CN101519654A - Preparation method for producing methane-oxidizing rhzomorph by utilizing methane-oxidizing bacteria - Google Patents
Preparation method for producing methane-oxidizing rhzomorph by utilizing methane-oxidizing bacteria Download PDFInfo
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- CN101519654A CN101519654A CN200910071723A CN200910071723A CN101519654A CN 101519654 A CN101519654 A CN 101519654A CN 200910071723 A CN200910071723 A CN 200910071723A CN 200910071723 A CN200910071723 A CN 200910071723A CN 101519654 A CN101519654 A CN 101519654A
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- methane
- rhzomorph
- oxidizing
- oxidizing bacteria
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Abstract
The invention relates to a preparation method for producing methane-oxidizing rhzomorph by utilizing methane-oxidizing bacteria. The method comprises the following steps: sectionally fermenting and culturing the methane-oxidizing bacteria in a culture medium with strictly limited copper to obtain the methane-oxidizing rhzomorph from zymotic fluid after being centrifugated, absorbed by macroporous resin of Dianion HP-20, frozen and dried. The method sectionally cultures the methane-oxidizing bacteria by enabling the methane-oxidizing bacteria to rapidly grow under the culture condition with higher copper content to reach a certain cell density and greatly releasing the methane-oxidizing rhzomorph under the low-copper culture condition so that the released methane-oxidizing rhzomorph incapable of combining with copper and incapable of being identified by cells to enter the cell interior can be deposited in a zymotic medium. The methane-oxidizing rhzomorph is obtained from the zymotic medium so as to solve the problem that the methane-oxidizing rhzomorph entering the cells is combined with an endomembrane system to cause separation and purification difficulties. Compared with a conventional production method, the method can greatly improve the yield of the methane-oxidizing rhzomorph and simplifies the separation and purification process of the methane-oxidizing rhzomorph, thereby playing a great role in the application of the methane-oxidizing rhzomorph.
Description
Technical field:
The present invention relates to a kind of preparation method who utilizes methane-oxidizing bacteria to produce the methane oxidation rhzomorph.
Background technology:
Superoxide-dismutase (SOD) can reach antioxidant effect by removing superoxide radical, allowed SOD to use at present as foodstuff additive, yet, SOD is a kind of protein enzyme, so it is very unstable in solution, when being subjected to the influencing of extraneous various physical chemical factors, the conformational change of depolymerization, sex change or the others of subunit also can take place as other zymoprotein molecule.The change that is accompanied by structure must cause the decline or the forfeiture of enzymic activity.In addition, because SOD content is low in the animals and plants, extracted amount is very little, and is therefore too expensive as the foodstuff additive price.Though and the SOD analogue enztme output that adopts chemical synthesis to produce may improve, but often active little, non-natural, may also have toxicity etc., comparatively speaking, because microorganism growth is fast, cultivation is easy, industrialization easily, therefore the technology of utilizing microorganisms producing to have the natural antioxidants of superoxide dismutase activity has broad application prospects.The methane oxidation rhzomorph is a kind of small molecules fluorescin, comes from methane-oxidizing bacteria.Have stronger superoxide dismutase activity with the methane oxidation rhzomorph after copper combines, molecular weight is little, and Stability Analysis of Structures is very potential antioxidant from natural food resource.Methane-oxidizing bacteria can finally be metabolized to carbonic acid gas and water with methane by the enzyme system that methane monooxygenase begins, and in this process, obtain the growth required carbon source and the energy, the synthetic adjusting that is subjected to copper of the growth of methane-oxidizing bacteria and methane monooxygenase, copper is being played the part of important biology role in methane-oxidizing bacteria.Methane-oxidizing bacteria can be secreted the methane oxidation rhzomorph that copper is had strong avidity and catch copper in the surrounding environment, thereby can be discerned by methane-oxidizing bacteria with copper bonded methane oxidation rhzomorph and come back to the effect of playing enriching Cu in the cell.When copper concentration was very low in the substratum, excretory methane oxidation rhzomorph by cell recognition, had the accumulation of methane oxidation rhzomorph owing to can't combine with copper in developing medium.But methane-oxidizing bacteria in the low copper developing medium is poor growth owing to lack copper, and the methane oxidation rhzomorph yields poorly, and causes in the fermentation media methane oxidation rhzomorph accumulation volume low, is unfavorable for separation and purification.When copper concentration is higher in the substratum, though the methane-oxidizing bacteria growth is fast, methane oxidation rhzomorph output height, but excretory methane oxidation rhzomorph accumulates in endomembrane system with can coming back in the cell after copper combines, the methane-oxidizing bacteria cellulose content is low in the fermentation media, and is incorporated into the methane oxidation rhzomorph separation and purification difficulty in the endomembrane system.These have become the bottleneck problem of restriction methane oxidation rhzomorph industrial applications.Therefore, make the secretion in a large number in fermentation media of methane oxidation rhzomorph, have important meaning to realizing the methane oxidation rhzomorph as natural antioxidants and aseptic applications so that adopt simple separation purification method to obtain the methane oxidation rhzomorph.
