CN102329757A - Catalyst used for synthesizing acrylamide and containing photosensitive nitrile hydratase strain and application of catalyst - Google Patents
Catalyst used for synthesizing acrylamide and containing photosensitive nitrile hydratase strain and application of catalyst Download PDFInfo
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- CN102329757A CN102329757A CN201110304076A CN201110304076A CN102329757A CN 102329757 A CN102329757 A CN 102329757A CN 201110304076 A CN201110304076 A CN 201110304076A CN 201110304076 A CN201110304076 A CN 201110304076A CN 102329757 A CN102329757 A CN 102329757A
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Abstract
The invention relates to a catalyst used for synthesizing acrylamide and containing a photosensitive nitrile hydratase strain and application of the catalyst. The catalyst is obtained by fermenting and culturing thalli and preparing catalyst suspension. The application of the catalyst comprises the following steps of: slowly adding acrylonitrile in an amount which is 20 weight percent based on the weight of the reaction system into the catalyst suspension, carrying out enzyme-catalyzed hydration reaction under illumination and normal pressure at the temperature of 25 DEG C at the stirring speed of 200r.min<-1>, and reacting for 10h to obtain an acrylamide solution, wherein the mass ratio of the catalyst suspension to an acrylonitrile reaction liquid is 1:100-1:200. The photosensitive nitrile hydratase Pseudomonas putida strain is subjected to traditional fermentation and hydration catalysis to synthesize the acrylamide; an acrylamide hydrated product synthesized by using the strain has high concentration, so that the burden of the concentration is reduced; and few byproducts are generated, the purity of the product is high, and the acrylamide can be industrially produced.
Description
Technical field
The invention belongs to the industrial microbial technology field, be specifically related to a kind of catalyzer and application thereof that synthesis of acrylamide contains the photosensitivity nitrile hydratase strain that be used for.
Background technology
Acrylic amide is a kind of broad-spectrum Organic Chemicals, is not following hundred kinds of monomer synthetic products with it, and is wherein the widest with the SEPIGEL 305 purposes.The main application of SEPIGEL 305 is to be used for oil field adjustment intake profile and " TOR "; In recent years; For adapting to the demand of crude oil in China " TOR " to super high molecular weight polyacrylamide (relative molecular mass >=2,000 ten thousand), the production of acrylic amide will have bigger development.
Acrylic amide be with the vinyl cyanide for the raw material hydration forms, from the 1950's so far, production technique has successively experienced the sulfuric acid hydration, copper is catalytic chemical hydration and 3 developmental stage of mikrobe Nitrile hydratase bio-transformation.1973, French investigator Galzy and study group thereof found that the hydration of a kind of ability catalyzing propone nitrile rest on the mikrobe BrevbacteriumR312 of acrylic amide, thereby have begun to become with mikrobe the research of product acrylic amide.1985, Japanese Ri Dong company built up the industrialness device that first microbial method is in the world produced acrylic amide.The Shanghai agricultural chemicals also from the soil in Mount Taishan, found a strain nitrile hydratase production in 1986 bacterial strain, and begun the research that microbial method is produced acrylic amide.With copper is that catalysis method is compared, and microbial method has many advantages: enzymic catalytic reaction carries out under normal temperature, normal pressure, has improved production security; The transformation efficiency of vinyl cyanide need not reclaim unreacting material near 100%; Owing to saved the copper centrifugal station, copper ions not in the product, product purity is high, is particularly suitable for the SEPIGEL 305 of production ultra-high molecular weight; Technological process is simple, and facility investment is few, and the production economic benefit is high.Microbial method catalytic production acrylic amide is first example of the key product utilization enzyme technology success of petrochemical complex, has epoch making significance.Nitrile hydratase is a kind ofly can carry out the itrile group of nitrile compound to change carboxamido-group into after the hydration, makes the enzyme of the mikrobe of corresponding amide compound, and Nitrile hydratase becomes the method for acrylic amide just becoming main flow as the catalyst acrylonitrile hydration.
Summary of the invention
The object of the present invention is to provide a kind of catalyzer and application thereof that synthesis of acrylamide contains the photosensitivity nitrile hydratase strain that be used for; Photosensitivity Nitrile hydratase pseudomonas putida bacterial classification of the present invention through traditional zymotic, hydration catalysis synthesis of acrylamide, utilizes bacterial strain synthetic acrylic amide hydrated product concentration of the present invention high; Reduced spissated burden; And by product is few, and product purity is high, is fit to the suitability for industrialized production acrylic amide.
