CN105002239A - Method for improving abamectin yield - Google Patents
Method for improving abamectin yield Download PDFInfo
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- CN105002239A CN105002239A CN201510441003.3A CN201510441003A CN105002239A CN 105002239 A CN105002239 A CN 105002239A CN 201510441003 A CN201510441003 A CN 201510441003A CN 105002239 A CN105002239 A CN 105002239A
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- avid kyowamycin
- power technology
- pulse power
- abamectin
- nanosecond pulse
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Abstract
The invention discloses a method for improving the abamectin yield. The method comprises the following steps that 1, a streptomyces avermitilis spore solution is processed through stimulation of the nanosecond pulse power technology; 2, the streptomyces avermitilis spore solution processed in the step (1) is placed into a culture medium for culture to improve the composite abamectin yield. The nanosecond pulse power technology is adopted for processing streptomyces avermitilis spores, expression of the key genes of aveR and malE generating abamectin is effectively improved, the biosynthesis process of the abamectin is promoted, meanwhile, the fermination period of the abamectin can be remarkably shortened, the abamectin yield is greatly improved, and the problems that existing abamectin technologies are long in period and low in titer are solved; meanwhile, according to the nanosecond pulse power technology, the device energy consumption is low, operation is easy and convenient, and the production cost is effectively decreased.
Description
Technical field
The present invention relates to a kind of method improving Avrmectin output, belong to biological technical field.
Background technology
Avrmectin (Avermectins, AVM) is the class sixteen-ring macrolide antibiotic produced by gram-positive bacteria Avid kyowamycin (Streptomycesavermitilis).According to the difference of linking group, Avrmectin is divided into 8 components and A1a, A2a, A1b, A2b, B1a, B2a, B1b and B2b, its textural difference mainly concentrates on 3 positions and C5, C22-C23 and C26, and wherein B1 composition activity is maximum, especially the strongest with the anthelmintic activity of B1a.Avrmectin has efficiently, safety, agriculture, herd, cure the features such as three use, and Avrmectin is one of the most effective harmless boilogical sterilant of generally acknowledging in the world at present, to person poultry safety under common dose, does not destroy the ecosystem.Along with people's living standard improve constantly and people to the demand of green food, biological pesticide gains great popularity in the market requirement.At present, the biosynthesizing research of Avrmectin on gene level has achieved breakthrough progress, its most of biosynthetic pathway is elucidated, and relevant Avrmectin biological synthesis gene cluster is by successful clone, and the function of most gene is also substantially clear and definite.Especially larger progress is also achieved to the generation key controlling gene of Avrmectin and the research of functional gene, found the corresponding key gene regulating and controlling Avrmectin output successively, as aveR, malE, sig25and orfX etc.Now, comprise about the technical way improving Avid kyowamycin fermentation titer and shortening fermentation period: utilize various mutagenic compound (ultraviolet (UV), nitrous acid HNO3) to process microorganism cells, improve cytogene mutation frequency, screening superior strain; Genetic engineering technique directional transformation cell, obtains superior strain; Optimize strain fermentation culture condition, improve thalline fermentation titer etc.But these technique means all also exist many shortcomings in actual applications.Such as, mutagenic compound easy inducible strain generation tolerance, thus cause negative sudden change to roll up, gain mutant sharply reduces and even disappears.The application of genetic engineering bacterium is mainly limited to the progress degree of people to Avid kyowamycin gene structure, because its gene structure is not also thoroughly elucidated completely now, therefore also cannot build mature and stable engineering bacteria so far; For the bacterial strain determined, when its extraneous factor such as somatomedin, inorganic salinity and temperature reaches optimum combination, then can not continue to improve its antibiotic yield.In a word, up to the present, above technique means can't be stablized, effectively shorten Avid kyowamycin fermentation period, Avrmectin output increased thus be restricted, and this present situation has become a Main Bottleneck problem of restriction Avrmectin industrial development.