Summary of the invention:
The object of the invention provide a kind of technology comparatively simple, with low cost, utilize methane-oxidizing bacteria to produce the preparation method of methane oxidation rhzomorph fast.The present invention seeks to realize like this: be in the liquid nutrient medium of routine cultivation methane-oxidizing bacteria, to add CuSO stage by stage
4, in fermented liquid, obtain the methane oxidation rhzomorph.At first add 5uM CuSO in the liquid medium within
4Cultivate methane-oxidizing bacteria, adopt the nutrient solution of low copper content to cultivate again after waiting to reach certain cell density, make methane-oxidizing bacteria under the culture condition of strictness limit copper in fermented liquid a large amount of secretion methane oxidation rhzomorphs, wherein, CuSO in the low copper nutrient solution
4Content range is 0-0.05uM.Adopt the culture condition segmentation of strict limit copper to cultivate methane-oxidizing bacteria, from fermented liquid by centrifugal, Dianion HP-20 macroporous resin adsorption and lyophilize acquisition methane oxidation rhzomorph.The present invention seeks to that in the different substratum of copper concentration methane-oxidizing bacteria is carried out stepwise fermentation and cultivate, from fermented liquid by centrifugal, Dianion HP-20 macroporous resin and lyophilize acquisition methane oxidation rhzomorph.For solving the problems of the technologies described above, the present invention takes following technical scheme: a kind of production method of methane oxidation rhzomorph comprises following 2 aspects: 1, (add 5uM CuSO at higher copper content substratum earlier
4) in methane-oxidizing bacteria is grown fast, wait to reach and put it into low copper substratum behind certain cell density again and (add 0-0.05uM CuSO
4) in make its a large amount of secretion methane oxidation rhzomorphs (mb), the methane oxidation rhzomorph that methane-oxidizing bacteria discharges under low copper bar spare can not combine with copper, the methane oxidation rhzomorph of debond copper is owing to can't be entered cell interior by cell recognition, thus accumulation in a large number in medium.2, from fermented liquid, obtain methane oxidation rhzomorph (mb) by centrifugal, Dianion HP-20 macroporous resin and lyophilize.When methane-oxidizing bacteria being carried out the segmentation cultivation with method of the present invention, methane-oxidizing bacteria growth fast under the culture condition of higher copper content earlier, reach certain cell density, under low copper bar spare, discharge the methane oxidation rhzomorphs then in a large number, the methane oxidation rhzomorph that discharges can not combine with copper, can't be entered cell interior by cell recognition, thereby in medium, accumulate, solved the problem that the methane oxidation rhzomorph yields poorly, and the method for separation and purification methane oxidation rhzomorph is simple from fermented liquid, has higher industrial applications feasibility.Compare with ordinary method, method of the present invention can improve the output of methane oxidation rhzomorph dramatically, and simplified the program of methane oxidation rhzomorph separation and purification, based on above-mentioned advantage, the present invention will play a great role in the application (as natural antioxidants and sanitas use etc.) of methane oxidation rhzomorph, have a extensive future.
Embodiment:
Be in the liquid nutrient medium of routine cultivation methane-oxidizing bacteria, to add CuSO stage by stage
4, in fermented liquid, obtain the methane oxidation rhzomorph.At first add 5uM CuSO in the liquid medium within
4Cultivate methane-oxidizing bacteria, adopt the nutrient solution of low copper content to cultivate again after waiting to reach certain cell density, make methane-oxidizing bacteria under the culture condition of strictness limit copper in fermented liquid a large amount of secretion methane oxidation rhzomorphs, wherein, CuSO in the low copper nutrient solution
4Content range is 0-0.05uM.Adopt the culture condition segmentation of strict limit copper to cultivate methane-oxidizing bacteria, from fermented liquid by centrifugal, Dianion HP-20 macroporous resin adsorption and lyophilize acquisition methane oxidation rhzomorph.The present invention seeks to that in the different substratum of copper concentration methane-oxidizing bacteria is carried out stepwise fermentation and cultivate, from fermented liquid by centrifugal, Dianion HP-20 macroporous resin and lyophilize acquisition methane oxidation rhzomorph.
