CN102321693A - Utilize natural oil body weight structure legal system to be equipped with immobilized lipase and be used for the production biofuel - Google Patents
Utilize natural oil body weight structure legal system to be equipped with immobilized lipase and be used for the production biofuel Download PDFInfo
- Publication number
- CN102321693A CN102321693A CN201110283115A CN201110283115A CN102321693A CN 102321693 A CN102321693 A CN 102321693A CN 201110283115 A CN201110283115 A CN 201110283115A CN 201110283115 A CN201110283115 A CN 201110283115A CN 102321693 A CN102321693 A CN 102321693A
- Authority
- CN
- China
- Prior art keywords
- oil body
- oil
- lypase
- lipase
- natural oil
- 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
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The present invention is a kind of method of utilizing synchronous purifying of natural oil body weight structure method and immobilized lipase; This method be utilize after the natural oil body disintegration can spontaneous reconstruct characteristics; Expression there is supersound process behind intestinal bacteria and the natural oil body proportional mixing of fusion rotein lypase oil body protein; Utilize the special hydrophobicity of oil body protein that fusion rotein is anchored on oil body half unit membrane of new composition; Formation contains the reconstruct oil body of lypase, thereby reaches lypase purifying and the synchronous purpose of accomplishing of immobilization.This invention provides a kind of brand-new lipase immobilization technology that can be used for the production biofuel, has solved high, the inefficient shortcoming of immobilized lipase cost in the current Production by Enzymes biofuel process, and preparation technology is simple; The present invention adopts the matrix of natural oil body as immobilized enzyme first, and is with low cost, substrate flexibility is extensive, and significantly improves the stability of lypase and to the tolerance of low unit alcohol.
Description
Technical field
The present invention relates to a kind ofly express lypase with engineered method, and use natural oil body as immobilized enzyme matrix to prepare the method that immobilized lipase produces thing diesel oil next life.
Background technology
1. biological enzyme prepares the overview of biofuel
It is to utilize the esterification and the transesterification reaction activity of lypase that biological enzyme prepares biofuel, and reaction generates biofuel to the catalysis grease with alcohol.Compare with chemical method with the physics method; Make biodiesel with lypase, advantage such as have applied range, material choice property is lower, catalytic efficiency (is high, reaction conditions is gentle and environmentally friendly, pure consumption is few, aftertreatment is simple, non-pollutant discharge, by-product glycerin are more easily separated.
At present; Natural lypase produces thing diesel oil as catalyzer and exist certain limitation next life; Mainly contain: (1) lypase is lower to the transformation efficiency of low chain alcohol, causes that the lypase consumption is excessive, reaction time is long, and the transesterification reaction activity of lypase remains further to be improved; (2) short chain alcohol particularly methyl alcohol lypase is had certain toxicity, work-ing life of enzyme shortens, production cost is too high, the methanol tolerance of lypase also must further improve.These factors are restricting the large-scale application of Production by Enzymes biofuel just.
Immobilized enzyme is in certain space, to be the enzyme that blocking exists, and can react continuously, and reacted enzyme can recycle and reuse.The free-fat enzyme is fixed on the synthetic needed product of catalyzed reaction on the carrier, makes expensive lypase be able to reuse, reduced the cost of reaction.And immobilized enzyme has and can reclaim, and reuses, and stability is high, the quality product advantages of higher.Therefore, the immobilized lipase application prospect is good by generally.
2. lipase immobilization method
The method of current immobilized lipase is a lot, can be summarized as physics method, chemical method and biological process.Because the differences such as source of solid support material, preparation method and immobilized enzyme, it is also different to cause preparing complexity, the fixed effect of immobilized enzyme, the stability of enzyme etc.
