CN101215531A - Method for separating extracellular cholesterol oxidase producing strain - Google Patents
Method for separating extracellular cholesterol oxidase producing strain Download PDFInfo
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- CN101215531A CN101215531A CNA2007100510273A CN200710051027A CN101215531A CN 101215531 A CN101215531 A CN 101215531A CN A2007100510273 A CNA2007100510273 A CN A2007100510273A CN 200710051027 A CN200710051027 A CN 200710051027A CN 101215531 A CN101215531 A CN 101215531A
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- cholesterol
- strain
- sample
- rco
- cholesterol oxidase
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Abstract
A method for separating extracellular cholesterol oxidase yield strain is disclosed, which is characterized in separating the yield strain of extracellular cholesterol oxidase from natural environment, meat products and fresh carnivore fecal. The method comprises the following step: a. gathering sample in the soil sample or water sample and the like, inoculating the sample in the liquid separation culture medium based on cholesterol as unique carbon source, enriching and culturing; b. using the dilute flat coating method to purify the strain on the solid separation culture medium; c. fermenting and culturing in the ferment and culture medium, using acetic ether to extract the fermentation liquor, adopting silica gel sheet chromatography to analyze the production of cholesteryl-4-olefin-3-ketone in the extract, sieving the high-yield strain of cholesteryl-4-olefin-3-ketone; d. selecting the high-yield strain of cholesteryl-4-olefin-3-ketone to ferment further, testing the cholesterol oxidase activity in the fermentation supernatant, and sieving the strain which can produce extracellular cholesterol oxidase.
Description
Technical field
The present invention relates to a kind of separation method of extracellular cholesterol oxidase producing strain, belong to the using microbe technical field.
Technical background
(cholesterol oxidase COD) can change cholesterol into courage steroid-4-alkene-3-ketone to rCO, produces hydrogen peroxide (H simultaneously
2O
2), H
2O
2Under the effect of peroxidase, decompose, can make 4-amino-quinizine and phenol form the compound that inferior quinones takes on a red color, at 500 nm places maximum absorption band is arranged, by measuring the absorbancy of reaction solution at the 500m place, but amount (the Richmond W. 1973 that quantitative analysis of cholesterol is oxidized, Preparation and properties of a cholesterol oxidase from Nocardia sp.and itsapplication to the enzymatic assay of total cholesterol in serum.Clin Chem19,1350-1356).In recent years, medical research proof serum hypercholesterolemia and relevant (the MacLachlan J of human diseases such as heart trouble, atherosclerosis, diabetes, liver and gall diseases, Wotherspoon AT, Ansell RO, Brooks CJ.2000, Cholesterol oxidase:sources, physical properties and analyticalapplications.J Steroid.Biochem Mol Biol.72:169-195).The cholesterol level that detects easily and accurately in blood and the food is extremely important clinically, prevention, cardiovascular and cerebrovascular diseases diagnosis and prevention etc. for diabetes monitoring and diabetic complication have important role (Aihara H, Watanabe K, Nakamura R.1 988, Degradation of cholesterol in Egg york by Rhodococcus equiNo.23.J Food Sci 53:659-660).Therefore, clinically, rCO is used for the detection of plasma cholesterol content in a large number.
RCO is as s-generation biotic pesticide, can kill the insect of Coleoptera, lepidopteran, Orthoptera effectively, have good environmental safety (Zhang Rui, Guo Sandui. engineering of insect-resistant plant progress [J]. biotechnology circular, 2001:8-12).In addition, rCO (the Aihara H that all has a good application prospect at aspects such as producing lower cholesterol content food and preparation pharmaceutical intermediate, Watanabe K, Nakamura R (1988) Degradation of cholesterol in Egg york by Rhodococcus equiNo.23.J Food Sci 53:659-660).
But the kind that can really can be applied to the rCO that cholesterol level clinically detects at present is single, costs an arm and a leg, and the strain enzyme-producing amount that major cause is to produce rCO is low, mostly is and produces enzyme in the born of the same parents, has strengthened production cost.And also only derive from a kind of streptomycete as the rCO of sterilant.
Summary of the invention:
The objective of the invention is to provide a kind of separation method of extracellular cholesterol oxidase producing strain, be characterized in from physical environment, meat product and carnivore fresh excreta, separating efficient extracellular cholesterol oxidase producing strain at the deficiencies in the prior art.
