CN102304502A - Preparation method for bacterial-originated defibrase - Google Patents
Preparation method for bacterial-originated defibrase Download PDFInfo
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- CN102304502A CN102304502A CN 201110266142 CN201110266142A CN102304502A CN 102304502 A CN102304502 A CN 102304502A CN 201110266142 CN201110266142 CN 201110266142 CN 201110266142 A CN201110266142 A CN 201110266142A CN 102304502 A CN102304502 A CN 102304502A
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Abstract
The present invention discloses a preparation method for bacterial-originated defibrase. According to the method, strains of enterococcus faecalis EF608 are adopted as the extract; the strains of the enterococcus faecalis EF608 are subjected to fermentation culture; the resulting fermentation broth is subjected to centrifugation to remove the precipitate; solid ammonium sulfate is added to the resulting supernatant until the saturation of the ammonium sulfate is 60-65%; the resulting salting-out component is subjected to dialysis and concentration to obtain a crude extracting solution; the resulting crude extracting solution is sequentially treated by a phenyl-agarose gel FF hydrophobic chromatography column, a DEAE-SephadexA-25 ion exchange chromatography column and a Sepharose-heparin affinity chromatography column to obtain the component having a high abundance fibrinolytic activity peak; the resulting component is subjected to treatments of dialysis and freeze drying to obtain the pure product, wherein the pure product is the bacterial-originated defibrase. With the preparation method provided by the present invention, the fibrinogenase having a molecular weight of 37.1 KD is firstly separated and purified from the fermentation broth of the strains of the enterococcus faecalis; the fibrinogenase is easy to be purified and prepared batchly; the fibrinogenase has strong fibrinolytic activity and no toxicity, and is an ideal raw material for preparing the fibrinogenase.
Description
Technical field
The present invention relates to a kind of biological technical field, relating in particular to a kind of is the preparation method that extract obtains fiber eliminating enzyme with the bacterial origin.
Background technology
Thrombus is the lump that the constituent of vascular system inner blood own forms, and the many pathologies that cause conduit occlusion to cause by thrombus all belong to thrombotic disease, like myocardial infarction, cerebral thrombosis, lung thrombus, venous blood embolism etc.Along with the prolongation of human longevity and the change of dietary structure; The thromboembolic states case is showed increased also; Show according to World Health Organization's investigation; Whole world thromboembolism property disease patient is no less than 1 500 ten thousand people; It is healthy that thrombotic disease has become current harm humans, causes one of the highest disease of mortality ratio.
At present, one of the most reliable and effective means of treatment thrombotic disease are thrombolytic therapies, promptly inject thrombolytics and make revascularization.Existing clinical used thrombolytics mainly is streptokinase, urokinase, staphylokinase, people's sense of organization plasminogen activator (tissue plasminogen activator; T-PA), Ahylysantinfarctase etc., but streptokinase, urokinase, Ahylysantinfarctase all have bigger side effect; Though t-PA has stronger special avidity with the thrombus stroma fibrin, can activate fibrinolytic system efficiently in the thrombus part, its transformation period in blood is very short, and the suppressor factor of the plasminogen activator in the blood plasma can suppress its activity fast.Staphylokinase also has very strong specificity to scleroproein, but because low and this bacterium itself of productive rate pathogenic limited its application.Having adopted engineered method t-PA to be transformed and produces third generation thrombolytic drug at present in the world---mutant and the mosaic of t-PA, its curative effect makes moderate progress, but costs an arm and a leg.Therefore, development relatively inexpensive, efficient, quick, prevent again embolism and lower the demand of novel thrombolytic drug of untoward reaction such as hemorrhage urgent.
Since Japanese scholar Sumi in 1987 etc. find that at first microorganism plasmin (fibrinolytic enzyme) has fibrinolytic; The microorganism plasmin has long half time in the body, the not available characteristics of other thrombolytics such as with low cost with it, receives various countries scientist's extensive concern and further investigation.
Divide from the mechanism of action; Microbe-derived thrombolytics can be divided into two types: one type is plasminogen activator; Like streptokinase, staphylokinase; Its action principle is that plasminogen activator can be plasmin with the Profibrinolysin activation; Plasmin with serine protease then can be degraded and constituted the scleroproein of thrombus skeleton, thereby causes the effect of thrombolysis; Another kind of is the fibrinolytic enzyme material; Plasminogen activation is not passed through in its effect; But the scleroproein in the clot of directly degrading; Thrombus; This type material has the Nattokinase that derives from subtilis, and (nattokinase NK) and respectively derives from the fibrinolytic material of genus bacillus, streptococcus faecium, chain enzyme bacteria etc.
