CN104878035A - Construction method for producing N-acetylneuraminic acid recombinant microorganisms and application of N-acetylneuraminic acid recombinant microorganisms - Google Patents
Construction method for producing N-acetylneuraminic acid recombinant microorganisms and application of N-acetylneuraminic acid recombinant microorganisms Download PDFInfo
- Publication number
- CN104878035A CN104878035A CN201510189018.5A CN201510189018A CN104878035A CN 104878035 A CN104878035 A CN 104878035A CN 201510189018 A CN201510189018 A CN 201510189018A CN 104878035 A CN104878035 A CN 104878035A
- Authority
- CN
- China
- Prior art keywords
- gene
- acetyl
- glucosamine
- pet
- nana
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a construction method for producing N-acetylneuraminic acid recombinant microorganisms and application of the N-acetylneuraminic acid recombinant microorganisms. Two recombinant escherichia colis of an N-acetylglucosamine-2-isomerase gene and an N-acetylneuraminic acid aldolase gene are respectively constructed through molecular biological techniques such as codon optimization and gene cloning; and the two recombinant microorganisms are subjected to induced cultivation and are mixed at a certain ratio, so as to efficiently produce the N-acetylneuraminic acid. With the N-acetylglucosamine and sodium pyruvate as substrates, and the two recombinant microorganisms as whole-cell catalysts, catalytic reaction is carried out, so that the N-acetylneuraminic acid yield can reach 260mM (80.5g/L); and the highest substrate conversion ratio reaches 43.3%.
Description
Technical field
The present invention relates to the recombinant microorganism and construction process thereof and application that produce N-acetyl-neuraminate.As far back as nineteen twenty-seven, the people such as the Landsteiner of the U.S. just find the component containing similar sugar in each thing of specific animal lipid system, and it can be purple with Bial ' s reagent react.Afterwards, method cracking cerebron and the submaxillary gland Saliva Orthana respectively that the people such as Klenk is gentle, therefrom isolates and Bial ' s reagent react is the material of purple, by its called after sialic acid (Sialic acid).Be extremely so far, found that the member of sialic acid family has more than 40 to plant, wherein most importantly N-acetyl-neuraminate (Neu5Ac), its content accounts for more than 99% of whole sialic acid family, and people are also more deep to its research.Prepared by the general said sialic technical scale being Neu5Ac of commercially producing, mainly comprise: from methods such as natural materials extraction, chemosynthesis, enzymatic clarification and fermentable.
Sialic acid is extensively present in zooblast surface with the form of saccharide complex, but content is quite low.Since discovery sialic acid, people extract respectively and obtain sialic acid from the natural matters such as bird's nest, milk, birds, beasts and eggs.Masskam Shimatanl extracts sialic acid (Masskma Shrinatnaletal.United States Patent, 5270462, Dee.14,1993) from casein.Martin etc. have extracted sialic acid (Martin JE, Tanenbaum SW.Carbohydrate Research, 1976.423-425) from bird's nest.2003, Koketsu is studied content sialic in Gallus Domesticus egg, be heated to 70 DEG C-90 DEG C under mildly acidic conditions and find that the sialic acid content extracted from single egg is about 218mg (Koketsu M.British Poultry Science, 2003,44:145-148).But sialic acid content in natural matter is extremely low, separation and purification process more complicated and yield is low, is thus difficult to the needs meeting suitability for industrialized production.
Chemical method sialic acid synthetase severe reaction conditions, need the noble metals such as indium as catalyzer, reactions steps is various.Such as in acidity alcohol solution, α-brooethyl acrylic acid carries out propylene glycosylation reaction to ManNAc under the katalysis of indium, again ozonization is carried out to the product obtained and can obtain N-acetyl-neuraminate (Chan T H, Lee M C.Journal of Organic Chemistry, 1995,60:4228-4232).But chemical synthesis relates to complicated radical protection and goes problem such as protection step and environment protection etc., and particularly productive rate is on the low side, therefore is restricted in application process.
