CN102776167A - Method for producing plasmin through microbial fermentation - Google Patents
Method for producing plasmin through microbial fermentation Download PDFInfo
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- CN102776167A CN102776167A CN201210106732XA CN201210106732A CN102776167A CN 102776167 A CN102776167 A CN 102776167A CN 201210106732X A CN201210106732X A CN 201210106732XA CN 201210106732 A CN201210106732 A CN 201210106732A CN 102776167 A CN102776167 A CN 102776167A
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Abstract
The invention relates to a method for producing plasmin through microbial fermentation. According to the method, microbes for generating plasmin are activated for 6 to 10 times and are subjected to product directional domestication for 4 to 6 times so that the microbes well grow under the environment condition being 24 to 30 DEG C; plasmin producing bacteria subjected to product directional domestication by a conventional method are subjected to step-by-step enlargement culture at 24 to 30 DEG C and are inoculated into a fermentation culture medium at the inoculation quantity being 3 to 9 percent of the fermentation liquid volume, the culture is carried out for 60 to 114 hours at 24 to 30 DEG C, and the plasmin production through microbial fermentation is completed; the fermentation liquid is centrifuged at 4000 to 8000 rpm, liquid is collected, and the collected liquid is crude enzyme liquid; and according to different requirements and use objects, the crude enzyme liquid can be further concentrated, separated and purified, and enzyme preparations with different activities, purities and preparation forms are prepared.
Description
Technical field
The present invention relates to fields such as microbiology, enzyme engineering, fermentation engineering, biological chemistry, be specifically related to the method that a kind of microbial fermentation is produced plasmin.The plasmin that this invention is produced is fibrin degradation directly; Be mainly used in medical treatment, health care and other plasmin application industries; The functional foodstuff and the newtype drug of treatment cardiovascular and cerebrovascular relative disease be can be developed as, treatment of diseases process, effect and securities such as cardiovascular and cerebrovascular embolism improved.
Background technology
Plasmin (plasmin, EC 3.4.21.7) is meant the proteolytic ferment of the single-minded fibrin degradation gel of ability, is an important component in the fibrinolytic system.Mainly through fibrin degradation and Fibrinogen, the multiple thrombin of hydrolysis (II.V.VII.VIII.X.XI) makes Profibrinolysin change plasmin into to plasmin, approach fibrin degradations (Yu Wangsheng etc., clinical department of internal medicine magazine, 1994) such as hydrolysis complement.Plasmin has been widely used in the industries such as medical treatment and health care as biological enzyme formulation; But the plasmin that uses at present mainly is a separation and Extraction from plant-animal; Though the research of the plasmin of microbial fermentation production both at home and abroad is a lot, the research of related microorganism fermentation plasmin is in the starting stage, mainly is basic works such as the cloning and expression (Wang Min etc. of strain improvement, condition of enzyme production optimization, zymologic property and genes involved; Acta Pharmaceutica Sinica, 1998; Gong Yong etc., mikrobe journal, 2001; Liu Chenguang, Qingdao Marine University's journal, 2001; Open few equality, South China Science & Engineering University's journal, 2010).Industrialization, mass-producing and the application of producing plasmin about microbial fermentation also do not appear in the newspapers.Microbial fermentation production plasmin is compared with separation and Extraction from plant-animal has very big advantage, from plant-animal, extracts plasmin and costs dearly, and need carry out strict detoxification, complex process, and raw material sources are limited, and waste is big; And microorganism fermentation process is simple, can be mass-produced the desired raw material wide material sources; Under the fermentation mode of normal temperature and pressure, production cost is low, and microbial metabolism is vigorous; Can improve raw material availability, the enzyme stability of acquisition is good, and output capacity is high; Clinical risk is little, aspect energy-conservation, sizable advantage is arranged; This will help promotion and application (Zhang Haifeng etc., Food Engineering Development, 2008 of mikrobe plasmin; Sun Yuee etc., China brewages, and 2010; The jade earrings bamboo tinkling of pieces of jade etc., Chinese Journal of Marine Drugs, 2003).Utilize the plasmin of microbial fermentation production can development functionality food and newtype drug be used to dissolve disease (Wang Gaoxue etc., Sichuan University's journal, 2009 such as arterial thrombus, therapeutic advance property cerebral infarction, unstable angina pectoris, cardiovascular and cerebrovascular thromboembolism; Fang Jing etc., Changjiang University's journal, 2010; Zhao Lianhao etc.; China's water power medical science, 2010), avoided the heavy dose of medication of traditional method to cause shortcomings such as vascular reocclusion behind angiorrbagia, the thrombolysis, drug allergy; Along with the development of modern biotechnology, clinical thrombolysis will more and more be paid close attention to microbial fermentation and produce plasmin.Therefore, the application of the plasmin that microbial fermentation is produced will produce far-reaching influence to traditional clinical thrombolysis field, have wide development potentiality and application prospect.
