CN113667709B - Fermentation method of recombinant humanized collagen - Google Patents
Fermentation method of recombinant humanized collagen Download PDFInfo
- Publication number
- CN113667709B CN113667709B CN202110999047.3A CN202110999047A CN113667709B CN 113667709 B CN113667709 B CN 113667709B CN 202110999047 A CN202110999047 A CN 202110999047A CN 113667709 B CN113667709 B CN 113667709B
- Authority
- CN
- China
- Prior art keywords
- feeding
- pichia pastoris
- collagen
- fermentation
- recombinant humanized
- 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.)
- Active
Links
- 238000000855 fermentation Methods 0.000 title claims abstract description 85
- 230000004151 fermentation Effects 0.000 title claims abstract description 85
- 238000000034 method Methods 0.000 title claims abstract description 52
- 102000008186 Collagen Human genes 0.000 title claims abstract description 48
- 108010035532 Collagen Proteins 0.000 title claims abstract description 48
- 229920001436 collagen Polymers 0.000 title claims abstract description 48
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 135
- 241000235058 Komagataella pastoris Species 0.000 claims abstract description 104
- 241000894006 Bacteria Species 0.000 claims abstract description 65
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 45
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 45
- 239000001301 oxygen Substances 0.000 claims abstract description 45
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 40
- 239000008103 glucose Substances 0.000 claims abstract description 40
- 238000012258 culturing Methods 0.000 claims abstract description 39
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 28
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 28
- 238000004519 manufacturing process Methods 0.000 claims abstract description 19
- 108090000623 proteins and genes Proteins 0.000 claims description 48
- 239000007788 liquid Substances 0.000 claims description 36
- 239000001963 growth medium Substances 0.000 claims description 31
- 239000000463 material Substances 0.000 claims description 25
- 239000002054 inoculum Substances 0.000 claims description 21
- 101150051118 PTM1 gene Proteins 0.000 claims description 18
- 102000012422 Collagen Type I Human genes 0.000 claims description 16
- 108010022452 Collagen Type I Proteins 0.000 claims description 16
- 102000000503 Collagen Type II Human genes 0.000 claims description 6
- 108010041390 Collagen Type II Proteins 0.000 claims description 6
- 239000001888 Peptone Substances 0.000 claims description 6
- 108010080698 Peptones Proteins 0.000 claims description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 6
- 235000019319 peptone Nutrition 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 239000007222 ypd medium Substances 0.000 claims description 6
- 239000012634 fragment Substances 0.000 claims description 5
- 239000011782 vitamin Substances 0.000 claims description 5
- 229940088594 vitamin Drugs 0.000 claims description 5
- 229930003231 vitamin Natural products 0.000 claims description 5
- 235000013343 vitamin Nutrition 0.000 claims description 5
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 5
- 102000001187 Collagen Type III Human genes 0.000 claims description 4
- 108010069502 Collagen Type III Proteins 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 3
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims description 2
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 2
- 239000007836 KH2PO4 Substances 0.000 claims 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims 1
- 239000002609 medium Substances 0.000 claims 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims 1
- 235000019796 monopotassium phosphate Nutrition 0.000 claims 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims 1
- 230000015556 catabolic process Effects 0.000 abstract description 9
- 238000006731 degradation reaction Methods 0.000 abstract description 9
- 238000009776 industrial production Methods 0.000 abstract description 4
- 230000002068 genetic effect Effects 0.000 abstract description 2
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 18
- 230000000052 comparative effect Effects 0.000 description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 11
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 10
- 230000006698 induction Effects 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 9
- 230000001965 increasing effect Effects 0.000 description 9
- 230000001502 supplementing effect Effects 0.000 description 6
- 229960002685 biotin Drugs 0.000 description 5
- 235000020958 biotin Nutrition 0.000 description 5
- 239000011616 biotin Substances 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000006052 feed supplement Substances 0.000 description 4
- 235000011187 glycerol Nutrition 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 230000000630 rising effect Effects 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 3
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 235000003642 hunger Nutrition 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 230000037353 metabolic pathway Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 108010025188 Alcohol oxidase Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012526 feed medium Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 230000037351 starvation Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 101710137510 Saimiri transformation-associated protein Proteins 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 235000011151 potassium sulphates Nutrition 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000003938 response to stress Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Mycology (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The application provides a fermentation method of recombinant humanized collagen, and relates to the technical field of high-density fermentation of genetic recombinant engineering bacteria. A method for fermenting recombinant humanized collagen, comprising the steps of: s1: inoculating Pichia pastoris genetically engineered bacteria into a seed tank for culture, and transferring to a production fermentation tank after dissolved oxygen is greatly risen; s2: culturing Pichia pastoris genetically engineered bacteria in a production fermentation tank until dissolved oxygen is greatly raised, and then using glucose solution and methanol for mixed carbon source feeding; s3: after the mixed carbon source feeding is finished, the methanol solution is used for feeding in a gradient pulse mode, and then the glucose solution and the methanol solution are used for constant-speed feeding until the fermentation is finished. The application can reduce degradation of the recombinant humanized collagen in the fermentation process and can carry out stable industrial production.
