CN106497957A - The method that recombination engineering bacterium fermentation synthesizes 2,5 furandicarboxylic acids - Google Patents
The method that recombination engineering bacterium fermentation synthesizes 2,5 furandicarboxylic acids Download PDFInfo
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- CN106497957A CN106497957A CN201610997507.8A CN201610997507A CN106497957A CN 106497957 A CN106497957 A CN 106497957A CN 201610997507 A CN201610997507 A CN 201610997507A CN 106497957 A CN106497957 A CN 106497957A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/02—Oxygen as only ring hetero atoms
- C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
Abstract
The invention discloses the method that recombination engineering bacterium fermentation synthesizes 2,5 furandicarboxylic acids, is by fusobacterium glycerol dehydrase gene gldABC, is integrated into autochthonal klebsiella spp gene dha β, form recombinant dna gene gldha；Expanded by PCR, and modified rear insertion coli expression carrier pTE28aT7, build 2,5 furandicarboxylic acid engineering bacteria pTE28aT7 gldha；In isopropylthiogalactoside（IPTG）Under induction, in the restriction culture medium containing fructose, through depth fermentation 2,5 furandicarboxylic acids of synthesis；2,5 furandicarboxylic acid of product is obtained by filtering fermentating liquid, organic solvent extractive crystallization and combination solvent recrystallization.The present invention recombination engineering bacterium fermentation synthesize 2,5 furandicarboxylic acids method simple and clear, yield is high, with low cost, 2,5 furandicarboxylic acids microbial method prepare in play an important role, have a extensive future.
Description
Technical field
The invention belongs to technical field of microbial fermentation, is related to recombination engineering bacterium fermentation synthesis FDCA
Method.
Background technology
FDCA (FDCA) is important organic synthesis intermediate, can be used to prepare various alkyl replace or
Esters furan derivatives.Alkyl replaces analog derivative to be widely used in synthesis of chiral catalyst, molecular recognition acceptor and macromolecule
In material;Ester derivative is important spices, is used primarily in food, cosmetic essence.Additionally, FDCA can
Terephthalic acid (TPA) is replaced to manufacture the plastic material of polyesters.In terms of pharmacology, FDCA diethylester have with can
The similar anesthetic effect of cacaine, FDCA calcium salt have the function of the growth for suppressing bacillus megaterium.
With diglycolic acid as raw material, dimethyl diglycolate is obtained with thionyl chloride and methyl alcohol reaction, made in potassium hydroxide
With under, FDCA is condensed to yield with two hydration trimerization glyoxals.This method raw material is difficult preparation, expensive, synthesis
In used the hazardous agents such as thionyl chloride, ether, chloroform, post-process frequently with column chromatography, be unfavorable for industrialized production.
With 5 hydroxymethyl furfural as raw material, gold closes ceria for catalyst, high pressure (1.01 × 106Pa) oxidation obtains 2,5- furans two
Formic acid.Although this method obtains preferable yield 99%, raw material and metallic catalyst are expensive, and condition of high voltage is to equipment requirement
Too high, high cost is industrialized, is unfavorable for production application.With furancarboxylic acid as initiation material, first react with methyl alcohol, through chloromethyl
Change, then hydrolyze, FDCA is obtained through potassium permanganate oxidation afterwards most, overall yield of reaction is 47.5%.The original of the method
Though material is more cheap and easy to get, operation technological requirement is high and environmental pollution is more serious.The present invention is by fusobacterium glycerol dehydratase base
Because of gldABC, autochthonal klebsiella spp gene dha β are integrated into, form recombinant dna gene gldha；Expanded by PCR, and through repairing
Coli expression carrier pTE28aT7 is inserted after decorations, FDCA engineering bacteria pTE28aT7-gldha is built；Different
Propyl dithiocarbamate galactoside（IPTG）Under induction, in the restriction culture medium containing fructose, through depth fermentation synthesis 2,5- furans
Dioctyl phthalate；Product 2,5- furandicarboxylic acids are obtained by filtering fermentating liquid, organic solvent extractive crystallization and combination solvent recrystallization.
