CN107815478A - Utilize the method for streptomyces microaureus BJX007 fermenting and producing kasugarnycin - Google Patents

Utilize the method for streptomyces microaureus BJX007 fermenting and producing kasugarnycin Download PDF

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Publication number
CN107815478A
CN107815478A CN201610817957.4A CN201610817957A CN107815478A CN 107815478 A CN107815478 A CN 107815478A CN 201610817957 A CN201610817957 A CN 201610817957A CN 107815478 A CN107815478 A CN 107815478A
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bjx007
fermentation
kasugarnycin
medium
fermenting
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CN107815478B (en
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周贤龙
石怀月
刘静
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MUDANJIANG BAIJIAXIN BIOLOGICAL TECHNOLOGY Co Ltd
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MUDANJIANG BAIJIAXIN BIOLOGICAL TECHNOLOGY Co Ltd
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/44Preparation of O-glycosides, e.g. glucosides
    • C12P19/46Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical bound to a cyclohexyl radical, e.g. kasugamycin

Abstract

The present invention provides a kind of method using streptomyces microaureus BJX007 fermenting and producing kasugarnycin, it is that proper amount of surfactant is added into fermentation medium, with streptomyces microaureus (Streptomyces microaureus) BJX007 (deposit number CGMCC No.11622) for fermentation strain, the fermenting and producing of kasugarnycin is carried out.Streptomyces microaureus BJX007 provided by the invention, the ability of its fermenting and producing kasugarnycin is high, yield is up to 25000 μ g/mL zymotic fluids, and it produces the ability of kasugarnycin and still keeps previous level after bacterial strain BJX007 continuous passages 5 times, show that its genetic stability is good, available for the production for industrializing extensive kasugarnycin.By adding surfactant into fermentation medium, so as to improve bacterial strain BJX007 cell leakages, fermentation yield can be made to be promoted to 28000 μ g/mL zymotic fluids.The present invention provides valuable microbial resources to produce kasugarnycin using biofermentation technique.

