CN107779402A - A kind of screening technique of ECB production bacterium superior strain - Google Patents
A kind of screening technique of ECB production bacterium superior strain Download PDFInfo
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Abstract
The present invention establishes a kind of screening technique of efficient ECB production bacterium superior strain, and has carried out preservation to screening obtained strains.First according to the characteristic of ECB producing strains product feedback inhibition, appropriate ECB is added in screening flat board as primary dcreening operation, filters out the bacterial strain for eliminating product feedback inhibition.Secondly, the mycelium of mutagenic strain is soaked in organic solvent and extracts ECB therein, recycle bacteriostasis of the ECB to Candida albicans, according to inhibition zone size, screen ECB superior strain again from primary dcreening operation bacterial strain, reach the purpose of secondary screening, the superior strain most filtered out at last investigates its production capacity with fermentation shake flask.The screening technique simple and effective, the screening cycle is greatly shortened, improves bacterial screening efficiency.
Description
Technical field
The invention belongs to field of biological pharmacy, is related to a kind of screening technique of ECB production bacterium superior strain.
Background technology
The 1970s, the mankind were found that echinocandin class medicine, were a kind of secondary metabolites of aspergillus nidulans, main
There are three types, wherein ECB (Echinocandin B) is mostly important type.One is included in its molecular structure
The individual cyclic hexapeptide core with lipid side chain, the lipid side chain can suppress fungal cell wall β -1,3- glucan noncompetitively
The activity of synzyme, so as to cause the emptying of cell wall glucan, osmotic transient fixed and the dissolving of fungal cell and play it
Antifungic action, show has a broad antifungal spectrum, the characteristics of activity is strong, be treatment immunosuppressed patient and immune normal patient fungi sense
The important selection medicine of dye, mainly acts on Mycotoruloides (Candidas) and Eurotium (Aspergillus).
The production producing strain of the ECB used now is relatively low, increases the difficulty of downstream drug extraction, product matter
Amount is not high.Therefore, the acquisition of superior strain has very important significance to the fermenting and producing of ECB.Conventional nature
The seed selection cycle is long, efficiency is low, randomness is big and a large amount of screenings, causes to be difficult to obtain good characteristic bacterial strain.And use different
Mutagens handle the cell colony of microorganism, then using the screening technique of simple and effective, therefrom select benign mutant strain, into
For the main method of strain improvement.
This method adds appropriate spine according to the characteristic of ECB producing strains product feedback inhibition in screening flat board
White rhzomorph B, as primary dcreening operation, filter out the bacterial strain for eliminating product feedback inhibition.Further, since ECB is intracellular metabolin,
Take the mycelium of mutagenic strain being soaked in organic solvent and extract ECB, recycle it to Candida albicans
Bacteriostasis, according to inhibition zone size, ECB superior strain is screened again in the bacterial strain selected from primary dcreening operation, reaches secondary screening
Purpose.The superior strain most filtered out at last investigates its production capacity with fermentation shake flask.The screening technique is simple and effective, significantly
Shorten screening the cycle, improve efficiency.
The content of the invention
The present invention is by lot of experiments, and it is an object of the present invention to provide a kind of ECB produces bacterium-aspergillus nidulans
The screening technique of (Aspergillus nidulans) superior strain.Finally screening has obtained a kind of white bacterium of spine to the screening technique
Plain B produces bacterium superior strain, and the strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, protects
It is CGMCC No.8987 to hide numbering, and address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences's microbe research
Institute, postcode 100101.
The screening technique is：Primary dcreening operation is carried out to mutagenic strain by eliminating product feedback inhibition, then according to white
The inhibition zone size of candida albicans reaches the purpose of secondary screening, finally investigates its life with fermentation shake flask to the strain excellent filtered out
Production capacity power.Comprise the following steps that：
(1) the aspergillus nidulans spore suspension of appropriate dilution processing is coated in screening flat board after mutagenic treatment,
It is incubated.According to product feedback principle, if mutagenic strain well-grown in the screening flat board containing ECB, says
It is bright to be possible to eliminate feedback inhibition, it may be superior strain, be carried out mark, half of bacterium colony of picking is dipped in organic solvent
In, the ECB of bacterial strain intracellular is extracted, and remaining half of bacterium colony does culture presevation.
