CN103215318A - Method for synthesizing PHA (Phytohemagglutinin) by using pseudomonas putida KT2442 - Google Patents
Method for synthesizing PHA (Phytohemagglutinin) by using pseudomonas putida KT2442 Download PDFInfo
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- CN103215318A CN103215318A CN2013101589305A CN201310158930A CN103215318A CN 103215318 A CN103215318 A CN 103215318A CN 2013101589305 A CN2013101589305 A CN 2013101589305A CN 201310158930 A CN201310158930 A CN 201310158930A CN 103215318 A CN103215318 A CN 103215318A
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- 241000589776 Pseudomonas putida Species 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 24
- 230000002194 synthesizing effect Effects 0.000 title abstract description 3
- 108010047620 Phytohemagglutinins Proteins 0.000 title abstract 6
- 230000001885 phytohemagglutinin Effects 0.000 title abstract 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 15
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 15
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 11
- 239000008103 glucose Substances 0.000 claims abstract description 11
- 239000001963 growth medium Substances 0.000 claims abstract description 8
- 239000012467 final product Substances 0.000 claims abstract description 7
- 238000007670 refining Methods 0.000 claims abstract description 5
- 238000001035 drying Methods 0.000 claims abstract description 4
- WLGDAKIJYPIYLR-UHFFFAOYSA-N octane-1-sulfonic acid Chemical compound CCCCCCCCS(O)(=O)=O WLGDAKIJYPIYLR-UHFFFAOYSA-N 0.000 claims description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 235000015097 nutrients Nutrition 0.000 claims description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 4
- 230000001580 bacterial effect Effects 0.000 claims description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 3
- 238000007792 addition Methods 0.000 claims description 2
- 239000003814 drug Substances 0.000 abstract description 8
- 239000000047 product Substances 0.000 abstract description 5
- 238000012258 culturing Methods 0.000 abstract description 4
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 238000002156 mixing Methods 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 230000002596 correlated effect Effects 0.000 abstract 2
- 239000011159 matrix material Substances 0.000 abstract 2
- 238000011218 seed culture Methods 0.000 abstract 2
- 239000002253 acid Substances 0.000 abstract 1
- 238000007710 freezing Methods 0.000 abstract 1
- 230000009477 glass transition Effects 0.000 abstract 1
- 230000028744 lysogeny Effects 0.000 abstract 1
- TVMXDCGIABBOFY-UHFFFAOYSA-N octane Chemical compound CCCCCCCC TVMXDCGIABBOFY-UHFFFAOYSA-N 0.000 abstract 1
- 239000002351 wastewater Substances 0.000 abstract 1
- 239000005014 poly(hydroxyalkanoate) Substances 0.000 description 47
- 229920000903 polyhydroxyalkanoate Polymers 0.000 description 46
- 208000037534 Progressive hemifacial atrophy Diseases 0.000 description 44
- 239000000463 material Substances 0.000 description 8
- 238000011160 research Methods 0.000 description 5
- 239000012620 biological material Substances 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- OMSUIQOIVADKIM-UHFFFAOYSA-N ethyl 3-hydroxybutyrate Chemical compound CCOC(=O)CC(C)O OMSUIQOIVADKIM-UHFFFAOYSA-N 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 239000000178 monomer Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 3
- 238000004806 packaging method and process Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000024287 Areas Species 0.000 description 1
- 229910016876 Fe(NH4)2(SO4)2 Inorganic materials 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 229920005601 base polymer Polymers 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000003519 biomedical and dental material Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- -1 molecular weight Substances 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 229920000520 poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Polymers 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 229920000098 polyolefin Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 108010046845 tryptones Proteins 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a method for synthesizing PHA (Phytohemagglutinin) by using pseudomonas putida KT2442. The method comprises the following steps of: 1, mixing a small quantity of pseudomonas putida KT2442 in an LB (lysogeny broth) culture medium, and culturing to obtain a seed culture solution containing strains; and 2, adding the seed culture solution obtained in the step 1 and a carbon source to the culture medium, culturing, separating, quick-freezing, drying, and refining to obtain a final product PHA. According to the method, the pseudomonas putida KT2442 in culture wastewater is used, the supply of the carbon source is adjusted, and correlated matrix octane acid and non-correlated matrix glucose are respectively added in the culturing process, thus the final product PHA is obtained. The product obtained by using the method has strong decomposability, low glass transition temperature, good flexibility and adhesion and can be applied to the medicine field, and therefore, the production has a bright application prospect.