Description
Technical Field
The application relates to the technical field of high-density fermentation of genetic recombinant engineering bacteria, in particular to a fermentation method of recombinant humanized collagen.
Background
Collagen is a product obtained by completely hydrolyzing collagen, is widely distributed in animal connective tissues, has an important function for maintaining normal physiological functions and injury repair of cells, tissues and organs, and has wide application in the fields of medicines, tissue engineering, foods, cosmetics and the like due to low immunogenicity, degradability, good biocompatibility and other biological characteristics. At present, collagen used in various fields is mainly extracted from animal skin, bones and other tissues by adopting an acid, alkali hydrolysis method or enzymolysis method, but the collagen prepared by the methods has poor water solubility and is difficult to separate into pure products, and pathogenic pollution is easy to cause clinically and rejection reaction occurs, so that the collagen is greatly limited in application as medicines, biomedical materials or medicine carriers and the like; there are also documents and patent reports for producing recombinant collagen by recombinant expression, and chinese patent application nos. CN200610098297.5, CN201110327865.5, CN201310033299.6, CN01106757.8 and CN201610388271.8 all produce recombinant collagen by recombinant expression, but these recombinant collagen sequences are modified or have tag proteins, belonging to recombinant collagens, not truly recombinant humanized collagen, which refers to full-length or partial amino acid sequence fragments encoded by specific genes of human collagen prepared by DNA recombinant technology, or a combination of functional fragments containing human collagen.
At present, the existing fermentation method of recombinant collagen is basically referred to the pichia pastoris fermentation manual of Invitrogen company, or is slightly modified on the basis, and is suitable for the recombinant collagen with stable structure; because the recombinant humanized collagen sequence is completely consistent with the natural collagen amino acid sequence, compared with the recombinant collagen-like protein structure modified by the sequence, the structure is more unstable, and if the fermentation method is used, degradation easily occurs in the fermentation process, so that the stable industrial production is not facilitated.
Disclosure of Invention
The application aims to provide a fermentation method of recombinant humanized collagen, which can reduce degradation of the recombinant humanized collagen in the fermentation process and can perform stable industrial production.
The embodiment of the application is realized by the following technical scheme:
a method for fermenting recombinant humanized collagen, comprising the steps of:
s1: inoculating Pichia pastoris genetically engineered bacteria into a seed tank for culture, and transferring to a production fermentation tank after dissolved oxygen is greatly risen;
s2: culturing Pichia pastoris genetically engineered bacteria in a production fermentation tank until dissolved oxygen is greatly raised, and then using glucose solution and methanol for mixed carbon source feeding;
s3: after the mixed carbon source feeding is finished, the methanol solution is used for feeding in a gradient pulse mode, and then the glucose solution and the methanol solution are used for constant-speed feeding until the fermentation is finished.
Further, the glucose solution flowing rate in the step S2 is 10-30 mL/h/L, and the methanol flowing rate is 0.1-1.5 mL/h/L.