Except that the Beta-hemolytic streptococcus (Streptococcus hemolytius) of the product streptokinase of early discovery with produce the streptococcus aureus (Staphylococcus aureus) of staphylokinase; In recent years, filter out successively again genus bacillus, actinomycetes, Penicillium chrysogenum bacterium, sharp fusarium, the fusarium that can produce plasmin activity, revolve spore mould, marine pseudomonas, micrococcus luteus etc.But there is bigger side effect in the yield of enzyme that has in these bacterial strains fiber eliminating enzyme that self exists bacterial strain pathogenic, that have to produce lower, that have after clinical application; These factors have restricted the development and application of microorganism plasmin to a certain extent; Thereby filter out no pathogenicity, be prone to cultivate, bacterial strain that yield of enzyme is high, and find out sophisticated processing condition and just seem particularly important.
Summary of the invention
It is the method that extract prepares fiber eliminating enzyme that technical problem to be solved by this invention is to provide a kind of enterococcus faecalis EF608 bacterial strain.
Technical scheme of the present invention is following: a kind of bacterial origin fiber eliminating enzyme preparation method; Its key is: enterococcus faecalis EF608 strain fermentation cultivated, and the centrifugal deposition of going of fermented liquid, supernatant liquor adds solid ammonium sulfate to saturation ratio and reaches 60%-65%; The component of saltouing is concentrated through dialysing, and gets crude extract.Crude extract is crossed phenyl sepharose gel FF hydrophobic chromatography post, DEAE-Sephadex A-25 ion exchange column and Sepharose-heparin affinity chromatography post successively; Obtain high abundance fibrinolytic peak; Dialysis, lyophilize obtain pure article and are the bacterial origin fiber eliminating enzyme.Separation of pure has dissolved the plasmin that a kind of molecular weight is 37.1KD from the enterococcus faecalis bacterial strain fermentation liquor.
Specifically carry out as follows:
(1) is extract with enterococcus faecalis EF608 bacterial strain, enterococcus faecalis EF608 strain fermentation is cultivated;
(2) ammonium sulfate precipitation: get fermented liquid, centrifugal, abandon deposition, add solid ammonium sulfate in the supernatant liquor, make its ammonium sulfate saturation concentration reach 60%-65%, the component of saltouing is concentrated through dialysing, crude extract;
(3) phenyl sepharose gel FF hydrophobic chromatography: crude extract is crossed phenyl sepharose gel FF hydrophobic chromatography post, with 50mmol/L Tris-HC1 and 1 mol/L (NH
4)
2SO
4Cocktail buffer, pH 7.5 carries out balance, using 50mmol/L Tris-HC1pH is 7.5 buffer solution elution; Collect the purpose peak, dialysis, lyophilize, 4 ℃ of preservations;
(4) DEAE-Sephadex A-25 ion exchange chromatography: purpose peak component is crossed DEAE-Sephadex A-25 ion exchange column; Use 50mmol/L Tris-HC1pH 7.5 to carry out balance; Using 50mmol/L Tris-HC1pH is that 7.5 Buffer increases NaCl gradually and improves salt concn in 0～0.5 mol/L scope, does linear gradient elution; Collection solution pipe to each peak is measured fibrinolytic, collects the purpose peak that fibrinolytic is arranged, dialysis, lyophilize, 4 ℃ of preservations;
(5) Sepharose-heparin affinity chromatography: purpose peak component is crossed Sepharose-heparin affinity chromatography post, and using 50mmol/L Tris-HC1pH is that 7.5 Buffer carries out balance; Using 50mmol/L Tris-HC1pH is that 7.5 Buffer increases NaCl gradually and improves salt concn in 0～0.5 mol/L scope, does linear gradient elution; Collection has the peak of fibrinolytic, and dialysis, lyophilize obtain pure article and be the bacterial origin fiber eliminating enzyme.
The centrifugal of fermented liquid is the centrifugal 10min of 8000 rpm in the above-mentioned steps (1).
