CN1295344C - Screening method for phophonomycin biological conversion strain - Google Patents
Screening method for phophonomycin biological conversion strain Download PDFInfo
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- CN1295344C CN1295344C CN 200510045858 CN200510045858A CN1295344C CN 1295344 C CN1295344 C CN 1295344C CN 200510045858 CN200510045858 CN 200510045858 CN 200510045858 A CN200510045858 A CN 200510045858A CN 1295344 C CN1295344 C CN 1295344C
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Abstract
The present invention relates to the biological conversion / biological catalysis technical field, particularly to a screening method for a phophonomycin biological conversion strain. The screening method for a phophonomycin biological conversion strain makes use of a cylindrical solid medium (agar blocks) to cultivate a bacterial strain to be detected, and uses colibacillus as an indicator bacterium for biological identification. Then, the agar blocks are reversely placed on a biological identification disk, and a fosfomycin conversion bacterial strain is sieved. According to different conversion capability of the bacterial strain to be selected, which grows on the agar blocks which have the same volumes and contain front body objects with the same quantity, the concentrations of produced fosfomycin are different. Furthermore, the dimensions of inhibition zones produced by the fosfomycin on an indicator bacterium flat plate are also different. At least, the conversion superiority bacterial strain of the fosfomycin is sieved. The method has the advantages of low equipment requirement and simple operation, and is very suitable for the bulk sieving of antibiotic bacterial conversion bacteria in the primary stage.
Description
Technical field
The present invention relates to bio-transformation/biocatalysis technology field, be specially a kind of novel screening methods of phosphonomycin bioconversion strain.
Background technology
Phosphonomycin [(-)-suitable-(1R, 2S)-1,2-phosphate epoxypropyl, FOM] be a kind of Broad spectrum antibiotics of bacteria growing inhibiting.FOM can (cis-propenylphosphonic acid cPA) obtains through microbial conversion by cis-propene phosphoric acid.Traditional method is to adopt the screening of shaking culture method to transform bacterial strain.This method adopts adds precursor cPA in liquid medium within, after shaking culture, be working sample with the centrifuged supernatant of fermented liquid.The shortcoming of this method is to need more labour, equipment and time.
Single bacterium colony agar block culture method is a kind of high efficiency production of antibiotics bacterial screening method.The main points of this method are: beat on nutrient agar plate with punch tool and get uniform agar block, and respectively they are moved in the empty plate of the bacterium of going out, inoculation bacterial strain to be measured keeps suitable temperature and humidity on each agar block, cultivates through certain hour.Long there is each the agar block of macrocolony to transfer on the agar plate that contains indication bacterial classification (being the antagonism object),, thereby gets it according to qualifications with the diameter of the microbiotic inhibition zone of measuring them respectively.The key of this method is that single bacterium colony agar block is cultivated respectively, in this case, the culture condition of each single bacterium colony agar block is basic identical, and the unlikely diffusion of meta-bolites that produces, therefore quite similar under data that record and the shaking culture condition, however working efficiency but greatly improves.
The limitation of this method is: but it only is fit to antibiotic actinomycetic screening of biosynthesizing itself, because actinomycetes have flourishing substrate mycelium (growing in agar block inside), in this case, its excretory microbiotic can be spread in the agar block equably, is convenient to detect.
Summary of the invention
The object of the invention provides a kind ofly screens FOM bio-transformation bacterial isolates fast, in batches, can tentatively distinguish the novel method of single colony transformation efficient again on the biological assay dish.
Technical scheme of the present invention is:
A kind of screening method of phosphonomycin bioconversion strain, inoculation strain culturing to be measured is inverted in agar block on the biological assay dish then on the agar block that contains the cis-propene phosphoric acid precursor, detects, filters out the bacterial strain with conversion capability.
The preparation of agar block and single bacterium colony agar block are cultivated and are comprised:
1) the agar block substratum is formed
Percentage ratio meter by weight, the agar block substratum consists of: cis-propene phosphoric acid 0.3%, extractum carnis 0.5%, peptone 1%, NaCl 0.5%, glycerine 3%, NaVO
30.02%, CoCl
20.05%, agar 1.8-2.0%, surplus is a tap water, sterilizes 30 minutes for 115 ℃.