Further, in the step S3, pulse gradients are set to be 1%, 1.5% and 2% of the volume of the fermentation broth, each gradient is respectively pulse fed for 2-5 times, and after each pulse fed, the next pulse fed is started after the dissolved oxygen is returned.
Further, in the step S3, when the constant-speed feeding is performed, the feeding rate of the glucose solution is 0.1-1 mL/h/L, and the feeding rate of the methanol solution is 4-10 mL/h/L.
Further, the mass fraction of the glucose solution is 40% -80%.
Further, PTM1 is added to the methanol solution used for the pulse and constant-speed feeding in the step S3, and the addition amount is 12-24 mL/L.
Further, the pichia pastoris engineering bacteria are pichia pastoris engineering bacteria for expressing recombinant humanized collagen; the recombinant humanized collagen is recombinant humanized type I collagen, recombinant humanized type II collagen, recombinant humanized type III collagen or a combination of functional region fragments of the recombinant humanized type I, type II and type III collagen.
Further, the specific method for culturing the pichia pastoris genetically engineered bacteria to the four-level seeds in the step S1 comprises the following steps: (1) Inoculating recombinant humanized type I collagen Pichia pastoris gene engineering bacteria into YPD culture medium in a shake flask according to the inoculum size of 0.1-0.3%, culturing at 29+ -1deg.C and 220rpm for 48-60 h until the wet weight of the Pichia pastoris gene engineering bacteria reaches 35-40 g/L, and culturing to obtain Pichia pastoris gene engineering bacteria first-stage seeds; (2) Inoculating the primary seed liquid of the pichia pastoris gene engineering bacteria into a bottom material culture medium in a 15L seed tank according to the inoculation amount of 6-12% by adopting a fire ring method, and culturing for 16-24 h at 29+/-1 ℃ and pH of 5.0 with the dissolved oxygen value range of 20-50% to obtain secondary seeds of the pichia pastoris gene engineering bacteria; (3) After dissolved oxygen of the secondary seed liquid of the pichia pastoris is greatly risen, inoculating the secondary seed liquid of the pichia pastoris into a bottom material culture medium in a 150L seed tank according to the inoculation amount of 6-12% by adopting a differential pressure method, and culturing for 16-24 hours at 29+/-1 ℃ and with the pH value of 5.0 and the dissolved oxygen value range of 20-50% to obtain the tertiary seed of the pichia pastoris genetically engineered bacterium; (4) After dissolved oxygen of the three-stage seed liquid of the pichia pastoris is greatly risen, inoculating the three-stage seed liquid of the pichia pastoris into a bottom material culture medium in a 1500L production fermentation tank according to an inoculum size of 6-12% by adopting a differential pressure method, and culturing for 16-24 hours at 29+/-1 ℃ and with a pH value of 5.0 and a dissolved oxygen numerical range of 20-50% to obtain four-stage seeds of the pichia pastoris genetically engineered bacteria; in the fermentation process, 1-5 mol/L KOH is adopted to adjust the pH of the fermentation liquid, so that the pH in the fermentation process is 3.0-7.0.
Further, the YPD medium comprises the following concentrations of components: yeast powder 0.8-1.2 g/L, peptone 1.8-2.2 g/L and glucose 1.8-2.2 g/L.
Further, the primer culture medium comprises the following components in concentration: KH (KH) 2 PO 4 21.5～43.0g/L，(NH 4 ) 2 SO 4 2.5～7.5g/L，CaSO 4 ·2H 2 O 0.2～1.0g/L，K 2 SO 4 7～14.3g/L，M gSO 4 ·7H 2 O6-11.7 g/L, glucose 80-120 g/L, PTM 1.2-8.4 mL/L and vitamin C0.01-0.1 g/L.