Description
Technical field
The present invention relates to PHA preparation method in the biological field, particularly, relate to method with the synthetic PHA of pseudomonas putida KT2442.
Background technology
Polyhydroxyalkanoate (PHA, polyhydroxyalkanoates): be the bioabsorbable polymer material that developed rapidly in nearly more than 20 years---polyhydroxyalkanoate (PHA), being polyester in a kind of cell of a lot of microorganism synthetic, is a kind of natural polymeric biomaterial.Because PHA has the hot workability of excellent biological compatibility energy, biodegradability and plastics simultaneously.Because can be used as bio-medical material and biodegradable wrapping material simultaneously, this has become in recent years technical field of biological material active research focus the most.PHA also has optically nonlinearity, piezoelectricity, a lot of high added value performances of being separated by property of gas.
The biodegradable high score material of natural or synthetic often has very high water vapor transmission, and this is disadvantageous in food fresh keeping.PHA then has good barrier properties for gases, makes in its aquatic foods product Freshkeeping Packaging that may be applied in the long period.Because penetrating of steam is important indicator in the Freshkeeping Packaging, PHA performance in this is can compare with present products such as PET, PP etc. fully.In addition-and the aspect, PHA also has stability to hydrolysis preferably, and PHA is always washed 20 circulations with 75 ℃ automatic dishwasher, and shape and molecular weight that PHA makes cup all do not change, and show that PHA can perform well in utensil production.With other polyolefins, polyaromatic base polymer ratio, PHA also has good ultraviolet stability in addition.PHA also can be used as the source of biodegradable environmentally friendly solvent, as the solvent of ethyl hydroxyl-sour EHB (ethyl3-hydroxy-butyrate) is water miscible, and poly-have low volatility, can be used for sanitising agent, glue) stick, dyestuff, ink.Just because of PHA has compiled these good performances, it can be applied in fields such as wrapping material, jointing material, spray material and dress material, utensil class material, electronic product, durable consumer goods, agricultural prods, automated production, chemical mediator and solvent.
PHA is bringing into play more and more important effect because of its favorable biological degradability and biocompatibility in the medicament slow release system.PHA the earliest is the research of nineteen eighty-three for PHB as the research of drug release parcel microballoon, and along with the development of PHBV, the medicine parcel research of PHA has brought very big progress afterwards.Studies show that and to realize that by monomer composition, molecular weight, medicine parcel amount, the enwrapped granule size of regulating PHA the controlled rate of medicine discharges.In addition, a lot of scholars also utilize other polymkeric substance such as PCL also to obtain certain achievement with the research of PHA mixing packaging medicine.
PHA is a kind of environment friendly biological plastics of excellent property, has many adjustable material properties again, and it is along with the further reduction of cost and the exploitation of value added applications, will become many Application Areass biomaterial that a kind of cost can be accepted by market.Because it is one and forms family widely that its performance from hard to elastomeric makes it go for different application needs.The structure variation of PHA and the mutability of performance make it become a member important in the biomaterial.With respect to PLA, the history of PHA development is very short, and the potentiality of development are bigger, and the space of its application is also bigger.
But the method complexity for preparing PHA in the prior art, cost are also than higher.
Summary of the invention
At defective of the prior art, the purpose of this invention is to provide a kind of method with the synthetic PHA of pseudomonas putida KT2442.
