CN101139570A - High-density fermentation method for HPV L1 protein prokaryotic expression - Google Patents
High-density fermentation method for HPV L1 protein prokaryotic expression Download PDFInfo
- Publication number
- CN101139570A CN101139570A CNA2007100902388A CN200710090238A CN101139570A CN 101139570 A CN101139570 A CN 101139570A CN A2007100902388 A CNA2007100902388 A CN A2007100902388A CN 200710090238 A CN200710090238 A CN 200710090238A CN 101139570 A CN101139570 A CN 101139570A
- Authority
- CN
- China
- Prior art keywords
- fermentation
- substratum
- hpv
- ytl
- air
- 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)
Abstract
The invention provides a high-density fermenting method and an improved culture medium for HPVL1 prokaryotic expression, also an engineering bacterial strain for efficiently expressing HPV L1 protein. The improved culture medium can fully meet the nutriment in fermentation of the engineering bacterial strain. The invention also controls efficiently the key parameters during fermentation. With the fermentation method in the invention, not only the retention rate of the expression plasmid is improved from 50-60% to more than 90%, but also the unit output of HPVL1 protein is improved to 3-4 times of that before, and the production cost is effectively reduced. The invention is of great importance for scaled industrial production.
Description
Technical field
The present invention relates to the fermentation field, relate to the fermentation process in high density of HPV L1 albumen pronucleus expression particularly.
Background technology
Human papillomavirus (Human papilloma virus; HPV) human multiple disease be can cause, optimum wart and cancer comprised.Therefore, the current hot research field of the preventative vaccine of HPV research becoming.L1 albumen is one of two capsid proteins of HPV, and 72 L1 albumen pentamers have just formed the shell of HPV virion, and this makes L1 albumen become important candidate's immunogen.
At present, adopt prokaryotic expression system to produce HPV L1 albumen and exist problems such as expression plasmid is easily lost, protein yield is low, production cost height, seriously restricted its industrial applications.The present invention starts with from the control fermentation parameter, by the improvement fermention medium, efficiently solves an above-mentioned difficult problem.
Summary of the invention
(1) technical problem that will solve
The purpose of this invention is to provide a kind of fermentation process in high density of HPV L1 albumen pronucleus expression and the substratum of improvement, another object of the present invention provides the proteic engineering bacteria of a plant height efficient expression HPV L1.
(2) technical scheme
The invention provides a kind of fermentation process in high density of HPV L1 albumen pronucleus expression, it is characterized in that it comprises the steps:
(1) seed activation: (this bacterial strain was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on February 9th, 2007 will to express the proteic engineering strain intestinal bacteria of HPV L1 (Escherichiacoli) CGMCC1944, be called for short CGMCC, deposit number is CGMCC No.1944) on solid 2YT substratum, activate and (refer under 25-37 ℃, on the 2YT flat board streak culture 12 hours), choose single bacterium colony then to 10ml YTL liquid nutrient medium, cultivated 24-36 hour down at 25-37 ℃; Again with the seed liquor that obtains by volume 5% inoculum size be seeded in the YTL substratum, cultivated 8-12 hour down at 30-37 ℃;
(2) inoculation: fill the YTL substratum in fermentor tank, the seed liquor that the inoculum size of 5%-10% is good with above-mentioned activation by volume is seeded to liquid YTL substratum then;
(3) fermentation: the gas mixture of bubbling air or air/oxygen in fermentor tank, air flow 4-8L/min; Leavening temperature 25-37 ℃, stir revolution 200-800rpm, dissolved oxygen is controlled at 30%-100%, and pH is controlled at 6.2-7.8, fermentation time 20-30 hour; Work as OD
600nmBegin feed supplement during=2-4, the flow acceleration of feed supplement is 25-400ml/h.
(4) induce the proteic expression of HPV L1, and carry out protein purification, obtain target protein.
In aforesaid method, used substratum consists of:
2YT: tryptone (import, Oxoid company produces) 16g, yeast extract (import, Oxoid company produces) 10g, NaCl 5g, H
2O 1L, silicon oil foam killer 0.2ml;
YTL (all raw materials are homemade): Na
2HPO
4.12H
2O 6-10g, KH
2PO
41-3g, tryptone 7-10g (magnificent biotech firm produces), yeast extract 3-6g (magnificent biotech firm produces), the Sodium.alpha.-hydroxypropionate 10-20g of 65-75%, MgSO
40.4-0.8g, NH
4Cl 2-3g, H
2O 1L, nitric acid defoamer 0.2ml, wherein Na
2HPO
4.12H
2O and KH
2PO
4Be dissolved in the 200mL water, separately sterilization.
