CN103468607B - Streptomyces clavuligerus for high-yield clavulanic acid and application of streptomyces clavuligerus - Google Patents
Streptomyces clavuligerus for high-yield clavulanic acid and application of streptomyces clavuligerus Download PDFInfo
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Abstract
The invention discloses a streptomyces clavuligerus for high-yield clavulanic acid, and application of the streptomyces clavuligerus. The streptomyces clavuligerus for high-yield clavulanic acid is specifically a streptomyces clavuligerus 16CF10 of which the preservation number is CGMCC No.7915 in the China General Microbiological Culture Collection Center. According to the invention, a streptomyces clavuligerus 70116 is taken as a starting strain, is subjected to metabolic engineering modification and is mutated to obtain the streptomyces clavuligerus 16CF10 CGMCC No.7915. Experimental results show that the amount of clavulanic acid produced by the strain cultivated through fermentation reaches 2.25 g/L of fermentation liquor, and is improved by about 51% compared with that the streptomyces clavuligerus 70116 serving as the starting strain, so that the streptomyces clavuligerus 16CF10 CGMCC No.7915 has a broad industrial application prospect.
Description
Technical field
The invention belongs to microorganism field, relate to clavuligerus and application thereof that a plant height produces clavulanic acid.
Background technology
Along with antibiotic a large amount of use, pathogenic bacteria resistance to drugs problem is day by day serious.The abuse of the β-lactam antibitics such as penicillin, cynnematin causes fraction of pathogens bacterium to produce β-lactamase, thus such microbiotic of degrading produces resistance.Clavulanic acid (clavulanic acid), also known as clavulanic acid, is the secondary metabolite that clavuligerus (Streptomyces clavuligerus) produces.During clavulanic acid independent role, anti-microbial activity is very weak, but it has unique 3R, and 5R steric conformation, makes it have spectrum, irreversible β-Nei acyl enzyme inhibition activity, be also first and be found and be successfully applied to clinical beta-lactamase inhibitor.In clinical application; normal and the β-lactam antibitics of clavulanic acid is as amoxycilline Trihydrate bp (amoxicillin), ticarcillin (ticarcillin) drug combination; make enzyme inhibitors-antibiotic combined preparation; inactivation can not decomposed by β-lactamase protecting in varying degrees β-lactam antibitics; thus improve this microbiotic to the anti-microbial activity producing enzyme resistant organism, improve clinical efficacy.Due to the clinical value that it is important, the market requirement of clavulanic acid is vast.The industrial production of current clavulanic acid mainly relies on fermentable.Therefore, transform clavulanic acid producing strains acquisition high production bacteria and there is important industrial application value.
The clavulanic acid output of wild-type clavuligerus is very low, can not meet the needs of industrial fermentation far away, so the bacterial strain of industrial application is all often the mutagenic fungi obtained by taking turns the mutagenesis screening such as physics, chemistry more at present.Along with going deep into clavulanic acid biosynthesizing fundamental research, multiple rationality metabolic engineering means successfully improve the clavulanic acid output of wild strain, but cannot surmount the mutagenic strain of industrial widespread use.Therefore, using the mutagenic strain of industrial application as starting strain, improve its clavulanic acid output by the means of metabolic engineering and there is important actual application value.
Summary of the invention
The object of this invention is to provide clavuligerus and application thereof that a plant height produces clavulanic acid.
Clavuligerus provided by the present invention is specially clavuligerus (Streptomyces clavuligerus) 16CF10.This bacterial strain be with clavuligerus (Streptomyces clavuligerus) 70116 for starting strain, metabolic engineering is carried out to it, the new strains obtained after mutagenesis.This bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (has been called for short CGMCC on 07 12nd, 2013, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC No.7915.
Another object of the present invention is to provide a kind of microbial inoculum.
The activeconstituents of microbial inoculum provided by the present invention is described clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915.
Described clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 or described microbial inoculum also belong to protection scope of the present invention preparing the application in clavulanic acid.
An also object of the present invention is to provide a kind of method preparing clavulanic acid.
The method preparing clavulanic acid provided by the present invention, can comprise the steps: clavuligerus described in fermentation culture (Streptomyces clavuligerus) 16CF10 CGMCC No.7915, collects tunning, obtains clavulanic acid.