Below in conjunction with specific embodiment the present invention is described in further detail.
Method therefor is ordinary method if no special instructions among the following embodiment, all percentage concentrations are mass/volume (W/V) percentage concentration or volume/volume (V/V) percentage concentration if no special instructions, and the solvent in all substratum is deionized water.
Minimal medium composition following (g/L):
K
2HPO
4：1.06；NaH
2PO
4·12H
2O：4.34；MgSO
4·7H
2O：0.074；CaCl
2·2H
2O：7.0；K
2SO
4：0.34；NaNO
3：1.7；FeSO
4·7H
2O：0.0224；MnSO
4·7H
2O：0.446；ZnSO
4·7H
2O：0.57；Na
2MoO
4·2H
2O：0.096；H
3BO
3：0.124；CoCl
2·6H
2O：0.096；KI：0.166；pH7.0。(methane-oxidizing bacteria M.trichosporium OB3b substratum)
NH
3Cl:0.5; K
2HPO
4: 0.49; KH
2PO
47H
2O:0.40; MgSO
47H
2O:0.3; CaCl
22H
2O:0.02; KNO
3: 1.6; NaCl:0.3; FeSO
47H
2O:0.004; MnSO
4H
2O:0.0004; ZnSO
47H
2O:0.00034; Na
2MoO
42H
2O:0.00024; PH7.0 (methane-oxidizing bacteria M.trichosporium3011 substratum)
The production of embodiment 1, methane oxidation rhzomorph of the present invention is example with methane-oxidizing bacteria M.trichosporium OB3b and M.trichosporium 3011, and detailed process is as follows:
Methane-oxidizing bacteria segmentation under different Cu concentration is cultivated
Methane-oxidizing bacteria M.trichosporium OB3b or Methylosinus trichosporiumIMV 3011 are connect the clock amount with 10% to be seeded in to contain and to add 5uM CuSO
4500 milliliters of airtight triangular flasks of 100 milliliters of aseptic liquid nutrient mediums in, adopting vacuumizing method is methane-oxygen (1:10 with air displacement, V/V) gas mixture, 32 degrees centigrade, cultivate for 300 rev/mins, carry out cell density every sampling in 24 hours and measure, when OD600 reaches 0.8-1.1 in the nutrient solution, with 8000 rev/mins of centrifugal thalline that obtain, add it to CuSO
4Concentration is to continue in the low copper fresh culture of 0.01uM to cultivate.When OD600 reached 0.8-1.2 in the nutrient solution, if developing medium presents yellow, harvested cell then, otherwise continue to replace with low copper substratum presented yellow in medium, showed that promptly the methane oxidation rhzomorph accumulates in fermentation media.Cell density is measured and is adopted optical densitometric method, measures the optical density value (OD600) of wavelength 600 nanometers on SP-756P type spectrophotometer.
Freeze-drying weighting method behind the quantitative assay employing Diaion HP-20 macroporous adsorption resin chromatography of methane oxidation rhzomorph.
Concrete steps are as follows:
With the centrifugal removal cell of cultured fermented liquid, remaining media was with 13000xg secondary centrifuging 20 minutes, supernatant liquor Diaion HP-20 macroporous adsorption resin chromatography, use the solution elution Diaion HP-20 of volume ratio 60% methyl alcohol and 40% water then, eluant carries out vacuum lyophilization, obtains methane oxidation rhzomorph solid.Since the methane oxidation rhzomorph with understand yellowing after copper combine, at the 394-422nm place maximum absorption is arranged, so also can in this wavelength region, adopt spectrophotometry to carry out quantitative assay.
The conventional cultural method of embodiment 2, methane-oxidizing bacteria cultural method of the present invention and methane-oxidizing bacteria obtains the comparison methane-oxidizing bacteria M.trichosporium OB3b of methane oxidation rhzomorph and Methylosinus trichosporium IMV 3011 and carries out cultural method of the present invention and methane-oxidizing bacteria routine cultural method obtain the methane oxidation rhzomorph in fermentation media comparison in fermentation media, detailed process is as follows:
1. the routine of methane-oxidizing bacteria M.trichosporium OB3b and Methylosinustrichosporium IMV 3011 is cultivated, with ordinary method methane-oxidizing bacteria M.trichosporium OB3b and Methylosinus trichosporium IMV 3011 are cultivated, concrete grammar is: methane-oxidizing bacteria Methylosinus trichosporium IMV 3011 is connect the clock amount with 10% be seeded in to contain and add 5uM or 0.01uM CuSO
4500 milliliters of airtight triangular flasks of 100 milliliters of aseptic liquid nutrient mediums in, adopting vacuumizing method is methane-oxygen (1:10 with air displacement, V/V) gas mixture, 32 degrees centigrade, cultivate for 300 rev/mins, carry out cell density every sampling in 24 hours and measure, and adopt vacuumizing method to replace into fresh methane-oxygen (1:10, V/V) gas mixture continues to cultivate under the same conditions, cultivates and ends after 72～96 hours to cultivate.