Synthesis route is simple and direct, energy-saving and environment-friendly, and the product quality that obtained is high and low cost, and gross mass yield reaches 65.5%
More than.
Content of the invention
It is an object of the invention to provide the method that recombination engineering bacterium fermentation synthesizes 2,5- furandicarboxylic acids.
The present invention is realized by following methods：
By fusobacterium glycerol dehydrase gene gldABC, autochthonal klebsiella spp gene dha β are integrated into, form recombinant dna gene
gldha；Expanded by PCR, and modified rear insertion coli expression carrier pTE28aT7, build FDCA work
Journey bacterium pTE28aT7-gldha；In isopropylthiogalactoside（IPTG）Under induction, in the restriction culture medium containing fructose,
Through depth fermentation synthesis 2,5- furandicarboxylic acids；Recrystallized by filtering fermentating liquid, organic solvent extractive crystallization and combination solvent
Obtain product 2,5- furandicarboxylic acids.
Plasmid and bacterial strain：
Cloning vector pTE28aT7-, Escherichia coli E.Coli JM109 are purchased from commercial company.
Restriction enzyme, Taq enzyme, T4DNA ligases are purchased from Dalian Bao Bio-Engineering Company, and other chemical reagent are
Domestic pure analysis pure chemical reagent.
Glycerol dehydratase isolation medium：Distilled water 1000mL, fructose 6g, NaCl 5g, 7 g of dusty yeast, glucose 20g,
Agar powder 15g, adjusts pH7.0,0.1MPa sterilizing 20min. to be cooled to 35 DEG C～45 DEG C, add Neil red（Nile Red）2mL/L
(0.30mg Nile Red are dissolved in 100mL dimethyl sulfoxide (DMSO)s）, pour culture dish under aseptic condition into, standby after cooling.
Eutrophy culture medium：Distilled water 1000mL, dusty yeast 10g, agar powder 10g, fructose 3g, (NH4)2SO45g, adjusts
PH7.0,0.1MPa sterilizing 10min.
Product fermentation medium：
The preparation of phosphate buffer：Distilled water 1000mL, NaH2PO4.12H2O 8.95g, KH2PO41.5g,
PH7.00.1MPa sterilizes, and 15min～20min is standby.
Genetic engineering bacterium enriched medium is LB culture mediums.Fermentation medium is the LB culture mediums for adding 15.0% fructose,
To blast air as genetic engineering bacterium oxidant；Cultivation temperature is 35 DEG C.
The acquisition of recombinant dna gene and the structure of recombinant plasmid：
According to the cDNA sequence of the gene gldABC of the fusobacterium glycerol dehydratase that has delivered, positive anti-primer is designed:Forward primer
Pl (5'-ACGAGGAATTCATGAATTGGCCTACTCG-3');Reverse primer P2 (5'-
ATATGCGGCCGCGTGAACAGTAGGCAGAG-3').Forward primer carries EcoRI restriction enzyme sites, and reverse primer carries Not works
Restriction enzyme site, with primer Pl, P2 obtains gldABC genetic fragments to recombinant plasmid pUC19-gldABC PCR amplifications.Equally,
The gene dha β of autochthonal Cray bacillus are designed, forward primer carries BamHI restriction enzyme sites, and reverse primer carries Xoh work digestions position
Point, with primer Pl ', P2 ' obtains dha beta gene fragments to recombinant plasmid pUCM-dha β PCR amplifications.By above-mentioned 2 kinds of gene pieces
Section, in the effect of ligase T4 ligase, is connected to become the gene gldha of recombinant DNA.Through the extraction of recombinant dna plasmid, height
After flux screening and sequencing, using CaCl2Plasmid recombinant technique, the gene gldha and carrier of recombinant DNA are proceeded to
In the competent cell of E.coli JM109, transformant is sequenced, and obtains expression plasmid pTE28aT7-gldha.
PCR is expanded：97 DEG C of denaturations 10min, 94 DEG C of deformation 60s, 58 DEG C of annealing 30s, 72 DEG C of extension 60-120s, 30
72 DEG C of extension 10min after circulation.
The preparation and conversion of competent escherichia coli cell：
Competent escherichia coli cell is prepared according to calcium method and is converted, conversion be followed by 1mlLB culture concentrate 36 DEG C,
150r/min shaken cultivations 2h, are spread evenly across screening recombination bacillus coli on the LB flat boards containing ampicillin.Screen
The bacterial strain for arriving is using bacterium colony PCR and digestion with restriction enzyme recombinant plasmid checking recon.