The enzymatic clarification of N-acetyl-neuraminate mainly with ManNAc and pyruvic acid for substrate, under the catalysis of Neu 5 Ac aldolase (Neu5Ac aldolase), generate N-acetyl-neuraminate.In order to overcome the expensive problem of ManNAc, people are by alkaline condition or use N-Acetyl-D-glucosamine-2-isomerase (GlcNAc 2-epimerase) to make cheap N-Acetyl-D-glucosamine be converted into ManNAc.
In recent years, researchist using two kinds of Bacillus coli cells of recombinant expressed N-Acetyl-D-glucosamine-2-isomerase and Neu 5 Ac aldolase as biocatalysis medium, transform GlcNAc and produce Neu5Ac (Isafomi Maru, Jua Ohnishi, Y.Journalof Bioscience and Bioscience, 2002,93 (3): 258-265).This method is without separation and purification enzyme, and simultaneously due to the protection of cytolemma, enzyme is relatively more stable, cofactor regenerating easily.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of method of recombinant microorganism catalytic production N-acetyl-neuraminate, and described recombinant bacterium is for being Neu 5 Ac aldolase nanA gene and derive from the N-Acetyl-D-glucosamine-2-isomerase slr975 gene of cytoalgae (Synechocystis sp.) and the restructuring E.coli that builds afterwards of codon optimization in clone's E.coliK12 genome.
Described N-Acetyl-D-glucosamine-2-isomerase gene derives from cytoalgae (Synechocystis sp.) PCC 6803, and nucleotide sequence is as GeneBank login sequence number: shown in NC_000911.1; Described Neu 5 Ac aldolase gene source is in E.coli K12, and nucleotide sequence is as GeneBank login sequence number: shown in NC_000913.3;
By Neu 5 Ac aldolase gene nanA and codon optimized after N-Acetyl-D-glucosamine-2-isomerase gene slr975 be connected respectively on carrier pET-28a (+).
Present invention also offers a kind of method building product N-acetyl-neuraminate genetic engineering bacterium, slr1975 gene segment and pET-28a (+) are carried out enzyme with restriction enzyme NdeI and EcoRI cut, and by ligation by between NdeI and the EcoRI site of slr1975 gene segment insertion plasmid pET-28a (+), obtain recombinant plasmid pET-28a (+)-slr; NanA gene segment and pET-28a (+) are carried out enzyme with restriction enzyme NdeI and EcoRI cut, and by ligation by between NdeI and the EcoRI site of nanA gene segment insertion plasmid pET-28a (+), obtain recombinant plasmid pET-28a (+)-nanA; By recombinant plasmid pET-28a (+)-slr and pET-28a (+)-nanA conversion E.coli respectively, obtain recombinant microorganism E.coli/pET-28a (+)-slr and E.coli/pET-28a (+)-nanA respectively.
Described recombinant bacterium abduction delivering condition: N-Acetyl-D-glucosamine-2-isomerase gene and Neu 5 Ac aldolase gene utilize IPTG to carry out abduction delivering respectively, it is characterized in that inducing culture is LB substratum, abduction delivering temperature is 28 DEG C, cell concn OD during abduction delivering
600be 0.6, the abduction delivering time is 10h;
The method of production N-acetyl-neuraminate provided by the invention, it is characterized in that described recombinant microorganism catalytic efficiency and enzyme activity exist specific relation, enzyme activity ratio after optimization is 3:1 (N-Acetyl-D-glucosamine-2-isomerase: Neu 5 Ac aldolase), described catalyzed reaction substrate N-Acetyl-D-glucosamine and the concentration of Sodium.alpha.-ketopropionate are respectively 0.6M and 1.6M, Triton X-100 concentration is 0.4%, temperature of reaction 30-35 DEG C, the reaction times is at 48-60h.
Accompanying drawing explanation
Fig. 1 is recombinant plasmid pET-28a (+)-nanA digestion verification agarose gel electrophoresis figure.
Fig. 2 is recombinant plasmid pET-28a (+)-slr digestion verification agarose gel electrophoresis figure.