Above-mentioned enterococcus faecalis EF608 strain fermentation is cultivated and specifically is divided into two steps:
(1) seed culture: seed culture medium is in g/100mL: take by weighing glucose 1.0g, peptone 1.0g, yeast extract 0.5g, K
2HP0
4 3H
20 1.0g, NaCl 0.2g, MgS0
47H
20 0.02 g transfer pH to 7.5, and 121 ℃ of autoclave sterilization 20 min were in 37 ℃, 180 rpm constant temperature culture 12 ~ 16 hours.
(2) liquid fermentation and culture: liquid fermentation medium is in g/100mL: take by weighing glucose 2.0 g, peptone 0.5 g, extractum carnis 0.5 g, yeast extract 0.5 g, K
2HP0
4 3H
20 1.0 g, NaCl 0.2 g, MgS0
47H
20 0.02g transfers pH to 7.5,121 ℃ of autoclave sterilization 20min; Seed culture medium by volume per-cent 6% access liquid amount is that culture condition is in 20 liters of fermentor tanks of 4 liters of liquid fermentation mediums: stirring velocity 180 rpm, culture temperature is 35 ~ 40 ℃, incubation time 10 ~ 12 hours.Through specific substratum, last resulting bacterial origin fiber eliminating enzyme amount is higher.
Beneficial effect: preparation method of the present invention first from the enterococcus faecalis bacterial strain fermentation liquor separation of pure dissolved the plasmin that a kind of molecular weight is 37.1KD, be easy to purifying and batch preparations, and fibrinolytic is strong, nontoxicity is an ideal plasmin preparation raw material comparatively.
Description of drawings
Fig. 1 is a phenyl sepharose gel FF hydrophobic chromatography collection of illustrative plates;
Fig. 2 is a DEAE-Sephadex A-25 ion exchange chromatography collection of illustrative plates;
Fig. 3 is a Sepharose-heparin affinity chromatography collection of illustrative plates;
Fig. 4 is the SDS-PAGE collection of illustrative plates of product plasmin of the present invention;
Fig. 5 measures plasmin of the present invention to the fibrinogenic degraded picture of ox blood for SDS-PAGE.
Embodiment
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is described further:
1, the preparation of enterococcus faecalis EF608 bacterial strain fiber eliminating enzyme
(1) bacterial classification: enterococcus faecalis EF608 bacterial strain
(2) seed culture: seed culture medium is in g/100mL: take by weighing glucose 1.0g, peptone 1.0g, yeast extract 0.5g, K
2HP0
4 3H
20 1.0 g, NaCl 0.2g, MgS0
47H
20 0.02g transfers pH to 7.5,121 ℃ of autoclave sterilization 20 min.In 37 ℃, 180 rpm constant temperature culture 12 hours.
(3) liquid fermentation and culture: liquid fermentation medium is in g/100mL: take by weighing glucose 2.0 g, peptone 0.5 g, extractum carnis 0.5 g, yeast extract 0.5 g, K
2HP0
4 3H
20 1.0 g, NaCl 0.2 g, MgS0
47H
20 0.02g transfers pH to 7.5,121 ℃ of autoclave sterilization 20min; It is that culture condition is in 20 liters of fermentor tanks of 4 liters of liquid fermentation mediums that seed culture medium 240 ml insert liquid amount: stirring velocity 180 rpm, culture temperature is 37 ℃, incubation time 12 hours.
(4) ammonium sulfate precipitation: get fermented liquid, 4 ℃, the centrifugal 10min of 8000 rpm abandon deposition, add solid ammonium sulfate in the supernatant liquor, make its ammonium sulfate saturation concentration reach 65%, and the component of saltouing is concentrated through dialysing, and get crude extract.
(5) phenyl sepharose gel FF hydrophobic chromatography: crude extract is crossed phenyl sepharose gel FF hydrophobic chromatography post, and (2.0cm * 20cm) is with 50mmol/L Tris-HC1 and 1 mol/L (NH
4)
2SO
4Cocktail buffer, pH 7.5 carries out balance, using 50mmol/L Tris-HC1pH is 7.5 buffer solution elution; Detect wavelength: 280nm; Flow velocity: 1mL/min; Collect: the 3ml/ pipe; Collect the purpose peak, dialysis, lyophilize, 4 ℃ of preservations.
Separating spectrum as shown in Figure 1, wash-out obtains 2 peaks, the active detection finds that the II peak is the purpose peak.