The extraction of recombination engineering bacteria：
Front culture is in L- test tubes, adds 5ml eutrophy culture mediums with sterile working, accesses Aerobacter aerogenes with sterile toothpick
Single bacterium colony.35 DEG C, 120r/min culture 15h.Front culture 0.5ml is seeded to containing 100ml eutrophy culture mediums
In 500ml triangular flasks, 30 DEG C, 110r/min shaken cultivations 24h.4 DEG C, 6000*g sterile centrifugation 10min abandon supernatant, in centrifugation
Mixed with sterile phosphate buffer vibration in pipe, then at 4 DEG C, 6000*g sterile centrifugation 12min abandon supernatant.By front culture
Thing 0.5ml is seeded in the 500ml triangular flasks containing 100ml eutrophy culture mediums, 35 DEG C, 110r/min shaken cultivations 24h.4
DEG C, 6000*g sterile centrifugation 10min abandon supernatant, are mixed with sterile phosphate buffer vibration in centrifuge tube, again at 4 DEG C,
6000*g sterile centrifugation 12min, abandon supernatant.Respectively by aseptic for the thalline for not containing eutrophy culture medium access 10 bottles contain
In the 500ml triangular flasks of 100m fermentation mediums, 31 DEG C～37 DEG C, 110r/min shaken cultivations 48h.
Recombination engineering bacterium fermentation produces 2,5- furandicarboxylic acids：
The recombination bacillus coli of -70 DEG C of preservations is activated on LB culture medium flat plates, is connected to sterilizing toothpick picking single bacterium colony
In 20mlLB culture mediums, 31 DEG C～37 DEG C overnight incubations are inoculated in fermentation medium with 2% inoculum concentration, add 1～
The IPTG of 2mmol/L induces the expression of gldhA, fermented and cultured 48h.
The separation of tunning：After fermentation harvest, centrifugation is extracted using butyl acetate with catalyzing enzyme, fermentation mother liquor is reclaimed
Take, by extraction gained organic condensing crystallizing after, using butyl acetate and petroleum ether（1:3～4 volumes）It is recrystallized to give product.
The present invention recombination engineering bacterium fermentation synthesis 2,5- furandicarboxylic acids method simple and clear, yield is high, low cost
Honest and clean.Above-mentioned advantage is based on, the engineering bacteria of the present invention plays an important role in preparing in the bioanalysis of FDCA,
Have a extensive future.
Specific embodiment
With reference to specific embodiment, the invention will be further elaborated, but is not limited to these specific embodiments,
And all of embodiment is operated by above-mentioned operating procedure.
Embodiment 1
Plasmid and bacterial strain：
Cloning vector pTE28aT7-, Escherichia coli E.Coli JM109 are purchased from commercial company.
Restriction enzyme, Taq enzyme, T4DNA ligases are purchased from Dalian Bao Bio-Engineering Company, and other chemical reagent are
Domestic pure analysis pure chemical reagent.
Glycerol dehydratase isolation medium：Distilled water 1000mL, fructose 6g, NaCl 5g, 7 g of dusty yeast, glucose 20g,
Agar powder 15g, adjusts pH7.0,0.1MPa sterilizing 20min. to be cooled to 35 DEG C～45 DEG C, add Neil red（Nile Red）2mL/L
(0.30mg Nile Red are dissolved in 100mL dimethyl sulfoxide (DMSO)s）, pour culture dish under aseptic condition into, standby after cooling.
Eutrophy culture medium：Distilled water 1000mL, dusty yeast 10g, agar powder 10g, fructose 3g, (NH4)2SO45g, adjusts
PH7.0,0.1MPa sterilizing 10min.
Product fermentation medium：
The preparation of phosphate buffer：Distilled water 1000mL, NaH2PO4.12H2O 8.95g, KH2PO41.5g,
PH7.00.1MPa sterilizes, and 15min～20min is standby.
