CN103923846A - Pichia pastoris culturing medium - Google Patents
Pichia pastoris culturing medium Download PDFInfo
- Publication number
- CN103923846A CN103923846A CN201310727410.1A CN201310727410A CN103923846A CN 103923846 A CN103923846 A CN 103923846A CN 201310727410 A CN201310727410 A CN 201310727410A CN 103923846 A CN103923846 A CN 103923846A
- Authority
- CN
- China
- Prior art keywords
- substratum
- pichia pastoris
- pastoris phaff
- fermentation
- culturing medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000235058 Komagataella pastoris Species 0.000 title claims abstract description 34
- 238000012258 culturing Methods 0.000 title abstract description 8
- 238000000855 fermentation Methods 0.000 claims abstract description 25
- 230000004151 fermentation Effects 0.000 claims abstract description 25
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 8
- 238000011218 seed culture Methods 0.000 claims description 6
- 101150051118 PTM1 gene Proteins 0.000 claims description 5
- 230000001186 cumulative effect Effects 0.000 claims description 5
- 235000011187 glycerol Nutrition 0.000 claims description 5
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 3
- 239000012533 medium component Substances 0.000 claims 1
- 230000001131 transforming effect Effects 0.000 claims 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 abstract description 7
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 abstract description 7
- 238000004519 manufacturing process Methods 0.000 abstract description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 abstract description 5
- 230000008901 benefit Effects 0.000 abstract description 2
- 235000011007 phosphoric acid Nutrition 0.000 abstract 1
- 230000006872 improvement Effects 0.000 description 19
- 239000002609 medium Substances 0.000 description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- 239000001963 growth medium Substances 0.000 description 11
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 10
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 10
- 238000000034 method Methods 0.000 description 10
- 229910052760 oxygen Inorganic materials 0.000 description 10
- 239000001301 oxygen Substances 0.000 description 10
- 241001052560 Thallis Species 0.000 description 7
- 239000002585 base Substances 0.000 description 7
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000002054 inoculum Substances 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 235000013343 vitamin Nutrition 0.000 description 3
- 239000011782 vitamin Substances 0.000 description 3
- 229940088594 vitamin Drugs 0.000 description 3
- 229930003231 vitamin Natural products 0.000 description 3
- 150000003722 vitamin derivatives Chemical class 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 101710194180 Alcohol oxidase 1 Proteins 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000000630 rising effect Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000009423 ventilation Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000008215 water for injection Substances 0.000 description 2
- 102100036826 Aldehyde oxidase Human genes 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000050051 Chelone glabra Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 101000928314 Homo sapiens Aldehyde oxidase Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 108091058545 Secretory proteins Proteins 0.000 description 1
- 102000040739 Secretory proteins Human genes 0.000 description 1
- 206010047571 Visual impairment Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000007797 corrosion Effects 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 230000024835 cytogamy Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 231100000206 health hazard Toxicity 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 231100000516 lung damage Toxicity 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 150000002897 organic nitrogen compounds Chemical class 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 238000011020 pilot scale process Methods 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 239000008057 potassium phosphate buffer Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000008786 sensory perception of smell Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 208000029257 vision disease Diseases 0.000 description 1
- 230000004393 visual impairment Effects 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a Pichia pastoris culturing medium. The culturing medium contains no KOH or H3PO4, so the destroy to the environment is reduced, and a fermentation apparatus is convenient to maintain; and when the culturing medium is used to carry out the mass culture of Pichia pastoris strains, each of the cell density and the recombinant protein expression level is high, so the fermentation production cost is reduced, and it is benefit for the industrial scale fermentation and production.
Description
Technical field
The invention belongs to biological fermentation field, be specifically related to a kind of for cultivating the substratum of pichia pastoris phaff.
