CN102864114B - Strain for highly yielding enramycin, and preparation method and application thereof - Google Patents
Strain for highly yielding enramycin, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a strain for highly yielding enramycin, simultaneously provides a screening method of the strain, and provides application of the strain in enramycin production. The preserving number of the strain DCS067 for highly yielding enramycin is CGMCCONO: 6220, the preserving unit is China General Microbiological Culture Collection Center (CGMCC), and the preserving date is June 14, 2012. The DCS067 strain enables production rate of enramycin to be improved by 47.80%.
Description
Technical field
The present invention relates to a kind ofly for the production of antibiotic bacterial strain, specifically, relate to bacterial strain of a kind of high yield enramycin and its preparation method and application.
Background technology
Enramycin is a kind of ring type polypeptide class microbiotic that is combined into by tens seed amino acids and unsaturated fatty acids that is produced by true sterilization streptomycete (Streptomyces fungicidious) fermentation.Mostly screened from wild strain for the production of the fungicidal streptomycete bacterial strain of enramycin in the past and obtain, its production level is lower.Chinese patent ZL 201010528367.2 discloses a kind of fungicidal streptomycete mutant strain FYFJ03(preserving number CGMCC No.4113), its screening method is: at first adopt 5-bromouracil to lure the starting strain sudden change into, produce mutant strain, then adopt streptococcus aureus, according to the biological value method, mutant strain is screened, obtain the bacterial strain of high yield enramycin.Although the method has improved the throughput of bacterial strain to a certain extent, owing to only having adopted chemomorphosis, and screening mode is single, so the raising of bacterial strain throughput is limited.
Summary of the invention
The bacterial strain that the purpose of this invention is to provide a kind of high yield enramycin provides the screening method of above-mentioned bacterial strains simultaneously, and the application of this bacterial strain in enramycin is produced is provided.
For realizing the object of the invention, the invention provides a kind of specific name is that fungicidal streptomycete (Streptomyces fungicidious) DCS067(is hereinafter to be referred as DCS067) the enramycin superior strain, its preserving number is CGMCC No.6220, depositary institution is China Committee for Culture Collection of Microorganisms (CGMCC), and preservation date is on June 14th, 2012.
Its microbial characteristic of DCS067 bacterial strain provided by the present invention is as follows:
The form of single bacterium colony is: colony diameter 2-3mm, and spore grey or white, plentiful.The microscopic examination spore is circular or oval.
The preparation method of DCS067 provided by the invention comprises the following steps:
The preparation of a, spore suspension I: get the starting strain of fungicidal streptomycete, be inoculated on slant medium, cultivated 5-8 days under 28.0 ± 0.5 ℃, relative humidity 40%-60% condition, then add sterilized water, spore is scraped, mixing obtains the spore suspension I;
B, organic solvent are processed: add aseptic organic solvent in the spore suspension I, and standing after vibration, layering, water intaking obtains the spore suspension II mutually; Described vibration can be adopted vortex oscillation device vibration 15-60 s, and time of repose is preferably 15-30min.
C, ultraviolet mutagenesis: the spore suspension II being placed in the culture dish of sterilization, is to shine 45-60s under the ultraviolet lamp of 254nm in wavelength; During irradiation, be preferably vertical range 30cm place's irradiation under the ultraviolet lamp of 30W at power.
d, resistance screening: the spore suspension II after mutagenic treatment is coated on contains on antibiotic isolation medium, cultivated 5-8 days under 28.0 ± 0.5 ℃, relative humidity 40%-60% condition, generate the some single bacterium colony with antibiotic resistance, during cultivation, best first day is just put, and second day rises to be inverted and cultivates, the above-mentioned single bacterium colony of difference picking, be inoculated on slant medium, at 28.0 ± 0.5 ℃, cultivate after 5-8 days under relative humidity 40%-60% condition, be inoculated on seed culture medium, at 28.0 ± 0.5 ℃, after cultivating 26-30h under 200 ~ 220 rpm conditions, be inoculated on fermention medium, at 28.0 ± 0.5 ℃, cultivated 9-12 days under 200 ~ 220 rpm conditions, obtain fermented liquid, in employing HPLC method mensuration fermented liquid, enramycin is tired, wherein, bacterial strain corresponding to the highest fermented liquid of tiring is fungicidal streptomycete of the present invention (Streptomyces fungicidious) DCS067.