The invention provides a kind of pseudomonas putida KT2442 synthetic PHA method of using, comprise the steps:
Step 1 is got pseudomonas putida KT2442 and is placed the LB substratum to mix, and cultivates, and obtains containing the kind nutrient solution of bacterial classification;
Step 2 adds described kind of nutrient solution, carbon source in the basal culture medium, cultivates, separate, and quick-frozen, drying, refining, promptly get final product PHA.
Preferably, in the step 1, the mass ratio of described pseudomonas putida KT2442 and LB substratum is 1:100.
Preferably, in the step 1, described incubation time is 24 hours, and culture temperature is 30 ℃.
Preferably, in the step 2, the mass ratio of described kind of nutrient solution, carbon source and basal culture medium is 1:2:100.
Preferably, in the step 2, described cultivation is specially: in the different steps of this cultivation, add octanesulfonic acid respectively in substratum, glucose is cultivated, and obtains PHA.
Preferably, in the step 2, the mass ratio of described octanesulfonic acid and glucose is 1:3.
Preferably, in the step 2, divide in the described octanesulfonic acid culturing process and add for three times, be respectively initial stage, 18 hours, 24 hours.
Preferably, in the step 2, three additions of described octanesulfonic acid are respectively: 0.15g, 0.2g, 0.15g.
Preferably, in the step 2, described refining comprising the steps:
(a) get dried PHA and put into chloroformic solution, filter, heating obtains rough PHA;
(b) rough PHA is added in acetone and the methyl alcohol mixed liquor, heating obtains final product PHA.
Preferably, in the step 2, described incubation time is 72 hours, and culture temperature is 30 ℃.
Compared with prior art, the present invention has following beneficial effect:
(1) PHA of the present invention has environmental protection, performance-adjustable, and elastomeric performance is widely used, and the potentiality of development are bigger, and the space of its application is also bigger.
(2) the inventive method is simple, operation easily, and cost is low, and its product decomposability is strong, and second-order transition temperature is low, has good flexibility and tackyness, can be used for field of medicaments, has application promise in clinical practice.
Embodiment
The present invention is described in detail below in conjunction with specific embodiment.Following examples will help those skilled in the art further to understand the present invention, but not limit the present invention in any form.Should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, can also make some distortion and improvement.These all belong to protection scope of the present invention.
Embodiment
Present embodiment provides a kind of method with the synthetic PHA of pseudomonas putida KT2442, comprises the steps:
Step 1 is got 1g pseudomonas putida KT2442 and is placed the 100gLB substratum to mix, and culture temperature is 30 ℃, cultivates 24 hours, obtains containing the kind nutrient solution of bacterial classification, and the composition of LB substratum is shown in Table 1:
Step 2, first group of 1g kind nutrient solution and 0.15g octanesulfonic acid and second group of 1g kind nutrient solution and 1.5g glucose are added the 100g basal culture medium respectively to be cultivated, add the 0.2g octanesulfonic acid after proceeding to 18 hours when first group, adding the 0.15g octanesulfonic acid after proceeding to 24 hours cultivates, separate, quick-frozen, drying is got dried 1gPHA and is put into the 60ml chloroform and dissolve, filter, heat 30 ℃, obtain rough PHA, the PHA that 1g is rough adds in 60ml acetone and the methyl alcohol mixed liquor, heat 30 ℃, cultivated 72 hours, and obtained final product PHA, wherein the composition of basal culture medium is shown in Table 2:
Table 1
| Tryptones | 10g/l |
| Yeast extract | 5g/l |
| NaCl | 5g/l |
Table 2
| KH 2PO 4 | 2.65g/l |
| Na 2HPO 4·12H 2O | 10.92g/l |
| MgSO 4·7H 2O | 0.1g/l |
