CN105200101B - A kind of method that Pichia yeast weight answers fermentation expression foreign protein - Google Patents
A kind of method that Pichia yeast weight answers fermentation expression foreign protein Download PDFInfo
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Abstract
The present invention provides a kind of methods that Pichia yeast weight answers fermentation expression foreign protein, this method directly utilizes the Pichia pastoris after normal fermentation, pass through the adjustment of parameter and step, so that the Pichia pastoris is reusable more than three times, the yield of foreign protein also can reach the standard of industrialized production simultaneously, this method can significantly reduce the cost of subsequent fermentation, shorten the duration of fermentation, have extensive promotional value.
Description
Technical field
The invention belongs to technical field of microbial fermentation, and in particular to a kind of Pichia yeast weight answers fermentation expression external source
The method of albumen is suitable for producing extracellular purpose product with pichia pastoris yeast engineering bacteria.
Background technique
Traditional Pichia pastoris fermenting and producing foreign protein method is mainly sent out by glycerol batch fermentation stage, glycerol feed supplement
Ferment stage and methanol feeding fermentation stage three parts composition.Entire fermentation process generally requires one week or so, sends out in glycerol batch
Ferment and fed-batch fermentation stage, Pichia pastoris will not express purpose product, only a large amount of proliferation, and glycerol price is significantly larger than first
Alcohol, so the stage improves the cost of entire technique；Methanol feeding fermentation stage, since the carbon source added in this early period is sweet
Oil, therefore in the early period for adding methanol, thallus needs the adaptation of a period of time, could utilize methanol, this also extend fermentation when
Between, reduce fermentation efficiency.After entire fermentation process, a large amount of bacterium for adapting to methanol environment completely will be obtained
The discarding of this thallus will be caused very big waste by body, so that the cost of production is increased, and about Pichia pastoris after one time fermentation
The report that thallus recycles just is to provide the Pichia pastoris thallus after a kind of recycling one time fermentation almost without, the present invention and lays equal stress on
The method utilized again.
Summary of the invention
It is an object of that present invention to provide a kind of methods that Pichia yeast weight answers fermentation expression foreign protein, and this method can
The cost for significantly reducing subsequent fermentation, shortens the duration of fermentation.
In order to achieve the above object, the present invention takes following technical measures:
A kind of method that Pichia yeast weight answers fermentation expression foreign protein, includes the following steps:
A, normal fermentation
Carry out conventional Pichia pastoris high density fermentation；
B, it repeats to ferment for the first time:
Fermentation liquid after B1, centrifugation normal fermentation, collects bacterium mud；
B2, repetition fermentation medium is prepared, is added in tank, the yeast thallus of collection is also added in tank after sterilizing；
The formula of the repetition fermentation medium other than without containing glycerol, send out by other components and constituent content and routine
Ferment culture medium is identical；
B3, stream plus 50% (mass ratio) glycerol containing 1.2% (volume ratio) Trace salts solution, increase thallus vigor；
The formula of the Trace salts solution includes: 6.0g cupric sulfate pentahydrate, 0.08g sodium iodide, 3.0g sulfuric acid monohydrate
Manganese, 0.2g Sodium Molybdate Dihydrate, 0.02g boric acid, 0.5g cobalt chloride, 20.0g zinc chloride, 65.0g ferrous sulfate, 0.2g biotin,
The 5.0ml concentrated sulfuric acid, adding water to total volume is 1L.
After B4, glycerol run out of, hungry 30min, start stream plus methanol, methanol feeding speed be 0.05ml/min/L~
0.2ml/min/L, for the flow acceleration of the methanol is compared to every liter culture medium；
Lower tank after B5, methanol consumption.
It in schemes described above, is collected by centrifugation and repeats the bacterium mud after fermenting for the first time, carry out second and repeat to ferment: weight
Multiple step B1~B5.
It in schemes described above, is collected by centrifugation second and repeats the bacterium mud after fermenting, carry out third time and repeat to ferment: weight
Multiple step B1~B5.