2. the limit copper segmentation of methane-oxidizing bacteria M.trichosporium OB3b and Methylosinus trichosporiumIMV 3011 is cultivated
Concrete grammar such as embodiment 1
Adopt the segmentation of limit copper to cultivate methane-oxidizing bacteria, the output of the methane oxidation rhzomorph in its fermentation media is higher than the output with normal copper content and the conventional methane oxidation rhzomorph of cultivating of low copper content substratum, this mainly is because segmentation limit copper cultural method has at first guaranteed the quick growth of methane-oxidizing bacteria under the normal copper content, reach the methane oxidation rhzomorph that under low copper, is secreted into behind certain cell density in the medium and can not combine by cell recognition with copper and enter cell interior, thereby in medium, accumulate in a large number.
The result is as shown in table 1:
| Cultural method | Limit copper segmentation cultural method | Initial copper content 5.0uM | Initial copper content 0.01uM |
| M.trichosporium OB3b cultivates and finishes back methane oxidation rhzomorph concentration (mg/litre) | 82 | 8 | 34 |
| Methylosinus trichosporium IMV 3011 cultivates and finishes back methane oxidation rhzomorph degree (mg/litre) | 44 | 3 | 18 |
Claims (3)
1, a kind of preparation method who utilizes methane-oxidizing bacteria to produce the methane oxidation rhzomorph is characterized in that: be to add CuSO stage by stage in the liquid nutrient medium of routine cultivation methane-oxidizing bacteria
4, in fermented liquid, obtain the methane oxidation rhzomorph.
2, a kind of preparation method who utilizes methane-oxidizing bacteria to produce the methane oxidation rhzomorph according to claim 1 is characterized in that: at first add 5uM CuSO in the liquid medium within
4Cultivate methane-oxidizing bacteria, adopt the nutrient solution of low copper content to cultivate again after waiting to reach certain cell density, make methane-oxidizing bacteria under the culture condition of strictness limit copper in fermented liquid a large amount of secretion methane oxidation rhzomorphs, wherein, CuSO in the low copper nutrient solution
4Content range is 0-0.05uM.
3, a kind of preparation method who utilizes methane-oxidizing bacteria to produce the methane oxidation rhzomorph according to claim 1, it is characterized in that: adopt the culture condition segmentation of strict limit copper to cultivate methane-oxidizing bacteria, from fermented liquid by centrifugal, Dianion HP-20 macroporous resin adsorption and lyophilize acquisition methane oxidation rhzomorph.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154441A (en) * | 2011-02-24 | 2011-08-17 | 广州安能特化学科技有限公司 | Method for quantitively detecting methane-oxidizing bacterium |
| CN113324959A (en) * | 2021-05-20 | 2021-08-31 | 哈尔滨商业大学 | Preparation method of self-assembled fluorescent probe for detecting nitrite, fluorescent probe prepared by preparation method and application of fluorescent probe |
| CN116333047A (en) * | 2022-12-22 | 2023-06-27 | 四川大学华西医院 | Antibacterial peptide and preparation method thereof |
-
2009
- 2009-04-03 CN CN200910071723A patent/CN101519654A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154441A (en) * | 2011-02-24 | 2011-08-17 | 广州安能特化学科技有限公司 | Method for quantitively detecting methane-oxidizing bacterium |
| CN102154441B (en) * | 2011-02-24 | 2012-12-26 | 广州安能特化学科技有限公司 | Method for quantitively detecting methane-oxidizing bacterium |
| CN113324959A (en) * | 2021-05-20 | 2021-08-31 | 哈尔滨商业大学 | Preparation method of self-assembled fluorescent probe for detecting nitrite, fluorescent probe prepared by preparation method and application of fluorescent probe |
| CN116333047A (en) * | 2022-12-22 | 2023-06-27 | 四川大学华西医院 | Antibacterial peptide and preparation method thereof |
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Open date: 20090902 |