Further, the PTM1 comprises the following concentration components: cuSO 4 ·5H 2 O 4～8g/L，N aI 0.3～0.5g/L，MnSO 4 ·H 2 O 4～6g/L，Na 2 MoO 4 ·2H 2 O 0.1～0.3g/L，H 3 BO 4 0.1～0.2g/L，CoCl 2 0.5～1.5g/L，ZnCl 2 25～35g/L，FeSO 4 ·7H 2 O75-95 g/L and H 2 SO 4 3-6 mL/L and biotin 0.1% to the whole0.3g/L。
Pichia pastoris can change the enzyme system of the Pichia pastoris according to the change of a carbon source and the stress of cells or organelles, so that the degradation of exogenous proteins is caused; according to the application, during carbon source feeding, high-concentration glucose is fed into the pichia pastoris cell, and a small amount of methanol is fed into the pichia pastoris cell to perform mixed carbon source feeding, so that the starting of alcohol oxidase is induced, no stress is exerted on pichia pastoris, the carbon source conversion from glucose to methanol is started in the carbon source feeding stage, then the feeding is performed in a gradient pulse mode in the induced expression feeding stage, the methanol metabolism pathway of the pichia pastoris is completely opened, the carbon source is changed in a mild mode, the stress reaction of pichia pastoris cells to external environment change is reduced, the protease secretion amount of the pichia pastoris cells is reduced, and the degradation of recombinant humanized collagen is effectively reduced.
The technical scheme of the embodiment of the application has at least the following advantages and beneficial effects:
1. in the application, when the carbon source is fed, high-concentration glucose is fed, and a small amount of methanol is fed at the same time to feed the mixed carbon source, so that the starting of alcohol oxidase is induced, and compared with the traditional starvation technology, the pichia pastoris has no stress effect.
2. The pichia pastoris starts the conversion from glucose to methanol from the carbon source in the feeding stage, then feeds the pichia pastoris in the induction expression feeding stage in a gradient pulse mode, and compared with the classical DO-Start method, the methanol metabolism pathway of the pichia pastoris is completely opened, the fluctuation condition of dissolved oxygen is not needed to be considered, the false image of dissolved oxygen rising formed when the concentration of methanol is too high in the production process of the DO-Start method to inhibit the growth of the thallus is avoided, and the operation method is simpler and the production operator can master the operation method more easily in the industrial amplification production.
3. The application greatly reduces the dosage of methanol by feeding glucose with a certain proportion at a constant speed in the late stage of the induced expression feeding stage, promotes the growth of pichia pastoris thallus, shortens the fermentation period and increases the expression quantity of recombinant humanized collagen.
4. According to the application, the metabolic pathway of the pichia pastoris is regulated in a special feeding mode, so that the stress effect of starvation on the growth of pichia pastoris cells is avoided, the stress response of the pichia pastoris cells to external environment change is reduced through a mild carbon source change method, and the protease secretion of the pichia pastoris cells is reduced, thereby effectively reducing the degradation of recombinant humanized collagen.
5. In the fermentation process, the pH value of the fermentation liquid is regulated by adopting 1-5 mol/L KOH, so that the use of volatile ammonia water is avoided, and the toxicity caused by volatilization of the ammonia water is avoided.
Drawings
FIG. 1 is a diagram showing the expression of recombinant humanized type I collagen by the recombinant humanized type I collagen Pichia pastoris gene engineering bacteria provided in example 1; wherein, before M-Marker, 1-induced expression feeding, 2-feeding, 3-feeding, 36h, 4-feeding, 42h, 5-feeding, 48h, 6-feeding, 54h and 7-feeding are respectively carried out, 60h;
FIG. 2 is a diagram showing the expression of recombinant humanized type I collagen by the recombinant humanized type I collagen Pichia pastoris gene engineering bacteria provided in the comparative example; wherein, M-Marker, 20h after 1-feeding, 30h after 2-feeding, 40h after 3-feeding, 50h after 4-feeding, 60h after 5-feeding, 70h after 6-feeding, 80h after 7-feeding, and 90h after 8-feeding.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the embodiments of the present application will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The following specifically describes a fermentation method of recombinant humanized collagen provided in the embodiment of the present application.