In the above-mentioned methods, substratum (fermention medium) composed as follows of described fermentation culture: analysis for soybean powder 20 ± 2g/L, dextrin 10 ± 1g/L, glycerine 15 ± 1mL/L, KH
2pO
40.6 ± 0.1g/L, 3-(N-morpholine) propanesulfonic acid (MOPS) 8 ± 1g/L, trace element solution 1 ± 0.2mL/L, surplus is water; PH is 7.0 ± 0.2; In described trace element, solvent is water, solute and concentration as follows: FeSO
47H
2o1.0 ± 0.2g/L, MnCl
24H
2o1.0 ± 0.2g/L, ZnSO
47H
2o1.0 ± 0.2g/L, CaCl
23H
2o1.3 ± 0.2g/L.
In the present invention, the composition of the substratum (fermention medium) of described fermentation culture is specific as follows: analysis for soybean powder 20g/L, dextrin 10g/L, glycerine 15mL/L, KH
2pO
40.6g/L, 3-(N-morpholine) propanesulfonic acid (MOPS) 8g/L, trace element solution 1mL/L, surplus is water; PH is 7.0 ± 0.2; In described trace element, solvent is water, solute and concentration as follows: FeSO
47H
2o1.0g/L, MnCl
24H
2o1.0g/L, ZnSO
47H
2o1.0g/L, CaCl
23H
2o1.3g/L.
In the above-mentioned methods, before fermentation culture, also comprise and utilize seed culture medium to cultivate described clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 to obtain the step of seed liquor, more described seed liquor is inoculated in above-mentioned fermention medium carries out fermentation culture.
Described seed culture medium is identical with the composition of described fermention medium.
Described " utilizing seed culture medium to cultivate described clavuligerus (Streptomyces clavuligerus) 16CF10CGMCC No.7915 ", culture temperature is 28 DEG C, incubation time is 48h, training method cultivates (rotating speed is 250rpm, and rotation radius is identical with the rotation radius of NBS Excella E25 model shaking table) for rotating concussion.
In the above-mentioned methods, described fermentation culture can be shake flask fermentation cultivation; The incubation time that described shake flask fermentation is cultivated is that 48-96h(is as 72h); The culture temperature that described shake flask fermentation is cultivated is 28 DEG C; The concussion rotating speed that described shake flask fermentation is cultivated is 200-300rpm, as 250rpm, (rotation radius is identical with the rotation radius of NBS Excella E25 model shaking table).
In one embodiment of the invention, when carrying out the cultivation of described shake flask fermentation, the model of shaking table used is NBSExcella E25.
In the above-mentioned methods, described fermentation culture also can be ferment tank cultivation; The incubation time that described ferment tank is cultivated is 6-7 days (as 6 days); The culture temperature that described ferment tank is cultivated is 28 DEG C; Air flow in described fermentation culture process is 0.5-1.0V/Vmin, and as described in 0.5V/Vmin(, air flow represents with the volume of air ratio by unit volume nutrient solution in per minute); Mixing speed in described fermentation culture process is 300-500rpm, as 400rpm.
In one embodiment of the invention, when carrying out the cultivation of described ferment tank, the model of fermentor tank used is NBS BioFlo/CelliGen 115.
Another object of the present invention is to provide the preparation method of described microbial inoculum.
The preparation method of described microbial inoculum, specifically can to comprise the steps: described clavuligerus (Streptomycesclavuligerus) 16CF10 CGMCC No.7915, as activeconstituents, to obtain described microbial inoculum.
The present invention for starting strain, carries out metabolic engineering to it with clavuligerus (Streptomyces clavuligerus) 70116, obtains clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 after mutagenesis.Ferment tank cultivates this bacterial strain 144h(6 days), find that the amount that it produces clavulanic acid reaches 2.25g/L fermented liquid, comparatively starting strain clavuligerus (Streptomyces clavuligerus) 70116 improves about 51%, and this bacterial strain has the prospect of wide industrial application.
Preservation explanation
Strain name: clavuligerus
Latin name: (Streptomyces clavuligerus)
Strain number: 16CF10
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on 07 12nd, 2013
Register on the books numbering in preservation center: CGMCC No.7915
Accompanying drawing explanation
Fig. 1 is the measurement result that shake flask fermentation cultivates clavulanic acid output in clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 fermentation 72h secondary fermentation liquid.Represent with the peak area of clavulanic acid chromatographic peak in fermented liquid.