Description

Utilize the method for streptomyces microaureus BJX007 fermenting and producing kasugarnycin
Technical field
The present invention relates to field of microbial fermentation, specifically, is related to one kind and is fermented using streptomyces microaureus BJX007 The method for producing kasugarnycin.
Background technology
Kasugarnycin (Kasugamycin) is also known as kasugarnycin, is the aminoglycoside antibiotics as caused by actinomyces, category Interior suction antibiotic, have treatment and prevention effect concurrently, sterling is white, needle-shaped crystals solid, is stablized at normal temperatures, acid and neutral Under the conditions of it is stable, easily decompose in the basic conditions.Aqua form is bottle green liquid, can be stored more than 2 years under normal temperature, to environment Close friend, it is comparatively ideal disinfectant use in agriculture, is used widely in agricultural production have excellent preventive effect to the rice blast on rice And therapeutic action, there is special efficacy to preventing and treating the diseases such as watermelon bacterial angular leaf spot, peach gummosis, shot hole, shothole disease in addition.
The content of the invention
It is an object of the invention to provide a kind of method using streptomyces microaureus BJX007 fermenting and producing kasugarnycin.
In order to realize the object of the invention, present invention firstly provides the streptomyces microaureus of plant height production kasugarnycin (Streptomyces microaureus) BJX007, it is general that the bacterium has been preserved in China Committee for Culture Collection of Microorganisms Logical microorganism center, address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode 100101, deposit number CGMCC No.11622, preservation date on November 5th, 2015.
The method that the present invention utilizes streptomyces microaureus BJX007 fermenting and producing kasugarnycin, it is into fermentation medium Proper amount of surfactant is added, using streptomyces microaureus BJX007 as fermentation strain, carries out the fermenting and producing of kasugarnycin.
The fermentative medium formula is as follows:Cornstarch 15-20g/L, beancake powder 45-50g/L, sodium chloride 2.5g-3/ L, potassium dihydrogen phosphate 0.2g-0.5/L, soya-bean oil 10-12mL/L, amylase 0.05-0.1g/L, pH value are natural;The amylase Enzyme activity is 10000U/g.
The surfactant includes at least one of Tween-20, neopelex and lecithin etc., preferably Tween-20.
Wherein, the content of Tween-20 is 0.5-3g/L, preferably 1g/L in the fermentation medium.
Fermentation condition is as follows:Bacterial strain BJX007 seed liquors are equipped with fermentation medium by 10% mass percent access In fermentation flask, the volume 250mL of the fermentation flask, liquid amount 35mL are (excellent in 28 DEG C of 220r/min concussion and cultivates 165-170h Select 7d), after fermentation ends, the isolated kasugarnycin from zymotic fluid.
Wherein, the preparation method of bacterial strain BJX007 seed liquors is as follows:The preparation method of the bacterial strain BJX007 seed liquors is such as Under:Take 1cm from bacterial strain BJX007 slant medium2With 250mL seed of the bacterium culture medium access equipped with fermentation medium In bottle, liquid amount 30mL, in 28 DEG C of 220r/min concussion and cultivate 48h, gained bacterium solution is filled by 10% mass percent access Have in the fermentation flask of fermentation medium, the volume 250mL of the fermentation flask, liquid amount 35mL, shaken in 28 DEG C of 220r/min 24h is cultivated, obtains seed liquor.
Streptomyces microaureus BJX007 provided by the invention, the ability of its fermenting and producing kasugarnycin is high, and yield is reachable 25000 μ g/mL zymotic fluids, and it produces the ability of kasugarnycin and still keeps previous level after bacterial strain BJX007 continuous passages 5 times, Show that its genetic stability is good, available for the production for industrializing extensive kasugarnycin.By adding table into fermentation medium Face activating agent, so as to improve bacterial strain BJX007 cell leakages, fermentation yield can be made to be promoted to 28000 μ g/mL zymotic fluids.This hair It is bright to provide valuable microbial resources to produce kasugarnycin using biofermentation technique.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment According to conventional laboratory conditions, as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J&Russell DW, Molecular Cloning:A Laboratory Manual, 2001), or the condition according to manufacturer's specification suggestion.
Amylase used is alpha-amylase in the present invention, enzyme activity 10000U/g.
The bacterial strain BJX007 of embodiment 1 separation and identification
1st, the separation identification of original strain
One plant of actinomyces is separated to from the vegetable field near of Mudanjiang Hailin City lateral road river, specific method is as follows:Will Suspension is made in soil, and soil supension is inoculated in isolation medium and cultivated, and picking single bacterium colony is diluted line separation Purifying, obtain pure culture bacterial strain.According to《Fungal identification handbook》Concordance list carries out strain morphology identification.
Identified, the bacterial strain is aerobic actinomyces, and twist, spore is spherical to oval, intermediate projections, table for fibrillae of spores Face is smooth, and aerial hyphae is canescence on synthetic media, and substrate mycelium is khaki.