The technical scheme that the present invention adopted is: this is used for the catalyzer that synthesis of acrylamide contains the photosensitivity nitrile hydratase strain:
Thalline fermentation culture: pseudomonas putida (Pseudomonas putida) thalline is inoculated in solid medium, picking lawn fermentative prepn Nitrile hydratase in shaking table after the activation, shaking speed 100～200rmin
-1, temperature is 25～40 ℃, uses 4molL in per 6 hours
-1NaOH solution regulate pH value, make it to maintain between 6.5～7.5, obtain having the fermented liquid of Nitrile hydratase vigor behind cultivation 50～100h, and total enzyme work of fermented liquid reaches 800～12,000,000 units per ml fermented liquids;
Catalyst suspension: fermented liquid in separation factor >=4000 spinnings, is removed supernatant, cell suspension in deionized water or 20mM, pH are 7.0～8.0 buffer solution of potassium phosphate, is made into the catalyst suspension that cell concn is 5% (wt).
Solid culture based formulas used in the such scheme is: glycerine 0.2～2%, yeast extract 0.5～1.5%, agar 0.2～2%, urea 0.1～1%, K
2HPO
40.1 KH～0.5%,
2PO
40.1～0.5%, NaCl 0.1～1% and MgSO
40.005～0.02%, de-salted water 10～15%, surplus is a starch; Fermentative medium formula is: glycerine 0.2～2%, yeast extract 0.5～1.5%, L-glutamic acid 0.2～2%, urea 0.2～2%, K
2HPO
40.1～0.5%, KH
2PO
40.1～0.5%, MgSO
40.005～0.02%, Fe
3+0.001～0.01%, pH value 6.5～7.5, surplus are de-salted water; Above-mentioned per-cent is mass percent.
The Application of Catalyst that contains the photosensitivity nitrile hydratase strain: comprise the steps: in catalyst suspension, slowly to add vinyl cyanide, at illumination, normal pressure, temperature is 25 ℃, 200rmin
-1Stirring velocity under carry out the enzyme catalysis hydration reaction, the addition of vinyl cyanide accounts for 20% (wt) of reaction system, through the reaction of 10h, obtains acrylamide soln; Catalyst suspension and acrylonitrile reactor liquid mass ratio are between 1: 100～1: 200.
The Fe that adds in the above-mentioned fermenting process
3+Be FeCl
3, Fe
2(SO
4)
3, Fe (NO)
3In any one; Acrylonitrile reactor liquid vinyl cyanide and water.
Above-mentioned said illumination condition is a fluorescent lamp irradiation fermentor tank.
The beneficial effect that the present invention had is: be through in the traditional zymotic process, adding Fe in the substratum
3+Production contains pseudomonas putida bacterial strain or its mutagenic strain cell of Nitrile hydratase; And this cell separated from nutrient solution process catalyst suspension; The hydration of catalyzing propone nitrile becomes acrylamide solution, under the traditional technology condition, adopts the illumination fermentor tank, and the transformation efficiency of vinyl cyanide can reach 99.99%; The acrylamide solution that is obtained can obtain highly purified acrylamide solution through operations such as nanofiltration, IXs.The present invention is through add Fe during the fermentation
3+, significantly improved the enzymic activity of Nitrile hydratase, when improving enzymic activity more than 50% the culture cycle of fermentation has been shortened greatly; In addition, the present invention has also increased illumination condition in the acrylonitrile hydration stage, thereby the enzymic activity of hydratase of acrylonitrile has been improved greatly, and the Nitrile hydratase transformation period has been prolonged 1.5 times.Utilize bacterial strain synthetic acrylic amide hydrated product concentration of the present invention high, reduced spissated burden, and by product is few, product purity is high, is fit to the suitability for industrialized production acrylic amide.
Bacterial strain, Fe among the present invention
3+, the mutual coordinative role of illumination condition could become acrylic amide by the efficient catalytic acrylonitrile hydration.The Nitrile hydratase that pseudomonas putida bacterial strain that the present invention uses and mutagenic strain thereof produce can efficiently change into acrylic amide with vinyl cyanide, and its operating process is simple, reaction conditions is gentle, environmental pollution is little, separation is purified simply, the vinyl cyanide transformation efficiency is high, product purity is high.
