CN103789245A - Oxytetracycline high-producing strain and high throughput screening method thereof - Google Patents
Oxytetracycline high-producing strain and high throughput screening method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganism strain screening and particularly relates to an oxytetracycline high-producing strain and a high throughput screening method thereof. The category name of the screened oxytetracycline high-producing strain is Streptomyces rimosus DC473 and is preserved at the CGMCC, the preservation number is 6218, and the preservation date is June 14th, 2012. The screening method of the high-producing strain comprises the following steps of test slant culturing; spore suspension preparing; spore suspension preprocessing; rationalization screening; high-producing strain screening; and strain retesting. The method has the advantages that according to the metabolic characteristic of Streptomyces rimosus, the high throughput screening method is designed reasonably, the high-producing strain can be screened out quickly, the method is easy to implement, related culture medium raw material sources are wide, cost is low, and the method is suitable for industrial production.
Description
Technical field
The present invention relates to industrial biotechnology, microorganism strains filtration technical field, especially a kind of terramycin superior strain and high-throughput screening method thereof.
Background technology
Terramycin (Spectniomycni) is the secondary metabolite generating after streptomyces rimosus (Streptomyces rimosus) aerobic fermentation, for Fourth Ring class Broad spectrum antibiotics, gram-positive microorganism and Gram-negative bacteria are all had to stronger inhibition ability, multiple coccus and bacillus are had to anti-microbial effect, rickettsia and loeschiasis protozoon are also had to restraining effect, being used for treating the sense of Shang Huxi road dyes the sense of ﹑ gi tract and dyes the cold ﹑ tsutsugamushi fever of ﹑ spot rash wound, the diseases such as Mycoplasma Infections and chlamydiaceae infection.
Terramycin is mainly used as livestock and poultry medication and fodder additives, in addition, good market prospect take terramycin as raw material production semisynthetic antibiotics, such as with its synthetic doxycycline (Doxycycline), its market value is 5-7 times of terramycin, the metacycline that is raw material production with it, clinical effectiveness is better than terramycin, a lot of enterprises are by composition compound oxytetracyclines such as terramycin, VITMAIN B1, vitamin B6s, can make terramycin curative effect increase, can also reduce digestive tube side reaction, be good animal feedstuff additive.Therefore, the seed selection to terramycin superior strain and research have good economic benefit and social benefit.
N-methyl-N'-nitro-N-nitrosoguanidine (NTG) is a kind of supramutagen, is widely used, and under alkaline condition, NTG can form diazomethane (CH2N2), and it is to cause major cause lethal and sudden change.Its effect is likely mutagenic compound and the one or more nucleic acid base generation chemical transformation that CH2N2 causes the alkanisation of DNA, base substitution while making DNA replication dna and cause variation.How from abundant transgenation storehouse, to obtain fast the key that object muton is selection by mutation.Traditional random screening method not only wastes time and energy, and also easily misses object mutant strain.
Summary of the invention
The object of this invention is to provide a kind of terramycin superior strain and high-throughput screening method thereof.
In order to complete above-mentioned purpose, the technical solution used in the present invention is:
A kind of terramycin superior strain, classification streptomyces rimosus (Streptomyces rimosus) DC473 by name, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number is CGMCC No.6216, and preservation date is on June 14th, 2012.
Described bacterial strain list bacterium colony is flat blossom type, spore white, substrate mycelium brown.