The separation method of extracellular cholesterol oxidase producing strain may further comprise the steps:
1. nature or artificial environment soil sample or the water sample gathering fish, chicken, pig, sheep, beef product, carnivore fresh excreta and be rich in cholesterol, be seeded in the cholesterol is in the liquid enrichment medium of sole carbon source, in temperature 30-37 ℃ of vibration enrichment, cultivated 3-4 days; Get 200 μ l nutrient solutions to the fresh liquid separation culture medium according to above-mentioned condition, repeats the interference of institute's carbonaceous sources in the elimination sample 3 times;
2. get above-mentioned cultivation suspension 200 μ l and be diluted to 10 with sterilized water
-5, 10
-6, 10
-7,, 10
-8, 10
-95 concentration gradients, separate application were cultivated cultivation preservation on the colony inoculation beef-protein medium inclined-plane that will occur 3-4 days in temperature 30-37 ℃ on solid separation culture medium;
3. the bacterial strain of preserving on the inclined-plane is activated the back in the 3ml fermention medium, in temperature 30-37 ℃ of shaking culture 48h, get fermented liquid, after adding isopyknic vinyl acetic monomer mixing, standing over night, get upper layer of extraction liquid, point sample on silica gel thin sheet, after launching seasoning under the room temperature, ultraviolet lamp in 254nm is observed down, occurring black on the silica gel thin sheet does not have fluorescence spot person and is courage steroid-4-alkene-3-ketone that rCO catalysis cholesterol produces, and chooses black and does not have the bigger bacterial strain of fluorescence spot and carry out next step screening;
4. choose courage steroid-4-alkene-further liquid fermentation and culture 48h of 3-ketone superior strain, detect the rCO enzymic activity of its fermented supernatant fluid, thereby screening obtains producing the bacterial strain of extracellular cholesterol oxidase.
In order effectively to screen cholesterol oxidase producing strain, the present invention is at first in the enrichment culture process, with the cholesterol is sole carbon source, and by repeating the liquid separation culture medium that nutrient solution is extremely fresh enrichment culture repeatedly, eliminate the interference of institute's carbonaceous sources in the sample, reach and really be enriched to the purpose that to decompose the microorganism that utilizes cholesterol; The prescription of used liquid enrichment medium is: cholesterol 0.1, NaNO
30.1, KH
2PO
40.025, MgSO
47H
2O 0.025, FeSO
40.0001 pH7.0 adds an amount of bromothymol blue as indicator, at temperature 115-121 ℃, and moist heat sterilization 20min.
For the hybrid bacterial strain that enrichment culture is obtained obtains purifying, adopt dilution to be coated with dull and stereotyped method, be 10 with the dilution of enrichment culture thing
-5, 10
-6, 10
-7,, 10
-8, 10
-95 concentration gradients, separate application are on solid separation culture medium, and described solid separation culture medium prescription is: yeast powder 0.5, cholesterol 0.1, tween-80 0.1, NaNO
30.1, KH
2PO
40.025, MgSO
47H
2O 0.025, FeSO
40.0001, agar 2%, pH7.0, at temperature 115-121 ℃, moist heat sterilization 20min.
Produce outside the rCO born of the same parents in the screening process of bacterium, at first adopt cholesterol in the silica gel thin sheet chromatographic analysis fermentation upper layer of extraction liquid to be converted into the output of courage steroid-4-alkene-3-ketone, preliminary screening produces bacterium to the rCO born of the same parents outside, with the simplification screening process.The prescription of described fermention medium is: yeast powder 0.5, cholesterol 0.1, tween-80 0.1, NaNO
30.1, KH
2PO
40.025, MgSO
47H
2O 0.025, FeSO
40.0001, pH7.0, at temperature 115-121 ℃, moist heat sterilization 20min.
According to season civilization etc. (colorimetric method for determining rCO enzyme is lived. Wuxi Light Industry Univ.'s journal, method 2000:251-254) is measured the rCO enzyme and is lived, concrete steps are as follows: 3mL solution A (4-amino-quinizine: 1mmol/L; Phenol: 6mmol/L; Sodium azide: 0.2g/L; Peroxidase: 7000U/L; Potassium phosphate buffer: 25mmol/L, pH7.5), 150 μ L solution B (cholesterol: 8.26mg/mL; Triton X-100: volume fraction 4.26%; Virahol is a solvent), 50 μ L enzyme liquid, 37 ℃, reaction 5min, boiling water bath 3min surveys absorbancy in 500nm.Enzyme (U/mL): 1.6832 * A500 alive, enzyme work is defined as at 37 ℃, and under the condition of pH7.5, catalysis 1 μ mol cholesterol is oxidized to courage steroid-4-alkene-needed enzyme of 3-ketone in the 1min.