In schemes described above, remaining fermentation parameter is identical as normal fermentation.
It is preferred in scheme described above:
In the B1 of step B, after consistent when first by the temperature for repeating fermentation medium, pH adjustment with fermentation, first part is trained
After feeding base is mixed with bacterium mud, then pour into fermentor.
In the B3 of step B, the amount of stream glycerol adding is determined according to the weight in wet base of yeast, when glycerol is exhausted, yeast
Weight in wet base culture to 250g/L~350g/L.
In schemes described above, it is preferred that after the normal fermentation that the biomass used is recycling that repeatedly ferments for the first time
3/4ths of bacterium mud total amount；
In schemes described above, it is preferred that second of biomass used that repeatedly ferments is to repeat the first time of recycling
The half of bacterium mud total amount after fermentation；
In schemes described above, it is preferred that third time, which is repeatedly fermented second that the biomass used is recycling, to be repeated
The one third of bacterium mud total amount after fermentation.
In schemes described above, in step A, the step of Pichia pastoris ferments for the first time are as follows: prepare Pichia pastoris fermentation training
Base is supported, sterilizing after inoculation, by way of flowing glycerol adding culture, accumulates rapidly the biomass of Pichia pastoris；Glycerol cultivation stage
After, Pichia pastoris starvation 30min or so is entered into methanol induction phase, methanol stream by way of feed supplement is added into fermentation
In tank, until terminating induction when exogenous protein expression amount reaches peak value, the normal fermentation stage terminates；
In schemes described above, it is preferred that the Pichia pastoris is pichia pastoris yeast MMpk-GS115
(Pichia pastoris MMpk-GS115), deposit number are as follows: CCTCC NO:M 2014171.
Protection content of the invention further includes that a kind of method that Pichia yeast weight answers fermentation expression foreign protein is dropping
Application in low fermentation costs；Or shortening the application in fermentation duration；Or the cost of fermentation is reduced simultaneously and shortens fermentation
Application in duration.
Compared with prior art, main advantages of the present invention:
(1) normal fermentation mode, glycerol cultivation stage probably need 40h~45h, and the present invention is repeating fermentation stage glycerol
Culture only needs 2h~8h, saves the plenty of time of glycerol cultivation stage；
(2) compared to usual manner, the present invention three that the usage amount for repeating fermentation stage glycerol is usual manner/
One or so, reduce the usage amount of glycerol, reduces costs；
(3) for the present invention in repetition fermentation stage, thallus has adapted to methanol completely, therefore in stream plus methanol, thallus can
Directly methanol to be utilized probably to can be shortened the fermentation time of 1h~3h compared to usual manner without the laundering period.
Specific embodiment
Invention is described in further detail below with reference to specific embodiment, but the scope of the present invention is not limited to down
State embodiment.Technical solution of the present invention, such as not special standby explanation, is the ordinary skill in the art.
Embodiment 1:
The present embodiment is with one plant of Pichia yeast engineering pichia pastoris yeast MMpk-GS115 (Pichia pastoris
MMpk-GS115), deposit number are as follows: be illustrated for CCTCC NO:M 2014171.
Normal fermentation:
It prepares 20L culture medium to pour into 50L fermentor, batch fermentation is carried out by carbon source of glycerol, using glycerol feed supplement
Fermentation, carbon source is hungry, then carries out methanol feeding fermentation；Its carbon source starvation time is 30min；To fermentor after carbon source is hungry
Middle methanol of the addition containing 12ml/L Trace salts solution.
