CN1556215A - Feed supplementing optimization method of saccharomyces pichia fermentation batch process - Google Patents
Feed supplementing optimization method of saccharomyces pichia fermentation batch process Download PDFInfo
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- CN1556215A CN1556215A CNA2004100156603A CN200410015660A CN1556215A CN 1556215 A CN1556215 A CN 1556215A CN A2004100156603 A CNA2004100156603 A CN A2004100156603A CN 200410015660 A CN200410015660 A CN 200410015660A CN 1556215 A CN1556215 A CN 1556215A
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Abstract
An optimized method for supplementing raw material in the Pichia yeast fementing procedure features that in the fermenting phase adding methanol in flowing mode, a linear increment method is first used to supplementing methanol to make the specific growth rate of cell reach a predefined value, and a parabolic method is then used to make the specific growth rate of cell contant. For the recombinant human serum albumin expression system with Pichia yeast GS115, it can obviously increase the output rate of albumin.
Description
Technical field
The present invention relates to a kind of optimization method, particularly a kind of pichia yeast fermentative production recombination human serum albumin is criticized the optimization of material makeup method of process, belongs to biological technical field.
Background technology
The polypeptide chain that human serum albumin is made up of 585 amino acid, molecular weight are 66.5kDa.Sero-abluminous concentration is up to 42gl in the human plasma
-1, human serum albumin mainly expands agent, the assisting therapy of rescue shock, burn and lose blood patient and cancer as plasma volume clinically.At present, the human serum albumin goods are separation of produced from human plasma or placenta mainly, and product is bigger by the probability that hemopathy provirus (as hepatitis B virus and HIV (human immunodeficiency virus)) pollutes.In recent years, along with the development of recombinant DNA technology, utilizing biotechnology to produce human serum albumin becomes an alternative route gradually.
From output, pichia yeast is the expression-secretion system of the best up to now recombination human serum albumin.Find by literature search, Invitrogen (U.S.) company set up the pichia yeast batch fermentation produce the technological specification of human serum albumin (
Http:// invitrogen.com/content/sfs/manuals / pichiaferm prot.pdf), this technology is decided to be 180 hours to whole fermentation period, is divided into three phases: glycerine batch fermentation phase, glycerine stream add yeast phase and methyl alcohol stream adds yeast phase.Fermentation time, medium component and the yeasting parameter (temperature, pH value and oxyty) in each stage have clearly been provided in the standard, provide glycerine stream simultaneously and added the feed supplement method that yeast phase and methyl alcohol stream add yeast phase, wherein, the feed supplement method that glycerine stream adds yeast phase is, with the constant rate of speed stream glycerol adding of 18.15 (milliliter is every liter of initial fermented liquids per hour); The feed supplement method that methyl alcohol stream adds yeast phase is that at first the data rate stream with 1 (milliliter is every liter of initial fermented liquid per hour) added methyl alcohol 2 hours; Then, the flow velocity of methyl alcohol was improved 10% in per 30 minutes and reach 3 (milliliter is every liter of initial fermented liquids per hour) up to flow velocity; At last, keep 3 (milliliter is every liter of initial fermented liquids per hour)-flow velocity to fermentation ends.The specific cell growth rate that the methyl alcohol stream of this method correspondence the adds yeast phase type that tapers off, for utilizing the system of pichia yeast GS115 recombinant Human Serum Albumin Expression, this feed supplement control method gained expressing quantity is lower.Also find in the retrieval, Ren et al., Macrokinetic model formethylotrophic Pichia pastoris based on stoichiometric balance, Journal ofBiotechnology, 106:53-68,2003 (appoint great waves etc., based on the pichia yeast macro-kinetic model of stoichiometric balance, biotechnology magazine, 106:53-68,2003) set up the macro-kinetic model of pichia yeast, but do not related to the output optimization problem in the article.
Summary of the invention
The present invention is directed to deficiency and defective that prior art exists, in keeping Invitrogen technology under the constant prerequisite of other processing condition, macro-kinetic model based on pichia yeast, a kind of optimization of material makeup method of pichia yeast fermentation batch process is provided, make it be applicable to that pichia yeast fermentative production recombination human serum albumin criticizes the methanol feeding method of process, this feed supplement method realized methanol induction phase-be methyl alcohol stream add fermentation initial stage-linear increment type feed supplement control and the control of constant specific growth rate subsequently, remedy the deficiency that methyl alcohol stream in the Invitrogen technology adds fermentation stage decrescendo specific growth rate feed supplement method, improved the albumen productive rate.
The present invention is achieved by the following technical solutions, at first makes specific cell growth rate induce the end of term to reach set(ting)value by linear increment type feed supplement method, and the time of inductive phase is specified by the user, is designated as t
InMake specific cell growth rate remain on set(ting)value consistently by Parabolic feed supplement method then, when linear increment type feed supplement method and parabolic type feed supplement method are changed, keep the stream rate of acceleration of methyl alcohol continuous.