Summary of the invention
The purpose of this invention is to provide a kind of microbial fermentation and produce the method for plasmin; This invention mainly is after mikrobe is tamed through the product orientation; In the method that 24～30 ℃ of liquid fermentings are produced plasmin, the microbial fermentation plasmin activity that this working method obtains can reach 150U/mL.As, can obtain the zymin of different concns and purity again through separating and purifying.The microbial fermentation plasmin preparation cost that this method is produced is low, and specificity is strong, good stability, and clinical risk is little, can fundamentally avoid utilizing shortcomings such as that plant-animal extract is expensive, the restriction of raw material, strict detoxification.
The method that a kind of microbial fermentation of the present invention is produced plasmin specifically may further comprise the steps:
The mikrobe that (1) will produce plasmin carries out 6～10 activation, through the directed domestication of product 4～6 times, makes its well-grown under 24～30 ℃ of envrionment conditionss again;
(2) by the fibrinolytic enzyme producing strain of ordinary method after with the directed domestication of product 24～30 ℃ of enlarged culturing step by step, be prepared into liquid first order seed and secondary seed;
(3) with liquid first order seed or secondary seed, 3～9% inoculum sizes of pressing fermentating liquid volume insert in the liquid fermentation medium, and when cultivating 60～114h for 24～30 ℃, promptly microbial fermentation is produced the plasmin end;
(4) with the fermented liquid of (3) 4,000～8, the centrifugal collection liquid of 000rpm is crude enzyme liquid;
(5) different with the use object according to different needs, the crude enzyme liquid that (4) are obtained further concentrates, separation and purification, is prepared into the zymin of different activities, purity and formulation.
The bacterial classification that uses among the present invention derives from China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), and initial stage activation and growth conditions are undertaken by the explanation that culture presevation unit provides.The fibrinolytic enzyme producing strain strain (for example; CGMCC numbering: 1.769 or 1.892); After the activation of bacterial strain elder generation, the directed domestication; By condition of enzyme production fermentative prodn plasmin of the present invention, the bacterial strain after the directed domestication can be preserved 2 months in 4 ℃ of environment, bacteria suspension, the preservation for a long time under subzero 80 ℃ of conditions of processing with 10～25% glycerine.
Embodiment
Embodiment one:
(1) medium preparation
1. strain activation and culture base: glucose 10.0g, scleroproein 20.0g, zero(ppm) water 1.0L, 7.0～7.2,121 ℃ of high pressure steam sterilization 30min of pH value.
2. the directed acclimation shaking culture base of bacterial classification: glucose 8.0～12.0g, scleroproein 15.0～25.0g, yeast powder 3.0～6.0g, NaCl 4.0～6.0g, agar powder 20.0g, zero(ppm) water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
3. liquid seed culture medium: glucose 5.0～15.0g, soy peptone 6.0～12.0g, K
2HPO
41.0～6.0g, KH
2PO
40.5～2.5g, CaCl
20.1～0.5g, MgSO
40.2～0.8g, pH value nature, tap water 1.0L, 121 ℃ of high pressure steam sterilization 30min.
4. enzymatic production substratum: glucose 15.0～25.0g, soya-bean milk 0.01～0.03L, yeast powder 1.0～6.0g, KH
2PO
40.5～2.0g, K
2HPO
41.5～6.5g, MgSO
47H
2O 0.2～0.8g, CaCl
20.01～0.05g, pH value nature, tap water 1.0L, 121 ℃ of high pressure steam sterilization 30min.
The mikrobe that (2) will produce plasmin through the directed domestication of product 4～6 times, makes its well-grown under 24～30 ℃ of envrionment conditionss again by 6～10 activation of bacterial strain explanation carrying out that Chinese common micro-organisms culture presevation administrative center (CGMCC) provides;
(3) by the fibrinolytic enzyme producing strain of ordinary method after with the directed domestication of product 24～30 ℃ step by step enlarged culturing 36～60h then carry out enlarged culturing step by step by 5% inoculum size, be prepared into liquid first order seed and secondary seed;
(4) 3%～5% inoculum size that the secondary seed that (3) is prepared is pressed fermentating liquid volume inserts in the enzymatic production substratum of 10L, and when cultivating 96～114h for 24～26 ℃, promptly microbial fermentation is produced the plasmin end;
(5) with the fermented liquid of (4) 4,000～8, the centrifugal collection liquid of 000rpm is crude enzyme liquid;
(6) different with the use object according to different needs, the crude enzyme liquid that (5) are obtained further concentrates, separation and purification, is prepared into the zymin of different activities, purity and formulation.
