CN104313109A - High-flux screening method of nosiheptide active streptomycete high-yield strain - Google Patents
High-flux screening method of nosiheptide active streptomycete high-yield strain Download PDFInfo
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- CN104313109A CN104313109A CN201410513049.7A CN201410513049A CN104313109A CN 104313109 A CN104313109 A CN 104313109A CN 201410513049 A CN201410513049 A CN 201410513049A CN 104313109 A CN104313109 A CN 104313109A
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Abstract
The invention provides a high-flux screening method of a nosiheptide active streptomycete high-yield strain. A novel ARTP breeding system is utilized to induce mutation of nosiheptide active streptomycete; a response surface process is utilized to optimize the 24-deep-hole plate fermentation conditions; and a 24-deep-hole plate culture process is utilized to establish the high-flux mutant screening method, thereby obtaining the high/stable-yield nosiheptide mutant strain. After verifying the relevance between the 24-deep-hole plate titer and shake flask titer, the screening method is very accurate and quick. Compared with the traditional shake flask fermentation screening process, the 24-deep-hole plate process can obviously enhance the screening efficiency of the nosiheptide high-yield strain.
Description
Technical field
The invention belongs to microorganism mutation breeding, field of fermentation engineering, particularly relate to a kind of high-throughput screening method of nosiheptide S.actuosus superior strain.
Background technology
Nosiheptide (Nosiheptide) is kind of a Thiopeptide antibiotics, also known as nosiheptide, mainly contains S.actuosus and produces.It has the growth promoting animal, the resistance against diseases improving efficiency of feed utilization, strengthen animal, and noresidue in animal body, to person poultry safety, the advantages such as environmental pollution is little are a kind of desirable feeding antibiotic updated articles.
At present, in areas such as European Union, Japan, Taiwan, nosiheptide is added at feed as unique feeding antibiotic.Domestic researcher has done large quantifier elimination in the seed selection and fermentation technology optimization etc. of nosiheptide S.actuosus kind, and the production technology of nosiheptide is greatly improved.But industrial fermentation per overall level is lower at present, the performance of producing bacterial classification still has very large room for promotion.
Utilize new A RTP breeding method mutagenesis nosiheptide S.actuosus fast and effeciently can obtain nosiheptide S.actuosus mutation library.In recent years medium optimization method turns to Statistic optimization gradually by traditional non-statistical optimized method, and namely design and interpretation of result by experiment, sets up relevant mathematical model, then the optimum value of solving model verifying.Wherein response surface optimization method achieves a series of good effect at biomedicine field.High flux screening is the committed step obtaining high productive mutant.
At present about screening method normally random screening method or the resistant panel sieve method of high yield nosiheptide S.actuosus strain, then adopt shaking flask carry out fermentation culture one by one and measure the target output of bacterial strain, the method screening operation amount is large, the cycle is long, efficiency is low and cost is high.
Summary of the invention
Method for current screening nosiheptide S.actuosus superior strain is consuming time, effort, poor efficiency, cost are higher, the present invention, by providing a kind of high-throughput screening method of nosiheptide S.actuosus superior strain, can solve above-mentioned Problems existing effectively; Optimize the fermentation condition of 24 deep-well plates simultaneously, verify 24 deep-well plates tire and shaking flask tire between dependency.
The present invention is achieved in that a kind of high-throughput screening method of nosiheptide S.actuosus superior strain, comprises the following steps:
(1) atmospheric pressure at room Cement Composite Treated by Plasma nosiheptide S.actuosus 150s is adopted;
(2) by nosiheptide S.actuosus dilution spread to be screened after mutagenesis in solid plate, at 37 ± 0.5 DEG C, cultivate 72h;
(3) access every hole with the single bacterium colony spore after the mutagenesis of toothpick picking to be equipped with in 24 deep-well plates of 4 ~ 6mL solid medium and to carry out solid culture, dig 0.2 ~ 0.3cm with inoculation shovel correspondingly simultaneously
2thalline access 24 deep-well plates that 2.55mL seed culture medium is housed carry out liquid culture; Seed culture terminates to transfer respectively and 24 deep-well plates of 0.93mL fermention medium is housed, and fermentation culture terminates survey and tires, and corresponding solid culture orifice plate bacterium colony is preserved, liquid culture 28 ± 0.5 DEG C, rotating speed 260r/min, seed culture 40h, fermentation 120h, solid culture 37 ± 0.5 DEG C, 72h;
(4) bacterium colony inoculating 24 deep-well plates solid culture is for shake flask fermentation after slant tube is cultivated, and survey is tired.