Below the inventive method is described specifically:
(1) make specific cell growth rate through t by linear increment type feed supplement method
InHour reach set(ting)value.
Be applicable to whole process inductive phase by linear increment type feed supplement control method, during methanol feeding speed be the linear function of time, the constant term of this function and scale-up factor are determined by following method:
(1) determines the constant term of linear function.The initial feed rate of methyl alcohol of the present invention is identical with the technological specification of Invitrogen company, therefore, the constant term of linear function equals 1 (milliliter is every liter of initial fermented liquid per hour) and multiply by initial fermentating liquid volume (liter), wherein, initial fermentating liquid volume is meant the fermentating liquid volume when methyl alcohol stream adds fermentation stage and begins.
(2) determine the scale-up factor of linear function.At first set the initial value of a scale-up factor, utilize golden section method to carry out linear search then, objective function is to make the model emulation value of inducing end of term specific cell growth rate and the square-error minimum of set(ting)value, and wherein, the model emulation value obtains by the pichia yeast macro-kinetic model.
(2) make specific cell growth rate remain on set(ting)value consistently by Parabolic feed supplement method.
Parabolic type feed supplement method is applicable to the remaining ferment time after finishing inductive phase, during feed rate be the quadratic function of time, the constant term of this function, once a coefficient and quadratic term coefficient are determined by following method:
(1) determines the constant term of quadratic function.The constant term of quadratic function is got the feed rate value of end's inductive phase, and it has kept the continuity of stream rate of acceleration.
(2) determine once the coefficient and the quadratic term coefficient of quadratic function.At first set once the initial value of a coefficient and quadratic term coefficient, utilize the simplicial method optimizing then, objective function is the fermentation period after inductive phase, makes the model emulation value of specific cell growth rate and the sum of the squares of errors minimum of set(ting)value.Wherein, the model emulation value is tried to achieve by the pichia yeast macro-kinetic model, and sum of the squares of errors is calculated at the time integral point.
Compared with prior art, on the pichia yeast macro-kinetic model basis of the present invention's great waves in office etc., linear feed supplement method and Parabolic feed supplement method are combined, and when two sections feed supplement method conversions, keep the stream rate of acceleration continuous, specific growth rate was close in inductive phase arrives set(ting)value evenly and make the specific cell growth rate of subsequent fermenting period remain on this set(ting)value consistently.This constant specific growth rate feed supplement method can improve the albumen productive rate than the highland, and protein concentration that fermentation time was located in 180 hours and ultimate production improve 40% than the feed supplement method that Invitrogen company provides.
Embodiment
Content in conjunction with the inventive method further provides following examples:
Object: pichia yeast GS115 recombination human serum albumin fermentation batch process.The fermentor tank volume is 10 (liters), and glycerine batch fermentation phase and glycerine stream add yeast phase to be undertaken by the working specification that Invitrogen company provides, and the initial fermentating liquid volume that methyl alcohol stream adds yeast phase is 4 (liters), the methanol induction phase be 20 (hour).
Controlled target: make methyl alcohol stream add the yeast phase specific cell growth rate and remain on 0.006 (per hour) consistently.
Concrete steps are as follows:
(1) add fermentation initial stage-at first implement linear feed supplement method at methanol induction phase-be positioned at methyl alcohol stream, its equation is:
F＝ω
1+ω
2*(t-t
f) (t
f≤t≤t
f+20) ①
Formula 1. in, F represents that the stream of methyl alcohol adds flow rate (milliliter per hour), t be with inoculation constantly be time zero fermentation time (hour), t
fBe methyl alcohol stream time of adding yeast phase and beginning to locate (hour), ω
1Be initial flow rate of acceleration (milliliter per hour), its value is made as 4 (milliliter per hour), and this is that fermentating liquid volume when being added yeast phase and begun by methyl alcohol stream multiply by 1 (milliliter is every liter of fermented liquid per hour) and draws ω
2Be scale-up factor, its initial value is made as 0.3, draws exact value by the golden section method optimizing again, and the optimizing objective function sees that formula 2..
Formula 2. in, J is a target function value, the implication of F and formula are identical in 1., F
MinAnd F
MaxBe the lower limit and the upper limit of methyl alcohol stream rate of acceleration, they are by the physical condition decision of fermentor tank, μ (t
f+ 20) be the model emulation value (per hour) that methyl alcohol stream adds fermentation specific cell growth rate after 20 hours.
(2) linear feed supplement method is implemented to implement Parabolic feed supplement method to fermentation ends after 20 hours, and its equation is:
F＝ω
3+ω
4*(t-t
f-20)+ω
5*(t-t
f-20)
2 (t
f+20＜t≤180) ③
F, t and the t of formula in 3.
fImplication and formula identical in 1., ω
3Be the initial value of parabolic type feed supplement method methyl alcohol stream rate of acceleration, the methyl alcohol stream rate of acceleration when it and linear feed supplement method finish is equal, i.e. ω
3=ω
1+ 20 ω
2, when this had just guaranteed two sections feed supplement method conversions, methyl alcohol flowed the continuous of rate of acceleration, ω
4And ω
5Be equation coefficient, its initial value is made as 0.0001 and 0.05 respectively, draws its exact value by the simplicial method optimizing again, and the optimizing objective function sees that formula 4..