Supplemented medium: peptone 72g, yeast extract 72g, the Sodium.alpha.-hydroxypropionate 100-200g of glycerine or 65-75%, NH
4Cl 2-3g, H
2O 1L.
Wherein, fermention medium (being the YTL substratum) and supplemented medium obtain for the present inventor improves on the basis of known substratum, do not see identical report at home and abroad.
Preferably, in the method for the present invention, stirring revolution in the step (3) is 400-600rpm, and pH is controlled at 6.5-7.5.
Preferably, in the method for the present invention, air in the gas mixture of the air/oxygen that feeds in the step (3): oxygen=1: 1-1: 10 (volume ratios).More preferably, at earlier fermentation (before referring to that dissolved oxygen is higher than 30%) bubbling air, the gas mixture of fermentation later stage (referring to that dissolved oxygen is higher than at 30% o'clock) bubbling air/oxygen, the control dissolved oxygen maintains 30%-100% all the time.
Preferably, in the method for the present invention, the flow acceleration control method of feed supplement is in the step (3): the controlling flow acceleration was 25ml/h in the 2-4 after feed supplement begins hour, and flow acceleration increased 25ml/h-100ml/h in every afterwards 2-4 hour.
In method of the present invention, inductor is selected from lactose in the step (4), and used lactose concn is 10g/L-30g/L.Inducing time of origin is fermentation beginning back 5-15 hour, induction time 6-16 hour.Adopt lactose to make inductor than using IPTG significantly to reduce production cost.
The present invention also provides the proteic engineering bacteria intestinal bacteria of plant height efficient expression HPV L1 (Escherichia coli) CGMCC1944.
(3) beneficial effect
The invention provides the substratum of improvement, this substratum can fully satisfy engineering bacteria nutritional requirement during the fermentation, and has effectively controlled the key parameter in the fermenting process.Adopt fermentation process of the present invention, not only the retention of expression plasmid has been brought up to more than 90% from 50%-60%, also make the proteic unit output of HPV L1 rise to the preceding 3-4 of improvement doubly, and effectively reduce production cost, significant to large-scale commercial production.
The proteic engineering bacteria intestinal bacteria of the HPV of efficiently expressing L1 of the present invention (Escherichiacoli) CGMCC1944 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on February 9th, 2007, be called for short CGMCC, deposit number is CGMCC No.1944.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
The high density fermentation of embodiment 1 HPV L1 albumen pronucleus expression
(1) seed activation: will express the proteic engineering strain intestinal bacteria of HPV L1 (Escherichiacoli) CGMCC1944 and on solid 2YT substratum, activate, promptly under 37 ℃ on the 2YT flat board streak culture 24 hours.Choose single bacterium colony then to 10ml YTL liquid nutrient medium, cultivated 10 hours down at 37 ℃; Again with the seed liquor that obtains by volume 5% inoculum size be seeded in the YTL substratum, cultivated 8 hours down at 37 ℃;
(2) inoculation: fill the YTL substratum in fermentor tank, the seed liquor that 5% inoculum size is good with above-mentioned activation by volume is seeded to liquid YTL substratum then;
(3) fermentation: bubbling air in fermentor tank, air flow 4L/min; 37 ℃ of leavening temperatures stir revolution 800rpm, and dissolved oxygen is controlled at 30%, and pH is controlled at 6.2, fermentation time 20 hours; Work as OD
600nmBegan feed supplement at=2 o'clock, the flow acceleration of feed supplement is 25ml/h.Used substratum is seen the record of technical scheme part.
(4) be the proteic expression of lactose-induced HPV L1 of 15g/L with concentration, and carry out protein purification (concrete grammar is referring to ZL02129070.9), obtain target protein 9.3mg/L nutrient solution.