A kind of terramycin superior strain high-throughput screening method, comprises the following steps:
A, test tube slant are cultivated: the strain switching test tube slant that sets out of sand tube preservation, and in 37.0 ± 0.5 ℃ of temperature, humidity 40%-60% thermostatic chamber is cultivated 3-5 days;
B, spore suspension preparation: in ripe test tube slant, add 10mL sterilized water, spore is scraped, mix, make spore suspension, with blood counting chamber counting, making spore concentration is 10
12-10
14individual/mL;
C, spore suspension pre-treatment: get the spore suspension preparing, add 0.5-1.5uL nitroso guanidine solution, making its final concentration is 0.1%-0.3%, vortex oscillation device vibration 30-60s, after mixing in 37 ± 0.5 ℃ of standing 2-3h;
D, rationalization screening: the spore suspension of nitrosoguanidine processing is diluted to 10
1-10
7doubly, diluent is coated on the Streptomycin sulphate isolation medium containing 10-15 μ g/mL terramycin and 0.2-0.4 μ g/mL, in 37.0 ± 0.5 ℃, and the dark 3-5 days that cultivates under 40%-60% humidity condition, when cultivation, first day is just put, and within second day, rises to be inverted and cultivates;
E, Screening of strain with high productivity: on isolation medium, the single colony inoculation of picking diameter more than 5mm is to F
1on slant medium, in 37.0 ± 0.5 ℃, under 40%-60% humidity condition, cultivate 3-5 days; After inclined-plane maturation, be inoculated in seed culture medium, in 30.0 ± 0.5 ℃, under 220rpm condition, 24-30h is cultivated in concussion; After planting liquid maturation, be inoculated in fermention medium, with 30.0 ± 0.5 ℃ of seed the same terms, under 220rpm condition, 5-7 days is cultivated in concussion, grows into all after date detections and tires, and choosing is tired and carried out sand drying preservation higher than the bacterial strain of 14000u/mL;
F, bacterial strain retrial: the bacterial strain switching test tube slant of getting sandy soil drying preservation, in 37.0 ± 0.5 ℃, under 40%-60% humidity condition, cultivate 3-5 days, inclined-plane maturation is forwarded in seed culture medium, in 37.0 ± 0.5 ℃, under 220rpm condition, 24-30h is cultivated in concussion, after planting liquid maturation, be forwarded in fermention medium, in 37.0 ± 0.5 ℃, under 220rpm condition, shake and cultivate 5-7 days, sampling detects and tires, and screening retains tires higher than the bacterial strain of 14000u/mL.
In described step D, terramycin content is 10-15 μ g/mL, and content of streptomycin is 0.2-0.4 μ g/mL.
Test tube slant medium component and content thereof used in described steps A, step e and step F are: 6.0%-7.0% wheat bran, 2.0%-2.5% agar.
In described step D solid separation culture medium, composition and content thereof are: 2.0%-3.0% W-Gum, 0.2%-0.5% corn steep liquor, 0.1%-0.3% ammonium sulfate, 0.1%-0.5% calcium carbonate, 0.3%-0.5% sodium-chlor, 2.0%-2.5% agar.
Seed culture based component and content thereof used in described step e and step F are: 2.0%-3.0% W-Gum, 0.2%-0.5% corn steep liquor, 2.0%-3.0% analysis for soybean powder, 0.1%-0.3% ammonium sulfate, 0.1%-0.5% calcium carbonate, 0.3%-0.5% sodium-chlor.
Fermentation culture based component and content thereof used in described step e and step F are: 10%-15% W-Gum, 0.2%-0.5% corn steep liquor, 2.0%-3.0% analysis for soybean powder, 1.0%-1.5% ammonium sulfate, 1.0%-1.5% calcium carbonate, 0.3%-0.5% sodium-chlor, 0.01%-0.05% potassium primary phosphate, 0.1%-0.3% polyether defoaming agent.
The invention has the beneficial effects as follows: when this bacterial strain shake flask fermentation 6 days, detect and tire as 18000U/mL, the strain of setting out (tiring as 14000U/mL under the same terms) has improved 28.6%.
Accompanying drawing explanation
Fig. 1 is screening method schema of the present invention.
Fig. 2 is the mono-colonial morphology figure of bacterial strain DC473 in the present invention.
Embodiment
The present invention is a kind of terramycin superior strain and high-throughput screening method thereof, when this bacterial strain shake flask fermentation 6 days, detects and tires as 18000U/mL, and the strain of setting out (tiring as 14000U/mL under the same terms) has improved 28.6%.
Below in conjunction with accompanying drawing, the present invention will be further described.
Specific embodiment 1, as shown in Figure 2, a kind of terramycin superior strain, classification streptomyces rimosus (Streptomyces rimosus) DC473 by name, this bacterial strain list bacterium colony is flat blossom type, spore white, substrate mycelium brown, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCC No.6216, and preservation date is on June 14th, 2012.
