CN101348809A - Method for producing L-aminothiopropionic acid by enzyme conversion - Google Patents
Method for producing L-aminothiopropionic acid by enzyme conversion Download PDFInfo
- Publication number
- CN101348809A CN101348809A CNA2007100581936A CN200710058193A CN101348809A CN 101348809 A CN101348809 A CN 101348809A CN A2007100581936 A CNA2007100581936 A CN A2007100581936A CN 200710058193 A CN200710058193 A CN 200710058193A CN 101348809 A CN101348809 A CN 101348809A
- Authority
- CN
- China
- Prior art keywords
- pseudomonas putida
- atc
- enzyme
- hours
- enzyme substratum
- 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
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a technology for transformation production of L-cysteine by the microbial enzyme method. Pseudomonas putida TS-1138 is taken as a strain to be tested; and after amplification culture and zymogencous culture, the Pseudomonas putida TS-1138 is converted into DL-2-amino-Delta2thiazolines-4carboxylic acid (DL-ATC for short) by taking whole cells as an enzyme source, wherein the concentration of substrate solution is between 0.05 and 5.0 percent, the substrate solution is added between 0 and 9 hours, the reaction temperature is between 36 and 42 DEG C, and the reaction time is between 2 and 12 hours. The technology has the advantages of simple process, low cost of raw materials for production, low energy consumption and no pollution.
Description
[technical field]
The invention belongs to amino acid whose production technical field, relate to the method that a kind of pseudomonas putida (Pseudomonas putida) enzymatic conversion method substrate is produced the L-halfcystine.
[background technology]
L-halfcystine (L-cysteine) is to constitute the amino acid that has active sulphur (sulfydryl) in proteinic 20 multiple amino acids, at present widespread use in medicine, foodstuff additive and makeup.Aspect medical, halfcystine and derivative thereof can be used for liver medicine and toxicide, antipyretic and analgesic, ulcer treatment medicine, fatigue recovery agent, transfusion and comprehensive amino acid preparation, particularly expectorant.Aspect food, halfcystine can be used as the bread fermentation auxiliary agent.Aspect makeup, be mainly used in hair-waving essence, sunscreen, hair care essence, hair lotion.Because in the most of microbe body, sulfydryl derives from sulfate ion, and sulfate ion reducible be certain sulfide, therefore very difficultly produce the L-halfcystine with microbe fermentation method.China extracts the L-Gelucystine after the production of L-halfcystine is mainly relied on the hair acid hydrolysis, then the L-Gelucystine is prepared the L-halfcystine through chemistry or electrolytic reduction, Gelucystine in the hair, content is about 14%, and the highest yield of domestic present hair-hydrolyzation method is about 4.5 to 5.5%.It is low to produce L-halfcystine yield with the hair acidolysis, the energy consumption height, and hydrolytic process produces unpleasant gas and a large amount of spent acid, and environmental pollution is serious; In addition, because hygienic issues, the product that extracts from hair does not meet medical use standard.Advantages such as and microbial enzyme method conversion production L-halfcystine has high specificity, and production technique is simple, and side reaction and by product are few, and the degree of environmental pollution is low will have good development prospect.
[summary of the invention]
It is low to the present invention seeks to solve the yield that exists in the prior art, the energy consumption height, and problem such as environmental pollution is serious provides a kind of microbial enzyme method conversion of substrate to produce the method for L-halfcystine.
Provided by the invention a kind of be the enzyme source with the full cell of pseudomonas putida (Pseudomonas putida) TS-1138, enzymatic conversion method DL-2-amino-Δ 2 thiazolines-4 carboxylic acid (DL-2-Amino-Δ 2thiazoline-4-Carboxylic Acid, DL-ATC) method of synthetic L-halfcystine comprises:
A. pseudomonas putida TS-1138 is carried out conventional enlarged culturing
(pseudomonas putida (Pseudomonas putida) TS1138 once delivered in " He'nan University of Technology's journal " the 28th the 1st phase of volume in " biotechnology communication " the 17th the 6th phase of volume and in February, 2007 in November, 2006, now be preserved in University Of Science and Technology Of Tianjin metabolic control fermentation research department, the public if needed, can be directly and this research department get in touch.)
