CN102517223A - High-density fermentation process and equipment for methanol type pichia pastoris - Google Patents
High-density fermentation process and equipment for methanol type pichia pastoris Download PDFInfo
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Abstract
The invention relates to a high-density fermentation process for methanol type pichia pastoris. The high-density fermentation process for the methanol type pichia pastoris comprises a batch fermentation stage, a material supplement fermentation stage and a methanol induced stage, wherein in the material supplement fermentation stage and the methanol induced stage, high-concentration oxygen prepared by an oxygen generator flows into an oxygen storage tank and is mixed with air entering a fermentation tank, so that the oxygen content is 40 to 60 percent; and the oxygen is introduced into the fermentation tank according to the flow quantity of 1.00 vvm, so that the fermentation liquid oxygen concentration of the fermentation tank is 30 percent higher than the solubility of the oxygen in the air in an initial culture solution at the same temperature under the same pressure. Yeast high-density culture equipment applied to the high-density fermentation process for the methanol type pichia pastoris comprises the oxygen generator, the oxygen storage tank, a gas water separator, a membrane filter and the fermentation tank which are connected with one another sequentially through pipelines. According to the process and the equipment, reduction of fermentation production rate caused by oxygen deficiency in the fermentation process is avoided and the utilization ratio of the oxygen is increased.
Description
Technical field
The invention belongs to technical field of biological fermentation, relate to a kind of high density fermentation Processes and apparatus of methanol type pichia spp.
Background technology
Along with the development of biology techniques, utilize genetic engineering bacterium to produce biologics and become a reality.In the microbial expression system; Yeast is as unicellular lower eukaryotes; Have be prone to cultivate, breeding is fast, be convenient to genetic manipulation, can translate post-treatment and modification to foreign protein, do not produce advantage such as toxic substance, become to be widely used in one of system that recombinant protein produces in the biology field.The methanol type pichia pastoris phaff
(Pichia pastoris) expression system is one of fitst water at present, most widely used heterologous gene expression system; It has not only overcome escherichia expression system can not complicated protein, the expressed proteins multiform of expression structure become defectives such as insoluble inclusion body, background albumen is many, expression yields poorly, and has remedied cells of mamma animals, insect cell expression system complicated operation, expression level is low, industrialization production involves great expense deficiency.Another characteristics of pichia pastoris phaff are in the non-selective substratum of cheapness, to grow, and can carry out the cell high-density culture, are beneficial to large-scale industrial production.Present many products all obtain to efficiently express
like Sumizyme PHY, zytase, mannase etc. in pichia pastoris phaff.
P. pastorisBe a kind of methyl yeast of having a liking for, can growth fast in the substratum that with the first ferment is the sole carbon source and the energy.The metabolic the first step of pichia methanolica is that oxidation methyl alcohol forms formaldehyde, this step be by alcohol oxidase (alcohol oxidase, Aox) catalytic, in pichia spp utilizes the pathways metabolism of methyl alcohol, have two alcohol oxidases, AOX1 and AOX2.When the methyl alcohol feeding culture, the AOX level sharply rises, and can reach more than 30% of cell soluble proteins.The strategy that adopts now is that foreign gene is replaced the AOXl gene, and the transformant that this replacement produced is the most stable.The synthetic of AOXl regulated and control on transcriptional level, and its gene promoter has the empty function of tangible accent, can be used for regulating and control expression of exogenous gene.This regulating and controlling effect is suppressed/separates to suppress by carbon source and the special double mechanism of inducing of carbon source is controlled, and promptly foreign gene is in non-expression status in the carbon source beyond the methyl alcohol, and after in nutrient solution, adding methyl alcohol, foreign gene can be by abduction delivering.
At 25 ℃ and 1.0 * 10
5The solubleness of Pa oxygen in water is merely about 0.25 mmol.L-1.In fermented liquid, contain a large amount of organism and inorganic salt, the solubleness of oxygen is about 0.21 mmol.L-1, and is littler 7000 times than the solubleness of sugar.Even the dissolved oxygen in the logarithmic phase fermented liquid can reach 100% saturation ratio, if end oxygen supply this moment, dissolved oxygen can in minutes just be exhausted in the fermented liquid, makes dissolved oxygen become limiting factor.