The present invention compared with prior art has following characteristics:
Prior art is used for the hydration of catalyzing propone nitrile and becomes the bacterial strain of acrylic amide to mainly contain the bacterium of Rhod, excellent bacillus and through the bacterial classification of mutagenesis.Bacterial classification of the present invention belongs to Rhodopseudomonas.This bacterial strain adds Fe during the fermentation
3+, when hydration stage had the condition of illumination, this bacterial classification enzymatic productivity increased substantially, and was up to 50 mg/ml fermented liquids, and total enzyme work of its unit volume fermented liquid reaches 1,200 ten thousand units per ml fermented liquids.
Embodiment:
Embodiment 1:
(1), producing of catalyst suspension:
Thalline fermentation culture: the pseudomonas putida thalline is inoculated in solid medium, picking lawn fermentative prepn Nitrile hydratase in shaking table after the activation, shaking speed 100rmin
-1, temperature is 30 ℃, uses 4molL in per 6 hours
-1NaOH solution regulate pH value, make it to maintain 7.5, obtain having the fermented liquid of Nitrile hydratase vigor behind the cultivation 72h.
Measure the strain enzyme-producing ability through weighing method.Getting the 10mL fermented liquid, is 5000 at separation factor, and supernatant is removed in spinning, behind distilled water wash three times, the cell that obtains is placed in the infrared drying oven, and 85 ℃ are dried to constant weight, and about 4h takes out and puts into moisture eliminator, cooling back weighing.The enzymatic productivity that is recorded by aforesaid method is 40.3 mg/ml fermented liquids.
Is 5000 with above-mentioned fermented liquid at separation factor, and supernatant is removed in spinning, and cell suspension in 20mM, pH are 7.5 buffer solution of potassium phosphate, is made into the catalyst suspension that cell concn is 5% (wt).
(2), utilize the catalyst suspension synthesis of acrylamide: in catalyst suspension, slowly add vinyl cyanide, at sun exposure fermented liquid, normal pressure, temperature is 25 ℃, 200rmin
-1Stirring velocity under carry out the enzyme catalysis hydration reaction; The addition of vinyl cyanide accounts for 20% (wt) of reaction system, and through the reaction of 10h, acquisition concentration is 26.74% acrylamide soln; Vinyl cyanide transformation efficiency 99.81%, vinylformic acid, propenal concentration 0.00005% in the solution.The mass ratio of fermented liquid and acrylonitrile reactor liquid is 1: 120.Adopt HPLC (HPLC) to analyze vinyl cyanide and converted product acrylic amide; Prepare both mixing solutionss and do typical curve; Quantitative through external standard method, Nitrile hydratase vigor in the assaying reaction liquid. the Nitrile hydratase vigor that is recorded by aforesaid method is 800u/mL.
Solid medium: glycerine 0.5%, yeast extract 1%, agar 1%, urea 0.3%, K
2HPO
40.3%, KH
2PO
40.3%, NaCl0.5%, MgSO
40.01%, de-salted water 10～15%, surplus is a starch; Fermention medium: glycerine 1.5%, yeast extract 1%, L-glutamic acid 1%, urea 1.2%, K
2HPO
40.3%, KH
2PO
40.3%, MgSO
40.01%, Fe
3+0.003%, pH value 7, surplus are de-salted water.
Embodiment 2:
(1), producing of catalyst suspension:
Thalline fermentation culture: the pseudomonas putida thalline is inoculated in solid medium, picking lawn fermentative prepn Nitrile hydratase in shaking table after the activation, shaking speed 100rmin
-1, temperature is 30 ℃, uses 4molL in per 6 hours
-1NaOH solution regulate pH value, make it to maintain 7.5, obtain having the fermented liquid of Nitrile hydratase vigor behind the cultivation 72h.The enzymatic productivity that is recorded by aforesaid method is 45.6 mg/ml fermented liquids.
Is 5000 with above-mentioned fermented liquid at separation factor, and supernatant is removed in spinning, and cell suspension in 20mM, pH are 7.5 buffer solution of potassium phosphate, is made into the catalyst suspension that cell concn is 5% (wt).