Example 1
The embodiment provides a fermentation method of recombinant humanized collagen, which comprises the following steps:
s1: (1) Inoculating recombinant humanized type I collagen Pichia pastoris gene engineering bacteria into YPD culture medium in a shake flask according to an inoculum size of 0.1%, culturing at 29 ℃ and 220rpm for 60 hours until the wet weight of the Pichia pastoris gene engineering bacteria reaches 35g/L, and culturing to obtain primary seeds of the Pichia pastoris gene engineering bacteria; YPD medium included the following concentrations of components: 1.0g/L yeast powder, 2.0g/L peptone and 2.0g/L glucose; (2) Inoculating the primary seed liquid of the pichia pastoris gene engineering bacteria into a bottom material culture medium in a 15L seed tank according to an inoculum size of 8% by adopting a fire ring method, and culturing for 20 hours at 29 ℃ and a pH value of 5.0 with a dissolved oxygen value of more than 30% to obtain secondary seeds of the pichia pastoris gene engineering bacteria; (3) After dissolved oxygen of the secondary seed liquid of the pichia pastoris is greatly risen, inoculating the secondary seed liquid of the pichia pastoris into a bottom material culture medium in a 150L seed tank according to an inoculum size of 8% by adopting a differential pressure method, and culturing for 19 hours at 29 ℃ and pH of 5.0, wherein the dissolved oxygen value is more than 30% to obtain the tertiary seed of the pichia pastoris genetically engineered bacterium; (4) After the dissolved oxygen of the three-stage seed liquid of the pichia pastoris is greatly risen, inoculating the three-stage seed liquid of the pichia pastoris into a bottom material culture medium in a 1500L production fermentation tank according to 10% of inoculum size by adopting a differential pressure method, and culturing for 21 hours at 29 ℃ and pH of 5.0, wherein the dissolved oxygen value is more than 30% to obtain four-stage seeds of the pichia pastoris genetically engineered bacteria; in the fermentation process, 5mol/L KOH is adopted to adjust the pH of fermentation liquor, so that the pH of the fermentation process is 7.0;
the substrate culture medium comprises the following components in concentration: KH (KH) 2 PO 4 42.9g/L，(NH 4 ) 2 SO 4 7.5g/L，CaSO 4 ·2H 2 O 1.0g/L，K 2 SO 4 14.3g/L，MgSO 4 ·7H 2 O11.7 g/L, glucose 120g/L, PTM 1.4 mL/L and vitamin C0.1 g/L; wherein PTM1 comprises the following concentration components: cuSO 4 ·5H 2 O 6g/L，NaI 0.4g/L，MnSO 4 ·H 2 O 5g/L，Na 2 MoO 4 ·2H 2 O 0.2g/L，H 3 BO 4 0.1g/L，CoCl 2 1g/L，ZnCl 2 30g/L，FeSO 4 ·7H 2 O90 g/L and H 2 SO 4 5mL/L and biotin 0.2g/L;
s2: culturing four-level seeds of the pichia pastoris gene engineering bacteria in the production fermentation tank until dissolved oxygen is greatly increased, using 60% glucose solution and methanol for mixed carbon source material supplementing, wherein the feeding rate of the 60% glucose solution is 10mL/h/L, and the feeding rate of the methanol is 0.1mL/h/L, and ending the mixed carbon source material supplementing when the wet bacterial weight of the pichia pastoris gene engineering bacteria reaches 200 g/L;
s3: after the mixed carbon source feeding is finished, feeding is firstly carried out by using a methanol solution added with 12mL/L PTM1 in a pulse mode, pulse gradients are respectively set to be 1%, 1.5% and 2% of the volume of the fermentation broth, each gradient is respectively subjected to pulse feeding for 3 times, and after each pulse feeding is finished, the next pulse feeding is started after waiting for the rising of dissolved oxygen; and then carrying out constant-speed feeding on the 60% glucose solution and the methanol solution added with 12mL/L PTM1, wherein the feeding rate of the 60% glucose solution is 0.1mL/h/L, and the feeding rate of the methanol solution added with 12mL/L PTM1 is 5.3mL/h/L, until the fermentation is finished, and finishing the whole fermentation process when the wet bacterial weight or the protein expression quantity of the pichia pastoris is not increased any more.