Embodiment two:
(1) medium preparation
1. strain activation and culture base: glucose 10.0g, scleroproein 20.0g, zero(ppm) water 1.0L, 7.0～7.2,121 ℃ of high pressure steam sterilization 30min of pH value.
2. the directed acclimation shaking culture base of bacterial classification: glucose 8.0～12.0g, scleroproein 15.0～25.0g, yeast powder 3.0～6.0g, NaCl 4.0～6.0g, agar powder 20.0g, zero(ppm) water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
3. liquid seed culture medium: glucose 5.0～15.0g, soy peptone 6.0～12.0g, K
2HPO
41.0～6.0g, KH
2PO
40.5～2.5g, CaCl
20.1～0.5g, MgSO
40.2～0.8g, pH value nature, tap water 1.0L, 121 ℃ of high pressure steam sterilization 30min.
4. enzymatic production substratum: glucose 15.0～25.0g, soya-bean milk 0.01～0.03L, yeast powder 1.0～6.0g, KH
2PO
40.5～2.0g, K
2HPO
41.5～6.5g, MgSO
47H
2O 0.2～0.8g, CaCl
20.01～0.05g, pH value nature, tap water 1.0L, 121 ℃ of high pressure steam sterilization 30min.
The mikrobe that (2) will produce plasmin through the directed domestication of product 4～6 times, makes its well-grown under 24～30 ℃ of envrionment conditionss again by 6～10 activation of bacterial strain explanation carrying out that Chinese common micro-organisms culture presevation administrative center (CGMCC) provides;
(3) by the fibrinolytic enzyme producing strain of ordinary method after with the directed domestication of product 24～30 ℃ step by step enlarged culturing 36～60h then carry out enlarged culturing step by step by 5% inoculum size, be prepared into liquid first order seed and secondary seed;
(4) 5%～7% inoculum size that the secondary seed that (3) is prepared is pressed fermentating liquid volume inserts in the enzymatic production substratum of 50L, and when cultivating 78～96h for 26～28 ℃, promptly microbial fermentation is produced the plasmin end;
(5) with the fermented liquid of (4) 4,000～8, the centrifugal collection liquid of 000rpm is crude enzyme liquid;
(6) different with the use object according to different needs, the crude enzyme liquid that (5) are obtained further concentrates, separation and purification, is prepared into the zymin of different activities, purity and formulation.
Embodiment three:
(1) medium preparation
1. strain activation and culture base: glucose 10.0g, scleroproein 20.0g, zero(ppm) water 1.0L, 7.0～7.2,121 ℃ of high pressure steam sterilization 30min of pH value.
2. the directed acclimation shaking culture base of bacterial classification: glucose 8.0～12.0g, scleroproein 15.0～25.0g, yeast powder 3.0～6.0g, NaCl 4.0～6.0g, agar powder 20.0g, zero(ppm) water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
3. liquid seed culture medium: glucose 5.0～15.0g, soy peptone 6.0～12.0g, K
2HPO
41.0～6.0g, KH
2PO
40.5～2.5g, CaCl
20.1～0.5g, MgSO
40.2～0.8g, pH value nature, tap water 1.0L, 121 ℃ of high pressure steam sterilization 30min.
4. enzymatic production substratum: glucose 15.0～25.0g, soya-bean milk 0.01～0.03L, yeast powder 1.0～6.0g, KH
2PO
40.5～2.0g, K
2HPO
41.5～6.5g, MgSO
47H
2O 0.2～0.8g, CaCl
20.01～0.05g, pH value nature, tap water 1.0L, 121 ℃ of high pressure steam sterilization 30min.