(2) from the leaching liquor in step (1), 10 microlitres are taken to add small filter paper.It is placed on long good Candida albicans
Flat board on, and carry out mark.After 28 DEG C are cultivated 24 hours, inhibition zone size is observed, is sieved again in the bacterial strain obtained from primary dcreening operation
The big bacterial strain of inhibition zone is selected, reaches the purpose of secondary screening, and enter half of the bacterium colony preserved in corresponding step (1) according to mark
Row Secondary Culture.
(3) bacterial strain screened in step (2) is prepared into seed liquor and is inoculated in fermentation shake flask after Secondary Culture.
After 26 DEG C of 280rpm are cultivated 6 days, fermentation unit is detected.
Above-mentioned Screening of strain with high productivity method, wherein described in step (1) method of mutagenesis be ultraviolet mutagenesis, NTG mutagenesis, from
Beamlet mutagenesis.
In above-mentioned Screening of strain with high productivity method, wherein step (1), plating medium component used is：Glucose 2-10g/
L, Dried Corn Steep Liquor Powder 3-10g/L, malt extract 4-10g/L, K2HPO4 0.5-5g/L、FeSO4.7H2O0.005-0.035g/
L、ZnSO4.7H2O0.005-0.045g/L、MnCL2.4H2O 0.005-0.015g/L, agar 20-25g/L, pH 6-8, add
ECB 0.05-1.5g/L.
Organic solvent is ethanol, acetone, methanol in above-mentioned Screening of strain with high productivity method, wherein step (1).
In above-mentioned Screening of strain with high productivity method, wherein step (1), the amount of organic solvent is 100-500ul.
The temperature control of constant incubator is 26-37 DEG C in above-mentioned Screening of strain with high productivity method, wherein step (1), time 2-8d.
Soak time when being extracted in above-mentioned Screening of strain with high productivity method, wherein step (1) is 0.20-4.0h.
Above-mentioned Screening of strain with high productivity method, wherein step (3) fermentation shake flask media components are glucose 2-10g/L, jade
Rice ＆ peanut milk dry powder 3-10g/L, malt extract 4-10g/L, K2HPO40.5-5g/L、FeSO4.7H2O 0.005-0.035g/L、
ZnSO4.7H2O 0.005-0.045g/L、MnCL2.4H2O 0.005-0.015g/L, pH 6-8.
Aspergillus nidulans CGMCC No.8987 category Deuteromycotina, Hyphomycetes, hyphomycetales, Moniliaceae, the one of aspergillus
Kind fungi, conidinphore shorter (75~100 μm), bends, 3.5~5.0 μm of diameter at nearly top capsule, brown, and surface is smooth.Point
Raw spore head short cylindrical, (40~80) μ m (25~40) μm.Top capsule hemispherical, 8~10 μm of diameter, there is metulae and stigma thereon
Each one layer.On stigma raw round and coarse conidium, 3.0~3.5 μm of diameter.Perfect stage forms spherical, dark violet red
Cleistothecium, 135~150 μm of diameter, faint yellow, spherical, heavy wall the hülle cell of one layer of outsourcing.It is ascospore biconvex mirror shape, purplish red
Color, above there is crista.Colony growth is fast, circular, green, velvet-like, is in yellowish-brown when producing a large amount of cleistotheciums, and the back side is purple
It is red.
It is diversified suitable for the culture medium for cultivating aspergillus nidulans CGMCC No.8987, as long as can be the bacterium
The culture medium of nutritional ingredient is all available necessary to strain provides growth and breeding.It is used to cultivate other in existing literature data
The culture medium of various aspergillus nidulanses is suitable for this paper aspergillus nidulans CGMCC No.8987 culture, for example can adopt
The culture medium made of glucose, peptone, starch, dusty yeast, peanut powder, oat, agar etc..