(2), utilize the catalyst suspension synthesis of acrylamide: in catalyst suspension, slowly add vinyl cyanide, at sun exposure fermented liquid, normal pressure, temperature is under 25 ℃, 200rmin
-1Stirring velocity under carry out the enzyme catalysis hydration reaction; The addition of vinyl cyanide accounts for 20% (wt) of reaction system, and through the reaction of 15h, acquisition concentration is 29.45% acrylamide soln; Vinyl cyanide transformation efficiency 99.93%, vinylformic acid, propenal concentration 0.00002% in the solution.The mass ratio of fermented liquid and acrylonitrile reactor liquid is 1: 100.The Nitrile hydratase vigor that is recorded by aforesaid method is 1100u/mL.
Solid medium: glycerine 0.5%, yeast extract 1%, agar 1%, urea 0.3%, K
2HPO
40.3%, KH
2PO
40.3%, NaCl0.5%, MgSO
40.01%, de-salted water 10～15%, surplus is a starch;
Fermention medium: glycerine 1.5%, yeast extract 1%, L-glutamic acid 1%, urea 1.2%, K
2HPO
40.3%, KH
2PO
40.3%, MgSO
40.01%, Fe
3+0.005%, pH value 7, surplus are de-salted water.
Embodiment 3:
(1), producing of catalyst suspension:
Thalline fermentation culture: the pseudomonas putida thalline is inoculated in solid medium, picking lawn fermentative prepn Nitrile hydratase in shaking table after the activation, shaking speed 100rmin
-1, temperature is 35 ℃, uses 4molL in per 6 hours
-1NaOH solution regulate pH value, make it to maintain 7.5, obtain having the fermented liquid of Nitrile hydratase vigor behind the cultivation 60h.The enzymatic productivity that is recorded by aforesaid method is 48.1 mg/ml fermented liquids.
Is 5000 with above-mentioned fermented liquid at separation factor, and supernatant is removed in spinning, and cell suspension in 20mM, pH are 7.5 buffer solution of potassium phosphate, is made into the catalyst suspension that cell concn is 5% (wt).
(2), utilize the catalyst suspension synthesis of acrylamide: in catalyst suspension, slowly add vinyl cyanide, at sun exposure fermented liquid, normal pressure, temperature is under 25 ℃, 200rmin
-1Stirring velocity under carry out enzyme catalysis hydration reaction vinyl cyanide addition account for 20% (wt) of reaction system; Reaction through 10h; Acquisition concentration is 26.72% acrylamide soln, vinyl cyanide transformation efficiency 99.75%, vinylformic acid, propenal concentration 0.00006% in the solution.The mass ratio of fermented liquid and acrylonitrile reactor liquid is 1: 150.The Nitrile hydratase vigor that is recorded by aforesaid method is 1000u/mL.
Solid medium: glycerine 0.5%, yeast extract 1%, agar 1%, urea 0.3%, K
2HPO
40.3%, KH
2PO
40.3%, NaCl 0.5%, MgSO
40.01%, de-salted water 10～15%, surplus is a starch;
Fermention medium: glycerine 1.5%, yeast extract 1%, L-glutamic acid 1%, urea 1.2%, K
2HPO
40.3%, KH
2PO
40.3%, MgSO
40.01%, Fe
3+0.008%, pH value 7.5, surplus are de-salted water.
Embodiment 4:
(1), thalline fermentation culture: the pseudomonas putida thalline is inoculated in solid medium, picking lawn fermentative prepn Nitrile hydratase in shaking table after the activation, shaking speed 100rmin
-1, temperature is 35 ℃, uses 4molL in per 6 hours
-1NaOH solution regulate pH value, make it to maintain 7.5, obtain having the fermented liquid of Nitrile hydratase vigor behind the cultivation 50h.The enzymatic productivity that is recorded by aforesaid method is 49.8 mg/ml fermented liquids.
Is 5000 with above-mentioned fermented liquid at separation factor, and supernatant is removed in spinning, and cell suspension in 20mM, pH are 7.5 buffer solution of potassium phosphate, is made into the catalyst suspension that cell concn is 5% (wt).