The high density fermentation of embodiment 2 HPV L1 albumen pronucleus expressions
(1) seed activation: will express the proteic engineering strain intestinal bacteria of HPV L1 (Escherichiacoli) CGMCC1944 and on solid 2YT substratum, activate, promptly under 30 ℃ on the 2YT flat board streak culture 24 hours.Choose single bacterium colony then to 10ml YTL liquid nutrient medium, cultivated 12 hours down at 30 ℃; Again with the seed liquor that obtains by volume 5% inoculum size be seeded in the YTL substratum, cultivated 10 hours down at 37 ℃;
(2) inoculation: fill the YTL substratum in fermentor tank, the seed liquor that 5% inoculum size is good with above-mentioned activation by volume is seeded to liquid YTL substratum then;
(3) fermentation: bubbling air dissolved oxygen is higher than 30% before the, (air in the gas mixture by volume: oxygen=1: 1), control dissolved oxygen at 30%-100%, air flow 6L/min of the gas mixture of bubbling air/oxygen when dissolved oxygen is higher than 30%; 37 ℃ of leavening temperatures stir revolution 400rpm, and pH is controlled at 6.5, fermentation time 24 hours; Work as OD
600nm=2.5 o'clock begin feed supplement, and the controlling flow acceleration was 25ml/h in the 2-4 after feed supplement begins hour, and flow acceleration increased 25ml/h in per afterwards 2 hours.Used substratum is seen the record of technical scheme part.
(4) be the proteic expression of lactose-induced HPV L1 of 10g/L with concentration, inducing time of origin is back 5 hours of fermentation beginning, induction time 16 hours.Carry out protein purification (concrete grammar is referring to ZL02129070.9) then, obtain target protein 15.2mg/L nutrient solution.
The high density fermentation of embodiment 3HPV L1 albumen pronucleus expression
(1) seed activation: will express the proteic engineering strain intestinal bacteria of HPV L1 (Escherichiacoli) CGMCC1944 and on solid 2YT substratum, activate, promptly under 37 ℃ on the 2YT flat board streak culture 24 hours.Choose single bacterium colony then to 10ml YTL liquid nutrient medium, cultivated 10 hours down at 30 ℃; Again with the seed liquor that obtains by volume 5% inoculum size be seeded in the YTL substratum, cultivated 12 hours down at 30 ℃;
(2) inoculation: fill the YTL substratum in fermentor tank, the seed liquor that 10% inoculum size is good with above-mentioned activation by volume is seeded to liquid YTL substratum then;
(3) fermentation: bubbling air dissolved oxygen is higher than 30% before the, (air in the gas mixture by volume: oxygen=1: 10), control dissolved oxygen at 30%-100%, air flow 8L/min of the gas mixture of bubbling air/oxygen when dissolved oxygen is higher than 30%; 30 ℃ of leavening temperatures stir revolution 600rpm, and pH is controlled at 7.5, fermentation time 26 hours; Work as OD
600nm=4 o'clock begin feed supplement, and 3 hours controlling flow acceleration after feed supplement begins are 25ml/h, and flow acceleration increased 100ml/h in per afterwards 4 hours.Used substratum is seen the record of technical scheme part.
(4) be the proteic expression of lactose-induced HPV L1 of 30g/L with concentration, inducing time of origin is back 15 hours of fermentation beginning, induction time 6 hours.Carry out protein purification (concrete grammar is referring to ZL02129070.9) then, obtain target protein 13.1mg/L nutrient solution.
The high density fermentation of embodiment 4HPV L1 albumen pronucleus expression
(1) seed activation: will express the proteic engineering strain intestinal bacteria of HPV L1 (Escherichiacoli) CGMCC1944 and on solid 2YT substratum, activate, promptly under 25 ℃ on the 2YT flat board streak culture 36 hours.Choose single bacterium colony then to 10ml YTL liquid nutrient medium, cultivated 12 hours down at 25 ℃; Again with the seed liquor that obtains by volume 5% inoculum size be seeded in the YTL substratum, cultivated 12 hours down at 25 ℃;
(2) inoculation: fill the YTL substratum in fermentor tank, the seed liquor that 5% inoculum size is good with above-mentioned activation by volume is seeded to liquid YTL substratum then;
(3) fermentation: (air in the gas mixture by volume: oxygen=1: 6), the control dissolved oxygen is at 30%-100%, air flow 5L/min for the gas mixture of bubbling air/oxygen; 25 ℃ of leavening temperatures stir revolution 200rpm, and pH is controlled at 7.8, fermentation time 30 hours; Work as OD
600nm=2 o'clock begin feed supplement, and 2 hours controlling flow acceleration after feed supplement begins are 25ml/h, and flow acceleration increased 50ml/h in per afterwards 2 hours.