Fig. 2 is the high-efficient liquid phase chromatogram of clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 bacterial strain fermentation liquor and clavulanic acid standard solution.Wherein, 1 represents clavuligerus (Streptomycesclavuligerus) 16CF10 CGMCC No.7915 bacterial strain fermentation liquor; 2 represent clavulanic acid standard solution.
Fig. 3 is the growing state measurement result of clavulanic acid output and clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 in the fermented liquid of different time points sampling in ferment tank culturing process.Wherein, A represents the peak area of clavulanic acid chromatographic peak in starting strain clavuligerus (Streptomyces clavuligerus) 70116 fermented liquids; B represents the peak area of clavulanic acid chromatographic peak in clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 fermented liquid; C represents that starting strain clavuligerus (Streptomycesclavuligerus) 70116 utilizes pentanoic method to measure the absorbance value (A595) of biomass; D represents that clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 utilizes pentanoic method to measure the absorbance value (A595) of biomass.
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.
Clavuligerus (Streptomyces clavuligerus) 70116: for " Li Jia .1. reporter gene method screening Clavulanic Acid High-Producing Strains 2. reporter gene method compares the activity of two kinds of actinomycete promoters. Capital Normal University; Master's thesis; 2009 " " clavuligerus (Streptomyces clavuligerus) industrial strain 70116 " recorded in a literary composition, for Haizheng Pharmaceutical Co is given.
Clavulanic acid standard substance: Sigma-Aldrich Products, its catalog number is 33454.
The acquisition of embodiment 1, clavuligerus (Streptomyces clavuligerus) 16CF10 and qualification
One, the acquisition of clavuligerus (Streptomyces clavuligerus) 16CF10
Utilize the erythromycin promotor (ermE*p) of constitutive expression in industrial strain clavuligerus (Streptomyces clavuligerus) 70116, to carry out process LAN to target gene glpF1 and ceas2 respectively by integrating vector, study its impact on clavulanic acid output.Result shows that the independent process LAN of these target genes is all conducive to the raising of clavulanic acid output in industrial strain.
Then, the present inventor builds the reporter plasmid that the two target spot of monitoring is simultaneously expressed, and carrier construction after target gene glpF1 and ceas2 combination of two is imported industrial strain clavuligerus (Streptomyces clavuligerus) 70116.Then by screening the mutagenic fungi of reporter gene simultaneously high expression level after NTG mutagenesis, a wherein strain recombinant bacterium is obtained, by its called after bacterial strain 16CF10.
Two, the qualification of clavuligerus (Streptomyces clavuligerus) 16CF10
Bacterial strain 16CF10 from the following aspects authentication step one obtains:
1, morphological feature qualification
Inoculation YD solid medium, observes metamorphosis in culturing process.Within about 3 days, grow substrate mycelium, within 5-7 days, produce white aerial hyphae, within 10-14 days, produce greyish-green spore.
2, Analysis of The Physiological And Biochemical Properties
Gram positive bacterium.
3, RpoD gene and gap1 gene homology are analyzed
Carry out sequencing to the rpoD gene of bacterial strain 16CF10 and gap1 gene, the sequencing result of its RpoD gene is sequence 1 in sequence table, and the sequencing result of its gap1 gene is sequence 2 in sequence table.The rpoD gene of starting strain clavuligerus (Streptomyces clavuligerus) 70116 and gap1 gene order are checked order simultaneously.
The result of online BLAST comparison shows, rpoD gene and the gap1 gene homology of the rpoD gene of bacterial strain 16CF10 and the sequence of gap1 gene and starting strain clavuligerus (Streptomyces clavuligerus) 70116 are 100%, and rpoD gene (GenBank:ADWJ01000053.1) and gap1 gene (GenBank:DQ178995.1) sequence homology of wild-type clavuligerus (Streptomyces clavuligerus) ATCC 27064 bacterial strain disclosed in simultaneously upper with NCBI are also 100%.
In view of above-mentioned form, analysis of physio biochemical characteristics and rpoD and gap1 gene homology analytical results, bacterial strain 16CF10 step one obtained is accredited as clavuligerus (Streptomyces clavuligerus).This bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City) on 07 12nd, 2013, deposit number is CGMCC No.7915.
Embodiment 2, fermentation clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 produce clavulanic acid
Solid YD substratum: solvent is water, solute and concentration as follows: yeast extract (Difco Products) 4.0g/L; Maltose extract (OXID Products) 10g/L; Glucose 4.0g/L; MgCl
22.0g/L; CaCl
21.5g/L; Agar 2.0g/L; PH7.5.