B. the bacterial classification of the enlarged culturing that obtains in the steps A is inserted and produce the enzyme substratum and cultivate: produce carbon source content in the enzyme substratum and should be 0.1%～30% butt and cultivate basic weight, carbon-nitrogen ratio N: C should be between 1: 100～50: 100, inorganic salt content should be 0.1～10%, and the addition of DL-ATC is 0.1%～0.9%.Produce initial p H6.0～9.0 of enzyme substratum, 25 ℃～33 ℃ of culture temperature, shaking culture or fermentor cultivation 12～36 hours, inoculum size are 0.01%～20% (V/V); Stream adds PH6.2～7.0 that ammoniacal liquor is regulated fermention medium in culturing process;
C. step B was cultivated 20～24 hours, centrifugation thalline 10min (4000r/min, 4 ℃), abandoning supernatant, with PBS damping fluid (Sodium phosphate dibasic-SODIUM PHOSPHATE, MONOBASIC-sodium-chlor damping fluid) the repetitive scrubbing thalline of pH 7.4 three times and resuspended, the wet thallus that in the PBS of pH 7.4 damping fluid, adds, the used cell suspension of preparation enzymatic reaction, cell concn is 100g/L～500g/L.
D. with the cell suspension of step C preparation, join in the substrate solution, carry out enzymatic reaction.Wherein the concentration of substrate solution is 0.05%～5.0%, adds at 0～9 hour, and temperature of reaction is 36～42 ℃, and the reaction times is 2～12 hours.After enzymatic reaction finished, the centrifugation thalline was got supernatant liquor, adopted the acid ninhydrin method to measure the L-cysteine content.
The carbon source that the above-mentioned B step produces in the enzyme substratum is: sugar, i.e. glucose, sucrose, lactose, maltose, sorbose; Or glycerine; Or low mass molecule alcohol, ethanol; The suitableeest scope of carbon source content is 10%～20%.
The nitrogenous source that the above-mentioned B step produces in the enzyme substratum is: ammonia or ammonium salt, i.e. ammonium chloride, ammonium sulfate, bicarbonate of ammonia, ammonium nitrate; Or organonitrogen, i.e. extractum carnis, peptone, yeast powder, corn steep liquor, urea.
The inorganic salt that the above-mentioned B step produces in the enzyme substratum are: potassiumphosphate, sal epsom, ferrous sulfate, sodium-chlor ammonia or manganous sulfate.
In example of the present invention, adopt the acid ninhydrin method to measure the content of L-halfcystine, concrete grammar is as follows: accurately take by weighing a certain amount of L-halfcystine standard substance, be dissolved among a certain amount of 0.05M HCl, the preparation final concentration is the standard mother liquor of 500mg/l, and further with distilled water above-mentioned mother liquor being diluted respectively is 10,20, ..., the standardized solution of 100mg/l.Get the diluent 0.2mL of a certain amount of standardized solution or testing sample solution, add the 0.2mL Glacial acetic acid, add 0.2mL acid ninhydrine reagent again, in boiling water bath, react 10min, cool off in cold water immediately then, add 2.4mL alcohol at last, making cumulative volume is 3mL.After placing 10min, measure its absorbancy down in 560nm, with concentration and absorbancy drawing standard curve.Calculate semicystinol concentration in the unknown solution by this typical curve.
Advantage of the present invention and positively effect: in the reaction of being undertaken by enzyme catalysis method, effect has two aspects to temperature to enzyme usually: on the one hand, temperature of reaction raises, and enzyme ' s reaction speeding is accelerated; On the other hand, along with temperature raises, the zymoprotein thermally denature is accelerated thereupon, and the easy inactivation of enzyme generally can not be reused; And immobilized method steps is more numerous and diverse, and more or less enzyme activity is had certain influence.And the inventive method is the enzyme source with pseudomonas putida TS-1138 cell, and enzymatic conversion method substrate DL-ATC obtains the L-halfcystine aqueous solution.This method directly utilizes cell to be the direct catalytic substrate in enzyme source, banned the method for immobilized enzyme, and by reducing the enzymatic reaction temperature, improve the stability of enzyme, current adding substrate DL-ATC in the enzymatic reaction process, make enzyme reusable, improved the utilising efficiency of enzyme source cell and the output of L-halfcystine.