Background technology
Pichia pastoris phaff (Pichiapastoris) is that the class in methyl alcohol nutritional type yeast can utilize methyl alcohol as the yeast of sole carbon source and the energy.Pichia pastoris phaff is unicellular eukaryote, and growth is fast, is easy to molecular genetics operation; Alcohol oxidase 1(Alcohol Oxidase1, the AOX1 of pichia pastoris phaff) promotor of gene has strong inducibility and strong startability, is suitable for the high-level abduction delivering of foreign gene; Goal gene is incorporated into has high stability on karyomit(e); Can high-level secretory expression recombinant protein, purifying is convenient; There is eukaryotic posttranslational modification function.Above advantage becomes in current biology field for expressing one of tool master of recombinant protein pichia pastoris phaff expression system.
Fermention medium is for growth, breeding and synthetic product.Energy ramp after it should make seed inoculate, reaches certain cell concentration, makes again the rapid synthetic product of thalline energy of having grown.Therefore, the composition of fermention medium, apart from outside the necessary element of thalli growth and compound, also will have the required element-specific of product, precursor and promotor etc.Aspect pichia pastoris phaff high density fermentation culture medium, the standard recipe that people adopt Invitrogen company to provide more: fermentation adopts BMGY/BMMY substratum on a small scale; Large scale fermentation adopts BMGY/BSM substratum.
In pichia pastoris phaff BMGY/BMMY substratum, main component is yeast extract (Yeast Extract), peptone (Peptone) and without amino yeast nitrogen (Yeast Nitrogen Base) etc.Yeast extract be yeast will be wherein after broken wall the extracting such as protein, nucleic acid, VITAMIN, again through the material of the active skull cap components such as the micromolecular amino acid of being rich in of biological enzymolysis, peptide, Nucleotide, VITAMIN, wherein aminoacids content more than 30%, total protein more than 50%, Nucleotide are more than 10%.Peptone is that meat, casein or gelatin are to flaxen pulvis by the dry outward appearance forming after acid or protease hydrolysis, is rich in organic nitrogen compound, also contains some VITAMIN and carbohydrate.BMGY/BMMY substratum is only applicable to the small-scale of pichia pastoris phaff and cultivates; During amplification culture, high cost due to organic nitrogen source unit volume, and above-mentioned composition organic, animal-origin exists possibility that external source is polluted, to downstream purification technique, bring multiple difficulty, so the large scale culturing of pichia pastoris phaff adopts BSM inorganic medium more.
In pichia pastoris phaff large scale fermentation BSM inorganic medium, a large amount of phosphoric acid and potassium hydroxide have been applied.Phosphoric acid (H
3pO
4) be a kind of middle strong acid, in air, easy deliquescence, larger to the Health hazard of human body.In fermentative production, due to its sour corrodibility, at the material of equipment with manufacture and all need to consider its impact.Potassium hydroxide (KOH), very easily moisture absorption and deliquescence in air, volatilization during high temperature and not decomposing, has strong corrosion, intense stimulus respiratory tract or cause and burn after sucking; Skin directly contacts and can cause and burn with eye; Long Term Contact can bring many adverse consequencess such as lung damage, visual impairment, sense of smell infringement.
Therefore, be necessary to provide a kind of pichia pastoris phaff substratum of environmental protection and economy more, the destructiveness of reduction to environment, be convenient to the maintenance of fermentation equipment, can access higher cell density and expression of recombinant proteins amount simultaneously, reduce the production cost of fermentation, thereby be beneficial to fermentation and the production of pichia pastoris phaff industrially scalable.
Summary of the invention
The object of the invention is to, provide a kind of pichia pastoris phaff substratum, to make up the deficiencies in the prior art.
Pichia pastoris phaff substratum provided by the invention is not containing KOH and H
3pO
4, contain NH
4h
2pO
4with KH
2pO
4.
Pichia pastoris phaff substratum provided by the invention is not containing KOH and H
3pO
4, the NH that contains 1.575～31.5g/L
4h
2pO
4kH with 1.155～23.1g/L
2pO
4.
The substratum of pichia pastoris phaff provided by the invention, by the cumulative volume of substratum, described substratum contains following composition:
According to a preferred embodiment of the present invention, by the cumulative volume of substratum, described substratum contains following composition:
The aforementioned substratum providing of the present invention, for cultivating pichia pastoris phaff.
According to a preferred embodiment of the present invention, the aforementioned substratum providing is used as fermention medium in the process of cultivating pichia pastoris phaff.