Preferably, in step (3), seed culture medium (%) is: soybean cake powder 2.0, glucose 3.0, corn steep liquor 2.0, MgSO
47H
2o 0.05, (NH
4) 2SO
40.3, light calcium carbonate 0.5,115 DEG C of sterilizing 25min.
Preferably, in step (3), described solid medium (%) is: Zulkovsky starch 2, soybean cake powder 0.5, KNO
30.1, NaCl 0.05, MgSO
47H
2o 0.05, K
2hPO
43H
2o 0.05, FeSO
47H
2o 0.001, agar 2.0-3.0,121 DEG C of sterilizing 20min; Seed culture medium (%) described in step (3): soybean cake powder 2.0, glucose 3.0, corn steep liquor 2.0, MgSO
47H
2o 0.05, (NH
4)
2sO
40.3, light calcium carbonate 0.5,115 DEG C of sterilizing 25min.
Preferably, in step (3), described fermention medium (%) is: starch 7.0, amylase (0.1% of starch), glucose 0.5, yeast powder 0.2, analysis for soybean powder (oil-containing) 4.0, (NH
4)
2sO
40.1, KNO
30.05, NaCl 0.4, light calcium carbonate 0.4,115 DEG C of sterilizing 25min.
The present invention overcomes the deficiencies in the prior art, a kind of high-throughput screening method of nosiheptide S.actuosus superior strain is provided, utilize new A RTP breeding system mutagenesis nosiheptide S.actuosus, 24 deep-well plates fermentation conditions are optimized by response phase method, and utilize 24 deep-well plates culture methods to set up the method for high flux screening mutant, obtain the nosiheptide mutant strain of high and stable yields.
Compared to the shortcoming and defect of prior art, the present invention has following beneficial effect: utilize new A RTP breeding method mutagenesis nosiheptide S.actuosus, porous plate culture method is utilized to set up the method for high flux screening mutant, the method verify 24 deep-well plates tire and shaking flask tire between dependency, make its screening method very accurately fast, compared with screening with traditional shake flask fermentation, 24 deep-well plates methods can significantly improve the screening efficiency of nosiheptide superior strain simultaneously.
Accompanying drawing explanation
Fig. 1 is that in the embodiment of the present invention 1, seed culture medium liquid amount cultivates the impact of tiring on 24 deep-well plates;
In Fig. 2 embodiment of the present invention 2, fermention medium liquid amount cultivates the impact of tiring on 24 deep-well plates;
In Fig. 3 embodiment of the present invention 3, fermentation time cultivates the impact of tiring on 24 deep-well plates;
Fig. 4 be in the embodiment of the present invention 5 mutant strain 24 deep-well plates tire and shaking flask tire between dependency;
Fig. 5 is the relative potency of mutant strain in the embodiment of the present invention 6;
The dependency that after the fermentation of Fig. 6 embodiment of the present invention 7 aperture plate, spectrophotometer and microplate reader gaging hole plate are tired;
In Fig. 7 embodiment of the present invention 8, after shake flask fermentation, spectrophotometer and microplate reader survey the dependency that shaking flask tires.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Bacterial strain: nosiheptide producing strains S.actuosus (Streptomyces actuosu) NXT-NA10 bacterial strain is by Anhui Polytechnic University's Laboratories Accession, this bacterial strain is at article " response phase method optimizes nosiheptide fermentation condition " (Chinese microbiotic magazine, 2013,38 (6): 430-433) open in.
