CN101029323B - Inter mass optimization during insulin precursor fermentation - Google Patents
Inter mass optimization during insulin precursor fermentation Download PDFInfo
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- CN101029323B CN101029323B CN2007100266838A CN200710026683A CN101029323B CN 101029323 B CN101029323 B CN 101029323B CN 2007100266838 A CN2007100266838 A CN 2007100266838A CN 200710026683 A CN200710026683 A CN 200710026683A CN 101029323 B CN101029323 B CN 101029323B
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- methyl alcohol
- acceleration
- time
- rate
- cell density
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Abstract
A method for supplementing and optimizing feed during fermentation of insulin precursor is carried out by supplementing methanol to determine its fluid acceleration ratio, computing while adjusting for specific growth speed ration by difference of final fermented strain density and real-time strain density and fermentation period and controlling growth speed ratio. It has more protein concentration.
Description
Technical field
The invention belongs to a kind of method of producing Regular Insulin, particularly the method for yeast fermentation production reorganization pork insulin precursor process.
Background technology
Insulin precurosor is a proinsulin, and molecular weight is about 9000, contains 86 amino acid, is a single chain polypeptide.Regular Insulin is to contain 51 amino acid whose small proteins, molecular weight about 6000.Pig and people's Regular Insulin only there are differences on an amino-acid residue of B chain C-terminal, and the former is a L-Ala, and the latter is a Threonine, but the physiological function of two kinds of Regular Insulin is in full accord.Regular Insulin is to promote anabolism, the stable major hormone of blood sugar regulation, is the specific medicament of treatment insulin-dependent diabetes mellitus.
At present, several production insulin human methods of industrial employing have: directly extract from people's pancreas (1), but the restriction of raw material supply.(2) synthetic by the single amino acids direct chemical, but the industrial production cost is very high.(3) being made the transition by the pork insulin chemistry is the insulin human, and cost is cheap relatively, so before the extensive industrialization of Recombulin, this semisynthesis is the production technique that quite a lot of biological producer adopts.In the process of yeast fermentation production reorganization pork insulin precursor method, Pichia pastoris (Methylotrophic yeastPichia Pastoris) has been acknowledged as one of outstanding expression system of external source secretory protein and intracellular protein production, and its powerful growth and some uniquenesses have been developed to the recombinant expression system of foreign protein commercial production, but prior art (Invitrogen yeast fermentation handbook) yeast fermentation exists the lower (0.8g.L only of concentration
-1) deficiency.
Summary of the invention
The objective of the invention is to: at the deficiency of prior art existence, in keeping Invitrogen yeast fermentation handbook technology under the constant prerequisite of other condition, based on the microorganism growth dynamical foundation, provide a kind of pichia spp fermentation inducement to express pork insulin precursor process optimization of material makeup method.
Inter mass optimization during insulin precursor fermentation method of the present invention, be achieved through the following technical solutions, at first determine initial methanol stream rate of acceleration by add methyl alcohol in batches, by final zymophyte volume density and the difference of test sample cell density and calculating and the adjustment that fermentation period carries out specific growth rate in real time, calculate methyl alcohol stream rate of acceleration in real time to be adjusted at last then.
Below the present invention is described specifically:
(1) determine initial methanol stream rate of acceleration by add methyl alcohol in batches, be specially:
(1) stop to add glycerine, the dissolved oxygen bounce-back, disposable benefit is gone into 0.3% (w/v) methyl alcohol, and thalline slowly utilizes, and adapts to methyl alcohol gradually.
(2) methanol consumption, dissolved oxygen rebounds for the second time, and the property benefit is gone into 0.3% (w/v) methyl alcohol again, and thalline utilizes rapidly, adapts to methyl alcohol comprehensively.
(3) methyl alcohol consumes once more, and dissolved oxygen rebounds for the third time, and the beginning Continuous Flow adds methyl alcohol, and increase methyl alcohol stream rate of acceleration, makes dissolved oxygen fall after rise to the preceding lower-most point of bounce-back, and the methyl alcohol stream rate of acceleration of this moment is initial methanol stream rate of acceleration.
(2) by final zymophyte volume density and the difference of test sample cell density and calculating and the adjustment that fermentation period carries out specific growth rate in real time, be specially:
(1) determines final zymophyte volume density and fermentation period.Specify according to concrete needs by the user.
(2) determine real-time test sample cell density.After methyl alcohol stream adds beginning, every the 2h sampling once, measure cell density.
(3) calculating of specific growth rate.According to difference and the fermentation period of final zymophyte volume density,, carry out the calculating of specific growth rate by microorganism growth kinetics with real-time test sample cell density.
(4) adjustment of specific growth rate.The specific growth rate of the specific growth rate that calculates as next moment.
(3) calculate methyl alcohol stream rate of acceleration in real time to be adjusted, be specially:
By next specific growth rate constantly, ignore the increase of interior thalline of unit time inductive phase and product, calculate methyl alcohol stream rate of acceleration in real time to be adjusted.