According to a preferred embodiment of the present invention, the aforementioned substratum providing is used as seed culture medium in the process of cultivating pichia pastoris phaff.
Pichia pastoris phaff of the present invention be wild-type, through transformation the recombinant strain that contains foreign gene in a kind of.
Substratum provided by the invention has been avoided acid, the harm of alkali to environment, personnel and fermentation equipment, has reduced the production cost of fermentation, also can access higher cell density and expression of recombinant proteins amount simultaneously.Substratum provided by the invention has broad application prospects in the production that utilizes pichia pastoris phaff expression system expression recombinant protein.
Accompanying drawing explanation
Fig. 1 adopts substratum of the present invention as the fermention medium of shaking flask level, the thalli growth curve of whole fermentation period.
Fig. 2 adopts substratum of the present invention as the fermention medium of 30L fermentor tank, the thalli growth curve of whole fermentation period.
Fig. 3 adopts improvement IV substratum of the present invention as seed culture medium and fermention medium, the thalli growth of whole fermentation period and the protein expression curve of 30L fermentor tank.
Embodiment
Below in conjunction with embodiment, further illustrate the present invention, following embodiment should not be construed as limitation of the present invention.Embodiment does not comprise the detailed description to traditional method, as those methods for carrier construction and plasmid, the gene of proteins encoded is inserted into the method for carrier and plasmid or plasmid is introduced to method and classical cytogamy and the method for purifying etc. of host cell.Such method is well-known for person having ordinary skill in the art, and all describe to some extent in many publications, comprise Sambrook, J., Fritsch, E.F.andManiais, T. (1989) Molecular Cloning:A Laboratory Manual, 2nd edition, Cold spring HarborLaboratory Press.
In following embodiment of the present invention, the pichia pastoris phaff using for culture presevation number be the bacterial strain of CGMCC No.1360, hereinafter referred to as HSA75-10, the recombination that this bacterium contains human serum albumin, can excreting and expressing recombinant human seralbumin (HSA) through induction.Pichia pastoris phaff (Pichia pastoris) HSA75-10 has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on 04 26th, 2005, register on the books and be numbered CGMCC No.1360, CGMCC is positioned at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City No. 3, Institute of Microorganism, Academia Sinica.
Substratum and solution in contrast in embodiment, all reagent and raw material are all purchased from Sigma company:
1) BMGY substratum:
2) BSM substratum:
3) PTM1 solution:
4) 100mM potassium phosphate buffer:
K
2HPO
4 61.5g/L
KH
2PO
4 8.5g/L
Adjust pH to 7.0, sterilizing, 4 ℃ of preservations.
5) supplemented medium: 50% glycerine; Methyl alcohol containing 1%PTM1.
The preparation of embodiment 1 substratum
In following embodiment of the present invention, the substratum of use, by the cumulative volume of substratum, contains following composition:
On the basis of above-mentioned substratum, by the formula of table 1, prepare respectively substratum, obtain four kinds of different modified form substratum, respectively called after improvement I, improvement II, improvement III and improvement IV.
Table 1
Process for preparation is as follows, and in showing with the water for injection of 80%～90% final volume, composition dissolves, and after fully stirring, measures pH, after adjusting pH, with water for injection, is settled to final volume with ammoniacal liquor.
Embodiment 2, the horizontal strain culturing of shaking flask and detection
1) on picking YPD flat board, the mono-colony inoculation of HSA75-10, in the 250ml shaking flask of 40ml BMGY is housed, is cultivated 24h in 200rpm, 30 ℃ of shaking tables, is prepared into first order seed;
2) by 5% inoculum size, first order seed is accessed respectively in the 1L shaking flask that 100ml BMGY, BSM, improvement I, improvement II, improvement III, improvement IV are housed, in 200rpm, 30 ℃ of shaking tables, continue to cultivate;
3) every 24 hours is 0.5% to methyl alcohol to the substratum final concentration adding containing 1%PTM1 in substratum;
4) by time point 0,6,12,24,36,48,60,72,84 and 96h, get respectively bacterium liquid sample, analyze thalline weight in wet base;
5) thalli growth comparative result as shown in Figure 1.Can find out, the growth tendency of HSA75-10 under various culture medium condition is consistent, and the difference of thalline weight in wet base is little;
6) 96h collects fermentation supernatant, carries out protein electrophoresis and detects HSA expression amount, the results are shown in Table 2.
