CN1737117A - Batch feeding method for cultivating bacillus coli - Google Patents
Batch feeding method for cultivating bacillus coli Download PDFInfo
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- CN1737117A CN1737117A CN 200510028037 CN200510028037A CN1737117A CN 1737117 A CN1737117 A CN 1737117A CN 200510028037 CN200510028037 CN 200510028037 CN 200510028037 A CN200510028037 A CN 200510028037A CN 1737117 A CN1737117 A CN 1737117A
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Abstract
The invention discloses a process for cultivating bacillus coli through batched feeding, wherein the fermentation procedure comprises five stages, in each stage, culture media with different carbon-nitrogen ratio are charged, thus realizing the equalization of real-time charge of carbon source and nitrogen source. The invention can realize a high thallus density.
Description
Technical field
The present invention relates to the colibacillary method of fermentation culture, relate in particular to batch feeding and cultivate colibacillary method.
Technical background
Trail protein, be that (tumor necrosis factor-related apoptosis-inducing ligand TRAIL), is one of TNF family member who finds recently to tumor necrosin relative death inducing ligand, claim again apo 2 ligand (apoptotin 2 ligand, Apo-2L).TRAIL is inducing apoptosis of tumour cell and insensitive to normal tissue cell optionally, therefore has good prospect in tumor treatment.
The nutritive ingredient that colibacillary genetic background is clear, growth velocity is fast, required is simple, is the most frequently used host bacterium of exogenous protein expression therefore.It is the essential method that realizes intestinal bacteria high-density culture and high expression level TRAIL that the stream of carbon source and nitrogenous source adds.Feed supplement stream at existing carbon source and nitrogenous source adds in the technology, because in the fermenting process, especially ferment the middle and later periods, thalline is bigger to the demand of carbon source, therefore the stream rate of acceleration of carbon source and stream add the emphasis that quality is consideration, but it also is very important that the stream of nitrogenous source adds, but also will consider carbon source and the different proportion relations of nitrogenous source at the different times of thalli growth.If ignored this point, for example, if it is too much to add carbon source, it is low to tend to cell density to occur, and the soluble proteins expression amount is few, and the big shortcoming of acetate accumulation volume, as Paalme T etc. at Biotech.Bioeng, 1990,35:312-319 and Shen Ya-Ling etc. are at Biotech.Lett.2004, the report of 26:981-984.And if it is too much to add nitrogenous source, it is too much often can to occur thalline self-dissolving, poor growth, fermented liquid foam again, shortcoming such as the exogenous protein expression amount of unit fermented liquid is low.
Intestinal bacteria are that change rather than unalterable to the demand of carbon source and nitrogenous source in the process of high density fermentation.For example, at the fermentation initial stage, intestinal bacteria often need more nitrogenous source, and excessive carbon source can suppress the growth of thalline on the contrary, and can produce a large amount of acetate.Ferment middle because bacterial metabolism is vigorous, needs more carbon source (glucose) in the body, with the growth of keeping thalline and the metabolism needs of energy, and more too to the demand of nitrogenous source.In the fermentation later stage, the part thalline begins self-dissolving, and thalline reduces the demand of nitrogenous source, and to the Requirement Increases of carbon source.Therefore, in the thalli growth stage of fermenting process, along with the growth of cell concentration, the required carbon-nitrogen ratio of thalline presents more and more higher trend.And the temperature-induced phase, owing to will produce foreign protein, thalline needs more nitrogenous source (being mainly each seed amino acid), also needs a certain amount of carbon source (glucose) in order to keep energy metabolism and thalli growth simultaneously.
Therefore, this area presses for the different times according to fermenting process, real-time monitoring carbon, nitrogenous source equilibrated feed supplement cultural method.
Summary of the invention
The purpose of this invention is to provide a kind of different times according to thalli growth, stream adds the batch feeding cultural method of the substratum of different carbon-nitrogen ratios, can not realize that to overcome prior art the real-time stream of carbon nitrogen source adds the equilibrated deficiency.
Design of the present invention is such, and according to the different times of thalli growth in the fermenting process, stream adds the carbon source and the nitrogenous source of different carbon-nitrogen ratios, rise along with the prolongation of incubation time in yeast culture stage carbon-nitrogen ratio, and the amplitude that raises increases gradually; Then be adjusted to than low value rapidly inductive phase, thereby the real-time stream of realizing carbon nitrogen source adds balance, finally obtains the foreign protein of highdensity thalline and high expression level.