Method provided by the present invention is easy to operate, and step is simple, substantially reduces the screening cycle, and pass through Double Selection
Add the probability for obtaining superior strain.Present invention screening gained aspergillus nidulans CGMCC No.8987 strain fermentation production spine
White rhzomorph B yield improves more than 720% than original strain.
Embodiment
Beneficial effects of the present invention now are further described by following examples, embodiment is only used for the purpose of illustration,
Do not limit the scope of the invention, while the obvious change and modification that those of ordinary skill in the art are made according to the present invention
It is also contained within the scope of the invention.
Embodiment 1
(1) the aspergillus nidulans spore suspension of appropriate dilution processing is coated on screening after ultraviolet mutagenesis is handled
On flat board.In constant incubator, 37 DEG C are cultivated 4 days, select well-grown 900 plants of bacterial strain, carry out mark, and picking respectively
Half of bacterium colony is soaked 2 hours in 150 microlitres of ethanol, and remaining half of bacterium colony is kept.
(2) from the leaching liquid in step (1), take 10 microlitres to add small filter paper, be placed on Candida albicans flat board
On.After 28 DEG C are cultivated 24 hours, inhibition zone size is observed, picks out 30 plants larger of bacterial strain of inhibition zone, and will be right according to mark
Half of the bacterium colony preserved in the step of should marking (1) carries out Secondary Culture.
(3) bacterial strain screened in step (2) is prepared into seed liquor and is inoculated in fermentation shake flask after Secondary Culture.
After 26 DEG C of 280rpm are cultivated 6 days, fermentation unit is detected, finally filters out 3 plants higher of bacterial strain of unit, its fermentation titer is respectively
1500mg/L, 1480mg/L, 1450mg/L, improve 750%, 740%, 725% compared with original strain respectively.
In step (1), plating medium composition used and content are：Glucose 2g/L, Dried Corn Steep Liquor Powder 3g/L, malt carry
Take thing 4g/L, K2HPO4 0.5g/L、FeSO4.7H2O 0.05g/L、ZnSO4.7H2O 0.005g/L、MnCL2.4H2O 0.005g/
L, agar 20g/L pH are 6.0, add ECB 0.5g/L.
In step (3), fermentation shake flask medium component and content are glucose 3g/L, Dried Corn Steep Liquor Powder 4g/L, malt carry
Take thing 5g/L, K2HPO4 0.6g/L、FeSO4.7H2O 0.005g/L、ZnSO4.7H2O 0.005g/L、MnCL2.4H2O
0.005g/L, pH 6.0.
Embodiment 2
(1) the aspergillus nidulans spore suspension of appropriate dilution processing is coated on screening flat board after NTG mutagenic treatments
On.In constant incubator, 26 DEG C are cultivated 8 days, select well-grown 750 plants of bacterium colony, carry out mark, and half of picking respectively
Bacterium colony is soaked 4 hours in 200 microlitres of acetone, and remaining half of bacterium colony is kept.
(2) from the leaching liquid in step (1), take 10 microlitres to add small filter paper, be placed on Candida albicans flat board
On.After 28 DEG C are cultivated 24 hours, inhibition zone size is observed, picks out 40 plants larger of bacterial strain of inhibition zone, and will be right according to mark
Half of the bacterium colony preserved in the step of should marking (1) carries out Secondary Culture.
(3) bacterial strain screened in step (2) is prepared into seed liquor and is inoculated in fermentation shake flask after Secondary Culture.
After 26 DEG C of 280rpm are cultivated 6 days, fermentation unit is detected, finally filters out 2 plants higher of bacterial strain of unit, its fermentation titer is respectively
1550mg/L, 1490mg/L, improve 775%, 745% compared with original strain respectively.
In step (1), plating medium composition used and content are：Glucose 3g/L, Dried Corn Steep Liquor Powder 3.5g/L, malt
Extract 5g/L, K2HPO40.6g/L、FeSO4.7H2O 0.07g/L、ZnSO4.7H2O 0.008g/L、MnCL2.4H2O
0.008g/L, agar 20g/L pH are 6.0, add Pneumocandin B0 .8g/L.