Physical method comprises absorption method and entrapping method.Absorption method is a kind of process for fixation that enzyme is fixed in fibrous carrier through physical adsorption.Physisorphtion has enzyme active center and is difficult for being destroyed the advantage with low uncertainty with the enzyme higher structure, and in addition, adsorption process can reach purifying and immobilized purpose simultaneously, and activating and regenerating again behind the enzyme deactivation.Thereby the enzyme activity loss is less, if can find appropriate carriers, this is good method.Entrapping method be with the enzyme physically trapping in polymer to reach immobilized purpose.Entrapping method generally need not carry out association reaction with the amino-acid residue of zymoprotein, seldom changes the higher structure of enzyme, so the enzyme recovery alive is higher.But chemical polymerization takes place when embedding, and the easy inactivation of enzyme must ingenious design reaction conditions, is unlikely to the reactive site and the active site of destructive enzyme when making it chemical reaction.
The investigator of Beijing University of Chemical Technology will have the lypase (Candida sp. 99-125) of strain fermentation extraction by oneself and fix with diatom; Adopt esterification process directly with lipid acid and methyl alcohol reaction; Oleic acid and methyl alcohol mol ratio are 1: 1.4; Add silica gel in the reaction process and make water-retaining agent, reaction 24h, esterification yield can reach 92%.People such as Yomi Watanabe use in fixed candiyeast (Candida antarctica) lipase-catalyzed soya-bean oil and the methyl alcohol reaction and have obtained 93.8% transesterification rate, and its immobilized enzyme circulates, and any change does not take place activity after 25 times.Official's spring clouds etc. carry out immobilization with zeyssatite to the dry enzyme powder of enterobacter agglomerans lypase that laboratory screening obtains, and the transesterification rate reaches 91.03%.
Chemical method mainly is to adopt the covalent attachment method.This method is through interconnecting with covalent linkage between the enzyme molecule or between the functional group of zymoprotein molecule and the reactive group on the carrier surface, forming immobilized enzyme.Luo Wen etc. are carrier with the sintered glass; Adopt covalent method that candiyeast 99-125 lypase is fixed; And be catalyzer with prepared immobilized enzyme, in the warm water system, utilize the rapeseed oil biodiesel synthesis, adopt 3 stream to add mode biodiesel synthesis in sherwood oil of methyl alcohol; Each batch reaction after-filtration goes out immobilized enzyme; Clean continued with the trimethyl carbinol and carry out the reaction of next batch, after the immobilized enzyme successive reaction 13 batches (every crowd 30 h), the biofuel reaction conversion ratio still maintains more than 70%.The transformation period of immobilized lipase is more than 390 h.
Thereby biological process is to adopt molecular biology method to fix the method for preparing immobilized enzyme with the goal gene clonal expression and with expressing protein.It mainly is to adopt full cell fixation method that current biological process prepares immobilized lipase.Full cell fixation method is that the mikrobe with yielding lipase directly is fixed on the upholder; Thereby preparation whole-cell catalyst; This method is more easier, economical than immobilized enzyme, and immobilization process can accomplish when mass ferments synchronously, has not only saved the process of separation and purification; And avoid again extracting the loss of enzyme in the purge process.According to the development course of whole-cell catalyst and the difference of fixing means, mainly be divided into three kinds of fungi whole-cell catalysts, yeast whole-cell catalyst, intestinal bacteria whole-cell catalyst.Seizaburo Shiraga etc. is fixed to the S. cervisiae surface with rhizopus oryzae lypase, compares with the free-fat enzyme, and the transesterification activity of fixed lipase catalyzed Palmiticacid of gained and n-amylalcohol (existence of 0.2 % water) has obtained huge raising.Hama etc. in urethane foam BSPs, carry out rhizopus Rhizopus oryzae cell fixation intermittence and cultivate in the packed bed bio-reactor of a 20L.Surpass 90%, the 10 all after date at first production cycle methyl esters content when flow velocity is 25L/h and can reach 80%.Kazuhiro etc. are fixed on rhizopus Rhizopus oryzae on the urethane foam particle, adopt to divide a mode that adds methyl alcohol for 3 times, and when to contain massfraction in the reaction system be 15% water, the methyl esters yield can be up to 90%.Through 6 reuses, the activity of intracellular lipase does not significantly decrease.Also find simultaneously to utilize different fatty acids as carbon source; The catalytic activity of whole-cell catalyst is also different with stability, and unsaturated fatty acids helps improving the permeability of cytolemma, and enzymatic activity is improved; And sfas helps improving the rigidity of cell, and enzyme stability is improved.Matsumoto etc. have made up the yeast strain MT821 of ability great expression Rhizopus oryzae lypase (Rhizopus oryzae lipase), and the activity of its intracellular lipase reaches 474.5 IU/L.Come catalysis VT 18 synthesizing fatty acid methyl ester with having strengthened infiltrative yeast cell through freeze thawing or air-dry method in advance, in the time of 37 ℃, react 165h with the mode of progressively adding methyl alcohol, the fatty acid methyl ester massfraction reaches 7l% in the final reaction liquid.This method has been saved the step of separation and external stabilityization.