The disclosed batch feeding of technical scheme of the present invention is cultivated colibacillary method, fermenting process is divided into five stages, each stage incubation time is 4-7 hour, the carbon-nitrogen ratio of each stage substratum, that is: the mass ratio of glucose/(peptone+yeast powder) is followed successively by 0.75-0.9,1-1.2,2.4-2.6,4.5-5.4,0.9-1.2
With volume ratio (seed liquor/fermented liquid) is 2～7% inoculations, and since the feed supplement of the 2nd stage, the feed supplement flow acceleration makes the ratio growth rate of thalli growth at 0.05-0.18h
-1Between; Whole fermentation process, dissolved oxygen maintain between the 20-30%, and the pH value is 6.8～7.5;
Wherein preceding four-stage, carbon-nitrogen ratio raises gradually, and the amplitude that raises increases gradually; In the 5th stage, adjust carbon-nitrogen ratio and fall after rise to 0.9-1.2.
The timed interval of each fermentation stage is:
(1) the batch culture stage time begins to count with inoculation, (OD till fermented 5-7 hour
600Value is between the 6-11);
(2) the feed supplement initial stage began to count with postvaccinal 5-7 hour, (OD till fermented 12-14 hour
600Value is between the 20-28);
(3) begin to count with postvaccinal 12-14 hour feed supplement mid-term, (OD till fermented 18-20 hour
600Value is between the 40-55);
(4) the feed supplement later stage began to count with postvaccinal 18-20 hour, (OD till fermented 23-25 hour
600Value is between the 70-90);
(5) 42 ℃ of temperature-induced phases began to count with postvaccinal 24-26 hour, (OD till fermented 28-30 hour
600Between 85-110).
In a preferred scheme of the present invention, the mass ratio of glucose/(peptone+yeast powder) is followed successively by 0.88,1.13,2.51,5,1.
In a preferred scheme of the present invention,, the composition and the ratio of substratum are as follows:
Peptone 10
Glucose X
NaH
2PO
4·12H
2O 2
Na
2HPO
4·3H
2O 7
K
2HPO
4·3H
2O 4
ZnSO
4·7H
2O 0.575
Wherein the ratio of various components is ratio of quality and the number of copies, and for example, when NaCl is 1 part, then yeast powder is 5 parts, and peptone is 10 parts; X represents the umber of glucose, is followed successively by at the concrete numerical value of five fermentation culture stage: 11.25-13.5,15-18,36-39,67.5-81,13.5-18.
In a preferred scheme of the present invention, be followed successively by 13.2,17,37.65,75,15 at the umber of five fermentation culture stage glucose.
In preferred scheme of the present invention, the 1-4 stage is the thalli growth stage, and the ratio growth rate of thalline is 0.1-0.18h
-1Between.
In preferred scheme of the present invention, the 5th stage was the abduction delivering stage, and the ratio growth rate of thalline is 0.05-0.1h
-1Between.
In a preferred scheme of the present invention, intestinal bacteria are selected for use and have expression plasmid pBV220-TRAIL intestinal bacteria C600, and the temperature in thalli growth stage is 30 ℃, and the temperature in abduction delivering stage is 42 ℃; Wherein TRAIL refers to tumor necrosin relative death inducing ligand (tumor necrosis factor-relatedapoptosis-inducing ligand, TRAIL) gene.
Compared with prior art, batch feeding cultural method characteristics of the present invention are as follows:
(1) meticulous segmentation regulation and control are tactful.With the fermenting process of escherichia coli expression trail protein meticulous be divided into five feed supplement stages, be respectively batch culture stage, feed supplement initial stage, feed supplement mid-term, feed supplement later stage and inductive phase.Such segmentation control methods make the fermentation condition in each stage adapt with the actual needs of thalline as much as possible, help the growth of thalline and efficiently expressing of foreign protein greatly.