Example 2
The embodiment provides a fermentation method of recombinant humanized collagen, which comprises the following steps:
s1: (1) Inoculating recombinant humanized II type collagen Pichia pastoris gene engineering bacteria into YPD culture medium in a shake flask according to an inoculum size of 0.3%, culturing at 29 ℃ and 220rpm for 56h until the wet weight of the Pichia pastoris gene engineering bacteria reaches 40g/L, and culturing to obtain primary seeds of the Pichia pastoris gene engineering bacteria; YPD medium included the following concentrations of components: yeast powder 0.8g/L, peptone 2.2g/L and glucose 2.2g/L; (2) Inoculating the primary seed liquid of the pichia pastoris gene engineering bacteria into a bottom material culture medium in a 15L seed tank according to 10% of inoculum size by adopting a fire ring method, and culturing for 16 hours at 29 ℃ and pH of 5.0 with a dissolved oxygen value of more than 30% to obtain secondary seeds of the pichia pastoris gene engineering bacteria; (3) After dissolved oxygen of the secondary seed liquid of the pichia pastoris is greatly risen, inoculating the secondary seed liquid of the pichia pastoris into a bottom material culture medium in a 150L seed tank according to 10% of inoculum size by adopting a differential pressure method, and culturing for 16 hours at 29 ℃ and pH of 5.0, wherein the dissolved oxygen value is more than 30% to obtain the tertiary seed of the pichia pastoris genetically engineered bacterium; (4) After the dissolved oxygen of the three-stage seed liquid of the pichia pastoris is greatly risen, inoculating the three-stage seed liquid of the pichia pastoris into a bottom material culture medium in a 1500L production fermentation tank according to 10% of inoculum size by adopting a differential pressure method, and culturing for 17 hours at 29 ℃ and with the pH value of 5.0 and the dissolved oxygen value of more than 30% to obtain four-stage seeds of the pichia pastoris genetically engineered bacteria; in the fermentation process, 3mol/L KOH is adopted to adjust the pH of fermentation liquor, so that the pH of the fermentation process is 5.0;
the substrate culture medium comprises the following components in concentration: KH (KH) 2 PO 4 21.5g/L，(NH 4 ) 2 SO 4 2.5g/L，CaSO 4 ·2H 2 O 0.2g/L，K 2 SO 4 7g/L，MgSO 4 ·7H 2 O6 g/L, glucose 80g/L, PT M1.2 mL/L and vitamin C0.01 g/L; wherein PTM1 comprises the following concentration components: cuS O 4 ·5H 2 O 6g/L，NaI 0.4g/L，MnSO 4 ·H 2 O 5g/L，Na 2 MoO 4 ·2H 2 O 0.2g/L，H 3 BO 4 0.1g/L，CoCl 2 1.0g/L，ZnCl 2 30g/L，FeSO 4 ·7H 2 O90 g/L and H 2 SO 4 5mL/L and biotin 0.2g/L;
s2: culturing four-level seeds of the pichia pastoris gene engineering bacteria in the production fermentation tank until dissolved oxygen is greatly increased, using 40% glucose solution and methanol for mixed carbon source material supplementing, wherein the flow rate of the 40% glucose solution is 20mL/h/L, and the flow rate of the methanol is 0.5mL/h/L, and ending the mixed carbon source material supplementing when the wet bacterial weight of the pichia pastoris gene engineering bacteria reaches 200 g/L;
s3: after the mixed carbon source feeding is finished, feeding is firstly carried out by using a methanol solution added with 24mL/L PTM1 in a pulse mode, pulse gradients are respectively set to be 1%, 1.5% and 2% of the volume of the fermentation broth, each gradient is respectively subjected to pulse feeding for 3 times, and after each pulse feeding is finished, the next pulse feeding is started after waiting for the rising of dissolved oxygen; and then carrying out constant-speed feeding on the 40% glucose solution and the methanol solution added with 24mL/L PTM1, wherein the feeding rate of the 40% glucose solution is 0.5mL/h/L, and the feeding rate of the methanol solution added with 24mL/L PTM1 is 5.2mL/h/L, until the fermentation is finished, and finishing the whole fermentation process when the wet bacterial weight or the protein expression quantity of the pichia pastoris is not increased any more.