Utilize screening method provided by the invention, screening obtains many strains extracellular cholesterol oxidase producing strain source and detailed being shown in Table 1 of product enzyme situation.
Therefore, screening method provided by the invention has following advantage: 1, have separation efficiency height, characteristics easy and simple to handle, and 2, can provide microbe-derived widely for medicine and industrial enzymes, have important use and value.
Embodiment:
Below by embodiment the present invention is carried out concrete description; be necessary to be pointed out that at this present embodiment only is used for the present invention is further specified; can not be interpreted as limiting the scope of the invention, the person skilled in the art in this field can make some nonessential improvement and adjustment according to the content of the invention described above.
Embodiment 1:
(1) gather fish, chicken, pig, sheep, beef product, getting that the 1g sample is seeded in the cholesterol is in the liquid enrichment medium of sole carbon source, in 36 ℃ of vibrations of temperature enrichment, cultivates 3 days; Get 200 μ l nutrient solutions to the fresh liquid separation culture medium according to above-mentioned condition, repeats the interference of institute's carbonaceous sources in the elimination sample 3 times;
(2) get above-mentioned cultivation suspension 200 μ l and be diluted to 10 with sterilized water
-5, 10
-6, 10
-7,, 10
-8, 10
-95 concentration gradients are got 200 μ l diluent separate application on solid separation culture medium, cultivate 3 days for 30 ℃ in temperature, preserve cultivating on the colony inoculation beef-protein medium inclined-plane that occurs;
(3) bacterial strain of preserving on the inclined-plane is activated the back in the 3ml fermention medium, in 30 ℃ of shaking culture 48h of temperature, get fermented liquid, after adding isopyknic vinyl acetic monomer mixing, standing over night, get upper layer of extraction liquid, point sample on silica gel thin sheet, after launching seasoning under the room temperature, ultraviolet lamp in 254nm is observed down, occurring black on the silica gel thin sheet does not have fluorescence spot person and is courage steroid-4-alkene-3-ketone that rCO catalysis cholesterol produces, and chooses black and does not have the bigger bacterial strain of fluorescence spot and carry out next step screening;
(4) choose courage steroid-4-alkene-further liquid fermentation and culture 48h of 3-ketone superior strain, detect the rCO enzymic activity of its fermented supernatant fluid, thereby screening obtains producing the bacterial strain of extracellular cholesterol oxidase.
Embodiment 2:
(1) gather the carnivore fresh excreta, getting that the 1g sample is seeded in the cholesterol is in the liquid enrichment medium of sole carbon source, in 30 ℃ of vibrations of temperature enrichment, cultivates 4 days; Get 200 μ l nutrient solutions to the fresh liquid separation culture medium according to above-mentioned condition, repeats the interference of institute's carbonaceous sources in the elimination sample 3 times;
(2) get above-mentioned cultivation suspension 200 μ l and be diluted to 10 with sterilized water
-5, 10
-6, 10
-7,, 10
-8, 10
-95 concentration gradients, separate application were cultivated 3 days for 30 ℃ in temperature on solid separation culture medium, preserved cultivating on the colony inoculation beef-protein medium inclined-plane that occurs;
(3) bacterial strain of preserving on the inclined-plane is activated the back in the 3ml fermention medium, in 30 ℃ of shaking culture 48h of temperature, get fermented liquid, after adding isopyknic vinyl acetic monomer mixing, standing over night, get upper layer of extraction liquid, point sample on silica gel thin sheet, after launching seasoning under the room temperature, ultraviolet lamp in 254nm is observed down, occurring black on the silica gel thin sheet does not have fluorescence spot person and is courage steroid-4-alkene-3-ketone that rCO catalysis cholesterol produces, and chooses black and does not have the bigger bacterial strain of fluorescence spot and carry out next step screening;
(4) choose courage steroid-4-alkene-further liquid fermentation and culture 48h of 3-ketone superior strain, detect the rCO enzymic activity of its fermented supernatant fluid, thereby screening obtains producing the bacterial strain of extracellular cholesterol oxidase.
Embodiment 3:
(1) enrichment of bacterial strain: gather the nature or the artificial environment soil sample that are rich in cholesterol, get the 1g sample in the test tube that 5ml liquid enrichment medium is housed,, cultivated 3 days in 37 ℃ of vibration enrichments down of temperature; Get 200 μ l nutrient solutions to the fresh liquid separation culture medium according to above-mentioned condition, repeats the interference of institute's carbonaceous sources in the elimination sample 3 times; Observe the colour-change of nutrient solution in each test tube, if the color of nutrient solution was by being that blueness changes substratum true qualities or light yellow into originally, the nutrient solution in this test tube is used for further experiment.