3. the evaluation and the development trend of lipase immobilization method in the existing production of biodiesel
Biofuel has mild condition, pure consumption is little, product is easy to advantages such as collection, non-pollution discharge, is a kind of very potential green production method though immobilized enzyme method prepares.But be not widely used yet in the actual production at present; Mainly be because they exist some severe defectives in the production of biofuel: the prepared by physical method immobilized lipase has advantage simple to operate, lower-cost; But do not reach the purpose of purifying, and entrapping method only is applicable to the substrate that molecular weight ratio is less and the enzymic catalytic reaction of product; The immobilized lipase of chemical method production have combine firmly, difficult drop-off, advantage such as duration of service is long continuously, but in fixation procedure, can cause the loss of enzyme part avtive spot inevitably, thus the vigor of reduction enzyme.Severe reaction conditions, complicated operation, the enzyme recovery of living is low; Even the Substratspezifitaet of enzyme also can change sometimes; And usually can cause that in operating process the zymoprotein higher structure changes; Thereby cause the active site to be damaged, its repeatability is not high, thereby is inappropriate for the production of biofuel.In addition, could obtain higher immobilization efficiency, make that the production cost of immobilized lipase enzyme process is still higher, limit its application in actual production because must carry out certain separation purification to lypase.Full cell fixation lypase is simple to operate, cost is low, and the problem that still has a maximum is that the amount of target protein (lypase) can't be improved fully.
To sum up analyze: immobilized lipase production biofuel research emphasis from now on; Still be to seek better immobilization material and process for fixation, further improve the performances such as relative vigor, thermostability, low-carbon alcohol tolerance and the catalytic substrate scope of application of immobilized enzyme.Go into background technology and describe paragraph.
Summary of the invention
The present invention utilizes synchronous purifying of natural oil body weight structure method and immobilized lipase; This invention provides a kind of brand-new lipase immobilization technology to can be used for the production biofuel, has solved high, the inefficient shortcoming of immobilized lipase cost in the current Production by Enzymes biofuel process.
The invention is characterized in that this method steps is following: utilize after the natural oil body disintegration can spontaneous reconstruct characteristics, be template with the reverse transcription product cDNA of the total mRNA of oil plant, adopt the oil body protein gene of 400 ~ 1000bp that the PCR method obtains, be labeled as O.The yielding lipase microbial genome that obtains with the method according to document " cheap grease favor type lypase produces the screening and the evaluation thereof of bacterial strain " is lypase and the chaperone gene ORF sequence thereof that template adopts PCR method picked up signal peptide disappearance, is labeled as Lipd25AB.Utilize " molecular cloning experiment guide 3 " to go up engineered method and make up oil body protein and lypase and chaperone expression vector pET32 (a+)-O-Lipd25AB thereof.This prokaryotic expression carrier is changed among the expressive host bacterium E.coli BL21 (DE3), obtain to be used for engineering strain E.coli BL21 (the DE3)-O-Lipd25AB of expressed fusion protein (oil body protein-lypase).With supersound process behind the engineering strain of abduction delivering and the natural oil body proportional mixing, utilize the special hydrophobicity of oil body protein that fusion rotein is anchored on oil body half unit membrane of new composition, forming the reconstruct oil body that contains lypase is immobilized lipase.Utilize the transesterification reaction of prepared immobilized lipase catalysis animal-plant oil to obtain biofuel.