(2) Du Te feed supplement mode.By adjusting and optimize the carbon-nitrogen ratio in each feed supplement stage, five feed supplement stages take different carbon-nitrogen ratios to carry out stream to add, along with the prolongation of incubation time, carbon-nitrogen ratio raises gradually, and the amplitude that raises increases gradually, last inductive phase, carbon-nitrogen ratio is fallen after rise to 0.9-1.2.This feed supplement mode can effectively be avoided owing to the excessive acetate accumulation that causes of certain stage nitrogenous source of fermentation and avoids because the excessive thalline autolysis that causes of certain stage nitrogenous source that ferments; And carbon-nitrogen ratio is fallen after rise to 0.9-1.2, then help the expression of solubility foreign protein TRAIL.
By segmentation regulation and control and feed supplement respectively, the ratio of soluble TRAIL protein expression amount of the present invention in total protein can be up to 6.7%, and trail protein content can reach 1.7g/L.
Description of drawings
Fig. 1 is the variation diagram of the carbon-nitrogen ratio (mass ratio) of each fermentation stage, and wherein C/N refers to carbon-nitrogen ratio, both glucose quality/(peptone+yeast powder) mass ratio.
Embodiment
The structure of expression plasmid pBV220-TRAIL is seen Chinese patent CN 02110880.3 among the present invention, and expression vector pBV220-TRAIL is CN 02110880.3 disclosed pBV-Apo-2L I
100PBV220-TRAIL transforms CaCl routinely
2Conversion method is transformed among the host bacterium C600, has obtained reorganization bacterium C600-TRAIL, and working method is seen " molecular cloning " (Science Press, second edition, 2002).
Peptone, yeast powder are OXOID company product, and glucose and other reagent are homemade analytical reagent.
The present invention is further illustrated below by embodiment, its objective is content for a better understanding of the present invention, and therefore, cited example does not limit protection scope of the present invention.
Initial fermentation volume 1.5L, the initial medium of employing (g/L) composed as follows [glucose: (peptone+yeast powder)=0.88: 1 (mass ratio)]:
Peptone 10
Glucose 13.23
NaH
2PO
4·12H
2O 2
Na
2HPO
4·3H
2O 7
K
2HPO
4·3H
2O 4
ZnSO
4·7H
2O 0.575
Each constituent concentration of feed supplement liquid at feed supplement initial stage [glucose: (peptone+yeast powder)=1.13: 1 (mass ratio)] is:
Peptone 100g/L
Yeast powder 50g/L
Glucose 170g/L
Each constituent concentration of feed supplement liquid [glucose: (peptone+yeast powder)=2.51: 1 (mass ratio)] in feed supplement mid-term is:
Peptone 200g/L
Yeast powder 100g/L
Glucose 753g/L
Each constituent concentration of feed supplement liquid in feed supplement later stage [glucose: (peptone+yeast powder)=5: 1 (mass ratio)] is:
Peptone 100g/L
Yeast powder 50g/L
Glucose 753g/L
Each constituent concentration of feed supplement liquid of 42 ℃ of temperature-induced phases [glucose: (peptone+yeast powder)=1: 1 (mass ratio)] is:
Peptone 100g/L
Yeast powder 50g/L
Glucose 150g/L
Insert the intestinal bacteria seed liquor with the inoculum size of 5% (volume ratio), 30 ℃, pH7.0 batch culture 7 hours are to OD
600Be 11.
Carry out the initial stage feed supplement then, the control feed rate makes that the specific growth rate of thalline is 0.13h
-1, be cultured to the 14th hour in the whole fermentation process, OD
600Be 28 o'clock, stop the initial stage feed supplement.
Change the feed supplement in mid-term into, the control feed rate makes that the specific growth rate of thalline is 0.11h
-1, be cultured to the 20th hour in the whole fermentation process, OD
600Be 55 o'clock, stop the feed supplement in mid-term.
Change the later stage feed supplement into, the control feed rate makes that the specific growth rate of thalline is 0.10h
-1, be cultured to the 25th hour in the whole fermentation process, OD
600Be 90 o'clock, stop the later stage feed supplement.
Change feed supplement inductive phase into, temperature is risen 42 ℃, the specific growth rate of thalline is controlled to be 0.05h
-1, induce 4 hours after, OD
600Be 110 o'clock, fermentation ends.
Whole fermentation process control dissolved oxygen is between 20-30%.Soluble TRAIL protein accounted for 6.7% of solubility total protein after fermentation finished, and content is 1.7g/L.Acetic acid content is 0.
The stream that uses adds the carbon-nitrogen ratio strategy and sees Fig. 1.