(2) separation and purification of bacterial strain: get above-mentioned cultivation suspension 200 μ l and be diluted to 10 with sterilized water
-5, 10
-6, 10
-7,, 10
-8, 10
-95 concentration gradients, separate application were cultivated 3 days down for 37 ℃ in temperature on solid separation culture medium, preserved cultivating on the colony inoculation beef-protein medium inclined-plane that occurs;
(3) primary dcreening operation: the bacterial strain of preserving on the inclined-plane is activated the back in the 3ml fermention medium, at 37 ℃ of following shaking culture 48h of temperature, get fermented liquid, after adding isopyknic vinyl acetic monomer mixing, standing over night, get upper layer of extraction liquid, point sample on silica gel thin sheet, after launching seasoning under the room temperature, ultraviolet lamp in 254 nm is observed down, occurring black on the silica gel thin sheet does not have fluorescence spot person and is courage steroid-4-alkene-3-ketone that rCO catalysis cholesterol produces, and chooses black and does not have the bigger bacterial strain of fluorescence spot and carry out next step screening;
(4) multiple sieve: choose courage steroid-4-alkene-further liquid fermentation and culture 48h of 3-ketone superior strain, detect the rCO enzymic activity of its fermented supernatant fluid, thereby screening obtains producing the bacterial strain of extracellular cholesterol oxidase.
Embodiment 4:
(1) enrichment of bacterial strain: gather the nature or the artificial environment water sample that are rich in cholesterol, get the 1ml sample in the test tube that 5ml liquid enrichment medium is housed,, cultivated 3 days in 37 ℃ of vibration enrichments down of temperature; Get 200 μ l nutrient solutions to the fresh liquid separation culture medium according to above-mentioned condition, repeats the interference of institute's carbonaceous sources in the elimination sample 3 times; Observe the colour-change of nutrient solution in each test tube, if the color of nutrient solution was by being that blueness changes substratum true qualities or light yellow into originally, the nutrient solution in this test tube is used for further experiment.
(2) separation and purification of bacterial strain: get above-mentioned cultivation suspension 200 μ l and be diluted to 10 with sterilized water
-5, 10
-6, 10
-7,, 10
-8, 10
-95 concentration gradients, separate application were cultivated 3 days down for 37 ℃ in temperature on solid separation culture medium, preserved cultivating on the colony inoculation beef-protein medium inclined-plane that occurs;
(3) primary dcreening operation: the bacterial strain of preserving on the inclined-plane is activated the back in the 3ml fermention medium, at 37 ℃ of following shaking culture 48h of temperature, get fermented liquid, after adding isopyknic vinyl acetic monomer mixing, standing over night, get upper layer of extraction liquid, point sample on silica gel thin sheet, after launching seasoning under the room temperature, ultraviolet lamp in 254nm is observed down, occurring black on the silica gel thin sheet does not have fluorescence spot person and is courage steroid-4-alkene-3-ketone that rCO catalysis cholesterol produces, and chooses black and does not have the bigger bacterial strain of fluorescence spot and carry out next step screening;
(4) multiple sieve: choose courage steroid-4-alkene-further liquid fermentation and culture 48h of 3-ketone superior strain, detect the rCO enzymic activity of its fermented supernatant fluid, thereby screening obtains producing the bacterial strain of extracellular cholesterol oxidase.
Adopt separation method of the present invention, can be from the artificial or physical environment that is rich in cholesterol, be separated to the various bacterial isolateses that can produce the high reactivity extracellular cholesterol oxidase effectively, thereby provide abundant rCO enzyme source for pharmaceutical sector, industry, agricultural.