Fig. 3 is the electrophoresis detection result of N-Acetyl-D-glucosamine-2-isomerase and Neu 5 Ac aldolase protein in the genetic engineering bacterium of product N-acetyl-neuraminate.
Fig. 4 is that whole-cell catalytic produces N-acetyl-neuraminate concentration of substrate optimum result.
Embodiment
The structure of embodiment 1, recombinant plasmid pET-28a (+)-slr
According to cytoalgae (Synechocystis sp.PCC 6803) AGE encoding gene slr1975 (ID:954945) sequence that American National Biotechnology Information center (NCBI) is announced, E.coli Codon Usage Analysis 2.0 software is utilized to carry out codon analysis and optimization to this sequence.Synthesize goal gene according to the DNA sequence dna after optimizing, this unnamed gene is slr1975*.NdeI and EcoRI restriction enzyme site is increased at two ends, after synthesis between insertion vector plasmid pET-28a (+) multiple clone site EcoRI and NdeI during goal gene synthesis.
Primer for the slr1975* gene that increases: SlrF:5'-GGAATTCCATATGATGATTGCCCATCGCCGTCA-3'; SlrR:5'-CCGGAATTCTTAAGAAACCGGCAGTTGC-3'
N-Acetyl-D-glucosamine-2-isomerase gene utilizes synthetic gene slr1975* carry out pcr amplification as masterplate and obtain.Slr1975* gene segment and pET-28a (+) are carried out enzyme with restriction enzyme NdeI and EcoRI cut, and by ligation by between NdeI and the EcoRI site of slr1975* gene segment insertion plasmid pET-28a (+), obtain recombinant plasmid pET-28a (+)-slr.
The structure of embodiment 2, recombinant plasmid pET-28a (+)-nanA
Primer for the nanA gene that increases: NanAF:5'-GGAATTCCATATG ATGGCAACGA
ATTTACGTGG-3'；NanAR：5'-CCGGAATTC TCACCCGCGCTCTTGCATCAA-3'
Neu 5 Ac aldolase gene nanA utilizes E.coliK12 genome (GeneBankNC_000913.3) carry out pcr amplification as masterplate and obtain, nanA gene segment and pET-28a (+) are carried out enzyme with restriction enzyme NdeI and EcoRI cut, and by ligation by between NdeI and the EcoRI site of nanA gene segment insertion plasmid pET-28a (+), obtain recombinant plasmid pET-28a (+)-nanA.
The conversion of embodiment 3, recombinant microorganism
By recombinant plasmid pET-28a (+)-slr and pET-28a (+)-nanA Transformed E .coli respectively, obtain recombinant microorganism E.coli/pET-28a (+)-slr and E.coli/pET-28a (+)-nanA respectively.
The induction of embodiment 4, recombinant microorganism
Respectively by single to E.coli/pET-28a (+)-slr and E.coli/pET-28a (+)-nanA colony inoculation to containing in the LB liquid medium of 50 μ g/mL kantlex, 37 DEG C of overnight shakings are cultivated, rotating speed 200rpm; Be the inoculum size inoculation of 1% with volume percent by overnight culture, work as OD
600to about 0.6 ~ 0.8 time, add 0.5mM IPTG in 28 DEG C of abduction delivering 8h.Detect the result (Fig. 3) of protein expression with SDS-PAGE, wherein slr (43.5kDa) and nanA (33kDa) two albumen have expression.
Embodiment 5, whole-cell catalytic prepare N-acetyl-neuraminate
Cell after E.coli/pET-28a (+)-slr and E.coli/pET-28a (+)-nanA is induced, respectively at 4 DEG C, 5000rpm, centrifugal 5min collect thalline, collect thalline with after 100mM pH7.5Tris-HCL buffer solution 2 times with identical centrifugal condition.Be resuspended in conversion of substrate liquid (the 100mM pH7.5Tris-HCL damping fluid of proper volume; 0.6M N-Acetyl-D-glucosamine; 0.8M pyruvic acid; In 0.2%TritonX-100 (concentration of volume percent), 30 DEG C, 250rpm, pH7.5, transform 72h, each 12h sampling and adjust ph to 7.5.
