CN104962495B - A kind of wheat anthropi of degradable lincomycin - Google Patents
A kind of wheat anthropi of degradable lincomycin Download PDFInfo
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- CN104962495B CN104962495B CN201510393769.9A CN201510393769A CN104962495B CN 104962495 B CN104962495 B CN 104962495B CN 201510393769 A CN201510393769 A CN 201510393769A CN 104962495 B CN104962495 B CN 104962495B
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Abstract
The invention discloses a kind of wheat anthropi of degradable lincomycin, the wheat anthropi is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center with deposit number CGMCC No. 10665.The wheat anthropi strain of the degradable lincomycin of the present invention, it is possible to use lincomycin grows for sole carbon source, the bacterial strain can reach 100% to the degradation capability of lincomycin within 24 hours.The bacterial strain can be used for the harmless treatment of fermentation bacteria residue in lincomycin industrial production, efficiently, environmental protection to economize on resources the processing method, and processing cost is low, completely eliminate adverse effect of the useless mycelia of lincomycin to environment.
Description
Technical field
The present invention relates to microbial technology field, more particularly, to a kind of wheat anthropi of degradable lincomycin.
Background technology
Lincomycin (lincomycin) is a kind of alkaline broad-spectrum antibiotic produced by streptomyces lincolnensis, and it is widely used in
Treat the inflammation triggered by gram-positive bacteria.
The production of industrial lincomycin is culture medium mainly with starch, corn steep liquor, beancake powder etc. at present, through streptomycete
Fermentation, extraction, purifying are formed.The preprocessed accessory substance mycelia part formed after separation of solid and liquid of the zymotic fluid is to produce
Contain lincomycin residual and crude protein, cellulose and crude fat etc. in waste residue, the production waste residue.Due to remaining woods in waste residue
Can mycin stable in physicochemical property, with long-term residual, bioconcentration, it, which enters after biological chain, easily causes biological resistance to
The various potential safety hazards such as the property of medicine.According to current international and domestic law rules and regulations, bacteria residue can only enter according to hazardous waste standard
Row is burned, and a certain degree of destruction so can be not only caused to environment, while the waste of grain resource and related enterprise can be caused
The raising of industry production cost.The lincomycin fungi residues that the country is produced every year can reach more than 15000 tons, waste residue (wet thallus) middle forest
Can mycin residual content be about 0.2-0.5g/kg.
And the method that harmless treatment is not carried out to the lincomycin remained in bacteria residue in the prior art, therefore, how
Using the means of environmental protection to remaining the appropriate harmless treatment of lincomycin progress in bacteria residue and effectively being utilized, as phase
Close the technical barrier of industry.
The content of the invention
It is an object of the invention to provide a kind of wheat anthropi of degradable lincomycin, it has efficient degradation woods can
The ability of mycin, can carry out harmless treatment to the fermentation strain of lincomycin, with environmental protection, save the energy, cost is low
Advantage.
In order to solve the above technical problems, the present invention provides a kind of wheat anthropi of degradable lincomycin, the wheat
Anthropi is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms with deposit number CGMCC No.10665
Center.
In one embodiment of the present of invention, the colony characteristicses that the wheat anthropi is formed on enriched medium are：
Circle, faint yellow, sticky, surface is smooth, neat in edge, a diameter of 0.5-0.7mm；
Thalline is characterized as：Gram-negative, thalline rod-short, size is about 0.6-1.5 μm of 0.4 μ m, paired or in heaps
Arrangement.
In one embodiment of the present of invention, oxidizing ferment, the urase of the wheat anthropi are positive；Ornithine, arginine
Decarboxylase is negative；Melibiose, arabinose fermentation are negative.
In one embodiment of the present of invention, the wheat anthropi can grow by sole carbon source of lincomycin.
The present invention provides a kind of wheat anthropi of degradable lincomycin, and it passes through the above-mentioned institute of mutagenesis or genetic transformation
The wheat anthropi stated is obtained.
In one embodiment of the present of invention, described wheat anthropi 16S rDNA whole base sequences are such as
Shown in SEQUENCE LISTING.
The present invention also provides a kind of cell fraction of degradable lincomycin, and it passes through wheat anthropi described above
Obtain.