Seed culture medium (identical with fermention medium): analysis for soybean powder 20g/L; Dextrin (Chemical Reagent Co., Ltd., Sinopharm Group's product, its catalog number is 69009992) 10g/L; Glycerine 15mL/L; KH
2pO
40.6g/L; 3-(N-morpholine) propanesulfonic acid (MOPS) 8.0g/L; Trace element 1mL/L; PH7.0 ± 0.2.Each concentration is respective components final concentration in the medium.Wherein, following (100ml): the FeSO of trace element formula
47H
2o 100mg; MnCl
24H
2o 100mg; ZnSO
47H
2o 100mg; CaCl
23H
2o 130mg; Surplus is water.
One, shake-flask culture clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 produces clavulanic acid
1, seed liquor is cultivated
Clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 embodiment 1 obtained is inoculated on solid YD substratum, cultivate in 28 DEG C and within 7 days, collect spore inoculating afterwards in seed culture medium, 250rpm(shaking table model NBS Excella E25), obtain seed liquor after 28 DEG C of cultivation 48h.Starting strain clavuligerus (Streptomyces clavuligerus) 70116 is set simultaneously in contrast.
2, shake-flask culture
Seed liquor step 1 obtained is inoculated in shaking flask (containing 50mL fermention medium in 250mL shaking flask) carries out fermentation culture, and inoculum size is 5%(volume fraction).
Fermentation condition: temperature 28 DEG C, rotates concussion and cultivates, rotating speed 250rpm(shaking table model NBS Excella E25), fermentation 72h.
Clavulanic acid output in fermented liquid is measured after fermentation ends.Test in triplicate, results averaged.
3, measuring method
Adopt the output of clavulanic acid in Fermentation Liquor by High Performance Liquid Chromatography, specific as follows:
A. chromatographic condition
Chromatographic condition: chromatographic column is anti-phase C18 post; Moving phase is 6%(volumn concentration) methyl alcohol+94%(volumn concentration) 0.1M biphosphate sodium water solution (Glacial acetic acid regulates pH3.68); Elution program is isocratic elution; Flow velocity is 1ml/min; Sampling volume is 50 μ L; Determined wavelength is 312nm.
B. the mensuration of clavulanic acid content in fermented liquid to be measured
Fermented sample derivation process: configuration imidazoles reagent (imidazoles reagent: dissolve 8.25g imidazoles in 65ml water, after adjusting pH to 6.8 ± 0.05 with 5M hydrochloric acid, with water increase-volume to 100ml), after fermented liquid to be measured and equal-volume imidazoles reagent mix, after 37 DEG C of reaction 30min, HPLC analyzes clavulanic acid output.
Fermented liquid to be measured through as above processing is carried out efficient liquid phase chromatographic analysis according to the chromatographic condition described in steps A, the clavulanic acid standard solution (preparing with methanol solution) determined using concentration is as external standard, according to concentration, the corresponding chromatographic peak peak area of clavulanic acid standard solution, and the peak area of the chromatographic peak that retention time is consistent with clavulanic acid standard substance in fermented liquid to be measured, calculate the clavulanic acid content in fermented liquid to be measured.
4, measurement result
In fermented liquid, the peak area of clavulanic acid (component consistent with clavulanic acid standard substance retention time, retention time is 12.5min) chromatographic peak as shown in Figure 1.The content results calculating clavulanic acid in gained fermented liquid is as shown in table 1.Result shows, clavulanic acid output in clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 fermented liquid is 1.36g/L fermented liquid, the output (0.79g/L fermented liquid) of this starting strain clavuligerus (Streptomyces clavuligerus) 70116 compared in contrast improves about 72.2%, through statistical analysis, both differences are extremely remarkable.
Table 1 shake flask fermentation cultivates clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915
Gained clavulanic acid yield result (unit: g/L fermented liquid)
| Bacterial strain | Repeat 1 | Repeat 2 | Repeat 3 | Mean value |
| 16CF10 | 1.30 | 1.40 | 1.38 | 1.36 |
| 70116 | 0.78 | 0.73 | 0.86 | 0.79 |
Two, fermentor cultivation clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 produces clavulanic acid
1, seed liquor is cultivated
Clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 embodiment 1 obtained is inoculated on solid YD substratum, cultivate in 28 DEG C and within 7 days, collect spore inoculating afterwards in seed culture medium, 250rpm(shaking table model NBS Excella E25), obtain seed liquor after 28 DEG C of cultivation 48h.Starting strain clavuligerus (Streptomyces clavuligerus) 70116 is set simultaneously in contrast.