[embodiment]
Embodiment 1: the preparation of pseudomonas putida TS1138 bacteria suspension
The lawn of picking one ring pseudomonas putida TS-1138 inserts 30mL seed culture medium (glucose 30g/L, DL-ATC3H is housed from the fresh inclined-plane
2O5g/L, yeast powder 15g/L, peptone 10g/L, urea 3g/L, MnSO
4H
2O 1g/L, K
2HPO
43H
2O3g/L, MgSO
47H
2O 0.5g/L, FeSO
47H
2O 0.01g/L, NaCl 1.5g/L, pH 7.5,0.1MPa in 500mL triangular flask 15min), 8 layers of gauze seal, in rotary shaking table 190r/min, behind 29 ℃ of shaking culture 14h, with 10% inoculum size 30mL is equipped with in the inoculum access and produces enzyme substratum (glucose 30g/L, DL-ATC3H
2O 4g/L, corn steep liquor 3g/L, urea 3g/L, MnSO
4H
2O 1g/L, K
2HPO
43H
2O 3.0g/L, MgSO
47H
2O 0.5g/L, FeSO
47H
2O 0.01g/L, NaCl3 g/L, pH 7.5,0.1MPa in 500mL triangular flask 15min), 8 layers of gauze seal, in rotary shaking table 170r/min, behind 29 ℃ of shaking culture 22h, centrifugation thalline 10min (4000r/min, 4 ℃), abandoning supernatant, with the PBS damping fluid repetitive scrubbing thalline of pH 7.4 three times and resuspended, the wet thallus that in the PBS of pH 7.4 damping fluid, adds, the used cell suspension of preparation enzymatic reaction, cell concn is 350g/L.
Embodiment 2: with pseudomonas putida TS1138 shake-flask culture, carry out enzymatic reaction at 42 ℃ and produce the L-halfcystine
The DL-ATC solution of adding 0.6% (contains 0.6%DL-ATC, 0.6%K in the 100ml triangular flask
2HPO
4, pH7.5 to 8.0) and 20ml, add the TS-1138 cell suspension 10ml of the foregoing description 1, mixing again.42 ℃ of constant temperature reacted after 2.5 hours, and the content of measuring halfcystine with the acid ninhydrin method is 1.65g/l.
Embodiment 3: with pseudomonas putida TS1138 shake-flask culture, carry out enzymatic reaction at 38 ℃ and produce the L-halfcystine
The DL-ATC solution of adding 0.6% (contains 0.6%DL-ATC, 0.6%K in the 100ml triangular flask
2HPO
4, pH7.5 to 8.0) and 20ml, add the TS-1138 cell suspension 10ml of the foregoing description 1, mixing again.38 ℃ of constant temperature reacted after 3 hours, and the content of measuring halfcystine with the acid ninhydrin method is 1.60g/l.
Embodiment 4: with pseudomonas putida TS1138 shake-flask culture, carry out enzymatic reaction at 38 ℃ of current adding substrates and produce the L-halfcystine
The DL-ATC solution of adding 0.6% (contains 0.6%DL-ATC, 0.6%K in the 100ml triangular flask
2HPO
4, pH7.5 to 8.0) and 20ml, add the TS-1138 cell suspension 20ml of the foregoing description 1, mixing again.38 ℃ of constant temperature, add the substrate solution that 10ml contains DL-ATC0.18g and 0.24g during respectively at enzymatic reaction 3h and 6h, the amount of ATC in the substrate solution is maintained about 0.6% substantially, and enzymatic reaction is after 9 hours, and the content of measuring halfcystine with the acid ninhydrin method is 5.70g/l.