At first the present invention processes the bacterial strain spore suspension through organic solvent, removed the dirt settlings such as glycocalix, protein matter of spore surface, is conducive to follow-up mutagenesis and screening.This be because strain cell in the growth and breeding process, can form the dirt settling that is mainly formed by glycocalix and protein matter around spore, this type of dirt settling can not only shielding of ultraviolet effect, can also the antibiotic infiltration of overslaugh, simultaneously, the Degradation of Antibiotics enzyme that also contains higher concentration in glycocalix has further stoped the impact of microbiotic on the deep layer spore.Remove above-mentioned dirt settling, spore is exposed in outside atmosphere better.The present invention is in subsequent step, and the mode that has adopted the screening of ultraviolet mutagenesis and antibiotics resistance to combine has overcome the deficiency of single mutagenesis in the past, has further improved the enramycin productive rate of mutant strain.Compare with starting strain, the enramycin productive rate of bacterial strain of the present invention has improved 47.80%.The described spore suspension I of a step of the present invention miospore concentration is preferably 10
11-10
13Individual/mL or absorbance A
600Be 1.0-2.0; Spore suspension under this concentration or absorbancy is easy to remove the dirt settling (glycocalix, protein matter) of spore surface, also has simultaneously good reproductive performance.The described organic solvent of b step of the present invention is the mixture of any one or a few in the phenol of sterilising treatment, trichloromethane, n-hexadecane, normal hexane, octane, n-dodecane, n-Octanol, dimethylbenzene preferably.Wherein, preferred combination is the mixture of phenol and trichloromethane; In mixture, the volume ratio of phenol and trichloromethane is preferably 25:4.When adopting said mixture that the spore suspension I is processed, can remove better glycocalix, the protein matter of spore surface.
Pressing mass volume ratio calculates, the composition of slant medium of the present invention is preferably: the 0.5-1.0% maltitol powder, the 0.05-0.10% dried yeast powder, 0.05-0.10% tuna cream, the 0.06-0.12% Tryptones, 0.1-0.5% sodium-chlor, 1.8-2.2% agar, all the other are water, preferred 0.1 ~ 0.7 μ g/mL of antibiotic content in described separation and Culture, all the other compositions and content are identical with slant medium, and it is that 30% the NaOH aqueous solution is regulated pH to 6.7 ± 0.1 that described slant medium and isolation medium all adopt the quality percentage composition.
Microbiotic preference chain mycin in isolation medium of the present invention.
Pressing mass volume ratio calculates, the described seed culture based component of d step of the present invention is preferably: the 2.5-4.5% Semen Maydis powder, the 0.2-0.8% Zein powder, 0.2-0.8% cottonseed meal, 1.5-2.5% light calcium carbonate, 0.15-0.35% ammonium sulfate, the 0.02-0.05% ferrous sulfate, 0.05-0.25% potassium primary phosphate, 1.5-3.5% corn steep liquor, all the other are water, and this substratum employing quality percentage composition is 30% NaOH aqueous solution adjusting pH to 6.7 ± 0.1.
pressing mass volume ratio calculates, the described fermentation culture based component of d step of the present invention is preferably: the 6-15% Semen Maydis powder, the 2-6% Zein powder, the 0.2-0.8% light calcium carbonate, 0.2-0.8% sodium-chlor, 0.1-0.5% ammonium sulfate, the 0.003-0.012% ferrous sulfate, the 0.007-0.027% potassium primary phosphate, 0.1-0.5% urea, the 0.005-0.015% zinc chloride, 0.05-0.25% potassium hydroxide, the 0.07-0.15% corn steep liquor, 0.1-0.5%L-lactic acid, the high temperature resistant α-amylase of 0.04-0.12%, all the other are water, this substratum employing quality percentage composition is 30% NaOH aqueous solution adjusting pH to 5.9 ± 0.1.
Bacterial strain of the present invention can be used in the production of enramycin.