By the conversion fluid that obtains in 12000rpm, centrifugal 5min, gets supernatant, after the membrane filtration of 0.45 μm, detects the output of N-acetyl-neuraminate with HPLC.HPLC adopts chromaster Hitachi III (DAD, RID).Chromatographic condition: AminexHPX-87H Column (300 × 7.8mm); Moving phase: 10mM H
2sO
4; Flow velocity: 0.50mL/min; Column temperature 65 DEG C; Sample size l0 μ L.N-acetyl-neuraminate (Neu5Ac) standard substance are purchased from sigma company.
Embodiment 6, whole-cell catalytic prepare the condition optimizing of N-acetyl-neuraminate
To the conversion of substrate concentration of whole-cell catalytic reaction, different N-acetylglucosamine 2-isomerase and Neu 5 Ac aldolase enzyme are lived and are compared, add tensio-active agent (Triton X-100, Tween-80, Tween-20, SDS) and show promoting agent and be optimized, the optimal conditions finally obtained is: N-Acetyl-D-glucosamine 2-isomerase and Neu 5 Ac aldolase enzyme are lived than being 3:1, the concentration of GlcNAc is 0.6M, the concentration of pyruvic acid is 1.6M, adds 0.4% tensio-active agent Triton X-100.
Claims (7)
1. a method for recombinant microorganism catalytic production N-acetyl-neuraminate, is characterized in that expressing N-Acetyl-D-glucosamine-2-isomerase gene slr1975* and Neu 5 Ac aldolase gene nanA respectively;
Described N-Acetyl-D-glucosamine-2-isomerase gene derives from cytoalgae (Synechocystis sp.) PCC 6803, and nucleotide sequence is as GenBank login sequence number: shown in NC_000911.1;
Described Neu 5 Ac aldolase gene source is in e. coli k12 (Escherichia coli), and nucleotide sequence is as GenBank login sequence number: shown in NC_000913.3;
Described microorganism host is E.coli BL21 (DE3).
2. N-Acetyl-D-glucosamine-2-isomerase gene slr1975* according to claim 1 is the DNA sequence dna after codon optimized.
3. recombinant microorganism according to claim 1, is characterized in that the recombinant microorganism of single expression N-Acetyl-D-glucosamine-2-isomerase gene and the recombinant microorganism of single expression Neu 5 Ac aldolase gene.
4. the recombinant microorganism described in claim 1-3, is characterized in that described N-Acetyl-D-glucosamine-2-isomerase gene and Neu 5 Ac aldolase gene adopt inducible expression carrier pET28a to express.
5. the construction process of the expression N-Acetyl-D-glucosamine-2-isomerase gene recombinant bacterium described in claim 1-4, concrete steps are as follows:
1) the slr1975* gene after codon optimized and pET-28a (+) carrier restriction enzyme NdeI and EcoRI are carried out enzyme to cut, and by ligation by between NdeI and the EcoRI site of slr1975* gene insertion plasmid pET-28a (+), obtain recombinant plasmid pET-28a (+)-slr;
2) nanA gene and pET-28a (+) are carried out enzyme with restriction enzyme NdeI and EcoRI to cut, and by ligation by between NdeI and the EcoRI site of nanA gene insertion plasmid pET-28a (+), obtain recombinant plasmid pET-28a (+)-nanA;
3) by plasmid pET-28a (+)-slr and pET-28a (+)-nanA Transformed E .coli respectively, recombinant microorganism E.coli/pET-28a (+)-slr and E.coli/pET-28a (+)-nanA is obtained respectively.
6. application rights requires recombinant microorganism described in 1, N-Acetyl-D-glucosamine-2-isomerase gene and Neu 5 Ac aldolase gene utilize IPTG to carry out abduction delivering respectively, it is characterized in that inducing culture is LB substratum, abduction delivering temperature is 28 DEG C, cell concn OD during abduction delivering
600be 0.6, the abduction delivering time is 10h.
