CN108034595A - A kind of culture medium of pichia pastoris and application thereof - Google Patents
A kind of culture medium of pichia pastoris and application thereof Download PDFInfo
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- CN108034595A CN108034595A CN201810081331.0A CN201810081331A CN108034595A CN 108034595 A CN108034595 A CN 108034595A CN 201810081331 A CN201810081331 A CN 201810081331A CN 108034595 A CN108034595 A CN 108034595A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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Abstract
The present invention provides a kind of culture medium of pichia pastoris, include the component of following concentration：20~30g/L of dusty yeast, 30~40g/L of soy peptone, 10~30g/L of glycerine, CaO250~100g/L, methanol 30~60g/L and PTM1 solution 0.5~5ml/L, pH are 6~8.In pichia pastoris yeast culture medium provided by the invention, CaO with the addition of2As dissolved oxygen carrier, it is possible to reduce the use cost of organic carrier of oxygen, reduces the production cost of fermentation, while can also obtain higher expression of recombinant proteins amount.Culture medium provided by the invention, has broad application prospects in the production using pichia pastoris yeast expression system expression recombinant protein.The present invention is remarkably improved the fermentation dissolved oxygen level of recombinant yeast and the expression of antibacterial peptide, and production process is simply easily controllable, fermentation period is short.
Description
Technical field
The present invention relates to a kind of culture medium of pichia pastoris and application thereof.
Background technology
In pichia pastoris yeast BMGY/BMMY culture mediums, main component be yeast extract (Yeast Extract),
Peptone (Peptone) and without amino yeast nitrogen (Yeast Nitrogen Base) etc..But this kind of culture medium is only applicable to small
Scale evaluation, after amplification, cost is excessive, and applies limited.At present in large scale fermentation culture medium, often containing substantial amounts of organic
The carrier of oxygen, adds production cost, and all bad for dissolved oxygen level and stabilization.CaO2Increase as common aqueous solution
Oxygen agent, is decomposed slowly, and does not have toxic action to yeast thalline, and accessory substance can also be used as fermentation salt ion, therefore, for
Add CaO2The dissolved oxygen level of fermentation can be not only improved, but also the use of the additives such as other organic carriers of oxygen can also be reduced
Cost, contributes to amplification and the industrialized production of fermentation.
The content of the invention
It is an object of the present invention to provide a kind of culture medium of pichia pastoris, to make up the deficiencies in the prior art.
To achieve the above object, the technical solution used in the present invention：A kind of culture medium of pichia pastoris, including following concentration
Component：20~30g/L of dusty yeast, 30~40g/L of soy peptone, 10~30g/L of glycerine, CaO250~100g/L, methanol
30~60g/L and PTM1 solution 0.5~5ml/L, pH are 6~8.
Preferably, the culture medium of pichia pastoris includes the component of following concentration：20~22g/L of dusty yeast, soybean protein
32~36g/L of peptone, 15~25g/L of glycerine, CaO250~70g/L, 3~4ml/L of methanol 35~40g/L and PTM1 solution, pH are
6~6.5.
Preferably, the culture medium of pichia pastoris includes the component of following concentration：Dusty yeast 20g/L, soy peptone
35g/L, glycerine 25g/L, CaO250g/L, methanol 35g/L and PTM1 solution 3ml/L, pH 6.5.
Preferably, the culture medium of pichia pastoris includes the component of following concentration：Dusty yeast 22g/L, soy peptone
32g/L, glycerine 15g/L, CaO270g/L, methanol 40g/L and PTM1 solution 4ml/L, pH 6.
The present invention provides a kind of method for producing antibacterial peptide pBD-1, the method is with Pichia pastoris described above
Culture medium carries out fermented and cultured as fermentation medium to Pichia pastoris, obtains the antibacterial peptide pBD-1.
Preferably, the Pichia pastoris is wild-type strain or the recombination engineering containing foreign gene by transformation
Strain.