In step (3), fermentation shake flask medium component and content are glucose 3g/L, Dried Corn Steep Liquor Powder 4g/L, malt carry
Take thing 5g/L, K2HPO4 0.6g/L、FeSO4.7H2O0.005g/L、ZnSO4.7H2O0.005g/L、MnCL2.4H2O 0.005g/
L, pH is 8.0.
Embodiment 3
(1) the aspergillus nidulans spore suspension of appropriate dilution processing is coated on screening after ion beam mutagenesis are handled
On flat board.In constant incubator, 35 DEG C are cultivated 2 days, select well-grown 750 plants of bacterium colony, carry out mark, and picking respectively
Half of bacterium colony is soaked 0.5 hour in 300 microlitres of methanol, and remaining half of bacterium colony is kept.
(2) from the leaching liquid in step (1), take 10 microlitres to add small filter paper, be placed on Candida albicans flat board
On.After 28 DEG C are cultivated 24 hours, inhibition zone size is observed, picks out 40 plants larger of bacterial strain of inhibition zone, and will be right according to mark
Half of the bacterium colony preserved in the step of should marking (1) carries out Secondary Culture.
(3) bacterial strain screened in step (2) is prepared into seed liquor and is inoculated in fermentation shake flask after Secondary Culture.
After 26 DEG C of 280rpm are cultivated 6 days, fermentation unit is detected, finally filters out 2 plants higher of bacterial strain of unit, its fermentation titer is respectively
1530mg/L, 1570mg/L, improve 792%, 739% compared with original strain respectively.
In step (1), plating medium composition used and content are：Glucose 8g/L, Dried Corn Steep Liquor Powder 6g/L, malt carry
Take thing 5g/L, K2HPO4 2.6g/L、FeSO4.7H2O 0.017g/L、ZnSO4.7H2O 0.028g/L、MnCL2.4H2O
0.008g/L, agar 20g/L pH are 7.0, add ECB 1.2g/L.
In step (3), fermentation shake flask medium component and content are glucose 6g/L, Dried Corn Steep Liquor Powder 8g/L, malt carry
Take thing 7g/L, K2HPO4 3g/L、FeSO4.7H2O0.025g/L、ZnSO4.7H2O0.025g/L、MnCL2.4H2O 0.010g/L、
PH is 7.0.
Claims (10)
1. a kind of ECB produces bacterium superior strain, the strain has been preserved in China Committee for Culture Collection of Microorganisms
Common micro-organisms center, deposit number are CGMCC No.8987, depositary institution：In China General Microbiological culture presevation management
Heart preservation (CGMCC), preservation address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, postal
Political affairs coding 100101, preservation date：On April 1st, 2014, Classification And Nomenclature：aspergillus nidulans.
2. the screening technique of bacterial strain described in a kind of claim 1, it is characterised in that it comprises the following steps：
(1) the aspergillus nidulans spore suspension of appropriate dilution processing is coated on containing ECB after mutagenic treatment
It is incubated in screening flat board；Half of bacterium colony of the well-grown bacterium colony of picking is dipped in organic solvent, by the spine of bacterial strain intracellular
White rhzomorph B extractions, and remaining half of bacterium colony is kept；
(2) from the leaching liquor in step (1), take 10 microlitres to add small filter paper, be placed on the flat of long good Candida albicans
On plate, and mark is carried out, after 28 DEG C are cultivated 24 hours, observe inhibition zone size, filtered out again in the bacterial strain obtained from primary dcreening operation
The big bacterial strain of inhibition zone, reach the purpose of secondary screening, and passed half of the bacterium colony preserved in corresponding step (1) according to mark
It is commissioned to train foster；
(3) bacterial strain screened in step (2) is prepared into seed liquor and is inoculated in fermentation shake flask after Secondary Culture；26℃
After 280rpm is cultivated 6 days, fermentation unit is detected.