Dissolved oxygen (DO) is that pichia spp growth institute is essential, and especially in recombinant yeast pichia pastoris high density fermentation technology, in the fed-batch fermentation stage, because the raised growth of thalline, oxygen consumption rate increases, and oxygen requirement is very big; At ensuing methanol induction expression phase, methyl alcohol carries out the oxygen participation that metabolism need be a large amount of through the alcohol oxidase (AOX1 and AOX2) of pichia spp, makes dissolved oxygen become the limiting factor of methanol type recombinant yeast pichia pastoris high density fermentation.Present zymotechnique and equipment can't reach the balance of oxygen supply and oxygen consumption in methanol type recombinant yeast pichia pastoris process of high-density fermentation.
Therefore; Methanol type recombinant yeast pichia pastoris high density fermentation to heavy industrialization; Need improve the supply and the utilization ratio of oxygen from the aspects such as optimization of reactor drum consequence devised, quality of filling gas, bacterial classification and zymotechnique, prevent the reduction of the fermentation production rate that fermenting process causes because of anoxic.
Summary of the invention
Technical problem to be solved by this invention is: the high density fermentation Processes and apparatus that a kind of methanol type pichia spp is provided; To overcome in the present methanol type recombinant yeast pichia pastoris process of high-density fermentation; Problems such as oxygen supply deficiency aspect zymotechnique and equipment prevent the reduction of the fermentation production rate that fermenting process causes because of anoxic.
The technical scheme that the present invention adopted is: a kind of high density fermentation technology of methanol type pichia spp; Comprise batch fermentation stage, fed-batch fermentation stage and methanol induction stage; In fed-batch fermentation stage and methanol induction stage; The high-concentration oxygen that is made by oxygen generator flows into oxygen storage tank, with the air mixed that gets into fermentor tank, makes it oxygen content at 40%-60% then; Feed fermentor tank with the flow of 1.00vvm, with the concentration of keeping fermentation liquid oxygen in the fermentor tank be higher than equality of temperature with depress airborne oxygen in initial incubation liquid solubleness 30%.
The further technical scheme of the present invention is: the yeast high-density culture equipment in a kind of high density fermentation technology that is applied to above-mentioned methanol type pichia spp is provided, and this equipment comprises oxygen generator, oxygen storage tank, air water separator, film filter and the fermentor tank that is connected successively by pipeline.Described fermentor tank comprises tank body, whipping appts and breather; Said whipping appts comprises agitator motor, is fixed on some groups of stirring arms on the stir shaft, and lower floor's stirring rake places tank base and is positioned at the breather top, and the middle level stirring rake places the middle part of tank body, and the upper strata stirring rake places the top of tank body; Said breather is the annular air sparger, settles the tubule of giving vent to anger of a plurality of miter angles on the annular air sparger, and the direction that the tubule of giving vent to anger is provided with a plurality of production well guiding gas rotations is opposite with the stir shaft sense of rotation.
The present invention and prior art relatively have following beneficial effect: 1. the oxygen that feeds high density in fed-batch fermentation stage of the high density fermentation technology of methanol type recombinant yeast pichia pastoris and methanol induction stage; Can increase the solubleness of oxygen in the fermented liquid; Strengthen the supply of oxygen; Eliminate the influence of the restriction of oxygen in the fermenting process, prevent the reduction of the fermentation production rate that fermenting process causes because of anoxic pichia spp growth and exogenous gene expression.2. owing to settled the tubule of giving vent to anger of miter angle on the annular air sparger, be arranged with production well on the tubule of giving vent to anger, the gas that breather is got into can rise by homodisperse, and has increased the gas-to-liquid contact face; Simultaneously because the direction of the rotation of the production well guiding gas on the tubule of giving vent to anger is opposite with the stir shaft sense of rotation, promoted mixing of air inlet and fermented liquid, help improving the utilization ratio of oxygen.