2, fermentation culture
Seed liquor step 1 obtained is inoculated in fermentor tank (7L fermentor tank contains 3L fermention medium, fermentor tank model NBS BioFlo/CelliGen115) carries out fermentation culture, and inoculum size is 5%(volume fraction).
Fermentation condition: temperature 28 DEG C, stir culture, mixing speed is 400rpm, and air flow 0.5V/Vmin(is to pass through the volume of air of unit volume nutrient solution than representing in per minute), ferment 6 days.
Once measure the growing state of clavulanic acid output and clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 in fermented liquid every 24h sampling in fermenting process.Test in triplicate, results averaged.
3, measuring method
(1) mensuration of clavulanic acid output in fermented liquid
Adopt the output of clavulanic acid in Fermentation Liquor by High Performance Liquid Chromatography, specific as follows:
A. chromatographic condition
Chromatographic condition: chromatographic column is anti-phase C18 post; Moving phase is 6%(volume fraction) methyl alcohol+94%(volume fraction) 0.1M biphosphate sodium water solution (Glacial acetic acid regulates pH3.68); Elution program is isocratic elution; Flow velocity is 1ml/min; Sampling volume is 50 μ L; Determined wavelength is 312nm.
B. the mensuration of clavulanic acid content in fermented liquid to be measured
Fermented sample derivation process: configuration imidazoles reagent (imidazoles reagent: dissolve 8.25g imidazoles in 65ml water, after adjusting pH to 6.8 ± 0.05 with 5M hydrochloric acid, with water increase-volume to 100ml), after fermented liquid to be measured and equal-volume imidazoles reagent mix, after 37 DEG C of reaction 30min, HPLC analyzes clavulanic acid output.
Fermented liquid to be measured through as above processing is carried out efficient liquid phase chromatographic analysis according to the chromatographic condition described in steps A, the clavulanic acid standard solution (preparing with methanol solution) determined using concentration is as external standard, according to concentration, the corresponding chromatographic peak peak area of clavulanic acid standard solution, and the peak area of the chromatographic peak that retention time is consistent with clavulanic acid standard substance in fermented liquid to be measured, calculate the clavulanic acid content in fermented liquid to be measured.
(2) growing state of clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 measures
Adopt pentanoic method to measure upgrowth situation, carry out absorbance measurement at 595nm place after culture and pentanoic reagent react, react the growing state of clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 with this.Concrete operations are civilian see " Zhao; Y.; et al. (2013) .A simplified diphenylamine colorimetricmethod for growth quantification.Appl Microbiol Biotechnol, 97 (11): 5069-5077 ".
4, measurement result
(1) clavulanic acid output in fermented liquid
The high-efficient liquid phase chromatogram of clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 bacterial strain fermentation liquor and clavulanic acid standard solution as shown in Figure 2, as can be seen from the figure, there is in clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 bacterial strain fermentation liquor the chromatographic peak of significantly consistent with clavulanic acid standard substance retention time (12.5min).
In the fermented liquid of different time points sampling, the peak area of clavulanic acid (component consistent with clavulanic acid standard substance retention time, retention time is 12.5min) chromatographic peak is as shown in A and B in Fig. 3.The content results calculating clavulanic acid in gained fermented liquid is as shown in table 2.Result shows, clavulanic acid content in clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 and starting strain clavuligerus (Streptomycesclavuligerus) 70116 fermented liquid in contrast all reaches maximum value when the 144h of fermentation culture, now, clavulanic acid output in clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 fermented liquid is 2.25g/L fermented liquid, the output (1.49g/L fermented liquid) of this starting strain clavuligerus (Streptomycesclavuligerus) 70116 compared in contrast improves about 51%, through statistical analysis, both difference is extremely remarkable.
Table 2 ferment tank cultivates clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915
Gained clavulanic acid yield result (unit: g/L fermented liquid)
(2) growing state of clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915
In the fermented liquid of different time points sampling, the growing state result of clavuligerus (Streptomyces clavuligerus) 16CF10CGMCC No.7915 and starting strain clavuligerus (Streptomyces clavuligerus) 70116 is as shown in C and D in Fig. 3, as can be seen from the figure, compared with starting strain clavuligerus (Streptomyces clavuligerus) 70116, difference is not remarkable with it for the growth curve of clavuligerus (Streptomyces clavuligerus) 16CF10CGMCC No.7915, and when fermentation culture 96h in fermented liquid the content of clavuligerus (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 and starting strain clavuligerus (Streptomyces clavuligerus) 70116 all reach maximum, after being maintained to 120h, bacterial strain content all starts again decline (but in fermented liquid, the content of clavulanic acid is still increasing).