| NH 4CL | 0.5g/l |
| CaCL 2·2H 2O | 0.01g/l |
| Fe(NH 4) 2(SO 4) 2 | 0.001g/l |
| KI | 0.5mg/l |
| ZnSO 4·7H 2O | 0.5mg/l |
| MnCl 2·4H 2O | 0.2mg/l |
| NiCl 6·6H 2O | 0.2mg/l |
| NaMoO 4·2H 2O | 0.03mg/l |
| CoCl 2·6H 2O | 0.01m?g/l |
| CuSO 4·5H 2O | 0.01m?g/l |
Implementation result: PHA analyzes and adopts gas chromatograph, and the operational condition of gas chromatograph is shown in Table 3:
Table 3
| Immovable bed | Thermon-3000on60/80SHINCARBON |
| Right cylinder | 1.6m x3.2mm i.d. glass cylinder |
| Mobile layer | He |
| Flow velocity | 60ml/min |
| Detector | Thermal conduction degree detector |
| Test portion inlet temperature | 230℃ |
| Detector temperature | 230℃ |
| The right cylinder temperature | Intensification to 8 ℃ of 80～230 ℃ of per minutes |
Operation steps:
Prepare and start → editor GC analytical procedure → sample analysis and image data → report
Output → shutdown
Test result is shown in Table 4:
Table 4
The result shows:
(1) with octanesulfonic acid and glucose as carbon source, it is feasible synthesizing PHA with pseudomonas putida.
(2) with octanesulfonic acid as carbon source, and compare as carbon source with glucose, the former thalline weight is about 1.5 times of the latter.
(3) with octanesulfonic acid as carbon source, and compare as carbon source with glucose, the former is 2.2 times of the latter for the weight of the interior contained PHA of dry back thalline.
(4) with octanesulfonic acid as carbon source, and compare as carbon source with glucose, the former is 3.3 times of the latter for the content of pure PHA.
(5) forming from the monomer of the PHA that produces gained, is that the monomer of carbon source synthetic PHA is formed based on 3HO with the octanesulfonic acid, forms then based on 3HD as carbon source synthetic PHA monomer with glucose.
More than specific embodiments of the invention are described.It will be appreciated that the present invention is not limited to above-mentioned specific implementations, those skilled in the art can make various distortion or modification within the scope of the claims, and this does not influence flesh and blood of the present invention.
Claims (10)
1. the method with the synthetic PHA of pseudomonas putida KT2442 is characterized in that, comprises the steps:
Step 1 is got pseudomonas putida KT2442 and is placed the LB substratum to mix, and cultivates, and obtains containing the kind nutrient solution of bacterial classification;
Step 2 adds described kind of nutrient solution, carbon source in the basal culture medium, cultivates, separate, and quick-frozen, drying, refining, promptly get final product PHA.
2. the method with the synthetic PHA of pseudomonas putida KT2442 as claimed in claim 1 is characterized in that in the step 1, the mass ratio of described pseudomonas putida KT2442 and LB substratum is 1:100.
3. the method with the synthetic PHA of pseudomonas putida KT2442 as claimed in claim 1 is characterized in that in the step 1, described incubation time is 24 hours, and culture temperature is 30 ℃.
4. the method with the synthetic PHA of pseudomonas putida KT2442 as claimed in claim 1 is characterized in that in the step 2, the mass ratio of described kind of nutrient solution, carbon source and basal culture medium is 1:2:100.
5. the method with the synthetic PHA of pseudomonas putida KT2442 as claimed in claim 1 is characterized in that in the step 2, described cultivation is specially: in the different steps of this cultivation, add octanesulfonic acid respectively in substratum, glucose is cultivated, and obtains PHA.
6. the method with the synthetic PHA of pseudomonas putida KT2442 as claimed in claim 5 is characterized in that in the step 2, the mass ratio of described octanesulfonic acid and glucose is 1:3.
7. the method with the synthetic PHA of pseudomonas putida KT2442 as claimed in claim 5 is characterized in that, in the step 2, divides three phases to add during described octanesulfonic acid is cultivated, and is respectively initial stage, 18 hours, 24 hours.