Description of drawings
Fig. 1 is that an embodiment of the present invention methanol type pichia spp high-density culture equipment is formed synoptic diagram.
Fig. 2 is a fermentor tank structural representation of the present invention.
Fig. 3 is the breather structural representation.
Fig. 4 is the A-A view of Fig. 3.
Embodiment
Embodiment as shown in Figure 1 is applied to the yeast high-density culture equipment in the high density fermentation technology of said methanol type pichia spp, comprises the oxygen generator 1, oxygen storage tank 2, air water separator 3, film filter 4 and the fermentor tank 5 that are connected successively by pipeline.
As shown in Figure 2, present embodiment fermentor tank 5 comprises tank body, whipping appts and breather; Said whipping appts comprises agitator motor 6, is fixed on three groups of stirring arms 8 on the stir shaft 7; Lower floor's stirring rake 8.1 places tank body 12 bottoms and is positioned at the breather top; Be the radial type stirring rake, middle level stirring rake 8.2 places the middle part of tank body, is the axial flow type stirring rake; Upper strata stirring rake 8.3 places the top of tank body, is the axial flow type stirring rake; Said breather is an annular air sparger 9, settles the tubule 10 of giving vent to anger of a plurality of miter angles on the annular air sparger 9, and the direction that is arranged with the rotation of production well guiding gas on the tubule 10 of giving vent to anger is opposite with stir shaft 8 sense of rotation.Like Fig. 2, shown in 3; Said breather is an annular air sparger 9; Settle the tubule 10 of giving vent to anger of a plurality of miter angles on the annular air sparger 9; The direction that the tubule 10 of giving vent to anger is provided with a plurality of production well guiding gas rotations is opposite with stir shaft 8 sense of rotation, and promotion entering gas mixes with fermented liquid, improves the utilization ratio of oxygen.Production well preferably is uniformly distributed on the tubule 10 of giving vent to anger, and the gas that breather is got into can rise by homodisperse, and increases the gas-to-liquid contact face.Mark 11 among Fig. 2, Fig. 3 is an inlet mouth.
It below is methanol type pichia spp in 15 liters of fermentor tanks
Pichia pastorisHigh-density (aerobic) zymotechnique.
Bacterial classification: methanol type pichia spp
Pichia pastorisATCC 76274, purchase in US mode culture collection warehousing (American type culture collection).
Fermentation culture and result: will from flat board, insert (1L contains: yeast powder 10g, peptone 20g, glucose 20g) 30 ℃ of following 220 r/min cultivation 20h the YPD seed culture medium (500mL Erlenmeyer flask liquid amount 125mL) by picking one single bacterium colony.Insert in the 15L fermentor tank that 10L batch fermentation substratum is housed (1L contains: glucose 30g, ammonium sulfate 15g, sal epsom 8g, vitriolate of tartar 7.5g, potassium primary phosphate 3.5g, calcium sulfate 1.0g, Pottasium Hydroxide 1.2g, PTM1 4mL) by 10% inoculum size then.30 ℃ of leavening temperatures utilize ammonia soln that pH is controlled to be 5.0, and fed-batch fermentation stage stream adds glucose and its concentration is maintained about 10.0g/L, adds methyl alcohol after 24 hours, and it is per hour to add 1.5～3.0 milliliters in every liter of nutrient solution that methyl alcohol adds speed.In high-concentration oxygen (about oxygen concentration 90%) the inflow gas storage tank 2 that makes by oxygen generator 1 (available from commercially available transformation adsorb oxygen producer); Then through air water separator 3 and film filter 4 and the air mixed that gets into fermentor tank 5; Make it oxygen content about 50%; Feed fermentor tank 5 with the flow of 1.00vvm, with the concentration of keeping fermentation liquid oxygen in the fermentor tank 5 be higher than equality of temperature with depress airborne oxygen in initial incubation liquid solubleness 30%.Through 60 hours cultivation, final cell concentration can reach 109.6g/L.With only with the universal ferment tank result (fermentation time 60 hour of air as source of the gas; Cell concn 64.2g/L) compares; Utilize the technology of increase bubbling air oxidation content and the fermentative prodn that fermentor tank of the present invention carries out the methanol type pichia spp, cell concn concentration has improved 1.71 times.