The sum of the squares of errors of formula in 4. calculated at the time integral point, wherein, and J, F
Min, F
Max, t and t
fImplication and formula 1. and identical 2., μ (t) is the integral point model emulation value of specific cell growth rate constantly.
The feed supplement method that the methyl alcohol stream that obtains as stated above adds fermentation stage can make specific cell growth rate remain on 0.006 (per hour) consistently, and final protein concentration and ultimate production improve 40% than the feed supplement method that Invitrogen company provides.
Claims (3)
1, a kind of optimization of material makeup method of pichia yeast fermentation batch process, it is characterized in that, linear feed supplement method and Parabolic feed supplement method are combined, at first make specific cell growth rate induce the end of term to reach set(ting)value by linear increment type feed supplement method, wherein, the time length of inductive phase is specified by the user, make specific cell growth rate remain on set(ting)value consistently by Parabolic feed supplement method then, when linear feed supplement method and parabolic type feed supplement method are changed, keep the stream rate of acceleration of methyl alcohol continuous.
2, the optimization of material makeup method of pichia yeast fermentation batch process according to claim 1 is characterized in that, and is described by linear increment type feed supplement method, is specially:
Linear feed supplement method is applicable to whole process inductive phase, during methanol feeding speed be the linear function of time, the constant term of this function and scale-up factor are determined by following method:
(1) determine the constant term of linear function: the constant term of linear function equal 1 milliliter per hour every liter of initial fermented liquid multiply by initial fermentating liquid volume, wherein, initial fermentating liquid volume is meant that methyl alcohol stream adds the fermentation stage fermentating liquid volume of the zero hour, unit rises;
(2) determine the scale-up factor of linear function: preset proportion coefficient initial value at first, utilize golden section method to carry out the one dimension optimizing then, the optimizing objective function is to make the model emulation value of inducing end of term specific cell growth rate and the square-error minimum of set(ting)value, wherein, the model emulation value obtains by the pichia yeast macro-kinetic model.
3, the optimization of material makeup method of pichia yeast fermentation batch process according to claim 1 is characterized in that described parabolic type feed supplement method is specially:
Parabolic type feed supplement method is applicable to the remaining ferment time after finishing inductive phase, during feed rate be the quadratic function of time, the constant term of this function, once a coefficient and quadratic term coefficient are determined by following method:
(1) determine the constant term of quadratic function: the constant term of quadratic function is got the feed rate value of end's inductive phase, and it has kept the continuity of stream rate of acceleration;
(2) determine once the coefficient and the quadratic term coefficient of quadratic function: the initial value of at first setting once a coefficient and quadratic term coefficient, utilize the simplicial method optimizing then, the optimizing objective function is in the fermentation period except that inductive phase, make the model emulation value of specific cell growth rate and the sum of the squares of errors minimum of set(ting)value, wherein, the model emulation value is tried to achieve by the pichia yeast macro-kinetic model, and sum of the squares of errors is calculated at the time integral point.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1854306B (en) * | 2005-04-29 | 2011-07-27 | 华北制药集团新药研究开发有限责任公司 | Production of recombinant human serum albumin HSA by fermentation |
| CN101419216B (en) * | 2008-10-16 | 2012-06-27 | 浙江工业大学 | Fermentation pharmacy product quality soft measurement method based on GP learning model building |
| CN109988803A (en) * | 2019-05-16 | 2019-07-09 | 齐智 | A kind of fermentation process of efficient production recombination human serum albumin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1103374C (en) * | 1999-03-04 | 2003-03-19 | 上海贸基生物工程科技有限公司 | Chemical synthesis, expression and recombinant protein production for human serum albumin reformed gene (I) |
| US20030119109A1 (en) * | 2001-07-26 | 2003-06-26 | Van Den Burg Harrold Alfred | Efficient 13C/15N double labeling of proteins in a methanol-utilizing strain (Mut+) of pichia pastoris |
| CN1384205A (en) * | 2002-05-22 | 2002-12-11 | 四川汉龙高新技术开发有限公司 | Large-scale protein process of recombinant human liver cell growth factor rhHGF |
| CN1231572C (en) * | 2003-03-27 | 2005-12-14 | 华东理工大学 | Fermentation process for reforming yeast of liking for carbinol |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1854306B (en) * | 2005-04-29 | 2011-07-27 | 华北制药集团新药研究开发有限责任公司 | Production of recombinant human serum albumin HSA by fermentation |
| CN101419216B (en) * | 2008-10-16 | 2012-06-27 | 浙江工业大学 | Fermentation pharmacy product quality soft measurement method based on GP learning model building |
| CN109988803A (en) * | 2019-05-16 | 2019-07-09 | 齐智 | A kind of fermentation process of efficient production recombination human serum albumin |
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