Nanosecond pulsed electric field technology is 21 century emerging physical technique, and it has pulsewidth short (ns), field intensity high (kV/cm), energy low (mJ/cc), can not produce numerous features such as thermal change before and after effect.In the recent period, nanosecond pulsed electric field is in oncotherapy, collaborative chemotherapeutics killing tumor cells, and numerous biomedical sectors such as intracellular calcium regulation and control, platelet gel, permeability of cell membrane regulation and control cause the extensive concern of scholars.Especially in recent research, find that pulsed electrical field effectively can promote the sprouting of haloxylon ammondendron seed, the growing and the cultivation effect of chondrocyte of Arabidopis thaliana, but mechanism need further research.
Summary of the invention
The object of this invention is to provide a kind of method improving Avrmectin output, the present invention adopts nanosecond pulse power technology process Avid kyowamycin spore effectively can raise the expression of key gene aveR and malE producing Avrmectin, promotes the biosynthetic process of Avrmectin; Meanwhile, obviously can also shorten the fermentation period of Avid kyowamycin, increase substantially Avrmectin output.
Raising Avid kyowamycin provided by the invention produces the method for the amount of Avrmectin, comprises the steps: 1) by Avid kyowamycin spores solution nanosecond pulse power technology stimulation process;
2) by step 1) the described Avid kyowamycin spores solution that processes is placed in substratum and cultivates, and the Avrmectin output of synthesis is improved.
In the present invention, described Avid kyowamycin spore metabolism produces Avid kyowamycin, Avid kyowamycin synthesis Avrmectin.
In the present invention, described nanosecond pulse power technology is the pulsed electrical field sequence of non-thermal effect.
In above-mentioned method, the pulse width of described nanosecond pulse power technology can be 30 ~ 100ns, specifically can be 100ns;
The strength of electric field of described nanosecond pulse power technology can be 5 ~ 20kV/cm, specifically can be 5kV/cm, 10kV/cm, 15kV/cm, 10 ~ 15kV/cm or 5 ~ 15kV/cm;
The pulse-repetition of described nanosecond pulse power technology can be 0.1 ~ 2Hz, specifically can be 1Hz.
In above-mentioned method, step 2) in, the cycle of described cultivation can be 9 ~ 11d, specifically can be 9d, and after nanosecond pulse power technology stimulates, Avrmectin output plateau time of occurrence did sth. in advance the 5th day till now from the original the 7th day.
In above-mentioned method, described nanosecond pulse power technology adopts nanosecond pulsed electric field producer.
In above-mentioned method, the preparation process of described Avid kyowamycin spores solution is have the plate culture medium PBS buffered soln of described Avid kyowamycin spore to rinse by long, filters out the mycelia of Avid kyowamycin, namely obtains pure described Avid kyowamycin spores solution.
In above-mentioned method, described Avid kyowamycin adopts ATCC31267 type bacterial strain.
In the present invention, described PBS buffered soln consist of NaCl 8g/L, KCl 0.2g/L, Na
2hPO
41.44g/L and KH
2pO
40.24g/L, the PH of described PBS buffered soln is 7.4; Need through high-temperature sterilization before using.
In the present invention, except the process of nanosecond pulse power technology, cultivate Avid kyowamycin and be separated the method obtaining Avrmectin, same as the prior art.
The present invention has the following advantages:
The present invention adopts nanosecond pulse power technology process Avid kyowamycin spore, effective expression of raising key gene aveR and malE producing Avrmectin, promote the biosynthetic process of Avrmectin, obviously can also shorten the fermentation period of Avid kyowamycin simultaneously, increase substantially Avrmectin output, overcome the problem of the cycle length of Avid kyowamycin existing fermentation technique existence, low deficiency of tiring, the power technology of nanosecond pulse simultaneously device energy-wasting is very low, easy and simple to handle, effectively reduce production cost.
Accompanying drawing explanation
Fig. 1 is the setting drawing of the nanosecond pulse power technology adopted in the embodiment of the present invention.
Fig. 2 is the detection to Avrmectin output in the embodiment of the present invention 1, wherein Fig. 2 (a) detects the characteristic feature absorption peak of Avrmectin at ultraviolet band OD=245nm place, and Fig. 2 (b) makes Avid kyowamycin output plateau shortening detected result histogram for the process of nanosecond pulse power technology.