Show based on the above results: transform through the present inventor its growing state of clavuligerus (Streptomycesclavuligerus) 16CF10 CGMCC No.7915 bacterial strain obtained substantially uninfluenced, clavulanic acid output obviously promotes, thus improves the ability that bacterial strain produces clavulanic acid.
Claims (8)
1. clavuligerus (Streptomyces clavuligerus) 16CF10, it is CGMCC No.7915 at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. a microbial inoculum, is characterized in that: the activeconstituents of described microbial inoculum is clavuligerus according to claim 1 (Streptomyces clavuligerus) 16CF10 CGMCC No.7915.
3. the application in clavulanic acid prepared by clavuligerus according to claim 1 (Streptomyces clavuligerus) 16CF10 CGMCC No.7915 or microbial inoculum according to claim 2.
4. prepare a method for clavulanic acid, comprise the steps: fermentation culture clavuligerus according to claim 1 (Streptomyces clavuligerus) 16CF10 CGMCC No.7915, collect tunning, obtain clavulanic acid.
5. method according to claim 4, is characterized in that: the substratum of described fermentation culture composed as follows: analysis for soybean powder 20 ± 2g/L, dextrin 10 ± 1g/L, glycerine 15 ± 1mL/L, KH
2pO
40.6 ± 0.1g/L, 3-(N-morpholine) propanesulfonic acid 8 ± 1g/L, trace element solution 1 ± 0.2mL/L, surplus is water; PH is 7.0 ± 0.2;
Described trace element solution, solvent is water, solute and concentration as follows: FeSO
47H
2o 1.0 ± 0.2g/L, MnCl
24H
2o 1.0 ± 0.2g/L, ZnSO
47H
2o 1.0 ± 0.2g/L, CaCl
23H
2o 1.3 ± 0.2g/L.
6. the method according to claim 4 or 5, is characterized in that: described fermentation culture is that shake flask fermentation is cultivated; The incubation time that described shake flask fermentation is cultivated is 48-96h; The culture temperature that described shake flask fermentation is cultivated is 28 DEG C; The concussion rotating speed that described shake flask fermentation is cultivated is 200-300rpm.
7. the method according to claim 4 or 5, is characterized in that: described fermentation culture is that ferment tank is cultivated; The incubation time that described ferment tank is cultivated is 6-7 days; The culture temperature that described ferment tank is cultivated is 28 DEG C; Air flow in described fermentation culture process is 0.5-1.0V/Vmin; The mixing speed that described ferment tank is cultivated is 300-500rpm.
8. the preparation method of microbial inoculum described in claim 2, to comprise the steps: clavuligerus according to claim 1 (Streptomyces clavuligerus) 16CF10 CGMCC No.7915, as activeconstituents, to obtain described microbial inoculum.
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| CN109913392A (en) * | 2019-03-30 | 2019-06-21 | 山东睿智医药科技有限公司 | The clavuligerus bacterial strain of high yield clavulanic acid, its stress selection and application thereof |
| CN111621434B (en) * | 2020-04-20 | 2022-05-06 | 成都大学 | Streptomyces clavuligerus, application, fermentation medium and preparation method of clavulanic acid |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101280336A (en) * | 2008-05-29 | 2008-10-08 | 鲁南制药集团股份有限公司 | Screening method for low-oxygen-consumption producer strain of clavulanic acid |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101280336A (en) * | 2008-05-29 | 2008-10-08 | 鲁南制药集团股份有限公司 | Screening method for low-oxygen-consumption producer strain of clavulanic acid |
Non-Patent Citations (2)
| Title |
|---|
| Coordination of glycerol utilization and clavulanic acid biosynthesis to improve clavulanic acid production in Streptomyces clavuligerus;Guo DeKun.et al;《Life Sciences》;20130731;第56卷(第7期);591-600 * |
| 报告基因法筛选克拉维酸高产菌 报告基因法比较两种放线菌启动子的活性;李佳;《中国优秀硕士学位论文全文数据库基础科学辑》;20091015(第10期);A006-96 * |
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