2) preparation of agar block
Above-mentioned substratum is melted, pours plate into, leave standstill solidify after, get agar block with punch tool, then at its surface seeding bacterial strain to be detected, place 30 ℃, cultivated 4-5 days under the 75%-85% relative humidity.
The screening that phosphonomycin transforms bacterial strain comprises:
1) preparation of indicator cultivation and suspension
Percentage ratio meter by weight, the indicator medium component: extractum carnis 0.5%, peptone 1%, NaCl 0.5%, agar 2%, surplus is a tap water, after the heating for dissolving, transfers pH 7.0-7.2, and 121 ℃ of moist heat sterilizations are after 20 minutes, and the pendulum inclined-plane is standby.
Indicator is cultivated: cultivate 24hr for 37 ℃, lawn is translucent to white.
The preparation of indicator suspension: the physiological saline of sterilization is poured in the eggplant type bottle, scraped with transfering loop and wash lawn, will scrape in the physiological saline that washing lotion pours sterilization into again, the mensuration absorbancy is 0.54-0.57.
2) bioassay substratum and dull and stereotyped preparation thereof
Percentage ratio meter by weight, substratum I composition: extractum carnis 0.5%, peptone 1%, NaCl 0.5%, agar 2.0%, surplus is a tap water, pH 7.0-7.2; Percentage ratio meter by weight, the medium ii composition: extractum carnis 0.5%, peptone 1%, NaCl 0.5%, agar 1.5%, surplus is a tap water, pH 7.0-7.2; Substratum I, medium ii are all 121 ℃ of sterilizations 20 minutes; Pour the substratum I that melts into the biological assay dish, make lower floor's flat board; Again the medium ii of melting is cooled to 55 ℃, adds the indicator bacteria suspension, pour the biological assay dish into behind the mixing rapidly, make upper panel.
3) phosphonomycin transforms the bacterial strain biological assay
Cultured single bacterium colony agar block is placed under the ultraviolet lamp sterilization 20 minutes, again agar block is inverted on the biological assay flat board, cultivate 16hr for 37 ℃, observe and also measure antibacterial circle diameter; Screen bacterial strain according to having or not of inhibition zone with phosphonomycin bio-transformation ability with the size of antibacterial circle diameter.
The used indicator of biological assay dish is intestinal bacteria or other phosphonomycin sensitive organism.
The present invention sieves again by the bacterial strain of shaker fermentation method to primary dcreening operation, and substratum is a cis-propene phosphoric acid 0.3%, extractum carnis 0.5%, and peptone 1%, NaCl 0.5%, glycerine 3%, NaVO
30.02%, CoCl
20.02%, surplus is a tap water, and 30 ℃, 160rpm cultivated 4 days, by the antibacterial circle diameter size, further filtered out the higher bacterial strain of transformation efficiency.
The present invention utilizes the thin layer chromatography analysis phosphonomycin, and developping agent is a propyl carbinol: acetate: water (volume ratio 3: 1: 1), and the chromatoplate after launching is air-dry, place on the biological assay flat board, take off after waiting to soak into, cultivate 16hr, contain phosphonomycin and partly produce inhibition zone for 37 ℃.
The invention has the beneficial effects as follows:
1, the present invention utilizes cylindric solid medium (agar block) to cultivate bacterial strain to be detected, utilizes the indicator of intestinal bacteria for biological assay, and the screening phosphonomycin transforms bacterial strain.According at equal-volume and contain the bacterial strain conversion capability difference to be selected of growing on the agar block of equivalent precursor, the phosphonomycin concentration difference that produces, and then the inhibition zone size that is produced on the indicator flat board by phosphonomycin is also different, and finishing screen is selected phosphonomycin and transformed dominant bacteria.
2, the equipment requirements of the inventive method is low, and is simple to operate, is highly suitable for the elementary batch screening of microbiotic bio-transformation bacterium.
Description of drawings
Fig. 1 is converted into phosphonomycin (FOM) synoptic diagram for cis-propene phosphoric acid of the present invention (cPA) under the katalysis of microorganism.