Fig. 3 is that the key gene after real-time quantitative RT-PCR technology for detection nanosecond pulse power technology stimulates Avid kyowamycin in the embodiment of the present invention 1 expresses changing conditions histogram, wherein Fig. 3 (a) is the result of aveR genetic expression, Fig. 3 (b) is the result of malE genetic expression, and Fig. 3 (c) is the result of sig25 genetic expression.
Avid kyowamycin before and after Fig. 4 position nanosecond pulse power technology of the present invention stimulates has cultivated the morphological change of 1,3,5,7 and 9 day respectively on solid medium, wherein C represents control group Avid kyowamycin, 5,10 and 15 field intensity (field intensity unit: kV/cm) representing pulse respectively.
In figure, mark is as follows:
1 nanosecond pulsed electric field producer, the pole cup of 2 Avid kyowamycin spores solution.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
In following embodiment, PBS buffered soln is by NaCl 8g/L, KCl 0.2g/L, Na
2hPO
41.44g/L and KH
2pO
40.24g/L forms, and PH is 7.4.
The method of embodiment 1, raising Avrmectin output
Experimental strain: Avid kyowamycin (ATCC31267)
Substratum and culture condition thereof:
Plate culture medium:
Starch 20g/L, yeast extract 4.0g/L, malt extract 10g/L, glucose 10g/L, agar 15g/L.
Seed culture medium:
Starch 30g/L, soybean cake powder 2g/L, yeast extract 4g/L, CoCl
26H
2o 0.01g/L.
Fermention medium:
Starch 70g/L, yeast extract 16.0g/L, glucose 5.0g/L, NaCl 0.2g/L, K
2hPO
43H
2o0.5g/L, MgSO
47H
2o 0.5g/L, CoCl
26H
2o 0.01g/L, KCl 4g/L, CaCO
32g/L.
Above substratum at 0.1MPa, 121 DEG C of sterilizings 30 minutes, culture temperature 28 DEG C, shaking speed 220 revs/min.
1, get 10ml PBS buffered soln be added on cover with Avid kyowamycin spore plate culture medium on, Avid kyowamycin spore is rinsed, then filter with 4 layers of gauze, Avid kyowamycin mycelia is filtered out, obtain purer Avid kyowamycin spores solution.
2, the stimulation process that 4 groups of (often organizing 4 parts) above-mentioned Avid kyowamycin spores solution of each 1ml carry out nanosecond short pulse power technology is respectively got in step 1.Open nanosecond pulsed electric field producer (i.e. nanosecond pulsed electric field sequencer), regulating impulse shooting parameter is that strength of electric field 4 groups uses 0kV/cm (control group), 5kV/cm, 10kV/cm and 15kV/cm respectively, pulse excitation number 20, pulse-repetition is 1Hz, pulse width 100 nanosecond.
3, after in step 2, the process of nanosecond pulse power technology terminates, Avid kyowamycin spores solution after collection and treatment, with PBS wash buffer twice, then 5000 revs/min centrifugal resuspended be 1ml spore liquid, this 1ml spore liquid is joined in the shaking flask that 20ml seed culture medium is housed, at 180 revs/min, cultivate in the shaking table incubator of 28 DEG C after 24 hours, the fermention medium getting the above-mentioned spores solution of 2ml and be placed in 100ml is fermented, 28 DEG C, 220 revs/min, fermentation period is 9 days, often organize 4 parts respectively 1, 3, 5, within 7 and 9 days, get 5ml bacterium liquid Detection and Extraction Avrmectin, carry out Avrmectin output and key gene detection of expression, experimental result as shown in Figures 2 and 3.