The present invention provides the application of culture medium of pichia pastoris described above in antibacterial peptide pBD-1 is produced.
The beneficial effects of the present invention are：
In pichia pastoris yeast culture medium provided by the invention, CaO with the addition of2As dissolved oxygen carrier, it is possible to reduce have
The use cost of the machine carrier of oxygen, reduces the production cost of fermentation, while can also obtain higher expression of recombinant proteins amount.
Culture medium provided by the invention, has in the production using pichia pastoris yeast expression system expression recombinant protein
Have broad application prospects.
The present invention is remarkably improved the fermentation dissolved oxygen level of recombinant yeast and the expression of antibacterial peptide, and production process
It is simple it is easily controllable, fermentation period is short.
Embodiment
The present invention is further explained with reference to embodiments, for specific method or material used in embodiment, sheet
Field technology personnel can carry out conventional replacement according to existing technology and select on the basis of the technology of the present invention thinking, and
It is not limited only to the specific record of the embodiment of the present invention.
Test method used in embodiment is accordingly to be regarded as conventional method unless otherwise specified；Used material, reagent
Deng unless otherwise instructed, being commercially available.
Embodiment 1
Contrast culture medium of the fermentation medium of the present embodiment as the present invention, component include as follows：
Dusty yeast 22g/L, soy peptone 36g/L, glycerine 24g/L, soya-bean oil 60g/L, methanol 37g/L, PTM1 solution
3ml/L, pH 6.5.
1st, fermentation process
Antibacterial peptide pBD-1 fermentations are carried out using the fermentation medium of the present embodiment component content, are comprised the following steps：
Step 1, takes the recombinant yeast pichia pastoris strain of expression antibacterial peptide pBD-1, in 100ml YPD culture mediums, in 27-
29 DEG C, when 250rpm/min shaken cultivations 18 are small；In 27-29 DEG C in 1000mL YPD culture mediums, 250rpm/min vibrations are trained
Support 18 it is small when after, obtain seed liquor.
Step 2, prepares 10L fermentation mediums, and the oxygen dissolving value that 10L fermentation tank dissolved oxygen electrodes are demarcated before inoculation is
100%.Seed liquor is fitted into above-mentioned fermentation medium by 5% inoculum concentration, starts to ferment.Ferment 12 it is small when, 24 it is small when,
36 it is small when, 48 it is small when, 60 it is small when, 72 it is small when, record dissolved oxygen DO actual monitoring values, as shown in table 1.
1 dissolved oxygen DO actual monitoring values of table
| Selecting time | The actually detected values of DO | Selecting time | The actually detected values of DO |
| 12 it is small when | 50% | 48 it is small when | 25% |
| 24 it is small when | 40% | 60 it is small when | 25% |
| 32 it is small when | 30% | 72 it is small when | 20% |
Step 3, ferment 80h, terminates fermentation, collects zymotic fluid.Supernatant is taken after centrifugation, with preparative high performance liquid chromatography,
Lyophilization, obtains pure protein.
In the present embodiment, the yield of antibacterial peptides that fermentation medium through this embodiment ferments out is in 160mg/L.
Embodiment 2
1st, fermentation medium
Included in the fermentation medium of the present embodiment：Dusty yeast 22g/L, soy peptone 36g/L, glycerine 24g/L, CaO2
20g/L, methanol 37g/L, PTM1 solution 3ml/L, pH 6.5.
1st, fermentation process
Antibacterial peptide pBD-1 fermentations are carried out using the fermentation medium of the present embodiment component content, are comprised the following steps：
Step 1, takes the recombinant yeast pichia pastoris strain of expression antibacterial peptide pBD-1, in 100ml YPD culture mediums, in 27-
29 DEG C, when 250rpm/min shaken cultivations 18 are small；In 27-29 DEG C in 1000mL YPD culture mediums, 250rpm/min vibrations are trained
Support 18 it is small when after, obtain seed liquor.