Claims (7)
1. the high density fermentation technology of a methanol type pichia spp; Comprise batch fermentation stage, fed-batch fermentation stage and methanol induction stage; It is characterized in that: in fed-batch fermentation stage and methanol induction stage; The high-concentration oxygen that is made by oxygen generator flows into oxygen storage tank, with the air mixed that gets into fermentor tank, makes it oxygen content at 40%-60% then; Feed fermentor tank with the flow of 1.00vvm, with the concentration of keeping fermentation liquid oxygen in the fermentor tank be higher than equality of temperature with depress airborne oxygen in initial incubation liquid solubleness 30%.
2. the high density fermentation technology of methanol type pichia spp according to claim 1 is characterized in that: said oxygen content is 50%.
3. the yeast high-density culture equipment in the high density fermentation technology that is applied to the said methanol type pichia spp of claim 1 is characterized in that: comprise the oxygen generator (1), oxygen storage tank (2), air water separator (3), film filter (4) and the fermentor tank (5) that are connected successively by pipeline.
4. yeast high-density culture equipment according to claim 3 is characterized in that: described fermentor tank (5) comprises tank body, whipping appts and breather; Said whipping appts comprises agitator motor (6), is fixed on the some groups of stirring arms (8) on the stir shaft (7); Lower floor's stirring rake (8.1) places tank base and is positioned at the breather top; Middle level stirring rake (8.2) places the middle part of tank body, and upper strata stirring rake (8.3) places the top of tank body; Said breather is annular air sparger (9); Annular air sparger (9) is gone up the tubule of giving vent to anger (10) of settling a plurality of miter angles, and the direction that the tubule of giving vent to anger (10) is provided with a plurality of production well guiding gas rotations is opposite with stir shaft (8) sense of rotation.
5. yeast high-density culture equipment according to claim 4 is characterized in that: said lower floor stirring rake (8.1) is the radial type stirring rake, and middle level stirring rake (8.2) is the axial flow type stirring rake, and upper strata stirring rake (8.3) is the axial flow type stirring rake.
6. yeast high-density culture equipment according to claim 4 is characterized in that: said production well is uniformly distributed on the tubule of giving vent to anger (10).
7. yeast high-density culture equipment according to claim 5 is characterized in that: said production well is uniformly distributed on the tubule of giving vent to anger (10).
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103045703A (en) * | 2013-01-05 | 2013-04-17 | 义马煤业集团煤生化高科技工程有限公司 | Recycling method of methanol protein fermentation filtrate |
| CN114807273A (en) * | 2022-05-23 | 2022-07-29 | 江苏熙壤农业科技有限公司 | Efficient low-carbon production system and method for methanol protein based on biomass synthesis |
| CN114836498A (en) * | 2022-05-23 | 2022-08-02 | 江苏熙壤农业科技有限公司 | System and method for efficient low-carbon production of methanol protein |
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| CN101570771A (en) * | 2009-06-09 | 2009-11-04 | 华东理工大学 | Method for producing S-adenosylmethionine through fermentation of recombinant pichia pastoris |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103045703A (en) * | 2013-01-05 | 2013-04-17 | 义马煤业集团煤生化高科技工程有限公司 | Recycling method of methanol protein fermentation filtrate |
| CN103045703B (en) * | 2013-01-05 | 2014-03-12 | 义马煤业集团煤生化高科技工程有限公司 | Recycling method of methanol protein fermentation filtrate |
| CN114807273A (en) * | 2022-05-23 | 2022-07-29 | 江苏熙壤农业科技有限公司 | Efficient low-carbon production system and method for methanol protein based on biomass synthesis |
| CN114836498A (en) * | 2022-05-23 | 2022-08-02 | 江苏熙壤农业科技有限公司 | System and method for efficient low-carbon production of methanol protein |
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Application publication date: 20120627 |