Embodiment
The present invention at first sneaks into the precursor cPA of equivalent in the agar block uniformly and in surface seeding bacterial strain to be measured, biotransformation at first needs thalline to absorb exogenous precursor, is translated into phosphonomycin by enzyme catalysis in the thalline.And because the bacterium of inoculation only grows in the agar block surface, can only absorb and transform the cPA on top layer and produce FOM, so FOM mainly accumulates in the agar block surface.Therefore, make screening sensitivity obtain to increase substantially, agar block is inverted in to do the inhibition zone experiment on the biological assay flat board be an effective way, and judge transformation efficiency by the inhibition zone size.
Concrete operations step of the present invention is as follows:
One, the preparation of indicator cultivation and suspension
Indicator is intestinal bacteria (E.coli), Shenyang Inst. of Applied Ecology, Chinese Academy of Sciences's preservation.
Percentage ratio meter by weight, the indicator substratum is formed: extractum carnis 0.5%, peptone 1%, NaCl 0.5%, agar 2%, surplus is a tap water.After the heating for dissolving, transfer pH 7.0-7.2.The eggplant type of packing into bottle 90mL, 121 ℃ of moist heat sterilizations are after 20 minutes, and the pendulum inclined-plane is standby.
Indicator is cultivated: cultivate 24hr for 37 ℃, lawn is translucent to white.
The preparation of indicator suspension: the physiological saline of 100mL sterilization is poured in the eggplant type bottle, scraped with transfering loop and wash lawn, will scrape in the empty conical flask that washing lotion pours sterilization into again, measuring absorbancy (O.D. value) is 0.54-0.57.
Two, the preparation of agar block and single bacterium colony agar block are cultivated
Percentage ratio meter by weight, the agar block substratum is formed: cPA 0.3%, extractum carnis 0.5%, peptone 1%, NaCl 0.5%, glycerine 3%, NaVO
30.02%, CoCl
20.05%, agar 1.8%, surplus is a tap water, sterilizes 30 minutes for 115 ℃.
The preparation of agar block: above-mentioned substratum is melted, pour 25mL into, leave standstill and solidify in each Φ 90mm plate.After solidifying, (Φ 6 * 6mm), then at its surface seeding bacterial strain to be detected, place 30 ℃, cultivate 4-5 days under the 75%-85% relative humidity to get agar block with punch tool.
Three, phosphonomycin transforms the screening of bacterial strain
Bioassay substratum and dull and stereotyped preparation thereof:
Percentage ratio meter by weight, substratum I composition: extractum carnis 0.5%, peptone 1%, NaCl 0.5%, agar 2%, surplus is a tap water, pH 7.0-7.2; The medium ii composition: extractum carnis 0.5%, peptone 1%, NaCl 0.5%, agar 1.5%, surplus is a tap water, pH 7.0-7.2; Substratum I, medium ii are all 121 ℃ of sterilizations 20 minutes.The substratum I that 200mL is melted pour into the biological assay dish (20 * 30cm), make lower floor's flat board; The medium ii that 100mL is melted is cooled to 55 ℃ again, adds indicator bacteria suspension 1mL, pours the biological assay dish into behind the mixing rapidly, makes upper panel.
Phosphonomycin transforms the bacterial strain biological assay:
Cultured single bacterium colony agar block is placed under the ultraviolet lamp (30W, 40cm) sterilization is 20 minutes, agar block is inverted on the biological assay flat board again, cultivates 16hr for 37 ℃.Observe and measure antibacterial circle diameter.Screen bacterial strain according to having or not of inhibition zone with phosphonomycin bio-transformation ability with the size of antibacterial circle diameter.
Embodiment:
The 210 strain microbionations that this laboratory is preserved are to agar block, and in 30 ℃, 75% relative humidity was cultivated 4 days.Will be through after the agar block ultraviolet sterilization after inoculation bacterial strain to be detected and the cultivation, inversion is affixed on the biological assay flat board.Observe having or not and diameter of inhibition zone behind 37 ℃ of cultivation 16hr, wherein have 12 strain bacteriums that tangible inhibition zone is arranged.