The present invention compared with prior art has following advantage and effect is: on the microorganism growth dynamical foundation, realized in time adjusting the no-load voltage ratio growth velocity control of real-time methyl alcohol stream rate of acceleration according to the thalli growth situation, specific growth rate and thalli growth, product expression are carried out parameter correlation, difference and fermentation period by final zymophyte volume density and real-time test sample cell density carry out specific growth rate calculating and adjustment, flow rate of acceleration thereby calculate methyl alcohol in real time to be adjusted.This no-load voltage ratio growth velocity regulation and control strategy can be adjusted the growth of thalline in time, make it to express coordination mutually with product, can improve proteic concentration significantly, the concentration and the ultimate production of reorganization pork insulin precursor have improved 50% than the feed supplement method that Invitrogen yeast fermentation handbook provides.
Embodiment
Below in conjunction with embodiment technical scheme of the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment:
Object: pichia spp GS115 reorganization pork insulin precursor fermenting process, the fermentor tank volume is 50L, and basestocks 4% (w/v) glycerine is as the batch fermentation stage, and Continuous Flow adds the glycerine of concentration 50% (w/w), treats cell density (OD
600) be about at 300 o'clock, stop glycerine and add, add 0.3% (w/v) methyl alcohol for twice in batches, determine initial methyl alcohol stream rate of acceleration according to dissolved oxygen, set final OD
600Be 520, fermentation period T is 100h.
Controlled target: final OD ferments
600Be 520.
Concrete steps are as follows:
(1) treats OD
600Be about at 300 o'clock, stop glycerine and add.Add 0.3% (w/v) methyl alcohol for twice in batches.
(2) methyl alcohol exhausts, and dissolved oxygen bounce-back begins stream and adds methyl alcohol, and increases and add speed, makes dissolved oxygen fall dissolved oxygen lower-most point to the bounce-back after rise, and this moment, methyl alcohol stream rate of acceleration was initial methanol stream rate of acceleration V
0=5.24g.L
-1.h
-1
(3) sampling, the time of establishing is t
0=33h, recording cell density is X
0(OD
600=321).
According to microorganism growth kinetics, μ=dx/dt/X, the unit time is 1h, specific growth rate is μ
0, X then
1=X
0(1+ μ
0), X
2=X
1(1+ μ
1) by that analogy ..., X
n=X
N-1(1+ μ
N-1).
Estimate t
0Back specific growth rate is steady state value, i.e. μ
0=μ
1=... μ
N-1, X then
n=X
0(1+ μ
0)
n, X
nBe set by the user n=T-t
0, can try to achieve μ
0
μ
0＝(X
n/X
0)
1/n-1＝(520/321)
1/(100-33)-1＝0.007226
In like manner, when next took a sample constantly, the time of establishing was t
0, measured cell density is X
0, can try to achieve next μ constantly
0
(4), adjust real-time methyl alcohol stream rate of acceleration according to the specific growth rate μ that calculates.
According to the base consumption equation, Δ S=m
1X+m
2μ X+m
3Δ P, wherein m
1, m
2, m
3Be scale-up factor, consider that interior product of unit time inductive phase and thalline increase are all very little, ignore, then Δ S=m
1X is because of time unit is 1h, so Δ S is methyl alcohol stream rate of acceleration.
If be t sample time
0=33h, measured cell density are X
0(OD
600=321), methyl alcohol stream rate of acceleration is Δ S
0=V
0=5.24g.L
-1.h
-1, the specific growth rate that calculates is μ
0=0.007226, next period Δ S then
1=m
1X
1So, Δ S
1/ Δ S
0=X
1/ X
0=(1+ μ
0), can try to achieve the Δ S of next period
1
ΔS
1＝ΔS
0(1+μ
0)＝5.24×(1+0.007226)＝5.28g.L
-1.h
-1
In like manner, when next took a sample the period again, the time of establishing was t
0, measured cell density is X
0, can try to achieve again the Δ S of next period
1
Annotate: because to ignoring that thalline and product in unit time inductive phase increase, cause slightly deviation of interior calculation result of unit time, but pass through real time sample, adjust methyl alcohol stream rate of acceleration in time, but can control thalli growth well generally, improve exogenous protein expression concentration, reach the purpose that thalli growth and protein expression are coordinated mutually.
Obtain the feed supplement method that methyl alcohol stream adds induction period according to the method described above and can coordinate thalli growth and proteic expression well.During cycle 100h, reach the final user and set cell density (OD
600=520) concentration that, and has significantly improved foreign protein (reaches 1.2g.L
-1), the concentration and the ultimate production of reorganization pork insulin precursor have improved 50% than the feed supplement method that Invitrogen yeast fermentation handbook provides.