Embodiment 5: cultivate at the 5L jar with pseudomonas putida TS1138, carry out enzymatic reaction at 42 ℃ and produce the L-halfcystine
The lawn of picking one ring stench ring pseudomonas TS-1138 inserts the seed culture medium from fresh inclined-plane, seed culture medium and seed culture condition are all with embodiment 1, inoculum size with 10% (seed culture fluid 300mL) is with good product enzyme substratum (glucose 50g/L, the DL-ATC3H of going out separately
2O 5g/L, corn steep liquor 3g/L, urea 3g/L, MnSO
4H
2O 1g/L, K
2HPO
43H
2O 3g/L, MgSO
47H
2O 0.5g/L, FeSO
47H
2O 0.01g/L, NaCl 3g/L, pH 7.0,0.1MPa 15min) the flame vaccination ways inserts 5L and controls in the fermentor tank liquid amount 3L in the 5L fermentor tank, 29 ℃ of temperature controls automatically, pH is about 6.7 for the control of auto-feeding ammoniacal liquor, ventilation 200L/h, initial mixing speed 200r/min adds bubble enemy froth breaking by stream.Stream adds the Glucose Liquid that concentration is 60g/L when residual sugar is lower than 5g/L.
After the fermentation culture 22 hours, get 5L jar cultured cells 2L, concentrate five times, join and contain 0.6% DL-ATC solution and (contain 0.6%DL-ATC, 0.6%K to 400ml
2HPO
4, pH7.5 to 8.0) and the 5L of 800ml controls in the fermentor tank mixing automatically.42 ℃ of constant temperature reacted after 2.5 hours, and the content of measuring halfcystine with the acid ninhydrin method is 1.81g/l.
Embodiment 6: to cultivate at the 5L jar with pseudomonas putida TS1138, carry out enzymatic reaction at 38 ℃ of current adding substrates and produce the L-halfcystine
Automatically the DL-ATC solution of controlling adding 0.6% in the fermentor tank at 5L (contains 0.6%DL-ATC, 0.6%K
2HPO
4, pH7.5 to 8.0) and 800ml, add the TS-1138 cell suspension 400ml of the foregoing description 5, mixing again.38 ℃ of constant temperature add the substrate solution that 30ml contains DL-ATC4.8g when enzymatic reaction 3h, the amount of ATC in the substrate solution is maintained about 0.6% substantially, and enzymatic reaction is after 6 hours, and the content of measuring halfcystine with the acid ninhydrin method is 2.95g/l.
Claims (4)
1, a kind of pseudomonas putida (Pseudomonas putida) TS-1138 enzymatic conversion method DL-2-amino-Δ 2 thiazolines-4 carboxylic acid (DL-2-Amino-Δ 2thiazoline-4-Carboxylic Acid that utilizes provided by the invention, DL-ATC) production technique of synthetic L-halfcystine comprises:
A. pseudomonas putida (Pseudomonas putida) TS-1138 is carried out conventional enlarged culturing
B. the bacterial classification of the enlarged culturing that obtains in the steps A is inserted and produce the enzyme substratum and cultivate: produce carbon source content in the enzyme substratum and should be 0.1%～30% butt and cultivate basic weight, carbon-nitrogen ratio N: C should be between 1: 100～50: 100, inorganic salt content should be 0.1～10%, and the addition of DL-ATC is 0.1%～0.9%.Produce initial p H6.0～9.0 of enzyme substratum, 25 ℃～33 ℃ of culture temperature, shaking culture or fermentor cultivation 12～36 hours, inoculum size are 0.01%～20% (V/V); Stream adds PH6.2～7.0 that ammoniacal liquor is regulated fermention medium in culturing process;
C. step B was cultivated 20～24 hours, centrifugation thalline 10min (4000r/min, 4 ℃), abandoning supernatant, with PBS damping fluid (Sodium phosphate dibasic-SODIUM PHOSPHATE, MONOBASIC-sodium-chlor damping fluid) the repetitive scrubbing thalline of pH 7.4 three times and resuspended, the wet thallus that in the PBS of pH 7.4 damping fluid, adds, the used cell suspension of preparation enzymatic reaction, cell concn is 100g/L～500g/L.
D. with the cell suspension of step C preparation, join in substrate (DL-ATC) solution, carry out enzymatic reaction.Wherein the concentration of substrate solution is 0.05%～5.0%, adds at 0～9 hour, and temperature of reaction is 36～42 ℃, and the reaction times is 2～12 hours.After enzymatic reaction finished, the centrifugation thalline was got supernatant liquor, adopted the acid ninhydrin method to measure the L-cysteine content.
2, production technique according to claim 1 is characterized in that the carbon source that B step produces in the enzyme substratum is: sugar, i.e. glucose, sucrose, lactose, maltose, sorbose; Or glycerine; Or low mass molecule alcohol, ethanol; The suitableeest scope of carbon source content is 10%～20%.
3, production technique according to claim 1 is characterized in that the nitrogenous source that B step produces in the enzyme substratum is: ammonia or ammonium salt, i.e. ammonium chloride, ammonium sulfate, bicarbonate of ammonia, ammonium nitrate; Or organonitrogen, i.e. extractum carnis, peptone, yeast powder, corn steep liquor, urea.
4, production technique according to claim 1 is characterized in that the inorganic salt in the B step product enzyme substratum are: potassiumphosphate, sal epsom, ferrous sulfate, sodium-chlor ammonia or manganous sulfate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100581936A CN101348809A (en) | 2007-07-18 | 2007-07-18 | Method for producing L-aminothiopropionic acid by enzyme conversion |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100581936A CN101348809A (en) | 2007-07-18 | 2007-07-18 | Method for producing L-aminothiopropionic acid by enzyme conversion |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101348809A true CN101348809A (en) | 2009-01-21 |
Family
ID=40267783
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007100581936A Pending CN101348809A (en) | 2007-07-18 | 2007-07-18 | Method for producing L-aminothiopropionic acid by enzyme conversion |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101348809A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948779A (en) * | 2010-08-31 | 2011-01-19 | 天津启仁医药科技有限公司 | Preparation and use of solid microbial enzyme preparation for producing L-cysteine through enzymatic conversion |
| CN102409033A (en) * | 2011-11-17 | 2012-04-11 | 天津启仁医药科技有限公司 | N-carbamyl-L-cysteine (L-NCC) amidohydrolase, encoding gene and application of recombinant expressed protein of L-NCC amidohydrolase |
| CN105177076A (en) * | 2015-10-08 | 2015-12-23 | 湖北工业大学 | Method for synthesizing L-cysteine through immobilized enzyme conversion of DL-ATC (DL-2-amino-delta<2>-thiazoline-4-carboxylic acid) |
| CN105200088A (en) * | 2015-10-08 | 2015-12-30 | 湖北工业大学 | Method for synthesizing L-cysteine by transforming DL-2-amino-delta<2>-thiazolinyl-4-carboxylic acid (DL-ATC) by enzyme process |
| CN107118986A (en) * | 2017-05-16 | 2017-09-01 | 浙江医药高等专科学校 | A kind of pseudomonas putida and the application in (R) 1 (2 trifluoromethyl) ethanol is prepared |
| CN114276947A (en) * | 2021-10-12 | 2022-04-05 | 湖北远大生物技术有限公司 | Method for preparing L-cysteine by enzymatic conversion and application |
-
2007
- 2007-07-18 CN CNA2007100581936A patent/CN101348809A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948779A (en) * | 2010-08-31 | 2011-01-19 | 天津启仁医药科技有限公司 | Preparation and use of solid microbial enzyme preparation for producing L-cysteine through enzymatic conversion |
| CN102409033A (en) * | 2011-11-17 | 2012-04-11 | 天津启仁医药科技有限公司 | N-carbamyl-L-cysteine (L-NCC) amidohydrolase, encoding gene and application of recombinant expressed protein of L-NCC amidohydrolase |
| CN105177076A (en) * | 2015-10-08 | 2015-12-23 | 湖北工业大学 | Method for synthesizing L-cysteine through immobilized enzyme conversion of DL-ATC (DL-2-amino-delta<2>-thiazoline-4-carboxylic acid) |
| CN105200088A (en) * | 2015-10-08 | 2015-12-30 | 湖北工业大学 | Method for synthesizing L-cysteine by transforming DL-2-amino-delta<2>-thiazolinyl-4-carboxylic acid (DL-ATC) by enzyme process |
| CN105200088B (en) * | 2015-10-08 | 2019-06-25 | 湖北工业大学 | A kind of enzymatic conversion method DL-2- amino-△2The method of thiazoline -4- carboxylic acid synthesis L-cysteine |
| CN105177076B (en) * | 2015-10-08 | 2019-06-25 | 湖北工业大学 | A kind of immobilised enzymes conversion DL-2- amino-△2The method of thiazoline -4- carboxylic acid synthesis L-cysteine |
| CN107118986A (en) * | 2017-05-16 | 2017-09-01 | 浙江医药高等专科学校 | A kind of pseudomonas putida and the application in (R) 1 (2 trifluoromethyl) ethanol is prepared |
| CN107118986B (en) * | 2017-05-16 | 2020-08-11 | 浙江医药高等专科学校 | Pseudomonas putida and application thereof in preparation of (R) -1- (2-trifluoromethylphenyl) ethanol |
| CN114276947A (en) * | 2021-10-12 | 2022-04-05 | 湖北远大生物技术有限公司 | Method for preparing L-cysteine by enzymatic conversion and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jones et al. | Fuel alcohol production: appraisal of nitrogenous yeast foods for very high gravity wheat mash fermentation | |
| CN109439701B (en) | Method for preparing ergothioneine by biosynthesis and fermentation medium | |
| CN101348809A (en) | Method for producing L-aminothiopropionic acid by enzyme conversion | |
| CN105802897B (en) | A kind of D-Psicose -3- epimerase production bacterial strain and its application | |
| CN100360679C (en) | Method for simultaneous production of ergosterol and glutathione by yeast fermentation | |
| CN102492633A (en) | Rhodotorula mucilaginosa capable of degrading urethane and its application in liquor products and food | |
| CN102703339B (en) | High-yield arginine deiminase bacterial strain and method for producing L-citrulline by same | |
| Kondo et al. | Preparation of high activity whole cell biocatalyst by permeabilization of recombinant flocculent yeast with alcohol | |
| CN102994405B (en) | Saccharomyces cerevisiae and application thereof | |
| CN104893989A (en) | Rhizopus microsporus root-shaped variant ZJPH1308 and application thereof in preparation of sitagliptin intermediate | |
| CN108624513A (en) | A kind of method of High Density Cultivation D-pantoyl lactone hydrolase producing strains and application | |
| CN101475974B (en) | Culture medium and method for fermentation production of adenosine using the same | |
| Phaff | Industrial microorganisms | |
| CN104762338A (en) | Method of producing nicotinamide by catalysis of rhodococcus | |
| CN102533607B (en) | Strain capable of producing beta-galactosidase and method for producing galactooligosaccharides by using beta-galactosidase | |
| CN102703542A (en) | Method for synthesizing lactobionic acid by ultrasound field assisted microbial transformation | |
| CN106011186B (en) | Method for producing succinic acid by fermentation of brevibacterium | |
| Brinkmann et al. | Simultaneous utilization of heterotrophic substrates by Hansenula polymorpha MH30 results in enhanced growth rates | |
| CN101603062B (en) | Method for preparing perylenequinone pigment under catalysis of Shiraia bambusicola | |
| CN103305495A (en) | Method for preparing glutamate decarboxylase (GAD) | |
| CN104830851A (en) | Recombinant bacterium of formate dehydrogenase and application of recombinant bacterium | |
| CN103757086A (en) | Method for synthesizing S-adenosylmethionine through coupling of escherichia coli and saccharomyces cerevisiae | |
| CN104789489A (en) | Arginine deiminase high-yielding Bacillus cereus and application thereof | |
| CN101407773B (en) | Pseudomonas putida GNA5 strain and method for preparing D-glucosamine acid by using same | |
| CN110305802A (en) | The culture medium and cultural method of high spore output monascus purpureus are bred in a kind of liquid state fermentation |
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 |
Open date: 20090121 |