It is wherein, described that to be separately cultured based formulas as follows:Tryptone 3g/L, beancake powder 10g/L, glycerine 10mL/L, chlorination Sodium 3g/L, calcium carbonate 2g/L, agar powder 25g/L, pH value are natural.The synthetic media formula is as follows:Cornstarch 20g/L, Beancake powder 45g/L, sodium chloride 2.5g/L, potassium dihydrogen phosphate 0.2g/L, soya-bean oil 12mL/L, amylase 0.1g/L, pH value are natural.
2nd, the mutagenic treatment of original strain
(1) ultraviolet mutagenesis is twice:Bacteria suspension 5ml is taken, is added in sterile petri dish, with 30W ultraviolet irradiation instrument in 30cm Highly locate vertical irradiation.It is respectively 60s, 90s to set irradiation time.After isolation medium sterilizing, addition final concentration 0.1 μ g/ml, The filtered degerming streptomysins of 0.2 μ g/ml, paved plate, 28 DEG C of culture 10d.Picking single bacterium colony carries out height according to the method described above Flux screening, shaking flask retrial.The high yield spring thunder tenebrarius strain of acquisition carries out next step mutagenesis.
(2) natrium nitrosum (NIT) mutagenesis
The bacterial strain that will be arrived by Uv-induced screening, prepares the bacteria suspension of the bacterial strain, takes 10mL bacteria suspensions to be separately added into It is silicate modified containing various concentrations (0,5,10,20,40,60 and 80mg/L) NIT equipped with 90mL in bottle in 250mL conical flasks Culture medium, 40min is handled under the conditions of 30 DEG C, 200r/min, be then centrifuged for separating thallus precipitation, and with sterile water washing 3 times, NIT is removed, terminates mutagenesis.Then each mutagenesis bacterium solution 1mL is taken to be coated on after diluting on modified medium agar flat board, at 30 DEG C Bacterium colony counting is carried out after cultivating 7d, it is determined that the bacterium colony under optimal Induced dosage and the picking Induced dosage carries out primary dcreening operation and secondary screening. The bacterial strain for obtaining high yield kasugarnycin continues mutagenesis processing.
(3) lithium chloride is handled
The bacterial strain that will be arrived by NIT mutagenesis screenings, prepares the spore suspension of the bacterial strain, takes 10mL spore suspension (spore counts 108Individual/mL), LiCl is added by 1% concentration, 10h, 12h, 15h, 20h are vibrated on shaking table, mutagenesis is terminated, takes each mutagenic bacteria Liquid 1mL dilution spreads carry out bacterium colony counting, it is determined that optimal mutagens on agar medium flat board after 10d is cultivated at 28 DEG C Measure the bacterium colony under simultaneously picking Induced dosage and carry out primary dcreening operation and secondary screening, the final bacterial strain for obtaining plant height production kasugarnycin BJX007。
3rd, bacterial strain BJX007 identification
Identified, bacterial strain BJX007 is aerobic actinomyces, and twist, spore is spherical to oval, middle convex for fibrillae of spores Rise, surface is smooth, and aerial hyphae is canescence on synthetic media, and substrate mycelium is khaki.
Bacterial strain BJX007 16SrDNA sequences such as SEQ ID NO:Shown in 1, its 16SrDNA sequence is entered in GenBank Row compares, and builds bacterial strain BJX007 systematic evolution tree.Wherein, for PCR amplification bacterial strain BJX007 16SrDNA sequences Primer is:
Forward primer:5′-AGAGTTTGATCCTGGCTCAG-3′
Reverse primer:5′-AAGTCGTAACAAGGTAGCCGTA-3′
Comprehensive strain morphology feature, 16SrDNA gene order phylogenetic analysis results, it is little Jin to identify bacterial strain BJX007 Color streptomycete (Streptomyces microaureus).
Applications of the bacterial strain BJX007 of embodiment 2 in fermenting and producing kasugarnycin
1st, seed liquor is prepared:Take 1cm from bacterial strain BJX007 slant medium2Band bacterium culture medium access is equipped with fermentation In the 250mL seed bottles of culture medium, liquid amount 30mL, in 28 DEG C of 220r/min concussion and cultivate 48h, gained bacterium solution is by 10% In fermentation flask of the mass percent access equipped with fermentation medium, the volume 250mL of the fermentation flask, liquid amount 35mL, in 28 DEG C of 220r/min concussion and cultivate 24h, obtain seed liquor.
2nd, fermented and cultured:Above-mentioned seed liquor is equipped with to the fermentation flask of fermentation medium by 10% mass percent access In, the volume 250mL of the fermentation flask, liquid amount 35mL, in 28 DEG C of 220r/min shaking table concussion and cultivate 7d, fermentation ends Afterwards, kasugarnycin content is up to 25000 μ g/mL in gained zymotic fluid, and then separation obtains kasugarnycin from zymotic fluid.
Wherein, the fermentative medium formula is as follows:Cornstarch 20g/L, beancake powder 45g/L, sodium chloride 2.5g/L, phosphorus Acid dihydride potassium 0.2g/L, soya-bean oil 12mL/L, amylase 0.1g/L, pH value are natural.Medium sterilization condition:121 DEG C of sterilizings 30min。
The method that embodiment 3 improves kasugarnycin fermentation yield
Surface active agent tween -20 are added into the fermentation medium of embodiment 2, make its final concentration of 1g/L.Then, exist Fermented and cultured is carried out under the same terms, kasugarnycin content can be promoted to 28000 μ g/mL in gained zymotic fluid.
Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.