Described lypase is with natural oil body weight structure technology fixed lypase, is used to prepare biofuel.
Described natural phant oil body derives from the oil plant seed like natural oil bodies such as jatropha curcas seed, Semen Brassicae campestris, Arabidopis thaliana seed, soybean, peanut, cottonseed, palm, coconuts.
Described natural oil body carries out the ultrasonication condition with the intestinal bacteria merging mixing that contains expression vector: power 20%-28%; Broken time: 5-10min in centrifugal 10 min of 10 000 g, collects the upper strata white mass with the mixture after the supersound process, is natural oil body immobilized lipase.
The oil body protein gene size that is obtained is at 400 ~ 1000bp.
The lipase gene source can be the different microorganisms according to the yielding lipase of the method acquisition of document " cheap grease favor type lypase produces the screening and the evaluation thereof of bacterial strain ".
Described transesterification reaction comprises that the natural oil body immobilized lipase with collection joins in the encloses container that contains fat-soluble organic solvent, animal-plant oil; Add then in the shaking table that is put in relevant temperature behind low first alcohol and the water and carry out transesterification reaction; The low unit alcohol that adds respective amount at set intervals again; Continuous several times obtains corresponding fatty ester and is biofuel after the transesterification.
Described grease is one or more animal-plant oil that are selected from jatropha curcas seed oil, rapeseed oil, lard, VT 18, peanut oil, Oleum Gossypii semen, plam oil, Oleum Cocois, edible waste oil, Chinese honey locust or acidifying wet goods.
Described low unit alcohol is methyl alcohol, ethanol, ETHYLE ACETATE etc., can add by one or many.
Described transesterification reaction parameter is: 15-45 ℃ of temperature, shaking speed 70-200 r/min, every separated 5-10 h add the low unit alcohol of respective amount, 3-5 time continuously again.
The present invention adopts fixing of natural oil body weight structure technology lypase to reach the purpose of fixing synchronously and efficiently purifying, has simplified the technology for preparing immobilized enzyme; Secondly, natural oil body immobilized enzyme is used for behind the transesterification reaction through simple processing or natural disintegration, and its staple (grease) can be used as the recycle of production of biodiesel substrate, has not only reached making full use of but also helping environmental protection of resource; Once more, the present invention adopts the basic material of natural oil body as immobilized enzyme first, and is not only with low cost, and significantly improved the stability of lypase and to the tolerance of low unit alcohol.
Claims (10)
- One kind use natural oil body as immobilized enzyme matrix to prepare the method that immobilized lipase produces thing diesel oil next life; It is characterized in that this method steps is following: utilize after the natural oil body disintegration can spontaneous reconstruct characteristics; Reverse transcription product cDNA with the total mRNA of oil plant is a template; Adopt the oil body protein gene of 400 ~ 1000bp of PCR method acquisition, be labeled as O; The yielding lipase microbial genome that obtains with the method according to document " cheap grease favor type lypase produces the screening and the evaluation thereof of bacterial strain " is lypase and the chaperone gene ORF sequence thereof that template adopts PCR method picked up signal peptide disappearance, is labeled as Lipd25AB; Utilize " molecular cloning experiment guide 3 " to go up engineered method and make up oil body protein and lypase and chaperone expression vector pET32 (a+)-O-Lipd25AB thereof; This prokaryotic expression carrier is changed among the expressive host bacterium E.coli BL21 (DE3), obtain to be used for engineering strain E.coli BL21 (the DE3)-O-Lipd25AB of expressed fusion protein (oil body protein-lypase); Destructing behind the engineering strain of abduction delivering and the natural oil body proportional mixing is handled, utilized the special hydrophobicity of oil body protein that fusion rotein is anchored on oil body half unit membrane of new composition, forming the reconstruct oil body that contains lypase is immobilized lipase; Utilize the transesterification reaction of prepared immobilized lipase catalysis animal-plant oil to obtain biofuel.
