented soya bean plasmin and cultivation method thereof |
| CN108836984A (en) * | 2018-07-26 | 2018-11-20 | 齐齐哈尔大学 | A kind of Cordyceps militaris leaching liquor and its preparation method and application and cordycepin beverage |
| CN110616151A (en) * | 2019-09-18 | 2019-12-27 | 华中农业大学 | Separated cordyceps sinensis and application thereof in production of plasmin |
| CN113025600A (en) * | 2021-05-08 | 2021-06-25 | 北京工商大学 | Fibrinolytic enzyme and preparation method and application thereof |
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| CN104152360B (en) * | 2014-08-15 | 2017-08-25 | 河南省科学院生物研究所有限责任公司 | A kind of Cordyceps militaris for producing the cordyceps silk powder with fibrinolytic and anti-oxidation function and its application |
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| CN1089369C (en) * | 1999-04-01 | 2002-08-21 | 孙启良 | Earthworm kinase prepn. tech. |
| KR20050021146A (en) * | 2003-08-26 | 2005-03-07 | 김승 | Fibrinolytic Enzyme from Cordyceps militaris |
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2008
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Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101962637A (en) * | 2009-12-04 | 2011-02-02 | 齐齐哈尔大学 | Cordyceps militaris plasmin and culturing method thereof |
| CN101962637B (en) * | 2009-12-04 | 2012-11-28 | 齐齐哈尔大学 | Cordyceps militaris plasmin and culturing method thereof |
| CN102604919A (en) * | 2012-04-16 | 2012-07-25 | 山东省农业科学院农业资源与环境研究所 | Method for fermenting and producing plasmin by use of cordyceps militaris liquid |
| CN102604919B (en) * | 2012-04-16 | 2013-10-16 | 山东省农业科学院农业资源与环境研究所 | Method for fermenting and producing plasmin by use of cordyceps militaris liquid |
| WO2013155862A1 (en) * | 2012-04-16 | 2013-10-24 | 济南亿安生物研究所 | Method for producing plasmin by liquid fermentation of cordyceps militaris |
| CN103333874A (en) * | 2013-05-02 | 2013-10-02 | 齐齐哈尔大学 | Actinomycete fibrinolytic enzyme and preparation method thereof |
| CN104911169A (en) * | 2015-04-07 | 2015-09-16 | 齐齐哈尔大学 | Method for culturing cordyceps militaris mycelium and plasmin |
| CN105219754A (en) * | 2015-09-30 | 2016-01-06 | 齐齐哈尔大学 | A kind of preparation method of Actinomycete fibrinolytic enzyme |
| CN106148308A (en) * | 2016-08-31 | 2016-11-23 | 武汉真福医药股份有限公司 | A kind of isolation and purification method of recombination bacillus subtilis fibrinolysin |
| CN106222151A (en) * | 2016-08-31 | 2016-12-14 | 武汉真福医药股份有限公司 | A kind of recombination bacillus subtilis fibrinolysin and the preparation method of enteric coated capsule thereof |
| CN106978411A (en) * | 2017-05-09 | 2017-07-25 | 江西师范大学 | Fermented soya bean plasmin and cultivation method thereof |
| CN108836984A (en) * | 2018-07-26 | 2018-11-20 | 齐齐哈尔大学 | A kind of Cordyceps militaris leaching liquor and its preparation method and application and cordycepin beverage |
| CN110616151A (en) * | 2019-09-18 | 2019-12-27 | 华中农业大学 | Separated cordyceps sinensis and application thereof in production of plasmin |
| CN110616151B (en) * | 2019-09-18 | 2020-12-22 | 华中农业大学 | Separated cordyceps sinensis and application thereof in production of plasmin |
| CN113025600A (en) * | 2021-05-08 | 2021-06-25 | 北京工商大学 | Fibrinolytic enzyme and preparation method and application thereof |
| CN113025600B (en) * | 2021-05-08 | 2021-09-28 | 北京工商大学 | Fibrinolytic enzyme and preparation method and application thereof |
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