8. the method with the synthetic PHA of pseudomonas putida KT2442 as claimed in claim 7 is characterized in that in the step 2, three additions of described octanesulfonic acid are respectively: 0.15g, 0.2g, 0.15g.
9. the method with the synthetic PHA of pseudomonas putida KT2442 as claimed in claim 1 is characterized in that, in the step 2, and described refining comprising the steps:
(a) get dried PHA and put into chloroformic solution, filter, heating obtains rough PHA;
(b) rough PHA is added in acetone and the methyl alcohol mixed liquor, heating obtains final product PHA.
10. the method with the synthetic PHA of pseudomonas putida KT2442 as claimed in claim 1 is characterized in that in the step 2, described incubation time is 72 hours, and culture temperature is 30 ℃.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2013101589305A CN103215318A (en) | 2013-05-02 | 2013-05-02 | Method for synthesizing PHA (Phytohemagglutinin) by using pseudomonas putida KT2442 |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2013101589305A CN103215318A (en) | 2013-05-02 | 2013-05-02 | Method for synthesizing PHA (Phytohemagglutinin) by using pseudomonas putida KT2442 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754471A (en) * | 2016-11-21 | 2017-05-31 | 天津大学 | A kind of method for producing PHA pseudomonad Primary Studies in activated sludge |
| CN109399604A (en) * | 2018-11-16 | 2019-03-01 | 中南大学 | A kind of porous carbon materials and its preparation method and application prepared using pseudomonas putida from modification |
| CN110760460A (en) * | 2019-09-30 | 2020-02-07 | 浙江工业大学 | Compound microbial inoculum capable of efficiently degrading kitchen waste oil components and application thereof |
| WO2022214922A1 (en) * | 2021-04-09 | 2022-10-13 | W.H.In S.R.L. | Process for producing polyhydroxyalkanoates for food packages |
-
2013
- 2013-05-02 CN CN2013101589305A patent/CN103215318A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 周新荣: "利用恶臭假单胞菌生物合成聚羟基脂肪酸酯均聚物", 《中国优秀硕士学位论文全文数据库工程科技I辑》 * |
| 姜岷等: "Pseudomonas putida KT2442制备中长链聚羟基烷酸酯", 《南京工业大学学报》 * |
| 王志雄等: "印染废水生化合成可降解mcl-PHA的可行性", 《印染》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754471A (en) * | 2016-11-21 | 2017-05-31 | 天津大学 | A kind of method for producing PHA pseudomonad Primary Studies in activated sludge |
| CN109399604A (en) * | 2018-11-16 | 2019-03-01 | 中南大学 | A kind of porous carbon materials and its preparation method and application prepared using pseudomonas putida from modification |
| CN109399604B (en) * | 2018-11-16 | 2022-04-05 | 中南大学 | Porous carbon material prepared by utilizing pseudomonas putida self-modification and preparation method and application thereof |
| CN110760460A (en) * | 2019-09-30 | 2020-02-07 | 浙江工业大学 | Compound microbial inoculum capable of efficiently degrading kitchen waste oil components and application thereof |
| WO2022214922A1 (en) * | 2021-04-09 | 2022-10-13 | W.H.In S.R.L. | Process for producing polyhydroxyalkanoates for food packages |
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Effective date of registration: 20130905 Address after: 200093 No. 101, Yingkou Road, Shanghai Applicant after: Shanghai Medical Instrumentation College Address before: 200010 room 6, No. 1586, Lane 402, Zhongshan South Road, Shanghai, Huangpu District Applicant before: Wang Zhixiong |
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| C53 | Correction of patent of invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Wang Zhixiong Inventor after: Yu Qianqian Inventor after: Jiu Weiwei Inventor before: Wang Zhixiong |
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| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: WANG ZHIXIONG TO: WANG ZHIXIONG YU QIANQIAN MIAO WEIWEI |
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| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20130724 |