- 2. method according to claim 1 is characterized in that described lypase is with natural oil body weight structure technology fixed lypase, is used to prepare biofuel.
- 3. method according to claim 1 is characterized in that described natural phant oil body derives from the oil plant seed like natural oil bodies such as jatropha curcas seed, Semen Brassicae campestris, Arabidopis thaliana seed, soybean, peanut, cottonseed, palm, coconuts.
- 4. the method for claim 1, the oil body protein gene that is obtained derive from oil plant as: Cortex jatrophae, rape, Arabidopis thaliana, soybean, peanut, cottonseed, palm, coconut etc., size is at 400 ~ 1000bp.
- 5. the method for claim 1, lipase gene source can be method according to document " cheap grease favor type lypase produces the screening and the evaluation thereof of bacterial strain " and obtain to produce the live different microorganisms of lypase of transesterification enzyme.
- 6. the method for claim 1; Its transesterification reaction comprises that the natural oil body immobilized lipase with collection joins in the encloses container that contains lipotropy organic solvent, animal-plant oil; Add then in the shaking table that is put in relevant temperature behind low first alcohol and the water and carry out transesterification reaction; The low unit alcohol that adds respective amount at set intervals again, continuous several times obtains corresponding fatty ester and is biofuel after the transesterification.
- 7. transesterification reaction as claimed in claim 7 is characterized in that described grease is one or more animal-plant oil that are selected from jatropha curcas seed oil, rapeseed oil, lard, VT 18, peanut oil, Oleum Gossypii semen, plam oil, Oleum Cocois, edible waste oil, Chinese honey locust or acidifying wet goods.
- 8. transesterification reaction as claimed in claim 7 is characterized in that described lipotropy organic solvent can be the normal hexane/sherwood oil/trimethyl carbinol/ether etc.
- 9. transesterification reaction as claimed in claim 7 is characterized in that described low unit alcohol is methyl alcohol, ethanol, ETHYLE ACETATE etc., can add by one or many.
- 10. transesterification reaction as claimed in claim 7 is characterized in that described transesterification reaction parameter is: 15-45 ℃ of temperature, shaking speed 70-200 r/min, every separated 5-10 h add the low unit alcohol of respective amount, 3-5 time continuously again.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110283115A CN102321693A (en) | 2011-09-22 | 2011-09-22 | Utilize natural oil body weight structure legal system to be equipped with immobilized lipase and be used for the production biofuel |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110283115A CN102321693A (en) | 2011-09-22 | 2011-09-22 | Utilize natural oil body weight structure legal system to be equipped with immobilized lipase and be used for the production biofuel |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102321693A true CN102321693A (en) | 2012-01-18 |
Family
ID=45449526
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110283115A Pending CN102321693A (en) | 2011-09-22 | 2011-09-22 | Utilize natural oil body weight structure legal system to be equipped with immobilized lipase and be used for the production biofuel |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102321693A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106676083A (en) * | 2016-11-14 | 2017-05-17 | 浙江大学 | Lipase extraction method |
| CN108251345A (en) * | 2017-12-28 | 2018-07-06 | 江苏世邦生物工程科技有限公司 | For the recombination engineering and its construction method containing lipase gene of sanitary sewage disposal |
| CN112760316A (en) * | 2021-04-07 | 2021-05-07 | 中国科学院天津工业生物技术研究所 | Method for producing tagatose by artificial oil body immobilized multienzyme |
-
2011
- 2011-09-22 CN CN201110283115A patent/CN102321693A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106676083A (en) * | 2016-11-14 | 2017-05-17 | 浙江大学 | Lipase extraction method |