Table 2
From table 2, data can be found out, using and improve I, improvement II, improvement III, improvement IV as the fermention medium of shaking flask level, compare with BSM with BMGY, and the expression amount of HSA does not have significant difference;
Embodiment 3, the horizontal strain culturing of 30L fermentor tank and detection
1) the kind liquid in the frozen seed pipe of HSA75-10 is inoculated in 5 1000ml shaking flasks that 200ml BMGY is housed, in 200rpm, 30 ℃, cultivates 24h, be prepared into first order seed.
2) by 5% inoculum size, first order seed is criticized to formula access and be equipped with in the 30L fermentor tank (purchased from B.BRAUN company) of 20L BMGY, BSM, improvement I, improvement II, improvement III, improvement IV, setting fermentation initial temperature and be 30 ℃, rotating speed 500rpm, ventilation 20L/min(air flow is 1V/Vmin), start fermentation after pH5.0.
3) the initial oxygen dissolving value 100% that ferments, fermentation started after 4-6 hour, and dissolved oxygen starts slow decreasing.Treat that the glycerine in fermention medium runs out of, dissolved oxygen is closely to 100% of rising rapidly, starts to add supplemented medium, and the speed of adding is controlled with oxygen dissolving value, and oxygen dissolving value is controlled between 25-35%.
4) by time point 0,24,48,72,96,120,144,168h, get respectively bacterium liquid sample, analyze thalline weight in wet base, and start to detect the expression amount of HSA in supernatant from 72h.
5) under various culture medium condition, thalli growth comparative result as shown in Figure 2.Can find out, adopt the fermention medium of different ingredients all can realize the high-density culture of bacterial strain simultaneously, wherein improve IV condition hypothallus weight in wet base the highest.
6) under each culture medium condition, expressing quantity the results are shown in Table 3.Can find out under above-mentioned all culture medium condition, equal energy highly effective expressing recombinant protein, wherein, under BMGY, BSM, three kinds of culture medium condition of improvement IV, protein expression difference is not remarkable.
Table 3
By analysis, application BMGY does seed culture medium, is adopting under the fermention medium condition of different ingredients, and the content of HSA is the more than 85% of secretory protein, and wherein each substratum difference is little.
Embodiment 4, using and improve IV as strain culturing and the detection of seed culture medium
1) HSA75-10 is inoculated in 5 1000ml shaking flasks that 200ml improvement IV is housed, in 200rpm, 30 ℃, cultivates 24h, be prepared into first order seed.
2) by 5% inoculum size, first order seed access is equipped with in the 30L fermentor tank of 20L improvement IV, setting fermentation initial temperature and be 30 ℃, rotating speed 500rpm, ventilation 20L/min(air flow is 1V/Vmin), start fermentation after pH5.0.
3) the initial oxygen dissolving value 100% that ferments, fermentation started after 4-6 hour, and dissolved oxygen starts slow decreasing.Treat that the glycerine in fermention medium runs out of, dissolved oxygen is closely to 100% of rising rapidly, starts to add supplemented medium, and the speed of adding is controlled with oxygen dissolving value, and oxygen dissolving value is controlled between 25-35%.
4) by time point 0,24,48,72,96,120,144,168,192,216h, get respectively bacterium liquid sample, analyze thalline weight in wet base.
5) expression amount of thalli growth situation and HSA is shown in Fig. 3, can find out fermentation period 216 hours (wherein methanol induction is expressed 192 hours), thalline weight in wet base is up to 56%, expressing quantity reaches 16g/L supernatant, with in embodiment 3, using BMGY and compare as seed culture medium, there is no marked difference, and slightly increase.
