CN107815426A - The special strain therefore of fermenting and producing kasugarnycin and its application - Google Patents

The special strain therefore of fermenting and producing kasugarnycin and its application Download PDF

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CN107815426A
CN107815426A CN201610817972.9A CN201610817972A CN107815426A CN 107815426 A CN107815426 A CN 107815426A CN 201610817972 A CN201610817972 A CN 201610817972A CN 107815426 A CN107815426 A CN 107815426A
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kasugarnycin
bjx007
bacterial strain
fermentation
medium
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CN107815426B (en
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周贤龙
石怀月
刘静
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Mudanjiang Bioseen Biological Co ltd
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/46—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical bound to a cyclohexyl radical, e.g. kasugamycin

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Abstract

The present invention relates to a kind of special strain therefore of fermenting and producing kasugarnycin and its application.The present invention is separated to one plant of aerobic actinomyces from the vegetable soil of Mudanjiang Hailin City, is handled by carrying out ultraviolet and mutagenesis to the actinomyces, and the final bacterial strain BJX007 for obtaining plant height production kasugarnycin, its deposit number is CGMCC No.11622.It is identified, bacterial strain BJX007 is streptomyces microaureus (Streptomyces microaureus), 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.The present invention provides valuable microbial resources to produce kasugarnycin using biofermentation technique.

Description

The special strain therefore of fermenting and producing kasugarnycin and its application
Technical field
The present invention relates to field of microbial fermentation, specifically, is related to a kind of special strain therefore of fermenting and producing kasugarnycin And its application.
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 special strain therefore of fermenting and producing kasugarnycin -- streptomyces microaureus BJX007.
It is a further object of the present invention to provide applications of the bacterial strain BJX007 in fermenting and producing kasugarnycin.
In order to realize the object of the invention, the present invention is separated to one from the vegetable field near of Mudanjiang Hailin City lateral road river Strain actinomyces, specific method are as follows：Suspension is made in soil, soil supension is inoculated in isolation medium and cultivated, is chosen Take single bacterium colony to be diluted line to isolate and purify, obtain pure culture bacterial strain.According to《Fungal identification handbook》Concordance list carries out bacterial strain Morphological 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.
Following mutagenic treatment is carried out to this plant of actinomyces：
(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。
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), the bacterium has been preserved in China Committee for Culture Collection of Microorganisms Common micro-organisms 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 present invention also provides applications of the streptomyces microaureus BJX007 in fermenting and producing kasugarnycin.
Comprise the following steps：
S1, prepare seed liquor：Take 1cm from bacterial strain BJX007 slant medium2Band bacterium culture medium access is equipped with hair In the 250mL seed bottles of ferment culture medium, liquid amount 30mL, in 28 DEG C of 220r/min concussion and cultivate 48h, gained bacterium solution presses 10% Mass percent access equipped with fermentation medium fermentation flask in, the volume 250mL of the fermentation flask, liquid amount 35mL, In 28 DEG C of 220r/min concussion and cultivate 24h, seed liquor is obtained；
S2, 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 cultivates 165-170h (preferably 7d), after fermentation ends, the isolated kasugarnycin from zymotic fluid.
Wherein, 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.
Preferably, 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.
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.The present invention is to utilize biofermentation technique Production kasugarnycin provides the microbial resources of preciousness.
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, according to《Fungal identification hand Volume》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.
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。
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 (4)

  1. Streptomyces microaureus 1. (Streptomyces microaureus) BJX007, its deposit number are CGMCC No.11622。
  2. 2. applications of the streptomyces microaureus BJX007 in fermenting and producing kasugarnycin described in claim 1.
  3. 3. application according to claim 2, it is characterised in that comprise the following steps：
    S1, prepare seed liquor：Take 1cm from bacterial strain BJX007 slant medium2Band bacterium culture medium access is equipped with fermented and cultured In the 250mL seed bottles of base, liquid amount 30mL, in 28 DEG C of 220r/min concussion and cultivate 48h, gained bacterium solution presses 10% quality 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 24h, obtain seed liquor；
    S2, fermented and cultured：Above-mentioned seed liquor is equipped with the fermentation flask of fermentation medium by 10% mass percent access, institute The volume 250mL of fermentation flask, liquid amount 35mL are stated, after 28 DEG C of 220r/min concussion and cultivate 165-170h, fermentation ends, from Isolated kasugarnycin in zymotic fluid；
    Wherein, 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.
  4. 4. application according to claim 3, 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.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109486881A (en) * 2018-12-27 2019-03-19 陕西麦可罗生物科技有限公司 A kind of fermentation medium and zymotechnique of kasugarnycin
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

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Publication number Priority date Publication date Assignee Title
CN105624240A (en) * 2014-11-28 2016-06-01 牡丹江佰佳信生物科技有限公司 Kasugamycin fermentation medium and fermentation method thereof

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CN105624240A (en) * 2014-11-28 2016-06-01 牡丹江佰佳信生物科技有限公司 Kasugamycin fermentation medium and fermentation method thereof

Non-Patent Citations (2)

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Title
CHENCHEN ZHU等: "Identification and engineering of regulation-related genes toward improved kasugamycin production", 《APPL MICROBIOL BIOTECHNOL》 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109486881A (en) * 2018-12-27 2019-03-19 陕西麦可罗生物科技有限公司 A kind of fermentation medium and zymotechnique of kasugarnycin
CN109486881B (en) * 2018-12-27 2021-07-13 陕西麦可罗生物科技有限公司 Fermentation medium and fermentation process of kasugamycin
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

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