| CN106676083B (en) * | 2016-11-14 | 2020-06-16 | 浙江大学 | Lipase extraction method |
| CN108251345A (en) * | 2017-12-28 | 2018-07-06 | 江苏世邦生物工程科技有限公司 | For the recombination engineering and its construction method containing lipase gene of sanitary sewage disposal |
| CN108251345B (en) * | 2017-12-28 | 2021-07-20 | 江苏世邦生物工程科技有限公司 | Recombinant engineering bacterium containing lipase gene for domestic sewage treatment and construction method thereof |
| CN112760316A (en) * | 2021-04-07 | 2021-05-07 | 中国科学院天津工业生物技术研究所 | Method for producing tagatose by artificial oil body immobilized multienzyme |
| WO2022213721A1 (en) | 2021-04-07 | 2022-10-13 | 中国科学院天津工业生物技术研究所 | Method for producing tagatose by immobilizing multiple enzymes by using artificial oil body |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wu et al. | Bioethanol production from taro waste using thermo-tolerant yeast Kluyveromyces marxianus K21 | |
| Du et al. | Perspectives for biotechnological production of biodiesel and impacts | |
| Jegannathan et al. | Production of biodiesel using immobilized lipase—a critical review | |
| US9249437B2 (en) | Process for preparing biodiesel from renewable oil and fat catalyzed by lipase with online dehydration | |
| Ganesan et al. | An overview on the recent advances in the transesterification of vegetable oils for biodiesel production using chemical and biocatalysts | |
| WO2001038553A1 (en) | Process for producing fatty acid lower alcohol ester | |
| Zhao et al. | Direct conversion of inulin and extract of tubers of Jerusalem artichoke into single cell oil by co-cultures of Rhodotorula mucilaginosa TJY15a and immobilized inulinase-producing yeast cells | |
| CN102453614B (en) | New method for comprehensively utilizing food wastes | |
| Singh et al. | Immobilized lipase from Schizophyllum commune ISTL04 for the production of fatty acids methyl esters from cyanobacterial oil | |
| Pitol et al. | Optimization studies to develop a low-cost medium for production of the lipases of Rhizopus microsporus by solid-state fermentation and scale-up of the process to a pilot packed-bed bioreactor | |
| Senthilraja et al. | Comparative analysis of bioethanol production by different strains of immobilized marine yeast | |
| Zhou et al. | Two-step biocatalytic process using lipase and whole cell catalysts for biodiesel production from unrefined jatropha oil | |
| CN101619329A (en) | Technical method for preparing biodiesel by biocatalysis one-pot method | |
| CN102321693A (en) | Utilize natural oil body weight structure legal system to be equipped with immobilized lipase and be used for the production biofuel | |
| CN104726477A (en) | Lipase coding gene and engineering strain thereof | |
| CN101418322A (en) | Method for preparing biodiesel through catalysis of renewable lipin by lipase recovered by membrane | |
| CN1453332A (en) | Biologically catalystic process of converting fat into ester to produce biological diesel oil | |
| CN101974573A (en) | Method for preparing microbial oil by fermenting cassava starch and preparing biomouldsel from microbial oil | |
| CN100347271C (en) | Process of producing biological diesel oil through catalysis of fatty acid and acid-containing grease with microbe cell | |
| CN104498544A (en) | Method for producing biodiesel by microalgae fermentation | |
| Liu et al. | Bioethanol production from potato starch by a novel vertical mass-flow type bioreactor with a co-cultured-cell strategy | |
| CN104450668A (en) | Biochar immobilized enzyme for converting waste kitchen oil into biodiesel and preparation method thereof | |
| Khamkeaw et al. | Hydrolysis of cassava starch by co-immobilized multi-microorganisms of Loog-Pang (Thai rice cake starter) for ethanol fermentation | |
| CN111378641A (en) | Immobilized enzyme carrier and immobilized enzyme | |
| CN101205474A (en) | Method for preparing biodiesel by grease biotransformation in solvent-free system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20120118 |