Beneficial effects of the present invention are：
The wheat anthropi strain of the degradable lincomycin of the present invention, it can be sole carbon source using lincomycin
Growth, the bacterial strain can reach 100% to the degradation capability of lincomycin within 24 hours.The bacterial strain can be used for lincomycin
The harmless treatment of fermentation bacteria residue in industrial production, the processing method is efficient, environmental protection, economizes on resources, processing cost is low, thorough
Bottom eliminates adverse effect of the useless mycelia of lincomycin to environment.
Brief description of the drawings
Fig. 1 is the thalli morphology figure of the wheat anthropi of the degradable lincomycin of the present invention.
Embodiment
The wheat anthropi strain (Ochrobactrum tritici) of the present invention, it is can efficient degradation antibiotic
The degradation bacteria of lincomycin, the strain number is KDSP-J0105, and the bacterial strain was preserved in China Microbiological on March 25th, 2015
Culture presevation administration committee common micro-organisms center, the address of depositary institution is：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number, Institute of Microorganism, Academia Sinica.Its preserving number is CGMCC No.10665.The wheat anthropi of the present invention
(Ochrobactrum tritici) bacterial strain, the ability with efficient degradation lincomycin, the bacterium that fermentation industry can be produced
Lincomycin in filament is effectively degraded, to eliminate antibiotic residue in the useless mycelia of lincomycin, available for lincomycin
The harmless treatment of fermentation bacteria residue, effective approach is provided for the harmless treatment of mycelia in industrial production.
Wherein, the degraded lincomycin mutant bacteria that wheat anthropi strain is obtained by mutagenesis or genetic transformation screening
Strain, the mutant strain at least remains the characteristic of degraded lincomycin, and it includes dashing forward by the insertion of wheat anthropi strain
Become, integrate the mutant that mutation etc. is obtained.Secondly, the present invention passes through wheat anthropi strain or mutagenesis, genetic transformation
The cell fraction that the mutant strain of the wheat anthropi strain of obtained degradable lincomycin obtains.The cell grade point includes
DNA prepared products or bacteria wall prepared product, the obtained nutrient solution of above-mentioned bacterial strains obtained from the culture of above-mentioned bacterial strains, this
The supernatant and the fraction of the supernatant of a little nutrient solutions.The cell fraction has the effect of degraded lincomycin.And combination
Thing includes wheat anthropi strain, the mutant strain of the wheat anthropi strain of degradable lincomycin and theirs is thin
The nutrient solution of born of the same parents' classification.
Below by specific embodiment, the detailed process that the present invention is realized is further illustrated.
The screening of the wheat anthropi strain of embodiment 1.
Step one：From the antibiotic enterprise periphery collection sludge 10g of Shijiazhuang one, it is inoculated into containing 20mg lincomycins
In 100mL minimal mediums, in 30 DEG C, 200rpm shaking table concussion and cultivates.Culture starts switching in latter every 7 days once, during switching
Be transferred to 10% (V/V) inoculum concentration in the culture medium of the Mycosporin containing woods of higher concentration, woods Mycosporin containing concentration according to
It is secondary for 20mg/L, 40mg/L, 80mg/L, 160mg/L, the like, to 640mg/L, obtain various concentrations containing thalline
Enrichment culture liquid.
Wherein, the composition of the minimal medium in the present invention and proportioning are (g/L)：K2HPO41.6g, KH2PO4
0.4g, MgSO4·7H2O 0.2g, CaCl2·2H2O 0.025g, FeCl3·6H2O 0.0023, NH4NO30.5g, agar
18.0g, pH value is adjusted to 7.0,121 DEG C of autoclaving 30min with 1mol/L HCL or NaOH.
The composition and proportioning of enriched medium in the present invention are (g/L)：Sucrose 20.0g, agar 18.0g, 20% Ma Ling
Potato leachate 1000mL, adjusts pH value to 7.0,121 DEG C of autoclaving 30min.
Step 2：Take and be applied to the corresponding selection containing concentration containing germy enrichment culture liquid in 0.1mL steps one and put down
On plate, 30 DEG C, cultivate 48 hours.Obtain one plant of speed of growth most fast and periphery of bacterial colonies and the bacterial strain of obvious transparent circle occur, by this
Inoculation is on the minimal medium containing 20mg/L lincomycins, and periphery of bacterial colonies may occur in which clearly transparent circle, by this
Strain number is KDSP-J-0105.