The mikrobe that (2) will produce plasmin through the directed domestication of product 4～6 times, makes its well-grown under 24～30 ℃ of envrionment conditionss again by 6～10 activation of bacterial strain explanation carrying out that Chinese common micro-organisms culture presevation administrative center (CGMCC) provides;
(3) by the fibrinolytic enzyme producing strain of ordinary method after with the directed domestication of product 24～30 ℃ step by step enlarged culturing 36～60h then carry out enlarged culturing step by step by 5% inoculum size, be prepared into liquid first order seed and secondary seed;
(4) 7%～9% inoculum size that the secondary seed that (3) is prepared is pressed fermentating liquid volume inserts in the enzymatic production substratum of 100L, and when cultivating 60～78h for 28～30 ℃, promptly microbial fermentation is produced the plasmin end;
(5) with the fermented liquid of (4) 4,000～8, the centrifugal collection liquid of 000rpm is crude enzyme liquid;
(6) different with the use object according to different needs, the crude enzyme liquid that (5) are obtained further concentrates, separation and purification, is prepared into the zymin of different activities, purity and formulation.
Claims (3)
1. a microbial fermentation is produced the method for plasmin, and it may further comprise the steps:
The mikrobe that (1) will produce plasmin carries out 6～10 activation, through the directed domestication of product 4～6 times, makes its well-grown under 24～30 ℃ of envrionment conditionss again;
(2) by the fibrinolytic enzyme producing strain of ordinary method after with the directed domestication of product 24～30 ℃ of enlarged culturing step by step, be prepared into liquid first order seed and secondary seed;
(3) with liquid first order seed or secondary seed, 3～9% inoculum sizes of pressing fermentating liquid volume insert in the liquid fermentation medium, and when cultivating 60～114h for 24～30 ℃, promptly microbial fermentation is produced the plasmin end;
(4) with the fermented liquid of (3) 4,000～8, the centrifugal liquid of collecting of 000rpm is crude enzyme liquid.
(5) different with the use object according to different needs, the crude enzyme liquid that (4) are obtained further concentrates, separation and purification, is prepared into the zymin of different activities, purity and formulation.
2. method according to claim 1 further comprises in step (5) afterwards: the crude enzyme liquid that step (5) is obtained further concentrates, separation and purification, is prepared into the zymin of different activities, purity and formulation.
3. method according to claim 1 and 2, wherein the directed acclimation shaking culture base of bacterial classification, liquid seed culture medium, enzymatic production substratum are respectively:
(1) the directed acclimation shaking culture base of bacterial classification: glucose 8.0～12.0g, scleroproein 15.0～25.0g, yeast powder 3.0～6.0g, NaCl 4.0～6.0g, agar powder 20.0g, zero(ppm) water 1.0L, pH value nature, 121 ℃ of high pressure steam sterilization 30min.
(2) liquid seed culture medium: glucose 5.0～15.0g, soy peptone 6.0～12.0g, K
2HPO
41.0～6.0g, KH
2PO
40.5～2.5g, CaCl
20.1～0.5g, MgSO
40.2～0.8g, pH value nature, tap water 1.0L, 121 ℃ of high pressure steam sterilization 30min.
(3) enzymatic production substratum: glucose 15.0～25.0g, soya-bean milk 0.01～0.03L, yeast powder 1.0～6.0g, KH
2PO
40.5～2.0g, K
2HPO
41.5～6.5g, MgSO
47H
2O 0.2～0.8g, CaCl
20.01～0.05g, pH value nature, tap water 1.0L, 121 ℃ of high pressure steam sterilization 30min.
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| CN201210106732XA CN102776167A (en) | 2012-04-12 | 2012-04-12 | Method for producing plasmin through microbial fermentation |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110616151A (en) * | 2019-09-18 | 2019-12-27 | 华中农业大学 | Separated cordyceps sinensis and application thereof in production of plasmin |
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| CN101113413A (en) * | 2007-07-04 | 2008-01-30 | 浙江大学 | Yellow-green halimasch fibrinolytic enzyme and production method thereof |
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2012
- 2012-04-12 CN CN201210106732XA patent/CN102776167A/en active Pending
Patent Citations (5)
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| CN1807610A (en) * | 2006-01-23 | 2006-07-26 | 迟乃玉 | Method for producing low temperature cellulase using microbe fermentation |
| CN101113413A (en) * | 2007-07-04 | 2008-01-30 | 浙江大学 | Yellow-green halimasch fibrinolytic enzyme and production method thereof |
| CN101948763A (en) * | 2009-10-13 | 2011-01-19 | 中国农业科学院饲料研究所 | High-efficiency compound microbial inoculum for fermenting beds, and preparation method and application thereof |
| CN102093992A (en) * | 2009-12-10 | 2011-06-15 | 大连大学 | Method for producing low-temperature protease by microbial fermentation |
| CN102093987A (en) * | 2009-12-11 | 2011-06-15 | 大连大学 | Method for producing low-temperature phytase by fermenting microorganisms |
Non-Patent Citations (1)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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Application publication date: 20121114 |