Detailed process includes:
Fermentation medium is prepared, is inoculated with, using glycerol as carbon source progress batch fermentation, fermentation 20~for 24 hours, when oxygen dissolving value is rapid
The mass ratio that stream plus 6L contain 12ml/L Trace salts solution is immediately begun to when rising to 100% as 50% glycerol, flow acceleration
For 24ml/min, when oxygen dissolving value rises very rapidly up to 100% again, the glycerol for showing that stream adds has been run out of, and thallus is wet at this time
It weighs to 350g/L, is all run out of from stream glycerol adding to glycerol and take around 20h.Then start stream after starvation 30min plus contain
There is the methanol of the Trace salts solution of 12ml/L, methanol feeding speed is 4ml/min, starts the initial stage of stream plus methanol, thallus has
One section of adaptation time, generally in 3h, in this time, thallus does not utilize substantially or slowly using methanol, methanol feeding 72h, to
When oxygen dissolving value rises very rapidly up to 100%, then shows that stream is added the methanol in tank and all run out of, tank can be played.
Conventional fermentation process fermentation total duration is about 125h, and the enzyme activity of protease is 10.5 × 10 in fermentation liquid5u/ml。
In the above conventional fermentation process, 28.5 ± 0.5 DEG C of temperature are controlled, pH adds tune by ammonia aqua stream for 5.0 ± 0.1, pH
Section, tank press 0.04MPa, speed of agitator 800rpm.
The fermentative medium formula are as follows: 26.7mL 85% (volume ratio) phosphoric acid, 0.93g calcium sulfate, 18.2g sulfuric acid
Potassium, 14.9g epsom salt, 4.13g potassium hydroxide, 40g glycerol, adding water to total volume is 1L.
The formula of the Trace salts solution are as follows: by 6.0g cupric sulfate pentahydrate, 0.08g sodium iodide, 3.0g sulfuric acid monohydrate
Manganese, 0.2g Sodium Molybdate Dihydrate, 0.02g boric acid, 0.5g cobalt chloride, 20.0g zinc chloride, 65.0g ferrous sulfate, 0.2g biotin,
The 5.0ml concentrated sulfuric acid, adding water to total volume is 1L.
A kind of method that Pichia yeast weight answers fermentation expression foreign protein, comprising the following steps:
It repeats to ferment for the first time:
(1) it by the fermentation liquid of normal fermentation, is centrifuged, revolving speed 6000rpm, is centrifuged 5 times through tube centrifuge, recycle bacterium
Mud；
(2) it prepares repetition fermentation medium 20L to pour into 50L fermentor, after sterilizing, 10L culture is released from fermentor
It after base is mixed with 3/4ths bacterium mud of bacterium mud total amount after the normal fermentation of recycling, together pours into tank, immediately stream plus 1L
The glycerol that mass ratio containing 12ml/L Trace salts solution is 50%, flow acceleration 24ml/min, the purpose is to increase thallus
Activity and weight in wet base, when oxygen dissolving value rises very rapidly up to 100%, the glycerol for showing that stream adds has been run out of, and thallus weight in wet base reaches at this time
To 350g/L, is all run out of from stream glycerol adding to glycerol and take around 3h.After glycerol consumption completely, hungry 30min, stream adds and contains
There is the methanol of the Trace salts solution of 12ml/L, methanol feeding speed is 4ml/min, this stage thallus can be once contact methanol at once
Utilize, do not need any adaptation process, methanol feeding 72h, when oxygen dissolving value rises very rapidly up to 100%, then show stream add into
Methanol in tank all runs out of, can play tank.
Repeating fermentation process fermentation duration for the first time is about 78h, and fermentation liquid proteinase activity is 6.5 × 105u/ml。
The formula for repeating fermentation medium includes: 26.7mL 85% (volume ratio) phosphoric acid, 0.93g calcium sulfate, 18.2g sulphur
Sour potassium, 14.9g epsom salt, 4.13g potassium hydroxide, adding water to total volume is 1L.
It is previously mentioned in the formula of Trace salts solution and normal fermentation identical.
The above first time repeats in fermentation process, controls 28.5 ± 0.5 DEG C of temperature, pH is that 5.0 ± 0.1, pH passes through ammonium hydroxide
Stream plus adjusting, tank press 0.04MPa, speed of agitator 800rpm.