When being used for enramycin production, be preferably in 28.0 ± 0.5 ℃, under 200 ~ 220 rpm conditions, the DCS067 bacterial strain fermented in fermention medium 9-12 days, make the fermented liquid that contains enramycin; The medium component that fermentation is adopted is preferably the 6-15% Semen Maydis powder, the 2-6% Zein powder, the 0.2-0.8% light calcium carbonate, 0.2-0.8% sodium-chlor, 0.1-0.5% ammonium sulfate, the 0.003-0.012% ferrous sulfate, the 0.007-0.027% potassium primary phosphate, 0.1-0.5% urea, 0.005-0.015% zinc chloride, 0.05-0.25% potassium hydroxide, the 0.07-0.015% corn steep liquor, 0.1-0.5%L-lactic acid, the high temperature resistant α-amylase of 0.04-0.12%, all the other are water, and this substratum employing quality percentage composition is 30% NaOH aqueous solution adjusting pH to 5.9 ± 0.1.
Fungicidal streptomycete DCS067 of the present invention through bacterial strain retrial and study on the stability result as can be known, it has good mitotic stability, during simultaneously for the production of enramycin, can effectively improve the productive rate of enramycin.Its screening method of the method for the invention, easy and simple to handle, reliable results.
Embodiment
The below further illustrates content of the present invention with specific embodiment, but and means never in any form and limit the invention.
The starting strain of the kabicidin streptomycete that following embodiment adopts is available from Chinese common micro-organisms culture presevation administrative center (being numbered 4.1312).
The screening of embodiment 1 enramycin superior strain
A, preparation slant medium: take the 8g maltitol powder, 0.8g dried yeast powder, 0.8g tuna cream, the 1g Tryptones, 3g sodium-chlor, 20g agar, adding water and be settled to 1000ml, is then 30% NaOH aqueous solution adjusting pH to 6.7 ± 0.1 with the quality percentage composition, obtains mixed liquor A.Get above-mentioned mixed liquor A, packing adds successively tampon, two-layer gauze, two-layer kraft paper and wraps to the test tube of some 30 mm * 200 mm, sterilization 20 min under 0.11 ± 0.05MPa, 121 ± 1 ℃ of conditions.Sterilizing shakes up after complete immediately, when being cooled to feel and not scalding, is paved into the inclined-plane, obtains slant medium after solidifying.
Preparation spore suspension I: the starting strain (4.1312) of getting the fungicidal streptomycete, be seeded on above-mentioned slant medium, cultivated 7 days under 28.0 ± 0.5 ℃, relative humidity 40-60% condition, then add the 3mL sterilized water, with the aseptic inoculation pin, spore is scraped, change in the aseptic triangular flask that contains several granulated glass spherees, vibration, the water intaking layer obtains the spore suspension I, adopts spectrophotometry, and its absorbancy is A
600=1.506, spore concentration is 10
12Individual/mL.
B, organic solvent are processed: after phenol, trichloromethane are mixed according to volume ratio 25:4, through the 0.22 organic filtering with microporous membrane degerming of μ m, obtain aseptic organic solvent.Get the spore suspension I that 5mL step a obtains, add the above-mentioned aseptic organic solvent of 5mL, after vibration 30 s on the vortex oscillation device, standing 30 min, layering; Water intaking obtains the spore suspension II mutually, adopts spectrophotometry, and its absorbancy is A
600=0.456.Compare with the spore suspension I, the absorbancy of spore suspension II obviously descends, and is also more clear and bright penetrating in appearance, and glycocalix, protein matter that spore surface is described are combined with organic solvent and have been transferred to organic phase from water afterwards.
C, ultraviolet mutagenesis: get the above-mentioned spore suspension II of 5mL, be placed in the sterile petri dish of diameter 6 cm, be placed with the sterilization rotor in culture dish, culture dish is placed on magnetic stirring apparatus, under agitation condition, vertical range 30cm place's irradiation 45s under the ultraviolet lamp of wavelength 254nm, 30W.
D, preparation contain the isolation medium of Streptomycin sulphate: get the described mixed liquor A of a step, divide by the consumption of 200 mL/ bottles and be filled in the triangular flask that volume is 500 mL, then add successively tampon, two-layer gauze, two-layer kraft paper packing, sterilization 20 min under 0.11 ± 0.05MPa, 121 ± 1 ℃ of conditions.Shake up immediately after sterilization, when being cooled to feel and not scalding, the addition according to every 1mL substratum 0.3 μ g Streptomycin sulphate adds aqueous streptomycin, is down flat plate after again shaking up, and obtains isolation medium after solidifying.