Instrument and reagent: ZHWY-C2112B fixed temperature and humidity incubator: Shanghai Zhi Cheng analytical instrument manufacturing company; HTS-T008 constant temperature oscillator: Shanghai Gan Wei Bioisystech Co., Ltd; Super clean bench: Purifying Equipment Co., Ltd., Suzhou, Sorvall ST 16R whizzer: Sai Mo flies generation that, and microplate reader: Sai Mo flies generation that; ARTP breeding machine: Beijing Siqingyuan Bioscience Co., Ltd..Pharmaceutical chemicals is domestic analytical pure.
The assay of nosiheptide: 723 spectrophotometric colo methods, 96 orifice plate microplate reader colorimetrys.
The impact that embodiment 1 24 deep-well plates seed culture medium liquid amount is tired on nosiheptide
It is 0.7mL, 1.0mL, 1.3mL, 1.6mL, 1.9mL, 2.2mL, 2.5mL, 2.8mL, 3.1mL that seed culture medium chooses initial loading liquid measure respectively, and compare its impact on nosiheptide output in 24 deep-well plates, result as shown in Figure 1.As seen from Figure 1, when initial liquid amount is at 2.5mL, the output of nosiheptide is the highest.
The impact that embodiment 2 24 deep-well plates fermention medium liquid amount is tired on nosiheptide
(%) is by starch 7.0 by mass percentage, amylase (0.1% of starch), glucose 0.5, yeast powder 0.2, analysis for soybean powder (oil-containing) 4.0, (NH
4)
2sO
40.1, KNO
30.05, NaCl 0.4, light calcium carbonate 0.4,115 DEG C of sterilizing 25min obtain fermention medium.
Under the fermention medium liquid amount of 0.7mL, 1.0mL, 1.3mL, 1.6mL, 1.9mL, 2.2mL, 2.5mL, 2.8mL, 3.1mL, 3.4mL, carry out fermentation test respectively, compare its impact on nosiheptide output, result as shown in Figure 2.When the output of 24 deep-well plates fermentations under the condition of fermention medium at 1.0mL in 24 deep-well plates is the highest.
Embodiment 3 fermentation time cultivates the impact of tiring on 24 deep-well plates
Select different fermentation times to measure the output of nosiheptide, result as shown in Figure 3.As seen from Figure 3, at earlier fermentation, along with the output of the prolongation nosiheptide of fermentation time is in increase, but after fermentation 120h, its output declines to some extent, and therefore, fermentation time selects 120h to be advisable.
The foundation of embodiment 4 response surface model and variance analysis
The basis of experiment of single factor result is designed Box-Behnken center combination design, tire as response value with the nosiheptide of 24 deep-well plates fermentations, response surface analysis test is designed to seed culture medium liquid amount (A), fermention medium liquid amount (B) and fermentation time (C).Result is as shown in table 1:
Table 1 Box-Behnken experimental design and result
Utilize the experimental data of SAS9.1 software his-and-hers watches 3 to carry out regression analysis and obtain quadratic regression equation:
Y1＝871.8507+26.957A-52.09825B+7.36475C-95.61708A
2+54.925AB+23.331AC-107.0856B
2-33.5345BC-141.3516C
2。
Variance interpretation of result is carried out according to this regression model, as shown in table 2 below:
The results of analysis of variance of table 2 regression model
As can be seen from Table 2, model Pr>F=0.000286, this model returns extremely remarkable, and it loses intends item Pr>F=0.06722, and mistake is intended remarkable, and model is more accurate.Coefficient R
2=0.9880 shows that fitting degree is good, has good dependency.Meanwhile, once item A is remarkable, and B item is extremely remarkable, quadratic term A
2, B
2, C
2be pole conspicuous level, mutual item AC is extremely remarkable, and BC item is remarkable, has obvious interaction between explanation factor.
The extreme point coordinate regression equation local derviation differential process obtained above being obtained to model is A=0.08176, B=-0.23173, C=0.06029.Namely the top condition of 24 deep-well plates fermentation product nosiheptide is: seed culture medium liquid amount 2.54906mL, fermention medium liquid amount 0.93048mL, fermentation time 121.4469h, according to actual experiment operation, optimal conditions is modified to: seed culture medium liquid amount 2.55mL, fermention medium liquid amount 0.93mL, fermentation time 121h.