3. screening technique according to claim 2, it is characterised in that：Method of mutagenesis lures for ultraviolet described in step (1)
Change, NTG mutagenesis, ion beam mutagenesis.
4. screening technique according to claim 2, it is characterised in that：Screening flat board media components described in step (1)
For：Glucose 2-10g/L, Dried Corn Steep Liquor Powder 3-10g/L, malt extract 4-10g/L, K2HPO4 0.5-5g/L、
FeSO4.7H2O0.005-0.035g/L、ZnSO4.7H2O0.005-0.045g/L、MnCL2.4H2O 0.005-0.015g/L, spine
White rhzomorph B 0.05-1.5g/L, agar 20-25g/L, medium pH 6-8, add ECB 0.05-1.5g/L.
5. screening technique according to claim 2, it is characterised in that：Organic solvent is ethanol, acetone, first in step (1)
One kind in alcohol.
6. screening technique according to claim 2, it is characterised in that：The amount of organic solvent is 100- in step (1)
500ul。
7. screening technique according to claim 2, it is characterised in that：Soak time when being extracted in step (1) is 0.2-
4.0h。
8. screening technique according to claim 2, it is characterised in that：Incubated temperature control is 26-37 in step (1)
DEG C, time 2-8d.
9. screening technique according to claim 2, it is characterised in that：Step (3) fermentation shake flask media components are：Grape
Sugared 2-10g/L, Dried Corn Steep Liquor Powder 3-10g/L, malt extract 4-10g/L, K2HPO40.5-5g/L、FeSO4.7H2O0.005-
0.035g/L、ZnSO4.7H2O 0.005-0.045g/L、MnCL2.4H2O 0.005-0.015g/L, pH 6-8.
10. the bacterial strain described in claim 1, it is characterised in that application of the described bacterial strain in ECB is produced.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114854603A (en) * | 2022-05-09 | 2022-08-05 | 中国科学院青岛生物能源与过程研究所 | Strain for high-yield echinocandin B and application thereof |
| CN114907989A (en) * | 2022-05-09 | 2022-08-16 | 中国科学院青岛生物能源与过程研究所 | High-yield echinocandin B strain as anidulafungin precursor and application thereof |
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| CN103289901A (en) * | 2012-02-22 | 2013-09-11 | 上海来益生物药物研究开发中心有限责任公司 | Echinocandin B high-producing strain and application thereof |
| CN103555591A (en) * | 2013-10-12 | 2014-02-05 | 浙江工业大学 | Method and bacterial strain for fermentation preparation of Echinocandin B |
| CN105734098A (en) * | 2016-03-25 | 2016-07-06 | 浙江工业大学 | Method for improving yield of echimocandins B |
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2016
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| CN103289901A (en) * | 2012-02-22 | 2013-09-11 | 上海来益生物药物研究开发中心有限责任公司 | Echinocandin B high-producing strain and application thereof |
| CN103555591A (en) * | 2013-10-12 | 2014-02-05 | 浙江工业大学 | Method and bacterial strain for fermentation preparation of Echinocandin B |
| CN105734098A (en) * | 2016-03-25 | 2016-07-06 | 浙江工业大学 | Method for improving yield of echimocandins B |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114854603A (en) * | 2022-05-09 | 2022-08-05 | 中国科学院青岛生物能源与过程研究所 | Strain for high-yield echinocandin B and application thereof |
| CN114907989A (en) * | 2022-05-09 | 2022-08-16 | 中国科学院青岛生物能源与过程研究所 | High-yield echinocandin B strain as anidulafungin precursor and application thereof |
| CN114854603B (en) * | 2022-05-09 | 2023-06-27 | 中国科学院青岛生物能源与过程研究所 | Strain for high-yield echinocandin B and application thereof |
| CN114907989B (en) * | 2022-05-09 | 2023-06-27 | 中国科学院青岛生物能源与过程研究所 | High-yield strain of anidulafungin precursor echinocandin B and application thereof |
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| CN107779402B (en) | 2021-07-02 |
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