Example 3
The embodiment provides a fermentation method of recombinant humanized collagen, which comprises the following steps:
s1: (1) Inoculating recombinant humanized I-type, II-type and III-type collagen functional region fragments combined pichia pastoris gene engineering bacteria into YPD culture medium in a shake flask according to an inoculation amount of 0.2%, culturing at 29 ℃ and 220rpm for 60 hours until the wet weight of the pichia pastoris gene engineering bacteria reaches 40g/L, and culturing to obtain pichia pastoris gene engineering bacteria primary seeds; YPD medium included the following concentrations of components: 1.0g/L yeast powder, 2.2g/L peptone and 2.2g/L glucose; (2) Inoculating the primary seed liquid of the pichia pastoris gene engineering bacteria into a bottom material culture medium in a 15L seed tank according to an inoculum size of 8% by adopting a fire ring method, and culturing for 17 hours at 29 ℃ and a pH value of 5.0 with a dissolved oxygen value of more than 30% to obtain secondary seeds of the pichia pastoris gene engineering bacteria; (3) After dissolved oxygen of the secondary seed liquid of the pichia pastoris is greatly risen, inoculating the secondary seed liquid of the pichia pastoris into a bottom material culture medium in a 150L seed tank according to an inoculum size of 8% by adopting a differential pressure method, and culturing for 18 hours at 29 ℃ and a pH value of 5.0, wherein the dissolved oxygen value is more than 30% to obtain the tertiary seed of the pichia pastoris genetically engineered bacterium; (4) After dissolved oxygen of the three-stage seed liquid of the pichia pastoris is greatly risen, inoculating the three-stage seed liquid of the pichia pastoris into a bottom material culture medium in a 1500L production fermentation tank according to an inoculum size of 8% by adopting a differential pressure method, and culturing for 19 hours at 29 ℃ and with a pH value of 5.0 and a dissolved oxygen value of more than 30% to obtain four-stage seeds of the pichia pastoris genetically engineered bacteria; in the fermentation process, 1mol/L KOH is adopted to adjust the pH of fermentation liquor, so that the pH of the fermentation process is 3.0;
the substrate culture medium comprises the following components in concentration: KH (KH) 2 PO 4 32.3g/L，(NH 4 ) 2 SO 4 4.8g/L，CaSO 4 ·2H 2 O 0.6g/L，K 2 SO 4 10.7g/L，MgSO 4 ·7H 2 O8.9 g/L, glucose 100g/L, PTM 1.3 mL/L and vitamin C0.05 g/L; wherein PTM1 comprises the following concentration components: cuSO 4 ·5H 2 O 6g/L，NaI 0.4g/L，MnSO 4 ·H 2 O 5g/L，Na 2 MoO 4 ·2H 2 O 0.2g/L，H 3 BO 4 0.1g/L，CoCl 2 1g/L，ZnCl 2 30g/L，FeSO 4 ·7H 2 O90 g/L and H 2 SO 4 5mL/L and biotin 0.2g/L;
s2: culturing four-level seeds of the pichia pastoris gene engineering bacteria in the production fermentation tank until dissolved oxygen is greatly increased, using 80% glucose solution and methanol for mixed carbon source material supplementing, wherein the feeding rate of the 80% glucose solution is 30mL/h/L, and the feeding rate of the methanol is 1.5mL/h/L, and ending the mixed carbon source material supplementing when the wet bacterial weight of the pichia pastoris gene engineering bacteria reaches 200 g/L;
s3: after the mixed carbon source feeding is finished, feeding is firstly carried out by using a methanol solution added with 18mL/L PTM1 in a pulse mode, pulse gradients are respectively set to be 1%, 1.5% and 2% of the volume of the fermentation broth, each gradient is respectively subjected to pulse feeding for 3 times, and after each pulse feeding is finished, the next pulse feeding is started after waiting for the rising of dissolved oxygen; and then carrying out constant-speed feeding on the 80% glucose solution and the methanol solution added with 18mL/L PTM1, wherein the flow rate of the 80% glucose solution is 1mL/h/L, the flow rate of the methanol solution added with 18mL/L PTM1 is 5.1mL/h/L, and the whole fermentation process is finished when the wet bacterial weight of the pichia pastoris or the protein expression quantity is not increased any more.