Second of repetition is fermented:
(1) fermentation liquid that first time is repeated to fermentation, is centrifuged, revolving speed 6000rpm through tube centrifuge, is centrifuged 5 times, returns
Receive bacterium mud；
(2) it prepares repetition fermentation medium 20L to pour into 50L fermentor, after sterilizing, 10L culture is released from fermentor
After base is mixed with the bacterium mud that the first time of recycling repeats the half of bacterium mud weight after fermenting, together pour into tank, immediately
Stream plus 2L contain the glycerol that the mass ratio of 12ml/L Trace salts solution is 50%, and flow acceleration 24ml/min, the purpose is to increase
Add microbial activity and weight in wet base, when oxygen dissolving value rises very rapidly up to 100%, the glycerol for showing that stream adds has been run out of, at this time thallus
Weight in wet base reaches 350g/L, all runs out of from stream glycerol adding to glycerol and takes around 6h.After glycerol consumption completely, hungry 30min,
The methanol of stream plus the Trace salts solution containing 12ml/L, methanol feeding speed are 4ml/min, this stage thallus is once contact methanol
It can utilize at once, not need any adaptation process, methanol feeding 72h then shows to flow when oxygen dissolving value rises very rapidly up to 100%
It adds the methanol in tank all to run out of, tank can be played.
It is about 85h that second, which repeats fermentation process fermentation duration, and fermentation liquid proteinase activity is 3.5 × 105u/ml。
It is identical this time to repeat formula and the first time repetition fermentative medium formula of fermentation medium.
It is previously mentioned in the formula of Trace salts solution and normal fermentation identical.
In above second of repetition fermentation process, 28.5 ± 0.5 DEG C of temperature are controlled, pH is that 5.0 ± 0.1, pH passes through ammonium hydroxide
Stream plus adjusting, tank press 0.04MPa, speed of agitator 800rpm.
Third time recycling fermentation:
(1) it by the fermentation liquid of second of fermentation, is centrifuged, revolving speed 6000rpm, is centrifuged 5 times through tube centrifuge, recycle bacterium
Mud；
(2) it prepares repetition fermentation medium 20L to pour into 50L fermentor, after sterilizing, 10L culture is released from fermentor
After base is mixed with second of bacterium mud for repeating the one third of bacterium mud weight after fermenting of recycling, together pour into tank, immediately
Stream plus 3L contain the glycerol that the mass ratio of 12ml/L Trace salts solution is 50%, and flow acceleration 24ml/min, the purpose is to increase
Add microbial activity and weight in wet base, when oxygen dissolving value rises very rapidly up to 100%, the glycerol for showing that stream adds has been run out of, at this time thallus
Weight in wet base reaches 350g/L, all runs out of from stream glycerol adding to glycerol and takes around 8h.After glycerol consumption completely, hungry 30min,
The methanol of stream plus the Trace salts solution containing 12ml/L, methanol feeding speed are 4ml/min, this stage thallus is once contact methanol
It can utilize at once, not need any adaptation process, methanol feeding 72h then shows to flow when oxygen dissolving value rises very rapidly up to 100%
It adds the methanol in tank all to run out of, tank can be played.
It is about 88h that third time, which repeats fermentation process fermentation duration, and fermentation liquid proteinase activity is 2.5 × 105u/ml。
It is identical this time to repeat formula and the first time repetition fermentative medium formula of fermentation medium.
It is previously mentioned in the formula of Trace salts solution and normal fermentation identical.
The above third time repeats in fermentation process, controls 28.5 ± 0.5 DEG C of temperature, pH is that 5.0 ± 0.1, pH passes through ammonium hydroxide
Stream plus adjusting, tank press 0.04MPa, speed of agitator 800rpm.