Embodiment 2
Initial fermentation volume 1.5L, the initial medium of employing (g/L) composed as follows [glucose: (peptone ten yeast powders)=0.8: the 1:(mass ratio)]:
Glucose 12
NaH
2PO
4·12H
2O 2
Na
2HPO
4·3H
2O 7
K
2HPO
4·3H
2O 4
ZnSO
4·7H
2O 0.575
Each constituent concentration of feed supplement liquid at feed supplement initial stage [glucose: (peptone+yeast powder)=1.07: 1 (mass ratio)] is:
Peptone 100g/L
Yeast powder 50g/L
Glucose 160g/L
Each constituent concentration of feed supplement liquid [glucose: (peptone+yeast powder)=2.47: 1 (mass ratio)] in feed supplement mid-term is:
Peptone 100g/L
Yeast powder 50g/L
Glucose 370g/L
Each constituent concentration of feed supplement liquid in feed supplement later stage [glucose: (peptone+yeast powder)=5.2: 1 (mass ratio)] is:
Peptone 100g/L
Yeast powder 50g/L
Glucose 780g/L
Each constituent concentration of feed supplement liquid of 42 ℃ of temperature-induced phases [glucose: (peptone+yeast powder)=1.1: 1 (mass ratio)] is:
Peptone 100g/L
Yeast powder 50g/L
Glucose 165g/L
Insert the intestinal bacteria seed liquor with the inoculum size of 5% (volume ratio), 30 ℃, pH7.0 batch culture 7 hours are to OD
600Be 8.
Carry out the initial stage feed supplement then, the control feed rate makes that the specific growth rate of thalline is 0.17h
-1, be cultured to the 13rd hour in the whole fermentation process, OD
600Be 22 o'clock, stop the initial stage feed supplement.
Change the feed supplement in mid-term into, the control feed rate makes that the specific growth rate of thalline is 0.12h
-1, be cultured to the 18th hour in the whole fermentation process, OD
600Be 40 o'clock, stop the feed supplement in mid-term.
Change the later stage feed supplement into, the control feed rate makes that the specific growth rate of thalline is 0.12h
-1, be cultured to the 23rd hour in the whole fermentation process, OD
600Be 74 o'clock, stop the later stage feed supplement.
Change feed supplement inductive phase into, temperature is risen to 42 ℃, the specific growth rate of thalline is controlled to be 0.06h
-1, induced 4 as a child, OD
600Be 94 o'clock, fermentation ends.
Whole fermentation process control dissolved oxygen is between 25%.Soluble TRAIL protein accounted for 6.0% of solubility total protein after fermentation finished, and content is 1.5g/L.Acetic acid content is 0.
Embodiment 3
Adopt traditional zymotic feed supplement method, purpose is to compare with example 1 and example 2, thereby finds out that the said stream of the present invention adds the advantage of strategy.
Initial fermentation volume 2L, the initial medium of employing (g/L) composed as follows:
NaH
2PO
4·12H
2O 2
Na
2HPO
4·3H
2O 7
K
2HPO
4·3H
2O 4
ZnSO
4·7H
2O 0.575
Each constituent concentration of feed supplement liquid is:
Feed supplement 1: glucose, 170g/L
Feed supplement 2: peptone, 100g/L; Yeast powder, 50g/L
At first, insert the intestinal bacteria seed liquor with the inoculum size of 5% (volume ratio), 30 ℃, pH7.0 batch culture 7 hours are to OD
600Be 6.8.
Carry out feed supplement then, to mend glucose (feed supplement 1), the specific growth rate of control thalline is 0.15h
-1Peptone and yeast powder adopt constant speed feed supplement (feed supplement 2) in whole feed supplement process, feed rate is: peptone 3.5 Grams Per Hours, yeast powder 1.75 Grams Per Hours.Cultivate and after 23 hours temperature is risen to 42 ℃, induce 4 hours OD
600Be 80 o'clock, fermentation ends.
Whole fermentation process control dissolved oxygen is between 20-30%.Soluble TRAIL protein accounted for 4.2% of solubility total protein after fermentation finished, and content is 0.98g/L.Acetic acid content is 4.6g/L.