The foundation of embodiment 5 mutant strain high flux screening model
In order to rapid screening is to the bacterial strain of high yield nosiheptide, the present invention establishes the high-throughput screening method utilizing 24 deep-well plates.The mutagenic treatment liquid dilution of acquisition will be operated by embodiment 1, coat in solid plate substratum and carry out cultivation 72h at 37 ± 0.5 DEG C, random picking 21 single bacterium colonies, access is equipped with in 24 deep-well plates of seed culture medium and solid medium respectively, seed culture terminates fermentation culture of transferring, survey after 120h and tire, corresponding solid culture orifice plate bacterium colony is preserved, liquid culture 28 ± 0.5 DEG C, rotating speed 260r/min, 72h, solid culture 37 ± 0.5 DEG C, 72h.Inoculate the bacterium colony of 24 deep-well plates solid culture after slant tube is cultivated, access is equipped with in the shaking flask of seed culture medium and is cultivated, temperature 28 ± 0.5 DEG C, humidity 40 ~ 50% in proportion, rotating speed 260r/min, seed culture 40h, turns to have access in the shaking flask of a certain amount of fermention medium and cultivates, temperature 28 ± 0.5 DEG C, humidity 40 ~ 50%, rotating speed 260r/min, cultivate 120h, survey is tired.Data origin 8.6 software recorded carries out regression analysis, with verify mutant strain 24 deep-well plates tire and shaking flask tire between dependency, result is as shown in Figure 4.Dependency between 21 plant mutant strain 24 deep-well plates are tired and shaking flask is tired is very high, and relation conefficient absolute value R is 0.88264.In general, when experiment sample amount is more than 9, as long as R value reaches 0.7, so just assert between two variablees it is really relevant.Sample size of the present invention is 21, and relation conefficient absolute value R reaches 0.88264, and therefore 24 deep-well plates culture methods can as the effective ways of rapid screening nosiheptide High yield Mutant.
The screening of embodiment 6 high productive mutant
The substratum liquid amount adopting embodiment 4 to optimize and fermentation time, carry out 24 deep-well plates culture method screenings to the mutant strain implementing to obtain by embodiment 1, survey the superior strain shake flask fermentation obtained and tire, result as shown in Figure 5.Seed selection is tired to 17 strains the mutant strain of raising more than 20%, wherein improves 5 strains of more than 30%, 3 strains of more than 40%.
The genetic stability analysis of embodiment 7 mutant strain
Solid plate activates bacterium, transfers in seed culture medium and carry out one-level cultivation, continuously transfer 8 times, then do shake flask fermentation and compare the stability that mutant strain produces nosiheptide ability.Result is as shown in table 3:
The genetic stability analysis of table 3 superior strain
The three strain High yield Mutants through plasma body mutagenesis all have good genetic stability.
The dependency that embodiment 8 microplate reader measures and spectrophotometric determination 24 deep-well plates is tired
The substratum liquid amount adopting embodiment 4 to optimize and fermentation time, 24 deep-well plates cultivation and fermentation are carried out to mutant strain, 24 deep-well plates are tired and adopts microplate reader and spectrophotometric determination respectively, as shown in Figure 6, the relation conefficient absolute value R that microplate reader measures and spectrophotometric determination 24 deep-well plates is tired is 0.99534 to result.