Make three groups of parallel controls simultaneously: promptly 1) do not contain the agar block contrast of cPA, in order to get rid of the interference of other antipathogenic composition in the substratum; 2) contain the agar block contrast of cPA, disturb in order to the abiotic conversion of getting rid of the cPA substrate; 3) do not contain the agar block contrast of cPA inoculating strain, in order to get rid of the interference of bacterial strain generation antibacterial substance itself.Experimental result shows that three groups of contrasts all do not have to produce inhibition zone on the evaluation flat board.Illustrate that the bacterial strain that filters out is that FOM presents inhibition zone by transforming cPA.
Sieve again by 12 bacterial strains of shaker fermentation method to primary dcreening operation, substratum is cPA 0.3%, extractum carnis 0.5%, and peptone 1%, NaCl 0.5%, glycerine 3%, NaVO
30.02%, CoCl
20.02%, surplus is a tap water, and 30 ℃, 160rpm cultivated 4 days, by the antibacterial circle diameter size, further filtered out the higher bacterial strain of transformation efficiency.
Utilize the thin layer chromatography analysis phosphonomycin, developping agent is a propyl carbinol: acetate: water (volume ratio 3: 1: 1).Chromatoplate after launching is air-dry, place on the biological assay flat board, take off after waiting to soak into, cultivate 16hr for 37 ℃.Contain phosphonomycin and partly produce inhibition zone.The Rf of comparative sample and phosphonomycin standard substance, the Rf value of sample is 3.52; The Rf value of standard substance is 3.53, and the microbiotic that checking produces is a phosphonomycin.
Claims (4)
1. the screening method of a phosphonomycin bioconversion strain is characterized in that: inoculate bacterium pearl to be measured and cultivate on the agar block that contains the cis-propene phosphoric acid precursor, then agar block is inverted on the biological assay dish, detect, filter out the bacterial strain with conversion capability;
The screening that described phosphonomycin transforms bacterial strain comprises:
1) preparation of indicator cultivation and suspension
Percentage ratio meter by weight, the indicator medium component: extractum carnis 0.5%, peptone 1%, NaCl 0.5%, agar 2%, surplus is a tap water, after the heating for dissolving, transfers pH 7.0-7.2, and 121 ℃ of moist heat sterilizations are after 20 minutes, and the pendulum inclined-plane is standby;
Indicator is cultivated: cultivate 24hr for 37 ℃, lawn is translucent to white;
The preparation of indicator suspension: the physiological saline of sterilization is poured in the eggplant type bottle, scraped with transfering loop and wash lawn, will scrape in the physiological saline that washing lotion pours sterilization into again, the mensuration absorbancy is 0.54-0.57;
2) bioassay substratum and dull and stereotyped preparation thereof
Percentage ratio meter by weight, substratum I composition: extractum carnis 0.5%, peptone 1%, NaCl 0.5%, agar 2.0%, surplus is a tap water, pH 7.0-7.2; Percentage ratio meter by weight, the medium ii composition: extractum carnis 0.5%, peptone 1%, NaCl 0.5%, agar 1.5%, surplus is a tap water, pH 7.0-7.2; Substratum I, medium ii are all 121 ℃ of sterilizations 20 minutes; Pour the substratum I that melts into the biological assay dish, make lower floor's flat board; Again the medium ii of melting is cooled to 55 ℃, adds the indicator bacteria suspension, pour the biological assay dish into behind the mixing rapidly, make upper panel;
3) phosphonomycin transforms the bacterial strain biological assay
Cultured single bacterium colony agar block is placed under the ultraviolet lamp sterilization 20 minutes, again agar block is inverted on the biological assay flat board, cultivate 16hr for 37 ℃, observe and also measure antibacterial circle diameter; Utilize the thin layer chromatography analysis phosphonomycin, the volume ratio of developping agent propyl carbinol, acetate, water is 3: 1: 1, chromatoplate after launching is air-dry, place on the biological assay flat board, take off after waiting to soak into, cultivate 16hr for 37 ℃, contain phosphonomycin and partly produce inhibition zone, screen bacterial strain according to the having or not of inhibition zone and the size of antibacterial circle diameter with phosphonomycin bio-transformation ability.