Genetic engineering bacterium enriched medium is LB culture mediums.Fermentation medium is the LB culture mediums for adding 15.0% fructose,
To blast air as genetic engineering bacterium oxidant；Cultivation temperature is 35 DEG C.
The acquisition of recombinant dna gene and the structure of recombinant plasmid：
According to the cDNA sequence of the gene gldABC of the fusobacterium glycerol dehydratase that has delivered, positive anti-primer is designed:Forward primer
Pl (5'-ACGAGGAATTCATGAATTGGCCTACTCG-3');Reverse primer P2 (5'-
ATATGCGGCCGCGTGAACAGTAGGCAGAG-3').Forward primer carries EcoRI restriction enzyme sites, and reverse primer carries Not works
Restriction enzyme site, with primer Pl, P2 obtains gldABC genetic fragments to recombinant plasmid pUC19-gldABCPCR amplifications.Equally, if
The gene dha β of autochthonal Cray bacillus are counted, forward primer carries BamHI restriction enzyme sites, and reverse primer carries Xoh work digestions position
Point, with primer Pl ', P2 ' obtains dha beta gene fragments to recombinant plasmid pUCM-dha β PCR amplifications.By above-mentioned 2 kinds of gene pieces
Section, in the effect of ligase T4 ligase, is connected to become the gene gldha of recombinant DNA.Through the extraction of recombinant dna plasmid, height
After flux screening and sequencing, using CaCl2Plasmid recombinant technique, the gene gldha and carrier of recombinant DNA are proceeded to
In the competent cell of E.coli JM109, transformant is sequenced, and obtains expression plasmid pTE28aT7-gldha.
PCR is expanded：97 DEG C of denaturations 10min, 94 DEG C of deformation 60s, 58 DEG C of annealing 30s, 72 DEG C of extension 60-120s, 30
72 DEG C of extension 10min after circulation.
The preparation and conversion of competent escherichia coli cell：
Competent escherichia coli cell is prepared according to calcium method and is converted, conversion be followed by 1mlLB culture concentrate 36 DEG C,
150r/min shaken cultivations 2h, are spread evenly across screening recombination bacillus coli on the LB flat boards containing ampicillin.Screen
The bacterial strain for arriving is using bacterium colony PCR and digestion with restriction enzyme recombinant plasmid checking recon.
The extraction of recombination engineering bacteria：
Front culture is in L- test tubes, adds 5ml eutrophy culture mediums with sterile working, accesses Aerobacter aerogenes with sterile toothpick
Single bacterium colony.35 DEG C, 120r/min culture 15h.Front culture 0.5ml is seeded to containing 100ml eutrophy culture mediums
In 500ml triangular flasks, 30 DEG C, 110r/min shaken cultivations 24h.4 DEG C, 6000*g sterile centrifugation 10min abandon supernatant, in centrifugation
Mixed with sterile phosphate buffer vibration in pipe, then at 4 DEG C, 6000*g sterile centrifugation 12min abandon supernatant.By front culture
Thing 0.5ml is seeded in the 500ml triangular flasks containing 100ml eutrophy culture mediums, 35 DEG C, 110r/min shaken cultivations 24h.4
DEG C, 6000*g sterile centrifugation 10min abandon supernatant, are mixed with sterile phosphate buffer vibration in centrifuge tube, again at 4 DEG C,
6000*g sterile centrifugation 12min, abandon supernatant.Respectively by aseptic for the thalline for not containing eutrophy culture medium access 10 bottles contain
In the 500ml triangular flasks of 100m fermentation mediums, 31 DEG C～37 DEG C, 110r/min shaken cultivations 48h.
Recombination engineering bacterium fermentation produces 2,5- furandicarboxylic acids：
The recombination bacillus coli of -70 DEG C of preservations is activated on LB culture medium flat plates, is connected to sterilizing toothpick picking single bacterium colony
In 20mlLB culture mediums, 31 DEG C～37 DEG C overnight incubations are inoculated in fermentation medium with 2% inoculum concentration, add 1～
The IPTG of 2mmol/L induces the expression of gldhA, fermented and cultured 48h.