Embodiment 2:
This time fermentor used is 100L unlike the first embodiment, and initial culture medium is 40L, and the stream of methanol accelerates
Degree also increases to one times of embodiment 1, and when repeating to ferment, early period, the amount of stream glycerol adding was also doubled, and recycling twice, terminates
Whole process.Repeating fermentation duration for the first time is about 80h or so, obtains fermentation liquid proteinase activity 5.5 × 105U/ml second
Secondary about 85h or so, obtains fermentation liquid proteinase activity 3.0 × 105u/ml。
Claims (2)
1. a kind of method that Pichia yeast weight answers fermentation expression foreign protein, includes the following steps:
A, normal fermentation
Carry out conventional Pichia pastoris high density fermentation；
B, it repeats to ferment for the first time:
B1, centrifugation normal fermentation after fermentation liquid, recycle normal fermentation after bacterium mud total amount 3/4ths；
B2, preparation repetition fermentation medium, are added in tank, after sterilizing, first will repeat the temperature of fermentation medium, pH is adjusted and normal
After rule are consistent when fermenting, after first mixing partial medium with bacterium mud, then pour into fermentor；
The formula of the repetition fermentation medium other than without containing glycerol, train by other components and constituent content and normal fermentation
It is same to support base phase；
B3, stream plus 50% glycerol containing 1.2% volume ratio Trace salts solution, increase thallus vigor；When glycerol is exhausted, ferment
Female weight in wet base culture is to 250g/L~350g/L；
The formula of the Trace salts solution includes: 6.0g cupric sulfate pentahydrate, 0.08g sodium iodide, 3.0g Manganous sulfate monohydrate,
0.2g Sodium Molybdate Dihydrate, 0.02g boric acid, 0.5g cobalt chloride, 20.0g zinc chloride, 65.0g ferrous sulfate, 0.2g biotin,
The 5.0ml concentrated sulfuric acid, adding water to total volume is 1L；
After B4, glycerol run out of, hungry 30min starts stream plus methanol, and methanol feeding speed is 0.05ml/min/L~0.2ml/
Min/L, for the flow acceleration of the methanol is compared to every liter culture medium；
Lower tank after B5, methanol consumption；
C, it repeats to ferment for second:
The half of bacterium mud total amount after repeating to ferment for the first time is collected by centrifugation, carries out second and repeats to ferment: repeating step B1
~B5；
D, third time repeats to ferment
Second of one third for repeating the bacterium mud total amount after fermenting is collected by centrifugation, carries out third time and repeats to ferment: repeating step
B1~B5；
The Pichia pastoris is pichia pastoris yeast MMpk-GS115 (Pichia pastoris MMpk-GS115), preservation
Number are as follows: CCTCC NO:M 2014171.
2. method described in claim 1 reduces the cost of fermentation at the same time and shortens the application in the duration fermented.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0911416A2 (en) * | 1997-10-01 | 1999-04-28 | F. Hoffmann-La Roche Ag | Protein production process |
| CN102242153A (en) * | 2011-05-03 | 2011-11-16 | 华中农业大学 | Method for repeatedly fermenting and utilizing culture medium of pichia pastoris |
| CN103981164A (en) * | 2014-05-30 | 2014-08-13 | 华中农业大学 | High-temperature-resistant protease, strain breeding method thereof and application method of high-temperature-resistant protease to enzymolysis |
-
2015
- 2015-10-19 CN CN201510678943.4A patent/CN105200101B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0911416A2 (en) * | 1997-10-01 | 1999-04-28 | F. Hoffmann-La Roche Ag | Protein production process |
| CN102242153A (en) * | 2011-05-03 | 2011-11-16 | 华中农业大学 | Method for repeatedly fermenting and utilizing culture medium of pichia pastoris |
| CN103981164A (en) * | 2014-05-30 | 2014-08-13 | 华中农业大学 | High-temperature-resistant protease, strain breeding method thereof and application method of high-temperature-resistant protease to enzymolysis |
Non-Patent Citations (1)
| Title |
|---|
| Continuous Production of Thrombomodulin from a Pichia pastoris Fermentation;Yinliang Chen et al.;《J. Chem. Tech. Biotechnol.》;19961031;第67卷(第2期);摘要第144页左栏第3-4段，第145页左栏第2段至右栏第1段，第146页左栏第1段至第148页左栏第2段，图5-6 |
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