(6) DEAE-Sephadex A-25 ion exchange chromatography: purpose peak component is crossed DEAE-Sephadex A-25 ion exchange column (2.0cm * 20cm); Use 50mmol/L Tris-HC1pH 7.5 to carry out balance; Using 50mmol/L Tris-HC1pH is that 7.5 Buffer increases NaCl gradually and improves salt concn in 0～0.5 mol/L scope, does linear gradient elution; Detect wavelength: 280nm; Flow velocity: 1mL/min; Collect: the 3ml/ pipe; Collection solution pipe to each peak is measured fibrinolytic, collects the strongest peak of fibrinolytic, dialysis, lyophilize, 4 ℃ of preservations.
Separating spectrum as shown in Figure 2, wash-out obtains 4 peaks, the active detection finds that IV peak activity is the strongest.
(7) Sepharose-heparin affinity chromatography: will go up the strongest component of step gained activity cross Sepharose-heparin affinity chromatography post (2.0cm * 20cm), using 50mmol/L Tris-HC1pH is that 7.5 Buffer carries out balance; Using 50mmol/L Tris-HC1pH is that 7.5 Buffer increases NaCl gradually and improves salt concn in 0～0.5 mol/L scope, does linear gradient elution; Detect wavelength: 280nm; Flow velocity: 1mL/min; Collect: the 3ml/ pipe; Collection has the peak of fibrinolytic, and dialysis, lyophilize obtain pure article.
Separating spectrum as shown in Figure 3, wash-out obtains 2 peaks, detect to find that the II peak has the activity of fibrin degradation.
(8) electrophoresis: the pure article of plasmin are done electrophoresis, use the SDS-polyacrylamide gel electrophoresis, resolving gel concentration is 12%.The result as shown in Figure 4, through above-mentioned purification step, sample is shown as a protein band, molecular weight is 37.1KD, this is a plasmin of the present invention.
2, present embodiment plasmin biological activity determination
(1) the plasmin fibrinolytic proves
A. agarose-fibrin plate method
Take by weighing Fibrinogen 16.0mg and be dissolved in the 8mL distilled water, be mixed with fibrinogen solution, water bath heat preservation in 37 ℃; Taking by weighing the 0.16g agar powder adds distilled water and is settled to 8mL and boiling; The fibrinogen solution that contains 10 U thrombin solutions and configure to about 50 ℃ adding 1ml to be cooled; Pour into after shaking up rapidly in the plate that diameter is 9cm; Room temperature keeps flat 30min, treats that flat board solidifies to be placed on 4 ℃ down refrigeration is subsequent use.Punch tool with direct 5mm punches on the fibrin plate for preparing, and every hole adds sample 20 microlitres, puts 37 ℃ of incubator insulations 18 hours, can see the transparent circle that solution fibrin forms
B. electrophoretic method
With plasmin of the present invention and 37 ℃ of reactions of ox blood Fibrinogen equal-volume mixing; Detect plasmin degraded ox blood Fibrinogen with SDS--PAGE, the degraded collection of illustrative plates is seen Fig. 2 (swimming lane is followed successively by and acts on 15min, 30min, 60min, 120min, 240min after ox blood Fibrinogen, plasmin of the present invention mix with Fibrinogen from left to right).Fiber eliminating enzyme of the present invention has stronger Degradation to ox blood Fibrinogen A α chain and B β chain, and the γ chain is not had Degradation.
(2) mouse subcutaneous hemorrhage toxicity test
Mouse back subcutaneous injection plasmin 0.1ml/ point of the present invention, enzyme activity is 400U/ml, peeling after 2 hours, the subcutaneous no extravasated blood spot of injection point occurs, and shows that this plasmin does not have hemorrhage toxicity.
Claims (4)
1. bacterial origin fiber eliminating enzyme preparation method; It is characterized in that: enterococcus faecalis EF608 strain fermentation is cultivated; The centrifugal deposition of going of fermented liquid; Supernatant liquor adds solid ammonium sulfate to saturation ratio and reaches 60%-65%; The component of saltouing is concentrated through dialysing; Get crude extract; Crude extract is crossed phenyl sepharose gel FF hydrophobic chromatography post, DEAE-Sephadex A-25 ion exchange column and Sepharose-heparin affinity chromatography post successively; Obtain high abundance fibrinolytic peak; Dialysis, lyophilize obtain pure article and are the bacterial origin fiber eliminating enzyme.