Used substratum is seen the record of technical scheme part.
(4) be the proteic expression of lactose-induced HPV L1 of 20g/L with concentration, inducing time of origin is back 10 hours of fermentation beginning, induction time 10 hours.Carry out protein purification (concrete grammar is referring to ZL02129070.9) then, obtain target protein 10.5mg/L nutrient solution.
The high density fermentation of embodiment 5 HPV L1 albumen pronucleus expressions
(1) seed activation: will express the proteic engineering strain intestinal bacteria of HPV L1 (Escherichiacoli) CGMCC1944 and on solid 2YT substratum, activate, promptly under 30 ℃ on the 2YT flat board streak culture 24 hours.Choose single bacterium colony then to 10ml YTL liquid nutrient medium, cultivated 9 hours down at 37 ℃; Again with the seed liquor that obtains by volume 5% inoculum size be seeded in the YTL substratum, cultivated 10 hours down at 37 ℃;
(2) inoculation: fill the YTL substratum in fermentor tank, the seed liquor that 10% inoculum size is good with above-mentioned activation by volume is seeded to liquid YTL substratum then;
(3) fermentation: (air in the gas mixture by volume: oxygen=1: 8), the control dissolved oxygen is at 30%-100%, air flow 7L/min for the gas mixture of bubbling air/oxygen; 37 ℃ of leavening temperatures stir revolution 300rpm, and pH is controlled at 7.0, fermentation time 25 hours; Work as OD
600nm=3 o'clock begin feed supplement, and the controlling flow acceleration was 25ml/h in the 2-3 after feed supplement begins hour, and flow acceleration increased 30ml/h in per afterwards 4 hours.Used substratum is seen the record of technical scheme part.
(4) be the proteic expression of lactose-induced HPV L1 of 25g/L with concentration, inducing time of origin is back 6 hours of fermentation beginning, induction time 12 hours.Carry out protein purification (concrete grammar is referring to ZL02129070.9) then, obtain target protein 12.6mg/L nutrient solution.
Effect before and after embodiment 6 fermention mediums change relatively
Used detection method is the known routine techniques of art technology in the present embodiment, and concrete operations can be referring to " molecular cloning " (publish in August, 2002 for the third edition, Science Press) and instrument specification sheets.
| Before fermention medium changes | After fermention medium changes | |
| The expression plasmid retention | 50％-60％ | More than 90% |
| Target protein output | The 3-4mg/L nutrient solution | The 9-15mg/L nutrient solution |
| Fermentation costs (unit/mg target protein) | 30.00～40.00 yuan | 3.00～6.00 yuan |
As can be seen from the above table, the present invention improve substratum and substitute IPTG with lactose make inductor after, effectively reduce production cost, and improved the output of target protein and the retention of expression plasmid.
Claims (10)
1. the fermentation process in high density of a HPV L1 albumen pronucleus expression is characterized in that it comprises the steps:
(1) seed activation: will express the proteic engineering strain intestinal bacteria of HPV L1 (Escherichiacoli) CGMCC1944 and on solid 2YT substratum, activate, and choose single bacterium colony then to 10ml YTL liquid nutrient medium, and cultivate 24-36 hour down at 25-37 ℃; Again with the seed liquor that obtains by volume 5% inoculum size be seeded in the YTL substratum, cultivated 8-12 hour down at 30-37 ℃;
(2) inoculation: fill the YTL substratum in fermentor tank, the seed liquor that the inoculum size of 5%-10% is good with above-mentioned activation by volume is seeded to liquid YTL substratum then;
(3) fermentation: the gas mixture of bubbling air or air/oxygen in fermentor tank, air flow 4-8L/min; Leavening temperature 25-37 ℃, stir revolution 200-800rpm, dissolved oxygen is controlled at 30%-100%, and pH is controlled at 6.2-7.8, fermentation time 20-30 hour; Work as OD
600nmBegin feed supplement during=2-4, the flow acceleration of feed supplement is 25-400ml/h.