Claims (1)
1. inter mass optimization during insulin precursor fermentation method, it is characterized in that: be pichia spp GS115 reorganization pork insulin precursor fermenting process with bacterial strain, the fermentor tank volume is 50L, basestocks 4%w/v glycerine is as the batch fermentation stage, Continuous Flow adds the glycerine of concentration 50%w/w, treats cell density OD
600Be 300 o'clock, stop glycerine and add, add 0.3%w/v methyl alcohol in batches for twice, determine initial methyl alcohol stream rate of acceleration, set final OD according to dissolved oxygen
600Be 520, fermentation period T is 100h;
Controlled target: the final OD600 that ferments is 520;
Concrete steps are as follows:
(1) treats OD
600Be 300 o'clock, stop glycerine and add, add 0.3%w/v methyl alcohol in batches for twice;
(2) methyl alcohol exhausts, and dissolved oxygen bounce-back begins stream and adds methyl alcohol, and increases and add speed, makes dissolved oxygen fall dissolved oxygen lower-most point to the bounce-back after rise; , this moment, methyl alcohol stream rate of acceleration was initial methanol stream rate of acceleration V
0=5.24g.L
-1.h
-1
(3) sampling, the time of establishing is t
0=33h, recording cell density is X
0OD
600=321; According to microorganism growth kinetics, μ=dx/dt/X, the unit time is 1h, specific growth rate is μ
0, X then
1=X
0(1+ μ
0), X
2=X
1(1+ μ
1) by that analogy ..., X
n=X
N-1(1+ μ
N-1);
Estimate t
0Back specific growth rate is steady state value, i.e. μ
0=μ
1=... μ
N-1, X then
n=X
0(1+ μ
0)
n, X
nBe set by the user n=T-t
0, can try to achieve μ
0
μ
0＝(X
n/X
0)
1/n-1＝(520/321)
1/(100-33)-1＝0.007226
In like manner, when next took a sample constantly, the time of establishing was t
0, measured cell density is X
0, can try to achieve next μ constantly
0
(4) according to the specific growth rate μ that calculates
0, adjust real-time methyl alcohol stream rate of acceleration;
According to the base consumption equation, Δ S=m
1X+rn
2μ X+m
3Δ P, wherein m
1, m
2, m
3Be scale-up factor, consider that interior product of unit time inductive phase and thalline increase are all very little, ignore, then Δ S=m
1X is because of time unit is 1h, so Δ S is methyl alcohol stream rate of acceleration;
If be t sample time
0=33h, measured cell density are X
0OD
600=321, methyl alcohol stream rate of acceleration is Δ S
0=V
0=5.24g.L
-1.h
-1, the specific growth rate that calculates is μ
0=0.007226, next period Δ S then
1=m
1X
1,, so Δ S
1/ Δ S
0=X
1/ X
0=(1+ μ
0), can try to achieve the Δ S of next period
1
ΔS
1＝ΔS
0(1+μ
0)＝5.24×(1+0.007226)＝5.28g.L
-1.h
-1
In like manner, when next took a sample the period again, the time of establishing was t
0, measured cell density is X
0, can try to achieve again the Δ S of next period
1
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| CN101029323B true CN101029323B (en) | 2011-12-21 |
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| CN106282274B (en) * | 2015-06-29 | 2021-05-11 | 宜昌东阳光长江药业股份有限公司 | High-density fermentation method of pichia pastoris for insulin precursor protein |
| CN107022591B (en) * | 2017-05-11 | 2018-06-12 | 宜昌东阳光长江药业股份有限公司 | A kind of Pichia pastoris fermentation process for improving insulin and the like precursor expression |
| CN111662836A (en) * | 2019-03-05 | 2020-09-15 | 上海医药工业研究院 | Genetically engineered bacterium for expressing insulin precursor and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1614019A (en) * | 2004-11-03 | 2005-05-11 | 马延高 | Secretory expression for human insulin gene in methyl alcohol yeast |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1614019A (en) * | 2004-11-03 | 2005-05-11 | 马延高 | Secretory expression for human insulin gene in methyl alcohol yeast |
Non-Patent Citations (6)
| Title |
|---|
| 姚钰舜，储炬，杭海峰，庄英萍，张嗣良.培养条件对重组毕赤酵母高密度表达猪胰岛素前体的影响.华东理工大学学报(自然科学版)32 4.2006,32(4),397-401. |
| 姚钰舜,储炬,杭海峰,庄英萍,张嗣良.培养条件对重组毕赤酵母高密度表达猪胰岛素前体的影响.华东理工大学学报(自然科学版)32 4.2006,32(4),397-401. * |
| 董鹏,陈劲春,冷雪.表达胰岛素的毕赤酵母生长动力学及诱导策略.北京化工大学学报33 3.2006,33(3),37-41. |
| 董鹏,陈劲春,冷雪.表达胰岛素的毕赤酵母生长动力学及诱导策略.北京化工大学学报33 3.2006,33(3),37-41. * |
| 陈文,郭美锦,储炬,庄英萍,张嗣良.重组毕赤酵母表达猪胰岛素前体代谢参数分析和动力学模型.华东理工大学学报(自然科学版)31 3.2005,31(3),300-304. |
| 陈文,郭美锦,储炬,庄英萍,张嗣良.重组毕赤酵母表达猪胰岛素前体代谢参数分析和动力学模型.华东理工大学学报(自然科学版)31 3.2005,31(3),300-304. * |
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