Preparation seed culture medium: take the 35g Semen Maydis powder, the 5g Zein powder, 5g cottonseed meal, 20g light calcium carbonate, 2.5g ammonium sulfate, 0.36g ferrous sulfate, the 1.25g potassium primary phosphate is after adding deionized water and stirring evenly, pour into and carry out gelatinization in boiling water, add the 28g corn steep liquor when being down to room temperature, then be settled to 1000mL with deionized water, regulate pH to 6.7 ± 0.1 with 30% sodium hydroxide solution at last.According to the consumption of 50 mL/ bottles, it is in the triangular flask of 500 mL that limit packing to some volumes are fully stirred on the limit, with tampon, two-layer gauze, the sealing of one deck kraft paper.Sterilization 30 min, obtain seed culture medium after cooling under 121 ± 1 ℃ of conditions of 0.11 ± 0.05MPa.
Preparation fermention medium: take the 120g Semen Maydis powder, 40g Zein powder, 5g light calcium carbonate, 5g sodium-chlor, 3g ammonium sulfate, 0.07g ferrous sulfate, 0.17g potassium primary phosphate, 3g urea, 0.1g zinc chloride, 1.5g potassium hydroxide, after adding the 400mL deionized water and stirring evenly, pour into and carry out gelatinization in 500mL boiling water, then add the high temperature resistant α-amylase of 0.7g to carry out liquefaction processing, add again 1g corn steep liquor and 3g Pfansteihl, add water and be settled to 1000mL; Regulate pH to 5.9 ± 0.1 with 30% sodium hydroxide solution at last.According to the consumption of 50 mL/ bottles, it is in the triangular flask of 500 mL that limit packing to some volumes are fully stirred on the limit, and with the sealing of eight layers of gauze, one deck kraft paper, 30 min that sterilize under 121 ± 1 ℃ of conditions of 0.11 ± 0.05MPa obtain fermention medium after cooling.
Resistance screening: the spore suspension II after c step ultraviolet mutagenesis is processed is diluted to every 1mL and contains 10
-4-10
-7Individual spore is coated on the above-mentioned isolation medium that contains Streptomycin sulphate, cultivates 7 days under 28.0 ± 0.5 ℃, relative humidity 40-60% condition, and first day is just put, and second day rises to be inverted and cultivates, and generates the some single bacterium colony with antibiotic resistance; Respectively the some above-mentioned single bacterium colonies of picking, be inoculated on the slant medium of a step preparation, cultivate 7 days under 28.0 ± 0.5 ℃, relative humidity 40-60% condition after, shovels with inoculation and take approximately 0.5 * 0.5cm
2Be inoculated on above-mentioned seed culture medium, at 28.0 ± 0.5 ℃, after cultivating 28h under 200 ~ 220 rpm conditions, be inoculated on above-mentioned fermention medium, at 28.0 ± 0.5 ℃, cultivated 10 days under 200 ~ 220 rpm conditions, obtain fermented liquid, in the mensuration fermented liquid, enramycin is tired, and tiring is up to 10617U/mL, with its corresponding bacterial strain called after fungicidal streptomycete DCS067.Measure the morphological feature of DCS067, the form of its single bacterium colony is: colony diameter 2-3mm, and spore grey or white, plentiful.The microscopic examination spore is circular or oval.
DCS067 is carried out the sand preservation: add 2 mL sterilized waters in the slant medium of preserving the DCS067 bacterial strain, with inoculating needle, spore is scraped, getting 0.2 mL after shaking up is transferred in blank sand pipe, indicate strain name, preparation date and validity period, be to vacuumize 4-6 h under-750 ~-760 mmHg posts at pressure, sealing is placed in the moisture eliminator that discolour silica gel is housed, in 5 ± 3 ℃ of preservations.Its depositary institution is China Committee for Culture Collection of Microorganisms (CGMCC), and preserving number is CGMCC No.6220, preservation date on June 14th, 2012.