2. according to the described screening method of claim 1, it is characterized in that the preparation of agar block and agar block are cultivated and comprised:
1) the agar block substratum is formed
Percentage ratio meter by weight, the agar block substratum consists of: cis-propene phosphoric acid 0.3%, extractum carnis 0.5%, peptone 1%, NaCl 0.5%, glycerine 3%, NaVO
30.02%, CoCl
20.05%, agar 1.8-2.0%, surplus is a tap water, sterilizes 30 minutes for 115 ℃;
2) preparation of agar block
Above-mentioned substratum is melted, pours plate into, leave standstill solidify after, get agar block with punch tool, then at its surface seeding bacterial strain to be detected, place 30 ℃, cultivated 4-5 days under the 75%-85% relative humidity, obtain single bacterium colony agar block.
3. according to the described screening method of claim 1, it is characterized in that: the used indicator of biological assay dish is intestinal bacteria or other phosphonomycin sensitive organism.
4. according to the described screening method of claim 1, it is characterized in that: sieve again by the bacterial strain of shaker fermentation method to primary dcreening operation, substratum is a cis-propene phosphoric acid 0.3%, extractum carnis 0.5%, and peptone 1%, NaCl 0.5%, glycerine 3%, NaVO
30.02%, CoCl
20.02%, surplus is a tap water, 30 ℃, 160rpm cultivated 4 days, utilize the thin layer chromatography analysis phosphonomycin, the volume ratio of developping agent propyl carbinol, acetate, water is 3: 1: 1, and the chromatoplate after launching is air-dry, place on the biological assay flat board, take off after waiting to soak into, cultivate 16hr, contain phosphonomycin and partly produce inhibition zone for 37 ℃, by the antibacterial circle diameter size, further filter out the higher bacterial strain of transformation efficiency.
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| CN1876830B (en) * | 2006-04-26 | 2012-11-14 | 中国科学院沈阳应用生态研究所 | Levo-phosphonomycin biotransformation strain screening method using dextro-phosphonomycin as substrate |
| CN1974786B (en) * | 2006-12-13 | 2010-06-16 | 中国科学院沈阳应用生态研究所 | Process of screening phosphonomycin producing strain |
| CN101153302B (en) * | 2007-08-24 | 2010-07-28 | 中国科学院沈阳应用生态研究所 | Method for conversion from dextro-phosphonomycin to levo-phosphonomycin |
| CN104977387A (en) * | 2015-05-03 | 2015-10-14 | 山西省农业科学院经济作物研究所 | Bacterial inhibition experiment method of propolis tincture |
| CN104911123B (en) * | 2015-05-19 | 2018-06-08 | 孙军 | One plant of Pediococcus acidilactici P3-4 and its screening and identification and the application in degradation erythromycin, roxithromycin |
| CN104946560B (en) * | 2015-05-19 | 2018-09-18 | 孙军 | One Enterococcus faecalis E9-10 and its screening and identification and the application in spiramvcin of degrading |
| CN104946559B (en) * | 2015-05-19 | 2018-06-05 | 孙军 | One plant of Pediococcus acidilactici P3-4 and its screening and identification and the application in spiramvcin of degrading |
| CN104946557B (en) * | 2015-05-19 | 2018-09-18 | 孙军 | One Enterococcus faecalis E9-10 and its screening and identification and the application in degradation erythromycin, roxithromycin |
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Effective date of registration: 20090703 Address after: No. 72, Wenhua Road, Shenhe District, Liaoning, Shenyang: 110016 Co-patentee after: Dongbei Pharmaceutical Factory Patentee after: Shenyang Institute of Applied Ecology, Chinese Academy of Sciences Address before: No. 72, Wenhua Road, Shenhe District, Liaoning, Shenyang: 110016 Patentee before: Shenyang Application Ecelogy Inst., Chinese Academy of Sciences |
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Effective date of registration: 20100415 Address after: 110016 Shenyang, Liaoning Province Cultural Road, No. 72, Shenhe Co-patentee after: Northeast Pharmaceutical Group Co., Ltd. Patentee after: Shenyang Application Ecelogy Inst., Chinese Academy of Sciences Address before: 110016 Shenyang, Liaoning Province Cultural Road, No. 72, Shenhe Co-patentee before: Dongbei Pharmaceutical Factory Patentee before: Shenyang Application Ecelogy Inst., Chinese Academy of Sciences |
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