(2), utilize the catalyst suspension synthesis of acrylamide: in catalyst suspension, slowly add vinyl cyanide, at sun exposure fermented liquid, normal pressure, temperature is under 25 ℃, 200rmin
-1Stirring velocity under carry out the enzyme catalysis hydration reaction; The addition of vinyl cyanide accounts for 20% (wt) of reaction system, and through the reaction of 20h, acquisition concentration is 29.47% acrylamide soln; Vinyl cyanide transformation efficiency 99.99%, vinylformic acid, propenal concentration 0.00003% in the solution.The mass ratio of fermented liquid and acrylonitrile reactor liquid is 1: 100.The Nitrile hydratase vigor that is recorded by aforesaid method is 1000u/mL.
Solid medium: glycerine 0.5%, yeast extract 1%, agar 1%, urea 0.3%, K
2HPO
40.3%, KH
2PO
40.3%, NaCl 0.5%, MgSO
40.01%, de-salted water 10～15%, surplus is a starch; Fermention medium: glycerine 1.5%, yeast extract 1%, L-glutamic acid 1%, urea 1.2%, K
2HPO
40.3%, KH
2PO
40.3%, MgSO
40.01%, Fe
3+0.01%, pH value 7.5, surplus are de-salted water.
Embodiment 5:
Present embodiment does not add Fe during the fermentation
3+, and at hydration stage illumination condition useless, to compare with the foregoing description 4.
(1), producing of catalyst suspension:
Thalline fermentation culture: the pseudomonas putida thalline is inoculated in solid medium, picking lawn fermentative prepn Nitrile hydratase in shaking table after the activation, shaking speed 100rmin
-1, temperature is 35 ℃, uses 4molL in per 6 hours
-1NaOH solution regulate pH value, make it to maintain 7, obtain having the fermented liquid of Nitrile hydratase vigor behind the cultivation 90h.
The enzymatic productivity that is recorded by aforesaid method is 15.7 mg/ml fermented liquids.
Is 5000 with above-mentioned fermented liquid at separation factor, and supernatant is removed in spinning, and cell suspension in 20mM, pH are 7.5 buffer solution of potassium phosphate, is made into the catalyst suspension that concentration is 5% (wt).The mass ratio of fermented liquid and acrylonitrile reactor liquid is 1: 100.
(1), utilize the catalyst suspension synthesis of acrylamide: in catalyst suspension, slowly add vinyl cyanide, at normal pressure, temperature is 25 ℃, 200rmin
-1Stirring velocity under carry out the enzyme catalysis hydration reaction, the addition of vinyl cyanide accounts for 22% (wt) of reaction system, through the reaction of 10h, obtains concentration and be 8.9% acrylamide soln, vinyl cyanide transformation efficiency 30.4%.
The Nitrile hydratase vigor that is recorded by aforesaid method is 106u/mL.
Solid medium: glycerine 0.8%, yeast extract 1%, agar 1%, urea 0.3%, K
2HPO
40.3%, KH
2PO
40.3%, NaCl0.5%, MgSO
40.01%, de-salted water 10%, surplus is a starch;
Fermention medium: glycerine 1.5%, yeast extract 1%, L-glutamic acid 1%, urea 1.2%, K
2HPO
40.3%, KH
2PO
40.3%, MgSO
40.01%, pH value 7, surplus are de-salted water.
Used per-cent is mass percent in the foregoing description.
Claims (3)
1. one kind is used for the catalyzer that synthesis of acrylamide contains the photosensitivity nitrile hydratase strain,
Thalline fermentation culture: pseudomonas putida (Pseudomonas putida) thalline is inoculated in solid medium, picking lawn fermentative prepn Nitrile hydratase in shaking table after the activation, shaking speed 100～200rmin
-1, temperature is 25～40 ℃, uses 4molL in per 6 hours
-1NaOH solution regulate pH value, make it to maintain between 6.5～7.5, obtain having the fermented liquid of Nitrile hydratase vigor behind cultivation 50～100h, and total enzyme work of fermented liquid reaches 800～12,000,000 units per ml fermented liquids;
Catalyst suspension: fermented liquid in separation factor >=4000 spinnings, is removed supernatant, cell suspension in deionized water or 20mM, pH are 7.0～8.0 buffer solution of potassium phosphate, is made into the catalyst suspension that cell concn is 5% (wt);
Solid culture based formulas used in the such scheme is: glycerine 0.2～2%, yeast extract 0.5～1.5%, agar 0.2～2%, urea 0.1～1%, K
2HPO
40.1 KH～0.5%,
2PO
40.1～0.5%, NaCl0.1～1% and MgSO
40.005～0.02%, de-salted water 10～15%, surplus is a starch; Fermentative medium formula is: glycerine 0.2～2%, yeast extract 0.5～1.5%, L-glutamic acid 0.2～2%, urea 0.2～2%, K
2HPO
40.1～0.5%, KH
2PO
40.1～0.5%, MgSO
40.005～0.02%, Fe
3+0.001～0.01%, pH value 6.5～7.5, surplus are de-salted water; Above-mentioned per-cent is mass percent.