The separation of tunning：After fermentation harvest, centrifugation is extracted using butyl acetate with catalyzing enzyme, fermentation mother liquor is reclaimed
Take, by extraction gained organic condensing crystallizing after, using butyl acetate and petroleum ether（1:3～4 volumes）It is recrystallized to give 2,5-
(308.5～309.2 DEG C of fusing point, content 99.32%, yield is 65.51%) for furandicarboxylic acid 8.57g.
Embodiment 2
The composition and condition of culture of each culture medium is ibid.
The acquisition of recombinant dna gene and the structure of recombinant plasmid：
According to the cDNA sequence of the gene gldABC of the fusobacterium glycerol dehydratase that has delivered, positive anti-primer is designed:Forward primer
Pl (5'-ACGAGGAATTCATGAATTGGCCTACTCG-3');Reverse primer P2 (5'-
ATATGCGGCCGCGTGAACAGTAGGCAGAG-3').Forward primer carries EcoRI restriction enzyme sites, and reverse primer carries Not works
Restriction enzyme site, with primer Pl, P2 obtains gldABC genetic fragments to recombinant plasmid pUC19-gldABC PCR amplifications.Equally,
The gene dha β of autochthonal Cray bacillus are designed, forward primer carries BamHI restriction enzyme sites, and reverse primer carries Xoh work digestions position
Point, with primer Pl ', P2 ' obtains dha beta gene fragments to recombinant plasmid pUCM-dha β PCR amplifications.By above-mentioned 2 kinds of gene pieces
Section, in the effect of ligase T4 ligase, is connected to become the gene gldha of recombinant DNA.Through the extraction of recombinant dna plasmid, height
After flux screening and sequencing, using CaCl2Plasmid recombinant technique, the gene gldha and carrier of recombinant DNA are proceeded to
In the competent cell of E.coli JM109, transformant is sequenced, and obtains expression plasmid pTE28aT7-gldha.
PCR is expanded：97 DEG C of denaturations 10min, 94 DEG C of deformation 60s, 58 DEG C of annealing 30s, 72 DEG C of extension 60-120s, 30
72 DEG C of extension 10min after circulation.
The preparation and conversion of competent escherichia coli cell：
Competent escherichia coli cell is prepared according to calcium method and is converted, conversion be followed by 1mlLB culture concentrate 36 DEG C,
150r/min shaken cultivations 2h, are spread evenly across screening recombination bacillus coli on the LB flat boards containing ampicillin.Screen
The bacterial strain for arriving is using bacterium colony PCR and digestion with restriction enzyme recombinant plasmid checking recon.
The extraction of recombination engineering bacteria：
Front culture is in L- test tubes, adds 5ml eutrophy culture mediums with sterile working, accesses Aerobacter aerogenes with sterile toothpick
Single bacterium colony.35 DEG C, 120r/min culture 15h.Front culture 0.5ml is seeded to containing 100ml eutrophy culture mediums
In 500ml triangular flasks, 30 DEG C, 110r/min shaken cultivations 24h.4 DEG C, 6000*g sterile centrifugation 10min abandon supernatant, in centrifugation
Mixed with sterile phosphate buffer vibration in pipe, then at 4 DEG C, 6000*g sterile centrifugation 12min abandon supernatant.By front culture
Thing 0.5ml is seeded in the 500ml triangular flasks containing 100ml eutrophy culture mediums, 35 DEG C, 110r/min shaken cultivations 24h.4
DEG C, 6000*g sterile centrifugation 10min abandon supernatant, are mixed with sterile phosphate buffer vibration in centrifuge tube, again at 4 DEG C,
6000*g sterile centrifugation 12min, abandon supernatant.Respectively by aseptic for the thalline for not containing eutrophy culture medium access 10 bottles contain
In the 500ml triangular flasks of 100m fermentation mediums, 31 DEG C～37 DEG C, 110r/min shaken cultivations 48h.