Claims (7)

1. utilize the method for streptomyces microaureus BJX007 fermenting and producing kasugarnycin, it is characterised in that into fermentation medium Proper amount of surfactant is added, using streptomyces microaureus (Streptomyces microaureus) BJX007 as fermentation strain, Carry out the fermenting and producing of kasugarnycin;
Wherein, the deposit number of the streptomyces microaureus BJX007 is CGMCC No.11622.
2. according to the method for claim 1, it is characterised in that the fermentative medium formula is as follows:Cornstarch 15- 20g/L, beancake powder 45-50g/L, sodium chloride 2.5g-3/L, potassium dihydrogen phosphate 0.2g-0.5/L, soya-bean oil 10-12mL/L, amylase 0.05-0.1g/L, pH value are natural;The enzyme activity of the amylase is 10000U/g.
3. according to the method for claim 2, it is characterised in that the fermentative medium formula is as follows:Cornstarch 20g/ L, beancake powder 45g/L, sodium chloride 2.5g/L, potassium dihydrogen phosphate 0.2g/L, soya-bean oil 12mL/L, amylase 0.1g/L.
4. according to the method described in claim any one of 1-3, it is characterised in that the surfactant includes Tween-20, ten At least one of dialkyl benzene sulfonic acids sodium and lecithin.
5. according to the method for claim 4, it is characterised in that the content of Tween-20 is 0.5- in the fermentation medium 3g/L。
6. according to the method described in claim any one of 1-5, it is characterised in that bacterial strain BJX007 seed liquors are pressed to 10% matter Measure in fermentation flask of the percentage access equipped with fermentation medium, the volume 250mL of the fermentation flask, liquid amount 35mL, in 28 DEG C 220r/min concussion and cultivate 165-170h, after fermentation ends, the isolated kasugarnycin from zymotic fluid.
7. according to the method for claim 6, it is characterised in that the preparation method of the bacterial strain BJX007 seed liquors is as follows: Take 1cm from bacterial strain BJX007 slant medium2With 250mL seed bottle of the bacterium culture medium access equipped with fermentation medium Interior, liquid amount 30mL, in 28 DEG C of 220r/min concussion and cultivate 48h, gained bacterium solution is equipped with by 10% mass percent access In the fermentation flask of fermentation medium, the volume 250mL of the fermentation flask, liquid amount 35mL, in 28 DEG C of 220r/min concussion trainings 24h is supported, obtains seed liquor.
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Cited By (4)

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Publication number Priority date Publication date Assignee Title
CN108477218A (en) * 2018-03-23 2018-09-04 吉林省八达农药有限公司 A kind of rice terrace fungicide and its application
CN108949642A (en) * 2018-08-31 2018-12-07 福建省微生物研究所 It is a kind of produce tacrolimus streptomycete high-efficiency fermenting culture medium and its application
CN111184030A (en) * 2020-01-09 2020-05-22 枣庄市杰诺生物酶有限公司 Kasugamycin raw powder and preparation method and application thereof
CN111705092A (en) * 2020-07-20 2020-09-25 山东齐发药业有限公司 Feed supplement for improving fermentation titer of spectinomycin and preparation method and use method thereof

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108477218A (en) * 2018-03-23 2018-09-04 吉林省八达农药有限公司 A kind of rice terrace fungicide and its application
CN108477218B (en) * 2018-03-23 2020-09-18 吉林省八达农药有限公司 Paddy field bactericide and application thereof
CN108949642A (en) * 2018-08-31 2018-12-07 福建省微生物研究所 It is a kind of produce tacrolimus streptomycete high-efficiency fermenting culture medium and its application
CN111184030A (en) * 2020-01-09 2020-05-22 枣庄市杰诺生物酶有限公司 Kasugamycin raw powder and preparation method and application thereof
CN111705092A (en) * 2020-07-20 2020-09-25 山东齐发药业有限公司 Feed supplement for improving fermentation titer of spectinomycin and preparation method and use method thereof

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