The dependency that embodiment 9 microplate reader measures and spectrophotometric determination shaking flask is tired
Carry out shake flask fermentation to the mutant strain implementing to obtain by embodiment 1, tiring to shaking flask adopts microplate reader and spectrophotometric determination respectively, and as shown in Figure 7, the relation conefficient absolute value R that microplate reader measures and spectrophotometric determination shaking flask is tired is 0.967 to result.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (4)
1. a high-throughput screening method for nosiheptide S.actuosus superior strain, is characterized in that, comprises the following steps:
(1) atmospheric pressure at room Cement Composite Treated by Plasma nosiheptide S.actuosus 150s is adopted;
(2) by nosiheptide S.actuosus dilution spread to be screened after mutagenesis in solid plate, at 37 ± 0.5 DEG C, cultivate 72h;
(3) access every hole with the single bacterium colony spore after the mutagenesis of toothpick picking to be equipped with in 24 deep-well plates of 4 ~ 6mL solid medium and to carry out solid culture, access with the thalline that inoculation shovel digs 0.2 ~ 0.3cm2 24 deep-well plates that 2.55mL seed culture medium is housed correspondingly simultaneously and carry out liquid culture; Seed culture terminates to transfer respectively and 24 deep-well plates of 0.93mL fermention medium is housed, and fermentation culture terminates survey and tires, and corresponding solid culture orifice plate bacterium colony is preserved, liquid culture 28 ± 0.5 DEG C, rotating speed 260r/min, seed culture 40h, fermentation 120h, solid culture 37 ± 0.5 DEG C, 72h;
(4) bacterium colony inoculating 24 deep-well plates solid culture is for shake flask fermentation after slant tube is cultivated, and survey is tired.
2. the high-throughput screening method of nosiheptide S.actuosus superior strain as claimed in claim 1, it is characterized in that, in step (3), seed culture medium (%) is: soybean cake powder 2.0, glucose 3.0, corn steep liquor 2.0, MgSO
47H
2o 0.05, (NH
4)
2sO
40.3, light calcium carbonate 0.5,115 DEG C of sterilizing 25min.
3. the high-throughput screening method of nosiheptide S.actuosus superior strain as claimed in claim 2, it is characterized in that, in step (3), described solid medium (%) is: Zulkovsky starch 2, soybean cake powder 0.5, KNO
30.1, NaCl 0.05, MgSO
47H
2o 0.05, K
2hPO
43H
2o 0.05, FeSO
47H
2o 0.001, agar 2.0-3.0,121 DEG C of sterilizing 20min; Seed culture medium (%) described in step (3): soybean cake powder 2.0, glucose 3.0, corn steep liquor 2.0, MgSO
47H
2o 0.05, (NH
4)
2sO
40.3, light calcium carbonate 0.5,115 DEG C of sterilizing 25min.
4. the high-throughput screening method of nosiheptide S.actuosus superior strain as claimed in claim 3, it is characterized in that, in step (3), described fermention medium (%) is: starch 7.0, amylase (0.1% of starch), glucose 0.5, yeast powder 0.2, analysis for soybean powder (oil-containing) 4.0, (NH
4)
2sO
40.1, KNO
30.05, NaCl 0.4, light calcium carbonate 0.4,115 DEG C of sterilizing 25min.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104719634A (en) * | 2015-02-10 | 2015-06-24 | 河北圣雪大成制药有限责任公司 | Method for preparing nosiheptide pre-mixing agent |
| CN105219658A (en) * | 2015-10-29 | 2016-01-06 | 无锡福祈制药有限公司 | A kind of method of rapidly and efficiently screening tacrolimus superior strain |
| CN106191030A (en) * | 2016-09-18 | 2016-12-07 | 江南大学 | A kind of high-throughput screening method of chlortetracycline superior strain |
| 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 |
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2014
- 2014-09-29 CN CN201410513049.7A patent/CN104313109A/en active Pending
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| 李霞: "含硫多肽类抗生素那西肽的菌种诱变及产量提高", 《工业微生物》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104719634A (en) * | 2015-02-10 | 2015-06-24 | 河北圣雪大成制药有限责任公司 | Method for preparing nosiheptide pre-mixing agent |
| CN104719634B (en) * | 2015-02-10 | 2018-08-24 | 河北圣雪大成制药有限责任公司 | A method of preparing Nosiheptide premixed agent |
| CN105219658A (en) * | 2015-10-29 | 2016-01-06 | 无锡福祈制药有限公司 | A kind of method of rapidly and efficiently screening tacrolimus superior strain |
| CN106191030A (en) * | 2016-09-18 | 2016-12-07 | 江南大学 | A kind of high-throughput screening method of chlortetracycline superior strain |
| 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 |
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