7. the method for production N-acetyl-neuraminate according to claim 1, it is characterized in that described recombinant microorganism catalytic efficiency and enzyme activity exist specific relation, enzyme activity ratio after optimization is 3:1 (N-Acetyl-D-glucosamine-2-isomerase: Neu 5 Ac aldolase), described catalyzed reaction substrate N-Acetyl-D-glucosamine and the concentration of Sodium.alpha.-ketopropionate are respectively 0.6M and 1.6M, Triton X-100 concentration is 0.4%, temperature of reaction 30-37 DEG C, the reaction times is at 48-60h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510189018.5A CN104878035A (en) | 2015-04-20 | 2015-04-20 | Construction method for producing N-acetylneuraminic acid recombinant microorganisms and application of N-acetylneuraminic acid recombinant microorganisms |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510189018.5A CN104878035A (en) | 2015-04-20 | 2015-04-20 | Construction method for producing N-acetylneuraminic acid recombinant microorganisms and application of N-acetylneuraminic acid recombinant microorganisms |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104878035A true CN104878035A (en) | 2015-09-02 |
Family
ID=53945711
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510189018.5A Pending CN104878035A (en) | 2015-04-20 | 2015-04-20 | Construction method for producing N-acetylneuraminic acid recombinant microorganisms and application of N-acetylneuraminic acid recombinant microorganisms |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104878035A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114196693A (en) * | 2021-10-25 | 2022-03-18 | 福州一诺维生物科技有限公司 | Preparation method of N-acetylneuraminic acid |
| CN114457133A (en) * | 2022-03-07 | 2022-05-10 | 江南大学 | Method for producing N-acetylneuraminic acid by whole-cell catalysis with N-acetylglucosamine fermentation broth as substrate |
| CN114507658A (en) * | 2022-04-02 | 2022-05-17 | 深圳瑞德林生物技术有限公司 | Enzyme coexpression system and application thereof in sialic acid synthesis |
| CN114874967A (en) * | 2022-06-17 | 2022-08-09 | 江南大学 | Recombinant escherichia coli for producing N-acetylneuraminic acid and construction method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060358A (en) * | 2011-10-20 | 2013-04-24 | 中国科学院微生物研究所 | Genetically engineered bacteria for producing N-acetylneuraminic acid as well as construction method and application thereof |
| CN103060300A (en) * | 2011-10-20 | 2013-04-24 | 中国科学院微生物研究所 | N-acetylneuraminic acid aldolase as well as coding gene and application thereof |
| CN103276001A (en) * | 2013-06-19 | 2013-09-04 | 南京工业大学 | Gene of codon optimized N-acetyl glucosamine isomerase and expression thereof |
-
2015
- 2015-04-20 CN CN201510189018.5A patent/CN104878035A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060358A (en) * | 2011-10-20 | 2013-04-24 | 中国科学院微生物研究所 | Genetically engineered bacteria for producing N-acetylneuraminic acid as well as construction method and application thereof |
| CN103060300A (en) * | 2011-10-20 | 2013-04-24 | 中国科学院微生物研究所 | N-acetylneuraminic acid aldolase as well as coding gene and application thereof |
| CN103276001A (en) * | 2013-06-19 | 2013-09-04 | 南京工业大学 | Gene of codon optimized N-acetyl glucosamine isomerase and expression thereof |
Non-Patent Citations (1)
| Title |
|---|
| ZHANG ET AL: "An efficient method for N-acetyl-D-neuraminic acid production using coupled bacterial cells with a safe temperature-induced system", 《APPL MICROBIOL BIOTECHNOL》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114196693A (en) * | 2021-10-25 | 2022-03-18 | 福州一诺维生物科技有限公司 | Preparation method of N-acetylneuraminic acid |
| CN114196693B (en) * | 2021-10-25 | 2023-11-24 | 福州一诺维生物科技有限公司 | Preparation method of N-acetylneuraminic acid |