Retrial and the study on the stability of embodiment 2 enramycin superior strains
Bacterial strain retrial: get the sand pipe of preservation DCS067 in embodiment 1, be seeded on the slant medium of embodiment 1 preparation, in 28.0 ± 0.5 ℃, cultivate under relative humidity 40-60% condition after 7 days, take approximately 0.5 * 0.5 cm with the inoculation shovel
2The bacterium layer is seeded on the seed culture medium of embodiment 1 preparation, in 28.0 ± 0.5 ℃, after under 220 rpm conditions, 26-30 h is cultivated in concussion, be forwarded on the fermention medium of embodiment 1 preparation, in 28.0 ± 0.5 ℃, 220 rpm concussions were cultivated 10 days, measured tiring of enramycin in fermented liquid, and result is 10079,11034,10604 U/mL, average 10572 U/mL.This result shows that the DCS067 bacterial strain is active good, and has the characteristic of high yield enramycin.
study on the stability: the sand pipe of getting preservation DCS067 in embodiment 1, be seeded on above-mentioned slant medium, in 28.0 ± 0.5 ℃, cultivate after 7 days under relative humidity 40-60% condition, again be inoculated on above-mentioned slant medium and cultivate, so repeat, after continuous switching three times, be forwarded on above-mentioned seed culture medium, in 28.0 ± 0.5 ℃, after under 220 rpm conditions, 26-30 h is cultivated in concussion, be forwarded in above-mentioned fermention medium, in 28.0 ± 0.5 ℃, 220 rpm concussions were cultivated 10 days, in the detection fermented liquid, terramycin tires, result is 10217, 9805, 10079U/mL, average 10034U/mL.This result shows that DCS067 has mitotic stability.
Embodiment 3 comparative examples
Take starting strain as fermentation strain, adopt the culture condition be equal to embodiment 2 retrials to ferment, detect tiring of enramycin in fermented liquid, result is 7114,7030,7315U/mL, average 7153U/mL.
In comparing embodiment 1, embodiment 3 gained fermented liquids, enramycin is tired as can be known, compares with the strain of setting out, and the productive rate of the enramycin of DCS067 bacterial strain of the present invention has improved 47.80%.
Claims (3)
1. enramycin superior strain, its feature is fungicidal streptomycete (Streptomyces fungicidious) DCS067 in described bacterial strain preservation title, the CGMCC of depositary institution, preserving number are CGMCC No.6220, preservation date on June 14th, 2012.
2. the application of the described bacterial strain of claim 1 in enramycin is produced.
3. the application of bacterial strain in enramycin is produced according to claim 2, is characterized in that, at 28.0 ± 0.5 ℃, under 200 ~ 220 rpm conditions, the DCS067 bacterial strain fermented in fermention medium 9-12 days, makes the fermented liquid that contains enramycin, described fermention medium is pressed mass volume ratio and is calculated, composition is: the 6-15% Semen Maydis powder, the 2-6% Zein powder, the 0.2-0.8% light calcium carbonate, 0.2-0.8% sodium-chlor, 0.1-0.5% ammonium sulfate, the 0.003-0.012% ferrous sulfate, the 0.007-0.027% potassium primary phosphate, 0.1-0.5% urea, the 0.005-0.015% zinc chloride, 0.05-0.25% potassium hydroxide, the 0.07-0.015% corn steep liquor, 0.1-0.5%L-lactic acid, the high temperature resistant α-amylase of 0.04-0.12%, all the other are water, this substratum employing quality percentage composition is 30% NaOH aqueous solution adjusting pH to 5.9 ± 0.1.
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| CN103289938B (en) * | 2013-06-19 | 2014-11-05 | 河北圣雪大成制药有限责任公司 | Streptomycin high-yield strain and screening method thereof |
| CN103740612A (en) * | 2013-12-19 | 2014-04-23 | 河北圣雪大成制药有限责任公司 | High-yield enramycin strain and screening method thereof |
| CN104195203B (en) * | 2014-08-29 | 2017-09-12 | 宁夏泰瑞制药股份有限公司 | A kind of antifungal streptomycete fermentation produces the culture medium and feed process of enramycin |
| CN106148460A (en) * | 2015-04-27 | 2016-11-23 | 牡丹江佰佳信生物科技有限公司 | A kind of fermentation medium improving enramycin B component yield and fermentation process |
| CN105524975A (en) * | 2015-12-24 | 2016-04-27 | 丽珠集团新北江制药股份有限公司 | Detection medium for determining microbiological potency of enramycin |
| MY194438A (en) * | 2016-12-06 | 2022-11-30 | Intervet Int Bv | Modified streptomyces fungicidicus isolates and their use |
| CN109628532A (en) * | 2018-12-30 | 2019-04-16 | 广东容大生物股份有限公司 | Fermentation medium and its application |
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