Fig. 2 (a) is the characteristic feature absorption peak of detection Avrmectin at ultraviolet band OD=245nm place, in this, as the High Throughput Screening Assay of high yield Avrmectin.From Fig. 2 (b), experimental result display millimicrosecond pulse technique of the present invention process makes Avid kyowamycin output plateau shorten, time did sth. in advance the 5th day till now from the original the 7th day, made the fermentation of the unit time of Avid kyowamycin take turns number and improve 40%.The Avrmectin yield increased group that experimental group Avid kyowamycin produces improves 42%.
Fig. 3 is that aveR gene, malE and the gene sig25 gene key gene after real-time quantitative RT-PCR technology for detection nanosecond pulse power technology of the present invention stimulates Avid kyowamycin expresses changing conditions.As shown in Figure 3, result shows the expression amount that the present invention can regulate and control key gene aveR and malE of Avrmectin output obtain significant rise after pulse sequence stimulates.
Fig. 4 is that the Avid kyowamycin before and after nanosecond pulse power technology of the present invention stimulates has cultivated the morphological change of 1,3,5,7 and 9 day respectively on solid medium, and wherein C represents control group Avid kyowamycin, 5,10,15 field intensity representing pulse respectively.Spore color and morphological change are the important biomolecule indexs whether evaluation bacterium morphs, as shown in Figure 4, we have cultivated 1,3,5,7 and 9 day respectively the Avid kyowamycin after nanosecond pulse sequence of stimuli on solid medium, find that the spore that the Avid kyowamycin before and after burst process is formed is consistent with control group in color and form, do not form mutant strain, prove that the present invention produces mutant strain to make Avrmectin output increased.
Claims (6)
1. improve the method that Avid kyowamycin produces the amount of Avrmectin, comprise the steps: 1) by Avid kyowamycin spores solution nanosecond pulse power technology stimulation process;
2) by step 1) the described Avid kyowamycin spores solution that processes is placed in substratum and cultivates, and Avrmectin output is improved.
2. method according to claim 1, is characterized in that: the pulse width of described nanosecond pulse power technology is 30 ~ 100ns;
The strength of electric field of described nanosecond pulse power technology is 5 ~ 20kV/cm;
The pulse-repetition of described nanosecond pulse power technology is 0.1 ~ 2Hz.
3. method according to claim 1 and 2, is characterized in that: step 2) in, the cycle of described cultivation is 9 ~ 11d.
4. the method according to any one of claim 1-3, is characterized in that: described nanosecond pulse power technology adopts nanosecond pulsed electric field producer.
5. the method according to any one of claim 1-4, it is characterized in that: the preparation process of described Avid kyowamycin spores solution is have the plate culture medium PBS buffered soln of described Avid kyowamycin spore to rinse by long, filter out the mycelia of Avid kyowamycin, namely obtain pure described Avid kyowamycin spores solution.
6. the method according to any one of claim 1-5, is characterized in that: described Avid kyowamycin adopts ATCC31267 type bacterial strain.
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| CN201510441003.3A CN105002239B (en) | 2015-07-24 | 2015-07-24 | A method of improving avermectin yield |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017146009A1 (en) * | 2016-02-24 | 2017-08-31 | 天野エンザイム株式会社 | Method for controlling enzyme productivity of microorganisms |
-
2015
- 2015-07-24 CN CN201510441003.3A patent/CN105002239B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| L.-Y. WANG等: "Novel mutation breeding method for Streptomyces avermitilis using an atmospheric pressure glow discharge plasma", 《JOURNAL OF APPLIED MICROBIOLOGY》 * |
| M. STACEY等: "Differential effects in cells exposed to ultra-short,high intensity electric fields: cell survival,DNA damage, and cell cycle analysis", 《MUTATION RESEARCH》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017146009A1 (en) * | 2016-02-24 | 2017-08-31 | 天野エンザイム株式会社 | Method for controlling enzyme productivity of microorganisms |
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Address after: 100086 Building-1 Floor-103-078, No. 25, Ningzhuang West Road, Haidian District, Beijing Patentee after: Beijing thirty-four Technology Co., Ltd. Address before: Room 313, Building No. 1, 538, Yongfeng Tun, Haidian District, Beijing 100094 Patentee before: Beijing thirty-four Technology Co., Ltd. |