A kind of terramycin superior strain high-throughput screening method, as shown in Figure 1, comprises the following steps:
A, test tube slant are cultivated: the strain switching test tube slant that sets out of sand tube preservation, in 37.0 ± 0.5 ℃ of temperature, humidity 40%-60% thermostatic chamber is cultivated 3-5 days.In slant medium, composition and content thereof are: 6.0%-7.0% wheat bran, 2.0%-2.5% agar;
B, spore suspension preparation: in ripe test tube slant, add a certain amount of sterilized water, spore is scraped, mix, make spore suspension, with blood counting chamber counting, making spore concentration is 10
12-10
14individual/mL;
C, spore suspension pre-treatment: get the spore suspension preparing, add a certain amount of nitroso guanidine solution, making its final concentration is 0.1%-0.3%, vortex oscillation device vibration 30-60s, after mixing in 37 ± 0.5 ℃ of standing 2-3h;
D, rationalization screening: the spore suspension of nitrosoguanidine processing is diluted, diluent is coated on the Streptomycin sulphate isolation medium containing 10-15 μ g/mL terramycin and 0.2-0.4 μ g/mL, in 37.0 ± 0.5 ℃, the dark 3-5 days that cultivates under 40%-60% humidity condition, when cultivation, first day is just put, and within second day, rises to be inverted and cultivates; In separation and Culture, composition and content thereof are: 2.0%-3.0% W-Gum, 0.2%-0.5% corn steep liquor, 0.1%-0.3% ammonium sulfate, 0.1%-0.5% calcium carbonate, 0.3%-0.5% sodium-chlor, 2.0%-2.5% agar;
E, Screening of strain with high productivity: on isolation medium, the single colony inoculation of picking diameter more than 5mm, to F1 slant medium, in 37.0 ± 0.5 ℃, cultivated 3-5 days under 40%-60% humidity condition; After inclined-plane maturation, be inoculated in seed culture medium, in 30.0 ± 0.5 ℃, under 220rpm condition, 24-30h is cultivated in concussion; After planting liquid maturation, be inoculated in fermention medium, with 30.0 ± 0.5 ℃ of seed the same terms, under 220rpm condition, 5-7 days is cultivated in concussion, seed culture based component and content thereof are: 2.0%-3.0% W-Gum, 0.2%-0.5% corn steep liquor, 2.0%-3.0% analysis for soybean powder, 0.1%-0.3% ammonium sulfate, 0.1%-0.5% calcium carbonate, 0.3-0.5% sodium-chlor; Fermentation culture based component and content thereof are: 10%-15% W-Gum, 0.2%-0.5% corn steep liquor, 2.0%-3.0% analysis for soybean powder, 1.0%-1.5% ammonium sulfate, 1.0%-1.5% calcium carbonate, 0.3%-0.5% sodium-chlor, 0.01%-0.05% potassium primary phosphate, 0.1%-0.3% polyether defoaming agent;
F, bacterial strain retrial: the bacterial strain switching test tube slant of getting sandy soil drying preservation, in 37.0 ± 0.5 ℃, under 40%-60% humidity condition, cultivate 3-5 days, inclined-plane maturation is forwarded in seed culture medium, in 37.0 ± 0.5 ℃, under 220rpm condition, 24-30h is cultivated in concussion, after planting liquid maturation, be forwarded in fermention medium, in 37.0 ± 0.5 ℃, under 220rpm condition, 5-7 days is cultivated in concussion, sampling detects and tires, and screening retains tires higher than the bacterial strain of 14000u/mL; Fermentation culture is to all after dates, carries out bioactivity by colorimetry, and screening obtains a strain and tires and reach the bacterial strain (tiring as 14000U/mL under the same terms) of 18000U/mL, and this bacterial strain carries out sandy soil drying preservation.This superior strain called after DC473, has been deposited in the common micro-organisms center C GMCC of China Committee for Culture Collection of Microorganisms, and deposit number is CGMCC No.6216, and preservation date is on 06 14th, 2012.