2. according to the said a kind of bacterial origin fiber eliminating enzyme preparation method of claim 1, it is characterized in that specifically carrying out as follows:
(1) is extract with enterococcus faecalis EF608 bacterial strain, enterococcus faecalis EF608 strain fermentation is cultivated;
(2) ammonium sulfate precipitation: get fermented liquid, centrifugal, abandon deposition, add solid ammonium sulfate in the supernatant liquor, make its ammonium sulfate saturation concentration reach 60%-65%, the component of saltouing is concentrated through dialysing, crude extract;
(3) phenyl sepharose gel FF hydrophobic chromatography: crude extract is crossed phenyl sepharose gel FF hydrophobic chromatography post, with 50mmol/L Tris-HC1 and 1 mol/L (NH
4)
2SO
4Cocktail buffer, pH 7.5 carries out balance, using 50mmol/L Tris-HC1pH is 7.5 buffer solution elution; Collect the purpose peak, dialysis, lyophilize, 4 ℃ of preservations;
(4) DEAE-Sephadex A-25 ion exchange chromatography: purpose peak component is crossed DEAE-Sephadex A-25 ion exchange column; Use 50mmol/L Tris-HC1pH 7.5 to carry out balance; Using 50mmol/L Tris-HC1pH is that 7.5 Buffer increases NaCl gradually and improves salt concn in 0～0.5 mol/L scope, does linear gradient elution; Collection solution pipe to each peak is measured fibrinolytic, collects the purpose peak that fibrinolytic is arranged, dialysis, lyophilize, 4 ℃ of preservations;
(5) Sepharose-heparin affinity chromatography: purpose peak component is crossed Sepharose-heparin affinity chromatography post, and using 50mmol/L Tris-HC1pH is that 7.5 Buffer carries out balance; Using 50mmol/L Tris-HC1pH is that 7.5 Buffer increases NaCl gradually and improves salt concn in 0～0.5 mol/L scope, does linear gradient elution; Collection has the peak of fibrinolytic, and dialysis, lyophilize obtain pure article and be the bacterial origin fiber eliminating enzyme.
3. according to the said a kind of bacterial origin fiber eliminating enzyme preparation method of claim 2, it is characterized in that: the centrifugal of fermented liquid is the centrifugal 10min of 8000 rpm in the said step (1).
4. according to the said a kind of bacterial origin fiber eliminating enzyme preparation method of claim 1, it is characterized in that: said enterococcus faecalis EF608 strain fermentation is cultivated and specifically is divided into two steps:
(1) seed culture: seed culture medium is in g/100mL: take by weighing glucose 1.0g, peptone 1.0g, yeast extract 0.5g, K
2HP0
43H
20 1.0g, NaCl 0.2g, MgS0
47H
20 0.02 g transfer pH to 7.5, and 121 ℃ of autoclave sterilization 20 min were in 37 ℃, 180 rpm constant temperature culture 12 ~ 16 hours;
(2) liquid fermentation and culture: liquid fermentation medium is in g/100mL: take by weighing glucose 2.0 g, peptone 0.5 g, extractum carnis 0.5 g, yeast extract 0.5 g, K
2HP0
43H
20 1.0 g, NaCl 0.2 g, MgS0
47H
20 0.02g transfers pH to 7.5,121 ℃ of autoclave sterilization 20min; Seed culture medium by volume per-cent 6% access liquid amount is that culture condition is in 20 liters of fermentor tanks of 4 liters of liquid fermentation mediums: stirring velocity 180 rpm, culture temperature is 35 ~ 40 ℃, incubation time 10 ~ 12 hours.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102899267A (en) * | 2012-08-13 | 2013-01-30 | 重庆师范大学 | Enterococcus faecalis |
| CN104498463A (en) * | 2014-12-23 | 2015-04-08 | 青岛康原药业有限公司 | Method for extracting defibrase from snake venom |
-
2011
- 2011-09-09 CN CN 201110266142 patent/CN102304502A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102899267A (en) * | 2012-08-13 | 2013-01-30 | 重庆师范大学 | Enterococcus faecalis |
| CN104498463A (en) * | 2014-12-23 | 2015-04-08 | 青岛康原药业有限公司 | Method for extracting defibrase from snake venom |
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Application publication date: 20120104 |