Table 1 is the detected result of the extracellular cholesterol oxidase of 13 strain different sources bacteriums
| The bacterial strain label | Enzyme activity (U/L) | The source | The bacterial strain label | Enzyme activity (U/L) | The source |
| 1-15 | 246 | Meat product | 7-8 | 54 | The carnivore fresh excreta |
| 3-3 | 198 | Meat product | 8-9 | 434 | The carnivore fresh excreta |
| 3-13 | 191 | Meat product | 9-9 | 174 | Soil |
| 3-14 | 251 | Meat product | 10-2 | 181 | Soil |
| 4-5 | 311 | Meat product | 10-4 | 237 | Soil |
| 4-8 | 266 | The carnivore fresh excreta | 12-9 | 329 | Soil |
| 5-6 | 300 | The carnivore fresh excreta |
Claims (4)
1. the separation method of an extracellular cholesterol oxidase producing strain strain is characterized in that, this separation method comprises the steps:
(1) nature or artificial environment soil sample or the water sample of gathering fish, chicken, pig, sheep, beef product, carnivore fresh excreta and being rich in cholesterol, be seeded to the cholesterol is in the liquid enrichment medium of sole carbon source, in temperature 30-37 ℃, the vibration enrichment was cultivated 3-4 days; Get 200 μ l nutrient solutions to the fresh liquid separation culture medium according to above-mentioned condition, repeats the interference of institute's carbonaceous sources in the elimination sample 3 times;
(2) get above-mentioned cultivation suspension 200 μ l and be diluted to 10 with sterilized water
-5, 10
-6, 10
-7,, 10
-8, 10
-95 concentration gradients, separate application were cultivated cultivation preservation on the colony inoculation beef-protein medium inclined-plane that will occur 3-4 days in temperature 30-37 ℃ on solid separation culture medium;
(3) with after the bacterial strain activation of preserving on the inclined-plane, in the 3ml fermention medium, 30-37 ℃ of shaking culture 48h, get fermented liquid, after adding isopyknic vinyl acetic monomer mixing, standing over night, get upper layer of extraction liquid, point sample on silica gel thin sheet, after launching seasoning under the room temperature, observe down in the 254nm ultraviolet lamp, occurring black on the silica gel thin sheet does not have fluorescence spot person and is courage steroid-4-alkene-3-ketone that rCO catalysis cholesterol produces, and chooses black and does not have the bigger bacterial strain of fluorescence spot and carry out next step screening;
(4) choose courage steroid-4-alkene-further liquid fermentation and culture 48h of 3-ketone superior strain, detect the rCO enzymic activity of its fermented supernatant fluid, thereby screening obtains producing the bacterial strain of extracellular cholesterol oxidase.
2. rCO born of the same parents according to claim 1 produce the separation method of bacterium outward, it is characterized in that, the recipe ingredient of described liquid enrichment medium is: cholesterol 0.1, NaNO
30.1, KH
2PO
40.025, MgSO
47H
2O 0.025, FeSO
40.0001 pH7.0 adds an amount of bromothymol blue as indicator, at temperature 115-121 ℃, and moist heat sterilization 20min.
3. rCO born of the same parents according to claim 1 produce the separation method of bacterium outward, it is characterized in that, described solid separation culture medium recipe ingredient is: yeast powder 0.5, cholesterol 0.1, tween-80 0.1, NaNO
30.1, KH
2PO
40.025, MgSO
47H
2O 0.025, FeSO
40.0001, agar 2%, pH7.0, at temperature 115-121 ℃, moist heat sterilization 20min.
4. rCO born of the same parents according to claim 1 produce the separation method of bacterium outward, it is characterized in that, the recipe ingredient of described fermention medium is: yeast powder 0.5, cholesterol 0.1, tween-80 0.1, NaNO
30.1, KH
2PO
40.025, MgSO
47H
2O 0.025, FeSO
40.0001, pH7.0, at temperature 115-121 ℃, moist heat sterilization 20min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100510273A CN101215531A (en) | 2007-12-29 | 2007-12-29 | Method for separating extracellular cholesterol oxidase producing strain |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100510273A CN101215531A (en) | 2007-12-29 | 2007-12-29 | Method for separating extracellular cholesterol oxidase producing strain |
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| Publication Number | Publication Date |
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| CN101215531A true CN101215531A (en) | 2008-07-09 |
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| CNA2007100510273A Pending CN101215531A (en) | 2007-12-29 | 2007-12-29 | Method for separating extracellular cholesterol oxidase producing strain |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110066849A (en) * | 2019-04-04 | 2019-07-30 | 大连大学 | A kind of screening technique producing cholesterol oxidase bacterial strain |
| CN117025467A (en) * | 2023-08-11 | 2023-11-10 | 中国农业大学 | Rabdosia for producing cholesterol oxidoreductase dependent on FAD, application, culture medium and preservation method |
-
2007
- 2007-12-29 CN CNA2007100510273A patent/CN101215531A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110066849A (en) * | 2019-04-04 | 2019-07-30 | 大连大学 | A kind of screening technique producing cholesterol oxidase bacterial strain |
| CN117025467A (en) * | 2023-08-11 | 2023-11-10 | 中国农业大学 | Rabdosia for producing cholesterol oxidoreductase dependent on FAD, application, culture medium and preservation method |
| CN117025467B (en) * | 2023-08-11 | 2024-03-29 | 中国农业大学 | Rabdosia for producing cholesterol oxidoreductase dependent on FAD, application, culture medium and preservation method |
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