Specific embodiment 2, bacterial strain retrial
A. test tube slant is cultivated: the superior strain DC473 that sets out strain and filter out of the sand tube preservation test tube slant of transferring respectively, in 37.0 ± 0.5 ℃ of temperature, humidity 40%-60% thermostatic chamber is cultivated 3-5 days, and in slant medium, composition and content thereof are: 6.0%-7.0% wheat bran, 2.0%-2.5% agar;
B. seed culture: inclined-plane maturation is switched in seed culture medium, in 30.0 ± 0.5 ℃, under 220rpm condition, 24-30h is cultivated in concussion; Seed culture based component and content thereof are: 2.0-3.0% W-Gum, 0.2%-0.5% corn steep liquor, 2.0%-3.0% analysis for soybean powder, 0.1%-0.3% ammonium sulfate, 0.1%-0.5% calcium carbonate, 0.3%-0.5% sodium-chlor;
C. fermentation culture: be inoculated into respectively in fermention medium after planting liquid maturation, in 30.0 ± 0.5 ℃, under 220rpm condition, 3-5 days is cultivated in concussion, and fermentation culture based component and content thereof are: 10%-15% W-Gum, 0.2%-0.5% corn steep liquor, 2.0%-3.0% analysis for soybean powder, 1.0%-1.5% ammonium sulfate, 1.0%-1.5% calcium carbonate, 0.3%-0.5% sodium-chlor, 0.01%-0.05% potassium primary phosphate, 0.1%-0.3% polyether defoaming agent;
Bioactivity: fermentation culture is used colorimetry to detect fermentation unit to all after dates.The experiment of more than implementing in example 2 repeats three times, and three the retrial fermentation units of strain that set out are 14105U/mL, 14240U/mL, 14010U/mL, and mean value is 14118U/mL; Three retrial fermentation units of DC473 bacterial strain are respectively 18120U/mL, 18502U/mL, 18370U/mL, and mean value is 18331U/mL..The relatively restraint of on average tiring of DC473 and starting strain, the fermentation unit of streptomyces rimosus DC473 of the present invention has improved 29.8% compared with the strain of setting out.
Claims (8)
1. a terramycin superior strain, it is characterized in that: classification streptomyces rimosus (Streptomyces rimosus) DC473 by name, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number is CGMCC No.6216, and preservation date is on June 14th, 2012.
2. a kind of terramycin superior strain according to claim 1, is characterized in that: described bacterial strain list bacterium colony is flat blossom type, spore white, substrate mycelium brown.
3. a terramycin superior strain high-throughput screening method, is characterized in that comprising the following steps:
A, test tube slant are cultivated: the strain switching test tube slant that sets out of sand tube preservation, and in 37.0 ± 0.5 ℃ of temperature, humidity 40%-60% thermostatic chamber is cultivated 3-5 days;
B, spore suspension preparation: in ripe test tube slant, add 10mL sterilized water, spore is scraped, mix, make spore suspension, with blood counting chamber counting, making spore concentration is 10
12-10
14individual/mL;
C, spore suspension pre-treatment: get the spore suspension preparing, add 0.5-1.5uL nitroso guanidine solution, making its final concentration is 0.1%-0.3%, vortex oscillation device vibration 30-60s, after mixing in 37 ± 0.5 ℃ of standing 2-3h;
D, rationalization screening: the spore suspension of nitrosoguanidine processing is diluted to 10
1-10
7doubly, diluent is coated on the Streptomycin sulphate isolation medium containing 10-15 μ g/mL terramycin and 0.2-0.4 μ g/mL, in 37.0 ± 0.5 ℃, and the dark 3-5 days that cultivates under 40%-60% humidity condition, when cultivation, first day is just put, and within second day, rises to be inverted and cultivates;
E, Screening of strain with high productivity: on isolation medium, the single colony inoculation of picking diameter more than 5mm is to F
1on slant medium, in 37.0 ± 0.5 ℃, under 40%-60% humidity condition, cultivate 3-5 days; After inclined-plane maturation, be inoculated in seed culture medium, in 30.0 ± 0.5 ℃, under 220rpm condition, 24-30h is cultivated in concussion; After planting liquid maturation, be inoculated in fermention medium, with 30.0 ± 0.5 ℃ of seed the same terms, under 220rpm condition, 5-7 days is cultivated in concussion, grows into all after date detections and tires, and choosing is tired and carried out sand drying preservation higher than the bacterial strain of 14000u/mL;
F, bacterial strain retrial: the bacterial strain switching test tube slant of getting sandy soil drying preservation, in 37.0 ± 0.5 ℃, under 40%-60% humidity condition, cultivate 3-5 days, inclined-plane maturation is forwarded in seed culture medium, in 37.0 ± 0.5 ℃, under 220rpm condition, 24-30h is cultivated in concussion, after planting liquid maturation, be forwarded in fermention medium, in 37.0 ± 0.5 ℃, under 220rpm condition, 5-7 days is cultivated in concussion, sampling detects and tires, and screening retains tires higher than the bacterial strain of 14000u/mL.
4. a kind of terramycin superior strain high-throughput screening method according to claim 3, is characterized in that: in described step D, terramycin content is 10-15 μ g/mL, content of streptomycin is 0.2-0.4 μ g/mL.