| CN114457133A (en) * | 2022-03-07 | 2022-05-10 | 江南大学 | Method for producing N-acetylneuraminic acid by whole-cell catalysis with N-acetylglucosamine fermentation broth as substrate |
| CN114457133B (en) * | 2022-03-07 | 2024-02-27 | 江南大学 | Method for producing N-acetylneuraminic acid by using N-acetylglucosamine fermentation broth as substrate through whole cell catalysis |
| CN114507658A (en) * | 2022-04-02 | 2022-05-17 | 深圳瑞德林生物技术有限公司 | Enzyme coexpression system and application thereof in sialic acid synthesis |
| CN114874967A (en) * | 2022-06-17 | 2022-08-09 | 江南大学 | Recombinant escherichia coli for producing N-acetylneuraminic acid and construction method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Tao et al. | Biotechnological production and applications of N-acetyl-D-neuraminic acid: current state and perspectives | |
| CN104878035A (en) | Construction method for producing N-acetylneuraminic acid recombinant microorganisms and application of N-acetylneuraminic acid recombinant microorganisms | |
| CN112625990B (en) | Recombinant escherichia coli for synthesizing 2' -fucosyllactose and construction method thereof | |
| Hehemann et al. | Aquatic adaptation of a laterally acquired pectin degradation pathway in marine gammaproteobacteria | |
| CN101633931A (en) | Hyaluronidase expression vector and application thereof | |
| CN106995811A (en) | A kind of algin catenase, its preparation method and application | |
| CN112662604B (en) | Recombinant escherichia coli for synthesizing 3-fucosyllactose and construction method thereof | |
| CN104673810A (en) | Malic dehydrogenase gene MIMDH1 and recombinant expression vector thereof | |
| CN105505899A (en) | Preparation method and application of endo-inulinase | |
| CN106434590A (en) | Fucosyltransferase, genetically engineered bacteria thereof and application | |
| Van Overtveldt et al. | Converting galactose into the rare sugar talose with cellobiose 2-epimerase as biocatalyst | |
| CN103290078B (en) | Method for preparing 5-aminovaleric acid by using L-lysine-2-monooxygenase and delta-valeramide hydrolase as catalysts | |
| CN104046586B (en) | One strain gene engineering bacterium and the application in producing (2R, 3R)-2,3-butanediol thereof | |
| Pittrof et al. | Products released from structurally different dextrans by bacterial and fungal dextranases | |
| Liu et al. | Characterization of two α-l-Arabinofuranosidases from Acetivibrio mesophilus and their synergistic effect in degradation of Arabinose-containing substrates | |
| CN104745547A (en) | Epoxide hydrolase mutant, engineering bacteria and application of epoxide hydrolase mutant | |
| CN102533801A (en) | Streptomyces griseus subsp.griseus trehalose synthetase gene and applications thereof | |
| CN104726435B (en) | A kind of beta-glucosidase enzyme mutant, its recombinant expression plasmid and the engineered strain of conversion | |
| CN102154326A (en) | Streptomyces coelicolor trehalose synthase enzyme gene and application thereof | |
| Mohamad Sobri et al. | In-silico characterization of glycosyl hydrolase family 1 β-glucosidase from Trichoderma asperellum UPM1 | |
| CN107603967B (en) | A kind of chitosan enzyme CSN4 and its encoding gene and application | |
| CN104004732B (en) | A kind of chitosanase and its preparation method and application | |
| Zhang et al. | Expression and characterization of a novel cold-adapted chitosanase from marine Renibacterium sp. suitable for chitooligosaccharides preparation | |
| Wang et al. | In vivo multienzyme complex coconstruction of N-acetylneuraminic acid lyase and N-acetylglucosamine-2-epimerase for biosynthesis of N-acetylneuraminic acid | |
| Silva et al. | Proteins encoded by Sphingomonas elodea ATCC 31461 rmlA and ugpG genes, involved in gellan gum biosynthesis, exhibit both dTDP-and UDP-glucose pyrophosphorylase activities |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150902 |