2. claim 1 is described is used for the catalyzer that synthesis of acrylamide contains the photosensitivity nitrile hydratase strain; It is characterized in that: thalline fermentation culture: thalline is inoculated in solid medium; Picking lawn fermentative prepn Nitrile hydratase in shaking table after the activation, shaking speed 100rmin
-1, temperature is 35 ℃, uses 4molL in per 6 hours
-1NaOH solution regulate pH value, make it to maintain 7.5, obtain having the fermented liquid of Nitrile hydratase vigor behind the cultivation 72h; And total enzyme work of fermented liquid reaches 800～1,200 ten thousand units per ml fermented liquids;
Is 5000 with above-mentioned fermented liquid at separation factor, and supernatant is removed in spinning, and cell suspension in 20mM, pH are 7.5 buffer solution of potassium phosphate, is made into the catalyst suspension that cell concn is 5% (wt);
Solid medium: glycerine 0.5%, yeast extract 1%, agar 1%, urea 0.3%, K
2HPO
40.3%, KH
2PO
40.3%, NaCl 0.5%, MgSO
40.01%, de-salted water 10～15%, and surplus is a starch;
Fermention medium: glycerine 1.5%, yeast extract 1%, L-glutamic acid 1%, urea 1.2%, K
2HPO
40.3%, KH
2PO
40.3%, MgSO
40.01%, Fe
3+0.003%, pH value 7, surplus are de-salted water.
3. claim 3 is described is used for the Application of Catalyst that synthesis of acrylamide contains the photosensitivity nitrile hydratase strain, comprises the steps: in catalyst suspension, slowly to add vinyl cyanide, and at illumination, normal pressure, temperature is 25 ℃, 200rmin
-1Stirring velocity under carry out the enzyme catalysis hydration reaction, the addition of vinyl cyanide accounts for 20% (wt) of reaction system, through the reaction of 10h, obtains acrylamide soln; Catalyst suspension and acrylonitrile reactor liquid mass ratio are between 1: 100～1: 200.
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|---|---|---|---|
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103834600A (en) * | 2014-03-17 | 2014-06-04 | 蔡建华 | Fermentation method of photosensitive nitrile hydratase bacterial strain for catalyzed synthesis of acrylamide |
| CN115558684A (en) * | 2022-09-27 | 2023-01-03 | 南通博亿化工有限公司 | Biological preparation method of amide compound and acrylamide prepared by biological method |
-
2011
- 2011-10-10 CN CN201110304076A patent/CN102329757A/en active Pending
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103834600A (en) * | 2014-03-17 | 2014-06-04 | 蔡建华 | Fermentation method of photosensitive nitrile hydratase bacterial strain for catalyzed synthesis of acrylamide |
| CN115558684A (en) * | 2022-09-27 | 2023-01-03 | 南通博亿化工有限公司 | Biological preparation method of amide compound and acrylamide prepared by biological method |
| CN115558684B (en) * | 2022-09-27 | 2023-08-18 | 南通博亿化工有限公司 | Biological preparation method of amide compound and acrylamide prepared by biological method |
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Owner name: CAI JIANHUA Free format text: FORMER OWNER: SUN ANSHUN Effective date: 20120608 |
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Effective date of registration: 20120608 Address after: 163000, No. three, 82 Village Road, Saertu District, Heilongjiang, Daqing Applicant after: Cai Jianhua Address before: 163000, No. three, 82 Village Road, Saertu District, Heilongjiang, Daqing Applicant before: Sun Anshun |
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Application publication date: 20120125 |