5. a kind of terramycin superior strain high-throughput screening method according to claim 3, is characterized in that: test tube slant medium component and content thereof used in described steps A, step e and step F are: 6.0%-7.0% wheat bran, 2.0%-2.5% agar.
6. a kind of terramycin superior strain high-throughput screening method according to claim 3, it is characterized in that: in described step D solid separation culture medium, composition and content thereof are: 2.0%-3.0% W-Gum, 0.2%-0.5% corn steep liquor, 0.1%-0.3% ammonium sulfate, 0.1%-0.5% calcium carbonate, 0.3%-0.5% sodium-chlor, 2.0%-2.5% agar.
7. a kind of terramycin superior strain high-throughput screening method according to claim 3, it is characterized in that: seed culture based component and content thereof used in described step e and step F are: 2.0%-3.0% W-Gum, 0.2%-0.5% corn steep liquor, 2.0%-3.0% analysis for soybean powder, 0.1%-0.3% ammonium sulfate, 0.1%-0.5% calcium carbonate, 0.3%-0.5% sodium-chlor.
8. a kind of terramycin superior strain high-throughput screening method according to claim 3, it is characterized in that: fermentation culture based component and content thereof used in described step e and step F are: 10%-15% W-Gum, 0.2%-0.5% corn steep liquor, 2.0%-3.0% analysis for soybean powder, 1.0%-1.5% ammonium sulfate, 1.0%-1.5% calcium carbonate, 0.3%-0.5% sodium-chlor, 0.01%-0.05% potassium primary phosphate, 0.1%-0.3% polyether defoaming agent.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104372058A (en) * | 2014-11-03 | 2015-02-25 | 金河生物科技股份有限公司 | Preparation method of oxytetracycline |
| CN104611280A (en) * | 2015-03-11 | 2015-05-13 | 河北圣雪大成制药有限责任公司 | Method for rapidly and selectively breeding terramycin strains |
| CN105821053A (en) * | 2015-01-04 | 2016-08-03 | 中国科学院微生物研究所 | Method for constructing recombinant strain by utilization of terramycin positive regulator gene and raising yield of terramycin |
| CN107574218A (en) * | 2017-09-28 | 2018-01-12 | 金河生物科技股份有限公司 | A kind of method of streptomyces rimosus fermenting and producing oxytetracycline calcium pre-mixing agent |
| CN108148884A (en) * | 2017-12-25 | 2018-06-12 | 安徽永生堂药业有限责任公司 | A kind of culture medium and fermentation process for producing terramycin |
| CN109136322A (en) * | 2018-09-19 | 2019-01-04 | 浙江浙大阳光科技有限公司 | A kind of high-throughput screening method for the streptomyces fradiae strain producing neomycin |
| CN111763667A (en) * | 2020-05-28 | 2020-10-13 | 河北圣雪大成制药有限责任公司 | Method for increasing yield of nortetracycline |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104372058A (en) * | 2014-11-03 | 2015-02-25 | 金河生物科技股份有限公司 | Preparation method of oxytetracycline |
| CN105821053A (en) * | 2015-01-04 | 2016-08-03 | 中国科学院微生物研究所 | Method for constructing recombinant strain by utilization of terramycin positive regulator gene and raising yield of terramycin |
| CN105821053B (en) * | 2015-01-04 | 2019-09-20 | 中国科学院微生物研究所 | Utilize method terramycin positive regulating gene building recombinant bacterium and improve oxytetracycline yield |
| CN104611280A (en) * | 2015-03-11 | 2015-05-13 | 河北圣雪大成制药有限责任公司 | Method for rapidly and selectively breeding terramycin strains |
| CN107574218A (en) * | 2017-09-28 | 2018-01-12 | 金河生物科技股份有限公司 | A kind of method of streptomyces rimosus fermenting and producing oxytetracycline calcium pre-mixing agent |
| CN108148884A (en) * | 2017-12-25 | 2018-06-12 | 安徽永生堂药业有限责任公司 | A kind of culture medium and fermentation process for producing terramycin |
| CN109136322A (en) * | 2018-09-19 | 2019-01-04 | 浙江浙大阳光科技有限公司 | A kind of high-throughput screening method for the streptomyces fradiae strain producing neomycin |
| CN111763667A (en) * | 2020-05-28 | 2020-10-13 | 河北圣雪大成制药有限责任公司 | Method for increasing yield of nortetracycline |
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