Recombination engineering bacterium fermentation produces 2,5- furandicarboxylic acids：
The recombination bacillus coli of -70 DEG C of preservations is activated on LB culture medium flat plates, is connected to sterilizing toothpick picking single bacterium colony
In 20mlLB culture mediums, 31 DEG C～37 DEG C overnight incubations are inoculated in fermentation medium with 2% inoculum concentration, add 1～
The IPTG of 2mmol/L induces the expression of gldhA, fermented and cultured 48h.
The separation of tunning：After fermentation harvest, centrifugation is extracted using butyl acetate with catalyzing enzyme, fermentation mother liquor is reclaimed
Take, by extraction gained organic condensing crystallizing after, using butyl acetate and petroleum ether（1:3～4 volumes）It is recrystallized to give 2,5-
(308.6～309.3 DEG C of fusing point, content 99.27%, yield is 69.35%) for furandicarboxylic acid 9.08g.
Claims (6)
1. the present invention relates to recombination engineering bacterium fermentation synthesize FDCA method, including：
（1）It is by fusobacterium glycerol dehydrase gene gldABC, is integrated into autochthonal klebsiella spp gene dha β, forms restructuring
DNA gene gldha；
（2）Expanded by PCR, and modified rear insertion coli expression carrier pTE28aT7, build FDCA
Engineering bacteria pTE28aT7-gldha；
（3）In isopropylthiogalactoside（IPTG）Under induction, high efficient strain is screened using high flux screening method；
（4）In the restriction culture medium containing fructose, through depth fermentation synthesis FDCA, by filtering fermentating liquid,
Organic solvent extractive crystallization and combination solvent recrystallization obtain product 2,5- furandicarboxylic acids.
2. method according to claim 1, it is characterised in that fusobacterium glycerol dehydrase gene gldABC and autochthonal Cray
Primary bacillus gene dha β, carry out restructuring and form recombinant dna gene gldha.
3. method according to claim 1, it is characterised in that PCR amplification conditions are：97 DEG C of denaturations 10min, 94 DEG C of changes
Shape 60s, 58 DEG C of annealing 30s, 72 DEG C of extension 60-120s, 72 DEG C of extension 10min after 30 circulations.
4. the method that recombination engineering fermentation according to claim 1 synthesizes FDCA, including：
（1）The recombination engineering is fermented in containing the restricted culture medium of fructose, using high flux screening；
（2）Filtering fermentating liquid, organic solvent extractive crystallization and combination solvent recrystallization obtains product 2,5- furandicarboxylic acids.
5. method according to claim 4, it is characterised in that recombination engineering bacterium fermentation synthesizes FDCA
Fermentation stage fermentation condition be 31 DEG C～37 DEG C of temperature, 110r/min shaken cultivations 48h.
6. according in accordance with the method for claim 4, it is characterised in that zymotic fluid first passes through filter and separates catalyzing enzyme reuse,
Filter after fermentation liquid using n-butyl acetate extraction, then by extract condensing crystallizing after, using butyl acetate and petroleum ether by volume
1：3～4 combination solvent recrystallization, obtains product FDCA.
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| CN109234217A (en) * | 2018-06-29 | 2019-01-18 | 安徽瑞赛生化科技有限公司 | Produce the immobilization and its application of 2,5- furandicarboxylic acid recombination engineering bacteria |
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| CN109234217A (en) * | 2018-06-29 | 2019-01-18 | 安徽瑞赛生化科技有限公司 | Produce the immobilization and its application of 2,5- furandicarboxylic acid recombination engineering bacteria |
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Inventor after: Hu Xiao Inventor after: Zhang Wenjuan Inventor after: Hu Wei Inventor after: Hu Manman Inventor after: Zhou Shun Inventor after: Wang Lixiang Inventor after: Mou Jie Inventor after: Sun Yongxian Inventor after: Zhang Wei Inventor after: Qu Zhirong Inventor after: Hu Danhong Inventor before: Hu Xiao Inventor before: Li Yuping Inventor before: Qu Zhirong Inventor before: Hu Wei Inventor before: Hu Manman Inventor before: Wang Lixiang Inventor before: Sun Yongxian Inventor before: Zhang Wei Inventor before: Yao Huanfeng Inventor before: Liu Xudong Inventor before: Zhao Hong Inventor before: Hu Danhong |
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Application publication date: 20170315 |