The present inventor also selects other 2 kinds of pichia pastoris phaff cell: KM71H and X33(purchased from Invitrogen company), with above-mentioned substratum, according to the method repeated authentication in above-described embodiment 2-4, all obtained similar experimental result.Illustrate that the substratum that provides in the present invention can be for other pichia pastoris phaffs on a small scale, or the application of the cultivation in pilot scale and industrial scale.
Claims (8)
1. a pichia pastoris phaff substratum, is characterized in that in medium component not containing KOH and H
3pO
4, contain NH
4h
2pO
4with KH
2pO
4.
2. substratum as claimed in claim 1, is characterized in that the NH that contains 1.575～31.5 g/L in substratum
4h
2pO
4kH with 1.155～23.1 g/L
2pO
4.
3. substratum as claimed in claim 2, by the cumulative volume of substratum, described substratum contains following composition:
NH
4H
2PO
4 1.575～31.5 g/L
KH
2PO
4 1.155～23.1 g/L
MgSO
4·7H
2O 1.49～14.9 g/L
K
2SO
4 0～18.2 g/L
CaSO
4 0.093～0.93 g/L
Glycerine 10～40 g/L
PTM1 solution 0.435～4.35ml/L
Ammoniacal liquor adjust pH to 4.5～6.2.
4. substratum as claimed in claim 3, by the cumulative volume of substratum, described substratum contains following composition:
NH
4H
2PO
4 15.75 g/L
KH
2PO
4 1.155 g/L
MgSO
4·7H
2O 1.49 g/L
K
2SO
4 1.82g/L
CaSO
4 0.093 g/L
Glycerine 10 g/L
PTM1 solution 0.435mL/L
Ammoniacal liquor adjust pH to 5.0.
5. the purposes of the substratum described in any one in cultivating pichia pastoris phaff in claim 1-4.
6. purposes as claimed in claim 5, wherein said cultivation is fermentation culture.
7. purposes as claimed in claim 5, wherein said cultivation is seed culture.
8. substratum as described in any one in claim 1-4, is characterized in that: described pichia pastoris phaff is wild type strain or the recombinant strain that contains foreign gene through transforming.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310727410.1A CN103923846B (en) | 2013-12-23 | 2013-12-23 | A kind of pichia pastoris phaff culture medium |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310727410.1A CN103923846B (en) | 2013-12-23 | 2013-12-23 | A kind of pichia pastoris phaff culture medium |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103923846A true CN103923846A (en) | 2014-07-16 |
| CN103923846B CN103923846B (en) | 2016-07-06 |
Family
ID=51142245
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310727410.1A Active CN103923846B (en) | 2013-12-23 | 2013-12-23 | A kind of pichia pastoris phaff culture medium |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103923846B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111662944A (en) * | 2019-03-05 | 2020-09-15 | 上海医药工业研究院 | Preparation method and purification method of human serum albumin |
| CN111909859A (en) * | 2019-05-07 | 2020-11-10 | 北京双鹭药业股份有限公司 | Pichia pastoris low-temperature culture medium |
| CN112501041A (en) * | 2020-12-03 | 2021-03-16 | 西安德诺海思医疗科技有限公司 | Pichia pastoris high-density fermentation medium and fermentation method thereof |
| CN117535164A (en) * | 2023-11-10 | 2024-02-09 | 江苏创健医疗科技股份有限公司 | Pichia pastoris fermentation medium and fermentation process suitable for recombinant collagen production |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101717737A (en) * | 2009-12-11 | 2010-06-02 | 中国科学院亚热带农业生态研究所 | Antibiotic peptide buforin II and porcine INF-alpha fusion expression pichia pastoris, and preparation method and applications thereof |
| CN101824444A (en) * | 2010-04-09 | 2010-09-08 | 上海化工研究院 | Method for preparing <13>C-labelled glucose |
| CN102586207A (en) * | 2012-03-01 | 2012-07-18 | 上海戴迪实业发展有限公司 | Method for preparing neutral bleaching xylanase |
-
2013
- 2013-12-23 CN CN201310727410.1A patent/CN103923846B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101717737A (en) * | 2009-12-11 | 2010-06-02 | 中国科学院亚热带农业生态研究所 | Antibiotic peptide buforin II and porcine INF-alpha fusion expression pichia pastoris, and preparation method and applications thereof |