Please refer to shown in Fig. 1, the numbering is that the Main Biological of KDSP-J-0105 bacterial strains is：The bacterial strain exists
The bacterium colony formed on enriched medium is circle, and faint yellow, sticky, surface is smooth, neat in edge, a diameter of 0.5-0.7mm；Should
The thalline of bacterial strain is characterized as：Gram-negative, thalline rod-short, size is about 0.6-1.5 μm of 0.4 μ m, row paired or in heaps
Row.
Determined through overtesting, the numbering is the oxidizing ferment of KDSP-J-0105 bacterial strains, the urase positive, and ornithine, arginine take off
Carboxylic acid is negative, and melibiose, arabinose fermentation are negative.
Step 3：By liquid chromatography for measuring in minimal medium, the bacterial strain has degraded energy to lincomycin
Power.The content of thalline is 10 in every milliliter of nutrient solution in nutrient solution8Individual, the concentration of lincomycin is 20mg/L, in 30 DEG C,
200rpm shaking tables concussion and cultivate 1 day, after measured, the degradation rate of lincomycin is 100%.Meanwhile, the bacterial strain is total to fermentation bacteria residue
Culture one week, using liquid chromatography for measuring, the bacterial strain can reach 100% to the degradation rate of lincomycin.It is demonstrated experimentally that degraded
Solution system is minimal medium, and KDSP-J-0105 bacterial strains are in pH 7.0-8.0,30 to 40 DEG C of well-growns of temperature, can be with
Lincomycin grows for sole carbon source, is one plant of very promising degradation bacteria.
The 16S rDNA of bacterial strain sequence is determined, is by the 16S rDNA sequences of the measure and Genebank accession number
KP341765 16S rDNA gene order carries out homology analysis.First, KDSP- is extracted using a small amount of extracting methods of DNA
The STb gene of J-0105 bacterial strains.Its 16S rRNA gene order is expanded using 16S rRNA universal primers 27F-1492R, will be expanded
(sequencing result is shown in SEQUENCE LISTING in sequence table) is directly sequenced in obtained PCR primer, by the 16S of acquisition
RRNA gene orders input GeneBank, and analysis is compared to all sequences in database by Blastn programs.This hair
The KDSP-J-0105 of bright bacterial strain 16S rRNA gene orders have higher phase with the existing type strain of Ochrobactrum
Like property.Wherein with Ochrobacterum tritici SCII24TSequence similarity be 99.93%.The bacterial strain belongs to
Ochrobactrum spp., in combination with its physiological and biochemical property, it is O.Tritici, i.e. wheat anthropi to identify the bacterial strain
Bacterial strain.
The screening of the wheat anthropi strain (Ochrobactrum tritici) of the present invention of embodiment 2 and can be mould to woods
The degradation of element.
Step one：Take 10g sludge to be contained in the 250mL conical flasks of sterilizing, add the hydrochloric acid that 100mL concentration is 2000mg/L
Lincomycin solution, in 30 DEG C of rotating speeds be 200r/min shaking table cultures.Culture start after, every 7 days switching once, during switching with
10% inoculum concentration is inoculated into fresh Lincomycin Hydrochloride solution domestication culture medium, and steps up Lincomycin Hydrochloride
Content.The degradation rate of Lincomycin Hydrochloride is detected, best one group is picked out so as to used in isolated strains.
Step 2：By the bacteria suspension (1 × 10 of above-mentioned bacterial strains in 1mL8Individual/mL) be linked into 100mL woodss containing 20mg/L can be mould
Element minimal medium in, take three times it is parallel, and with not inoculated bacteria but the lincomycin containing same concentrations inorganic salts training
It is control to support base.30 DEG C, 200rpm shaking table cultures, cultivate 8 respectively, 16,24,32 hours, liquid chromatography detection lincomycin
Content, experimental result is shown in Table one.Culture 24 hours, selected bacterial strain can be almost degradable by lincomycin.By obtained bacterium
Strain numbering is KDSP-J-0105.
Degradation rate of the KDSP-J-0105 bacterial strains of table one in minimal medium to lincomycin
Step 3：The 16S rDNA for the bacterial strain that numbering is KDSP-J-0105 sequence is determined, by the 16S rDNA sequences of measure
Row carry out homology analysis with Genebank accession number for KP341765 16S rDNA gene order, it is believed that KDSP-J-
0105 bacterial strain belongs to Ochrobactrum spp., in combination with its physiological and biochemical property, and it is O.Tritici to identify the bacterial strain, i.e.,
Wheat anthropi.
Embodiment 3：Degradation of the wheat anthropi strain to lincomycin in bacteria residue.
Lincomycin wet mycelia of giving up is crossed into 0.2mm sieves, 121 DEG C of sterilizing 30min are placed in.Weigh the useless bacterium after 10g sterilizings
Silk, and be put into 100mL water, the wheat anthropi thalline 5g cultivated 48 hours is added into the wet mycelia solution that gives up (dry
Weight), if 3 groups of parallel tests, using add mycelia but be not added with wheat anthropi fermentation culture for control, respectively effect 16,32,
48th, sample within 60 hours, high-efficient liquid phase technique detects the residual quantity of lincomycin.As a result show, the bacterial strain is after 48 hours of incubation, right
The degradation rate of lincomycin can reach 100%.
Degradation effect of the wheat anthropi strain of table two to lincomycin in bacteria residue
From the foregoing, it will be observed that the bacterial strain of the wheat anthropi strain of the present invention to be one plant be capable of efficient degradation lincomycin, its
It can be grown using lincomycin for sole carbon source, the bacterial strain can reach to the degradation capability of lincomycin within 24 hours
100%.The present invention utilizes wheat anthropi strain to be used for the harmless treatment of fermentation bacteria residue in lincomycin industrial production, should
Processing method is efficient, environmental protection, and economize on resources the energy, and cost is low, and wheat anthropi strain of the invention eliminates woods can
Mycin gives up mycelia to environment adverse effect.Meanwhile, the wheat anthropi strain of the invention gene related to degraded and key
Enzyme has extensive effect in biological prosthetic and purification and industry and biomedicine field.
Presently preferred embodiments of the present invention is these are only, this is not able to and limits the protection domain that the present invention is implemented, therefore all ginsengs
The simple equivalent changes and modifications that the description of the present invention is made is examined, still belongs to protection scope of the present invention.
Claims (6)
1. a kind of wheat anthropi of degradable lincomycin, it is characterised in that
The wheat anthropi（Ochrobactrum tritici）China is preserved in deposit number CGMCC No. 10665 micro-
Biological inoculum preservation administration committee common micro-organisms center.
2. the wheat anthropi of degradable lincomycin according to claim 1, it is characterised in that
The colony characteristicses that the wheat anthropi is formed on enriched medium are：Circle, faint yellow, sticky, surface is smooth,
Neat in edge, a diameter of 0.5-0.7mm；
Thalline is characterized as：Gram-negative, thalline rod-short, size is 0.6-1.5 μm of 0.4 μ m, paired or stack array.
3. the wheat anthropi of degradable lincomycin according to claim 1, it is characterised in that
Oxidizing ferment, the urase of the wheat anthropi are positive；Ornithine, arginine decarboxylase are negative；Melibiose, arabinose
Fermentation is negative.
4. the wheat anthropi of degradable lincomycin according to claim 1, it is characterised in that
The wheat anthropi can grow by sole carbon source of lincomycin.
5. wheat anthropi according to claim 1, it is characterised in that
Described wheat anthropi 16S rDNA whole base sequences are as shown in SEQUENCE LISTING.
6. a kind of cell fraction of degradable lincomycin, it is characterised in that
It is obtained by the wheat anthropi any one of claim 1 to claim 5.
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|---|---|---|---|---|
| CN101338284A (en) * | 2007-07-06 | 2009-01-07 | 中国农业科学院植物保护研究所 | Degradation strain capable of high-efficiency degrading bactericide chlorothalonil and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101338284A (en) * | 2007-07-06 | 2009-01-07 | 中国农业科学院植物保护研究所 | Degradation strain capable of high-efficiency degrading bactericide chlorothalonil and use thereof |
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| GenBank: GU345782.1;Wu, R. J. et al.;《GenBank》;20100127;第1页 * |
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