Step 2, prepares 10L fermentation mediums, and the oxygen dissolving value that 10L fermentation tank dissolved oxygen electrodes are demarcated before inoculation is
100%.Seed liquor is fitted into above-mentioned fermentation medium by 5% inoculum concentration, starts to ferment.Ferment 12 it is small when, 24 it is small when,
36 it is small when, 48 it is small when, 60 it is small when, 72 it is small when, record dissolved oxygen DO actual monitoring values, as shown in table 2.
2 dissolved oxygen DO actual monitoring values of table
| Selecting time | The actually detected values of DO | Selecting time | The actually detected values of DO |
| 12 it is small when | 60% | 48 it is small when | 28% |
| 24 it is small when | 55% | 60 it is small when | 25% |
| 32 it is small when | 30% | 72 it is small when | 20% |
Step 3, ferment 80h, terminates fermentation, collects zymotic fluid.Supernatant is taken after centrifugation, with preparative high performance liquid chromatography,
Lyophilization, obtains pure protein.
In the present embodiment, the yield of antibacterial peptides that fermentation medium through this embodiment ferments out is in 180mg/L.
Embodiment 3
1st, fermentation medium
Included in the fermentation medium of the present embodiment：Dusty yeast 20g/L, soy peptone 35g/L, glycerine 25g/L, CaO2
50g/L, methanol 35g/L, PTM1 solution 3ml/L, pH 6.5.
2nd, fermentation process
Antibacterial peptide pBD-1 fermentations are carried out using the fermentation medium of the present embodiment component content, are comprised the following steps：
Step 1, takes the recombinant yeast pichia pastoris strain of expression antibacterial peptide pBD-1, in 100ml YPD culture mediums, in 27-
29 DEG C, when 250rpm/min shaken cultivations 18 are small；In 27-29 DEG C in 1000mL YPD culture mediums, 250rpm/min vibrations are trained
Support 18 it is small when after, obtain seed liquor.
Step 2, prepares 10L fermentation mediums, and the oxygen dissolving value that 10L fermentation tank dissolved oxygen electrodes are demarcated before inoculation is
100%.Seed liquor is fitted into above-mentioned fermentation medium by 5% inoculum concentration, starts to ferment.Ferment 12 it is small when, 24 it is small when,
36 it is small when, 48 it is small when, 60 it is small when, 72 it is small when, record dissolved oxygen DO actual monitoring values, as shown in table 3.
3 dissolved oxygen DO actual monitoring values of table
| Selecting time | The actually detected values of DO | Selecting time | The actually detected values of DO |
| 12 it is small when | 65% | 48 it is small when | 55% |
| 24 it is small when | 63% | 60 it is small when | 50% |
| 32 it is small when | 58% | 72 it is small when | 50% |
Step 3, ferment 80h, terminates fermentation, collects zymotic fluid.Supernatant is taken after centrifugation, with preparative high performance liquid chromatography,
Lyophilization, obtains pure protein.
In the present embodiment, the yield of antibacterial peptides that fermentation medium through this embodiment ferments out is in 500mg/L.
Embodiment 4
Included in the fermentation medium of the present embodiment：Dusty yeast 22g/L, soy peptone 32g/L, glycerine 15g/L, CaO2
70g/L, methanol 40g/L, PTM1 solution 4ml/L, pH 6..
2nd, fermentation process
Restructuring pBD-1 fermentations are carried out using the fermentation medium of the present embodiment component content, are comprised the following steps：
Step 1, takes the recombinant yeast pichia pastoris strain of expression antibacterial peptide pBD-1, in 100ml YPD culture mediums, in 27-
29 DEG C, when 250rpm/min shaken cultivations 18 are small；In 27-29 DEG C in 1000mL YPD culture mediums, 250rpm/min vibrations are trained
Support 18 it is small when after, obtain seed liquor.