(4) induce the proteic expression of HPV L1, and carry out protein purification, obtain target protein.
2. method according to claim 1 is characterized in that used substratum consists of:
2YT: tryptone 16g, yeast extract 10g, NaCl 5g, H
2O 1L, silicon oil foam killer 0.2ml;
YTL:Na
2HPO
4.12H
2O 6-10g, KH
2PO
41-3g, tryptone 7-10g, yeast extract 3-6g, the Sodium.alpha.-hydroxypropionate 10-20g of 65-75%, MgSO
40.4-0.8g, NH
4Cl 2-3g, H
2O 1L, nitric acid defoamer 0.2ml, wherein Na
2HPO
4.12H
2O and KH
2PO
4Be dissolved in the 200mL water, separately sterilization.
Supplemented medium: peptone 72g, yeast extract 72g, the Sodium.alpha.-hydroxypropionate 100-200g of 65-75%, NH4Cl 10-30g, H2O 1L.
3. method according to claim 1 is characterized in that stirring revolution in the step (3) is 400-600rpm.
4. method according to claim 1 is characterized in that pH is controlled at 6.5-7.5 in the step (3).
5. method according to claim 1 is characterized in that air in the gas mixture of the air/oxygen that feeds in the step (3): oxygen=1: 1-1: 10.
6. method according to claim 5 is characterized in that the earlier fermentation bubbling air, the gas mixture of fermentation later stage bubbling air/oxygen.
7. method according to claim 1, it is characterized in that the flow acceleration control method of feed supplement in the step (3) is: the controlling flow acceleration was 25ml/h in the 2-4 after feed supplement begins hour, flow acceleration increased 25ml/h-100ml/h in every afterwards 2-4 hour.
8. method according to claim 1 is characterized in that inductor is selected from lactose in the step (4).
9. method according to claim 8, the concentration that it is characterized in that used lactose in the step (4) is 10g/L-30g/L.
10. the proteic engineering bacteria intestinal bacteria of plant height efficient expression HPV L1 (Escherichia coli) CGMCC1944.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100902388A CN101139570A (en) | 2007-04-16 | 2007-04-16 | High-density fermentation method for HPV L1 protein prokaryotic expression |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007100902388A CN101139570A (en) | 2007-04-16 | 2007-04-16 | High-density fermentation method for HPV L1 protein prokaryotic expression |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101139570A true CN101139570A (en) | 2008-03-12 |
Family
ID=39191673
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007100902388A Pending CN101139570A (en) | 2007-04-16 | 2007-04-16 | High-density fermentation method for HPV L1 protein prokaryotic expression |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101139570A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101845453A (en) * | 2010-05-11 | 2010-09-29 | 浙江普康生物技术股份有限公司 | Optimized expression method of type 16 human papillomavirus L2E7 genes in colon bacillus |
| CN102643888A (en) * | 2012-04-27 | 2012-08-22 | 天津生机集团股份有限公司 | High density fermentation method for prokaryotic expression of chicken interferon alpha |
| CN104293668A (en) * | 2013-07-17 | 2015-01-21 | 南京朗恩生物科技有限公司 | Recombinant escherichia coli high-density fermentation method |
| CN105543144A (en) * | 2016-02-02 | 2016-05-04 | 武汉爱博泰克生物科技有限公司 | Culture medium of Escherichia coli suitable for expressing crybb2 antigen protein, and fermentation method and application thereof |
| US9994618B2 (en) | 2012-07-30 | 2018-06-12 | Hong-Jin Kim | High efficiency method for purifying human papillomavirus virus-like particles |
| CN114045251A (en) * | 2021-12-02 | 2022-02-15 | 四川百诺吉科技有限公司 | High-density fermentation medium for genetically engineered escherichia coli and fermentation process thereof |
-
2007
- 2007-04-16 CN CNA2007100902388A patent/CN101139570A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101845453A (en) * | 2010-05-11 | 2010-09-29 | 浙江普康生物技术股份有限公司 | Optimized expression method of type 16 human papillomavirus L2E7 genes in colon bacillus |
| CN101845453B (en) * | 2010-05-11 | 2012-12-26 | 浙江普康生物技术股份有限公司 | Optimized expression method of type 16 human papillomavirus L2E7 genes in colon bacillus |
| CN102643888A (en) * | 2012-04-27 | 2012-08-22 | 天津生机集团股份有限公司 | High density fermentation method for prokaryotic expression of chicken interferon alpha |
| CN102643888B (en) * | 2012-04-27 | 2015-07-01 | 天津生机集团股份有限公司 | High density fermentation method for prokaryotic expression of chicken interferon alpha |
| US9994618B2 (en) | 2012-07-30 | 2018-06-12 | Hong-Jin Kim | High efficiency method for purifying human papillomavirus virus-like particles |
| CN104507956B (en) * | 2012-07-30 | 2018-11-06 | 普瓦丝株式会社 | The process for effectively purifying of human papilloma virus virus-like particle |
| CN104293668A (en) * | 2013-07-17 | 2015-01-21 | 南京朗恩生物科技有限公司 | Recombinant escherichia coli high-density fermentation method |
| CN105543144A (en) * | 2016-02-02 | 2016-05-04 | 武汉爱博泰克生物科技有限公司 | Culture medium of Escherichia coli suitable for expressing crybb2 antigen protein, and fermentation method and application thereof |
| CN114045251A (en) * | 2021-12-02 | 2022-02-15 | 四川百诺吉科技有限公司 | High-density fermentation medium for genetically engineered escherichia coli and fermentation process thereof |
| CN114045251B (en) * | 2021-12-02 | 2022-08-02 | 四川百诺吉科技有限公司 | High-density fermentation medium for genetically engineered escherichia coli and fermentation process thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103881954B (en) | Gamma-polyglutamic acid-genetic engineering bacterium and high yield gamma-polyglutamic acid-method thereof are produced in one strain | |
| CN101139570A (en) | High-density fermentation method for HPV L1 protein prokaryotic expression | |
| CN104561180B (en) | A kind of culture medium and feed process for being mutated Avid kyowamycin fermenting and producing doractin | |
| CN103740629B (en) | The genetically engineered acetic bacteria of overexpression coenzyme PQQ synthetic proteins and application thereof | |
| CN107988291A (en) | The methanol feeding zymotechnique of Pichia anomala expression recombination human source collagen | |
| CN102268466A (en) | Method for fermentation production of lipstatin and culture medium components thereof | |
| CN102925502A (en) | Industry method for producing arachidonic acid grease by using mortierella alpine | |
| CN105062947A (en) | Production method for bacillus licheniformis with high sporation rate | |
| CN103882081B (en) | A kind of Continuous Flow adds the method that fed-batch fermentation raising bacitracin is tired | |
| CN103898181A (en) | Method for producing nosiheptide by virtue of fermentation | |
| CN101182499B (en) | Method for preparing phytase taking glycerol as carbon source | |
| CN102586381A (en) | Production process for improving fermentative strength of 2-keto-L-gulonic acid | |
| CN102660596B (en) | Medium of producing pleuromutilin by fermenting Pleurotus sp and its fermentation method | |
| CN102093990B (en) | Method for producing low temperature amylases through microbial fermentation | |
| CN104560743B (en) | A kind of pichia farinose high cell density fermentation method | |
| CN105420265A (en) | Genetic engineering acetic bacteria for over-expressing ATP (Adenosine Triphosphate) enzyme as well as construction method and application thereof | |
| CN107201383B (en) | D-lactic acid production method capable of improving production strength of D-lactic acid | |
| CN102080031A (en) | Production method of beer drink | |
| CN105316371B (en) | A method of for improving tryptophan fermentation yield | |
| CN105779328A (en) | Method for culturing clostridium thermocellum | |
| CN104745545B (en) | A kind of method of efficiently production L dglutamic oxidases | |
| CN106148255B (en) | Engineering bacteria lacking organic acid production way and application thereof in co-production of 1, 3-propylene glycol, 2, 3-butanediol and ethanol | |
| CN103627775B (en) | Improve the method for KGA fermentation production efficiency | |
| CN101580823A (en) | Method for obtaining lipase by continuous fermentation or semi-continuous fermentation | |
| CN103966273B (en) | A kind of dino flagellate fermenting and producing DHA method |
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: 20080312 |