Claims (7)
1, a kind of batch feeding is cultivated colibacillary method, it is characterized in that, fermenting process is divided into five stages, each stage incubation time is 4-7 hour, the carbon-nitrogen ratio of each stage substratum, that is: the mass ratio of glucose/(peptone+yeast powder) is followed successively by 0.75-0.9,1-1.2,2.4-2.6,4.5-5.4,0.9-1.2;
Wherein initial inoculum size is 2-7% by volume, begins feed supplement from subordinate phase, and the feed supplement flow acceleration makes the ratio growth rate of thalli growth at 0.05-0.18h
-1Between; The whole fermentation process dissolved oxygen maintains between the 20-30%, and the pH value is 6.8-7.5.
2, the method for claim 1 is characterized in that, the carbon-nitrogen ratio of five stage substratum of fermenting process is: 0.88,1.13,2.51,5,1.
3, the method for claim 1 is characterized in that, the composition and the ratio of substratum are as follows:
Peptone 10
Yeast powder 5
NaCl 1
Glucose X
NaH
2PO
4·12H
2O 2
Na
2HPO
4·3H
2O 7
K
2HPO
4·3H
2O 4
ZnSO
4·7H
2O 0.575
MgSO
4 1
Wherein the ratio of various components is ratio of quality and the number of copies; X represents the umber of glucose, is followed successively by at the concrete numerical value of five fermentation culture stage: 11.25-13.5,15-18,36-39,67.5-81,13.5-18.
4, method as claimed in claim 3 is characterized in that, is followed successively by 13.2,17,37.65,75,15 at the umber of five fermentation culture stage glucose.
5, method as claimed in claim 3 is characterized in that, the 1-4 stage of fermenting process is the thalli growth stage, and the ratio growth rate of thalline is at 0.1-0.18h
-1Between.
6, method as claimed in claim 3 is characterized in that, the 5th stage of fermenting process is the abduction delivering stage, and the ratio growth rate of thalline is at 0.05-0.1h
-1Between.
As each the described method among the claim 1-6, it is characterized in that 7, said intestinal bacteria are selected for use and have expression plasmid pBV220-TRAIL intestinal bacteria C600, the temperature in thalli growth stage is 30 ℃, and the temperature in abduction delivering stage is 42 ℃.
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|---|---|---|---|
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| CN1318572C CN1318572C (en) | 2007-05-30 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104160016A (en) * | 2012-03-12 | 2014-11-19 | 韩美科学株式会社 | Method of culturing e. coli cells for high density |
| CN106442685A (en) * | 2016-10-25 | 2017-02-22 | 山东大学 | Method for rapidly and inexpensively screening protein expression conditions |
| CN109053867A (en) * | 2018-08-24 | 2018-12-21 | 艾美汉信疫苗（大连）有限公司 | A kind of cultural method of high density fermentation expression recombinant rotavirus Δ VP8* antigen |
| CN110777100A (en) * | 2019-11-29 | 2020-02-11 | 宁波酶赛生物工程有限公司 | Escherichia coli fermentation method |
| CN114672531A (en) * | 2020-12-24 | 2022-06-28 | 江苏万邦医药科技有限公司 | Method for improving escherichia coli protein expression quantity through stage dissolved oxygen control |
-
2005
- 2005-07-22 CN CNB2005100280376A patent/CN1318572C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104160016A (en) * | 2012-03-12 | 2014-11-19 | 韩美科学株式会社 | Method of culturing e. coli cells for high density |
| CN106442685A (en) * | 2016-10-25 | 2017-02-22 | 山东大学 | Method for rapidly and inexpensively screening protein expression conditions |
| CN109053867A (en) * | 2018-08-24 | 2018-12-21 | 艾美汉信疫苗（大连）有限公司 | A kind of cultural method of high density fermentation expression recombinant rotavirus Δ VP8* antigen |
| CN109053867B (en) * | 2018-08-24 | 2022-04-12 | 艾美诚信生物制药有限公司 | Culture method for high-density fermentation expression of recombinant rotavirus delta VP8 antigen |
| CN110777100A (en) * | 2019-11-29 | 2020-02-11 | 宁波酶赛生物工程有限公司 | Escherichia coli fermentation method |
| CN114672531A (en) * | 2020-12-24 | 2022-06-28 | 江苏万邦医药科技有限公司 | Method for improving escherichia coli protein expression quantity through stage dissolved oxygen control |
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| Publication number | Publication date |
|---|---|
| CN1318572C (en) | 2007-05-30 |
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