Step 2, prepares 10L fermentation mediums, and the oxygen dissolving value that 10L fermentation tank dissolved oxygen electrodes are demarcated before inoculation is
100%.Seed liquor is fitted into above-mentioned fermentation medium by 5% inoculum concentration, starts to ferment.Ferment 12 it is small when, 24 it is small when,
36 it is small when, 48 it is small when, 60 it is small when, 72 it is small when, record dissolved oxygen DO actual monitoring values, as shown in table 4.
4 dissolved oxygen DO actual monitoring values of table
Step 3, ferment 80h, terminates fermentation, collects zymotic fluid.Supernatant is taken after centrifugation, with preparative high performance liquid chromatography,
Lyophilization, obtains pure protein.
In the present embodiment, the yield of antibacterial peptides that fermentation medium through this embodiment ferments out is in 630mg/L.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, those of ordinary skill in the art should
Understand, can be to technical scheme technical scheme is modified or replaced equivalently, without departing from the essence of technical solution of the present invention
And scope.
Claims (7)
1. a kind of culture medium of pichia pastoris, it is characterised in that include the component of following concentration：20~30g/L of dusty yeast, soybean egg
30~40g/L of white peptone, 10~30g/L of glycerine, CaO250~100g/L, 0.5~5ml/ of methanol 30~60g/L and PTM1 solution
L, pH are 6~8.
2. culture medium of pichia pastoris according to claim 1, it is characterised in that include the component of following concentration：Dusty yeast
20~22g/L, 32~36g/L of soy peptone, 15~25g/L of glycerine, CaO250~70g/L, 35~40g/L of methanol and
3~4ml/L of PTM1 solution, pH are 6~6.5.
3. culture medium of pichia pastoris according to claim 1, it is characterised in that include the component of following concentration：Dusty yeast
20g/L, soy peptone 35g/L, glycerine 25g/L, CaO250g/L, methanol 35g/L and PTM1 solution 3ml/L, pH 6.5.
4. culture medium of pichia pastoris according to claim 1, it is characterised in that include the component of following concentration：Dusty yeast
22g/L, soy peptone 32g/L, glycerine 15g/L, CaO270g/L, methanol 40g/L and PTM1 solution 4ml/L, pH 6.
A kind of 5. method for producing antibacterial peptide pBD-1, it is characterised in that the method is with as described in claim 1-4 is any
Culture medium of pichia pastoris as fermentation medium to Pichia pastoris carry out fermented and cultured, obtain the antibacterial peptide pBD-1.
6. according to the method described in claim 5, it is characterized in that, the Pichia pastoris is wild-type strain or passes through transformation
The recombinant strain containing foreign gene.
7. application of the culture medium of pichia pastoris in antibacterial peptide pBD-1 is produced as described in claim 1-4 is any.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103403171A (en) * | 2010-12-20 | 2013-11-20 | 纳幕尔杜邦公司 | Use of a nitrogen- free peroxygen- releasing compound to reduce growth of contaminant microorganisms in ethanol fermentation |
| CN106947724A (en) * | 2017-05-22 | 2017-07-14 | 中国科学院成都生物研究所 | A kind of method of increase γ polyglutamic acid zymotic fluid dissolved oxygens |
| CN107475139A (en) * | 2017-09-30 | 2017-12-15 | 广州大学 | A kind of pH stable types fermentation medium and its application |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103403171A (en) * | 2010-12-20 | 2013-11-20 | 纳幕尔杜邦公司 | Use of a nitrogen- free peroxygen- releasing compound to reduce growth of contaminant microorganisms in ethanol fermentation |
| CN106947724A (en) * | 2017-05-22 | 2017-07-14 | 中国科学院成都生物研究所 | A kind of method of increase γ polyglutamic acid zymotic fluid dissolved oxygens |
| CN107475139A (en) * | 2017-09-30 | 2017-12-15 | 广州大学 | A kind of pH stable types fermentation medium and its application |
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