| CN101824444A (en) * | 2010-04-09 | 2010-09-08 | 上海化工研究院 | Method for preparing <13>C-labelled glucose |
| CN102586207A (en) * | 2012-03-01 | 2012-07-18 | 上海戴迪实业发展有限公司 | Method for preparing neutral bleaching xylanase |
Non-Patent Citations (1)
| Title |
|---|
| 郑海英等: "内切中性纤维素酶EGⅡ的发酵条件优化", 《微生物学杂志》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111662944A (en) * | 2019-03-05 | 2020-09-15 | 上海医药工业研究院 | Preparation method and purification method of human serum albumin |
| CN111909859A (en) * | 2019-05-07 | 2020-11-10 | 北京双鹭药业股份有限公司 | Pichia pastoris low-temperature culture medium |
| CN111909859B (en) * | 2019-05-07 | 2022-07-19 | 北京双鹭药业股份有限公司 | Low-temperature culture medium for pichia pastoris |
| CN112501041A (en) * | 2020-12-03 | 2021-03-16 | 西安德诺海思医疗科技有限公司 | Pichia pastoris high-density fermentation medium and fermentation method thereof |
| CN117535164A (en) * | 2023-11-10 | 2024-02-09 | 江苏创健医疗科技股份有限公司 | Pichia pastoris fermentation medium and fermentation process suitable for recombinant collagen production |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103923846B (en) | 2016-07-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2018014453A1 (en) | Fermentation technique to improve production level of recombinant human collagen | |
| CN103923846A (en) | Pichia pastoris culturing medium | |
| CN103074315B (en) | Lipase LIP, gene and application thereof | |
| CN104164367A (en) | Dried silkworm cordyceps militaris and culture method thereof | |
| CN101948784A (en) | Bdellovibrio bacteriovorus preparation and fermentation method and application thereof | |
| CN107937311B (en) | Streptococcus thermophilus for high yield of gamma-aminobutyric acid, preservation and culture method and method for preparing fermented milk by using streptococcus thermophilus | |
| CN114107073B (en) | Method for producing hypha protein by utilizing molasses | |
| CN102229902A (en) | Bacillus pumilus, feed additive thereof, premix, and feed mildewproof agent | |
| CN105543147A (en) | Pseudomonas aeruginosa strain and application thereof in producing proteinase | |
| CN105255771A (en) | Staphylococcus succinus for generating collagenase and application thereof | |
| CN103725738B (en) | The method preparing collagen polypeptide with Carnis Pseudosciaenae leftover bits and pieces | |
| CN103947825B (en) | A kind of feed for pet additive and production method thereof | |
| CN104031862A (en) | High-yield gamma-aminobutyric acid (GABA) strain and application thereof | |
| CN115851497A (en) | Bovine bile resistant type bezoar transformation bacterium and application thereof | |
| CN104046583A (en) | Bacillus thuringiensis liquid culture medium and preparation method thereof | |
| CN103563649A (en) | Cultivation method of cordyceps militaris | |
| CN102286411B (en) | Lactobacillus plantarum and application thereof in fermenting cabbage wrapper leaf | |
| CN106889373A (en) | A kind of large yellow croaker phagostimulant with squid processing byproduct as primary raw material | |
| CN103667111B (en) | A kind of bacillus megaterium and application thereof microcystic aeruginosa to solvency action | |
| CN102796731B (en) | A kind of rape bee pollen nucleic acid extraction method | |
| CN103865865B (en) | A kind of sea cucumber enteron aisle produces Sumizyme MP bacterial strain and application thereof | |
| CN112877217B (en) | Montania fulva strain and application thereof in degrading chicken feather | |
| CN102246659A (en) | Method for producing selenium-enriched cordyceps | |
| CN102229895A (en) | Method for producing organic nitrogen source of yeast culture medium by using water-bloom microalgae biomasses | |
| CN102206588B (en) | Strain generating antioxidant |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |