CN103509762A - Method for increasing liquid state laccase storage stability - Google Patents
Method for increasing liquid state laccase storage stability Download PDFInfo
- Publication number
- CN103509762A CN103509762A CN201210211312.8A CN201210211312A CN103509762A CN 103509762 A CN103509762 A CN 103509762A CN 201210211312 A CN201210211312 A CN 201210211312A CN 103509762 A CN103509762 A CN 103509762A
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- laccase
- enzyme
- liquid state
- liquid
- storage stability
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- 108010029541 Laccase Proteins 0.000 title claims abstract description 31
- 239000007788 liquid Substances 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 14
- 238000003860 storage Methods 0.000 title claims abstract description 8
- 108090000790 Enzymes Proteins 0.000 claims abstract description 31
- 102000004190 Enzymes Human genes 0.000 claims abstract description 31
- 238000004519 manufacturing process Methods 0.000 claims abstract description 12
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims abstract description 7
- 235000007164 Oryza sativa Nutrition 0.000 claims abstract description 7
- 235000009566 rice Nutrition 0.000 claims abstract description 7
- 239000010902 straw Substances 0.000 claims abstract description 7
- 238000010563 solid-state fermentation Methods 0.000 claims abstract description 6
- 230000014759 maintenance of location Effects 0.000 claims abstract description 4
- 239000002994 raw material Substances 0.000 claims abstract description 4
- 238000011160 research Methods 0.000 claims abstract description 4
- 241000008502 Pycnoporus sp. SYBC-L3 Species 0.000 claims abstract description 3
- 240000007594 Oryza sativa Species 0.000 claims abstract 2
- 230000001580 bacterial effect Effects 0.000 claims description 3
- 238000000855 fermentation Methods 0.000 claims description 2
- 230000004151 fermentation Effects 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 9
- 239000001963 growth medium Substances 0.000 abstract description 5
- 230000002779 inactivation Effects 0.000 abstract description 2
- 229940088598 enzyme Drugs 0.000 abstract 4
- 229940079919 digestives enzyme preparation Drugs 0.000 abstract 1
- 238000004321 preservation Methods 0.000 abstract 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 230000002255 enzymatic effect Effects 0.000 description 7
- 238000011218 seed culture Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 230000003068 static effect Effects 0.000 description 6
- 241000209094 Oryza Species 0.000 description 5
- 238000001816 cooling Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 239000012531 culture fluid Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000007639 printing Methods 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 239000004753 textile Substances 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 238000004065 wastewater treatment Methods 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000004922 lacquer Substances 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0061—Laccase (1.10.3.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03002—Laccase (1.10.3.2)
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention provides a method for increasing liquid state laccase storage stability, wherein a high laccase yield strain Pycnoporus sp.SYBC-L3 preserved in the China Center for Type Culture Collection on September 15, 2010 and having a preservation number of CCTCC M 2010235 is adopted as a starting strain, liquid state laccase prepared by adopting rice straws as a main raw material through solid state fermentation production is adopted as a main research object, storage stability of the prepared liquid state laccase is significantly increased after ammonium nitrate with a certain concentration is added to a culture medium for producing laccase through solid state fermentation, enzyme activity retention rate is up to more than 90% after the enzyme solution is stored for 90 days at a temperature of 30 DEG C, enzyme stability is significantly increased, enzyme activity is increased by more than 10%, the problem that the liquid state laccase is easily subjected to inactivation is solved with the method, and the method belongs to the technical field of enzyme preparations.
Description
Technical field
The present invention is a kind of method that improves liquid laccase storage stability, belongs to zymin technical field.
Background technology
Laccase has substrate widely, can not only the multiple aromatics of catalyzed oxidation, can also lignin degrading, remove the toxicity of many poisonous aldehydes matters, can also make multiple dye decoloredly and remove the toxicity etc. of oil industry wastewater, therefore in fields such as textile printing and dyeing, papermaking, lawn reparation, energy regeneration, feed, food, wastewater treatments, have compared with large using value and paid close attention to widely.
Now there are some researches show, the preparation majority of laccase is confined to use for laboratory all ingredients and is made into substratum and prepares, prepared high expensive, we work out take rice straw and as main raw material solid state fermentation, prepares laccase and succeed, in application on January 12nd, 2012 national patent, number of patent application: 201210007885.9, the method has significantly reduced production cost and the simple and easy to do less investment of laccase, but the prepared easy inactivation of liquid laccase, this impels us to go exploratory development to improve the method for liquid laccase stability, the present invention is exactly under such guiding theory, through test of many times, succeed.
Summary of the invention
The present invention works out a kind of highly effective method of liquid towards laccase storage stability---in culture medium, add certain density ammonium nitrate solution, thereby the storage stability of prepared liquid laccase is improved greatly, and the activity of enzyme also increase.
The wild strain Pycnoporus sp.SYBC-L3 that technical scheme of the present invention: be with our deposit number that is stored in Chinese Typical Representative culture collection center of patent applied for: CCTCC M 2010235 produces the samguineus genus of laccase sends out a bacterial strain for producing, the liquid laccase that is main raw material solid state fermentation manufacture by rice straw is research object, in the substratum of fermentation lacquer producing enzyme, add certain density ammonium nitrate, concrete technology is:
(1) seed culture
Seed culture medium: in mass body volume concentrations: 2% glucose, 20% potato.
Potato need be cut into slices or piece, adds poach 20-30 minute, by filtered through gauze, gets its juice constant volume.
Inoculating process: get slant strains one ring point on PDA flat board and connect mycelia, cultivate 7 days for 30 ℃, cutting the bacterium colony that 3 diameter are 0.5CM accesses in the triangular flask of the 500ml specification that 50ml seed culture medium is housed, add again granulated glass sphere 10-20 grain, prepare some parts, be placed in shaking table, culture condition: rotating speed 200rpm, 30 ℃ of temperature, constant temperature culture 2 days, is the seed liquor of enzymatic production.
(2) enzymatic production substratum
Rice straw is cut into the segment of 1 centimetre of left and right, claims 20 grams of triangular flasks of putting into 500ml, add the water 40ml that adjusts PH=3 with hydrochloric acid and sodium hydroxide, then to add concentration be the ammonium nitrate solution 5ml of 20g/L, in 121 ℃ of sterilizings 20 minutes, cooling rear stand-by.
(3) enzymatic production and enzyme liquid extract
After sterilizing is cooling, in enzymatic production substratum, add 20m1 seed culture fluid and fully mix with stalk, and be placed in 30 ° of static cultivations of environment after 8 days with 8 layers of gauze sealing, first again add 400ml water, the stalk bacterium piece clumping together is stirred after loose and stirs or be placed in shaking table at 30 ℃, under 200rpm condition, continue to cultivate 3 hours, then static 12-24 hour in 30 ° of environment, finally centrifugal with whizzer or remove by filter stalk and thalline obtains laccase crude enzyme liquid by suction filtration method or with gauze, by DMP method, surveying enzyme lives, and calculate total enzyme and live except the weight of stalk, obtain the U/g. of unit that every gram of stalk produces laccase
(4) measuring method that enzyme is lived
3mL reaction system comprises: 2 of 0.5ml10mmol/L, 6-dimethoxy phenol; 0.1mol/L Sodium phosphate dibasic-vitamin P damping fluid of 2.4ml pH=3.0; 0.1ml crude enzyme liquid---fermented liquid obtains supernatant liquor in centrifugal 10 minutes and looks enzyme activity dilution through high-speed refrigerated centrifuge 10000g, using deionized water place of crude enzyme liquor as blank, at 469nm place, survey light absorption value and read absorbance one time every 5s, generally continue to carry out to stop measuring after 1min, with the linearity range of reacting, calculate enzyme and live.Enzyme activity definition: the required enzyme amount of catalysis in 1 minute one micromole's substrate, enzymic activity is with UL
-1represent.
Beneficial effect of the present invention:
The present invention has following features:
1, proferment material price is cheap, and can turn waste into wealth, and environmental protection, does not have waste.Because bacterial classification is medicinal fungi, therefore the residue remnant after laccase filters can be used for animal-feed, papermaking and bio-feritlizer.
2, fermentative production equipment is simple, and fund input is few.
3, thick enzymatic property is stable, take DMP during as substrate, under sour environment, can bring into play maximum catalysis effect, and optimum temperuture is 55 degree, more heat-resisting.
4, effect substrate is extensive, has broad application prospects.
5, manufacture cost is low, is extremely conducive to be in full swing, and meets national policy.
Because institute of the present invention manufacture laccase cost is out low and can be widely used in the aspects such as textile printing and dyeing, papermaking, lawn reparation, energy regeneration, feed, food, wastewater treatment.Therefore the present invention has industrial application to be widely worth and significant economic benefit prospect.
Embodiment
Control Example
Get slant strains one ring point on PDA flat board and connect mycelia, cultivate 7 days for 30 ℃, cutting the bacterium colony that 3 diameter are 0.5CM accesses in the triangular flask of the 250ml specification that 50ml seed culture medium is housed, add again 10 of granulated glass spherees, prepare some parts, be placed in shaking table, culture condition: rotating speed 200rpm, 30 ℃ of temperature, constant temperature culture 2 days, is the seed liquor of enzymatic production.
Rice straw is cut into the segment of 1 centimetre of left and right, claim 20 grams of triangular flasks of putting into 500ml, add the water 20ml that adjusts PH=3 with hydrochloric acid and sodium hydroxide, 121 ℃ of sterilizings 20 minutes, after cooling, add 20ml seed culture fluid and fully mix, 30 ° of static cultivations 8 days, add again 400ml water, with rod fully stir loose after, it is to shake 3 hours on 30 ° of rotating speeds shaking table that is 200rpm that 500ml triangular flask is placed in to temperature, static 24 hours again, filtration obtains enzyme liquid, survey enzyme every gram of stalk alive and can produce laccase 81U/g, owing to producing in enzyme cultivation, do not add ammonium nitrate solution, prepared liquid crude enzyme liquid is placed 30d and 90d under 30 ℃ of conditions, enzyme retention rate alive is respectively 63.12% and 26.25%.
Embodiment 1
The seed liquor of enzymatic production is cultivated the same reference examples of operation.
Rice straw is cut into the segment of 1 centimetre of left and right, claim 20 grams of triangular flasks of putting into 500ml, add the water 40ml that adjusts PH=3 with hydrochloric acid and sodium hydroxide, adding concentration is the ammonium nitrate solution 5ml of 20g/L again, 121 ℃ of sterilizings 20 minutes, after cooling, add 20ml seed culture fluid and fully mix, 30 ° of static cultivations 8 days, add again 400ml water, with rod fully stir loose after, it is to shake 3 hours on 30 ° of rotating speeds shaking table that is 200rpm that 500ml triangular flask is placed in to temperature, static 24 hours again, filtration obtains enzyme liquid, survey enzyme every gram of stalk alive and can produce laccase 90U/g, enzyme activity improves 10%, and prepared liquid crude enzyme liquid is placed 30d and 90d under 30 ℃ of conditions, enzyme retention rate alive is respectively 97.24% and 90.6%.
Claims (1)
1. a method that improves liquid laccase storage stability, it is characterized in that it take the samguineus of the high yield laccase that preserving number is CCTCC M2010235, to belong to bacterial strain Pycnoporus sp.SYBC-L3 be starting strain, using rice straw be the liquid laccase of main raw material solid state fermentation manufacture as research object, in the substratum of Produced by Solid-state Fermentation laccase, by 1/12 of substratum gross weight, add liquid laccase prepared by ammonium nitrate solution secondary fermentation that concentration is 20g/L:
(1) enzyme liquid is stored after 90 days in 30 ℃ of environment, and enzyme is lived retention rate up to more than 90%, has improved the stability of enzyme.
(2) vigor of prepared liquid laccase has improved more than 8%, has strengthened the result of use of enzyme.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210211312.8A CN103509762B (en) | 2012-06-26 | 2012-06-26 | A kind of method improving liquid state laccase storage stability |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210211312.8A CN103509762B (en) | 2012-06-26 | 2012-06-26 | A kind of method improving liquid state laccase storage stability |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103509762A true CN103509762A (en) | 2014-01-15 |
| CN103509762B CN103509762B (en) | 2016-01-20 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210211312.8A Active CN103509762B (en) | 2012-06-26 | 2012-06-26 | A kind of method improving liquid state laccase storage stability |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103509762B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102080046A (en) * | 2010-09-25 | 2011-06-01 | 江南大学 | High-yield laccase strain and method for producing laccase through fermentation |
-
2012
- 2012-06-26 CN CN201210211312.8A patent/CN103509762B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102080046A (en) * | 2010-09-25 | 2011-06-01 | 江南大学 | High-yield laccase strain and method for producing laccase through fermentation |
Non-Patent Citations (2)
| Title |
|---|
| 刘家扬 等: "菌种L3深层发酵产漆酶及其对废水脱色的研究", 《工业水处理》 * |
| 王欣 等: "培养条件对Trametes versicolor SYBC L3固态发酵产漆酶的影响", 《西北农业学报》 * |
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| Publication number | Publication date |
|---|---|
| CN103509762B (en) | 2016-01-20 |
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Effective date of registration: 20190110 Address after: 415400 Jiashan New Industrial Zone, Jin City, Changde City, Hunan Province Patentee after: Hunan Hongying Biological Science & Technology Co., Ltd. Address before: 415400 No. 362 Beida Road, Jinshi City, Changde City, Hunan Province Patentee before: Hongyingxiang Biological Engineering Co., Ltd., Hunan |
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