Comparative example
The comparative example provides a fermentation process for recombinant humanized collagen comprising the steps of:
s1: (1) Inoculating recombinant humanized type I collagen Pichia pastoris gene engineering bacteria into YPD culture medium in a shake flask according to an inoculum size of 0.1%, culturing at 29 ℃ and 220rpm for 60 hours until the wet weight of the Pichia pastoris gene engineering bacteria reaches 35g/L, and culturing to obtain primary seeds of the Pichia pastoris gene engineering bacteria; YPD medium included the following concentrations of components: yeast powder 0.8g/L, peptone 2.0g/L and glucose 2.0g/L; (2) Inoculating the primary seed liquid of the pichia pastoris gene engineering bacteria into a base material culture medium in a 15L seed tank according to an inoculum size of 8% by adopting a flame method, and culturing for 23h at 30 ℃ and pH of 5.0 with a dissolved oxygen value of more than 30% to obtain secondary seeds of the pichia pastoris gene engineering bacteria; (3) After dissolved oxygen of the secondary seed liquid of the pichia pastoris is greatly risen, inoculating the secondary seed liquid of the pichia pastoris into a bottom material culture medium in a 150L seed tank according to an inoculum size of 8% by adopting a differential pressure method, and culturing for 22 hours at 29 ℃ and pH of 5.0, wherein the dissolved oxygen value is more than 30% to obtain the tertiary seed of the pichia pastoris genetically engineered bacterium; (4) After dissolved oxygen of the three-stage seed liquid of the pichia pastoris is greatly risen, inoculating the three-stage seed liquid of the pichia pastoris into a bottom material culture medium in a 1500L production fermentation tank according to an inoculum size of 8% by adopting a differential pressure method, and culturing for 21 hours at 29 ℃ and a pH value of 5.0, wherein the dissolved oxygen value is more than 30% to obtain four-stage seeds of the pichia pastoris genetically engineered bacteria; in the fermentation process, 1mol/L ammonia water is adopted to adjust the pH of the fermentation liquid, so that the pH of the fermentation process is 5.0;
the substrate culture medium comprises the following components in concentration: 26.7mL/L of 85% phosphoric acid, 0.9g/L of calcium sulfate, 18.2g/L of potassium sulfate, 14.9g/L of magnesium sulfate heptahydrate, 4.13g/L of potassium hydroxide, 40g/L of glycerol and 4.35mL/L of PT M; wherein PTM1 comprises the following concentration components: cuSO 4 ·5H 2 O 6g/L，NaI0.4g/L，MnSO 4 ·H 2 O 5g/L，Na 2 MoO 4 ·2H 2 O 0.2g/L，H 3 BO 4 0.1g/L，CoCl 2 1g/L，ZnCl 2 30g/L，FeSO 4 ·7H 2 O90 g/L and H 2 SO 4 5mL/L and biotin 0.2g/L;
s2: culturing four-level seeds of the pichia pastoris gene engineering bacteria in the production fermentation tank until dissolved oxygen is greatly risen, and then feeding a carbon source by using a glycerol feed supplement culture medium at a feeding rate of 18.2mL/h/L until the wet bacterial weight of the pichia pastoris gene engineering bacteria reaches 200g/L, and ending the carbon source feed supplement; the glycerol feed medium included the following concentration components: glycerin 500mL/L, PTM1 mL/L, deionized water 488mL/L;
s3: starving for 40min after the mixed carbon source feeding is finished, feeding by using a methanol feeding culture medium, starting feeding at a feeding speed of 3.6mL/L/h, increasing the feeding speed to 7.2mL/L/h after 3h, increasing the feeding speed to 10.9mL/L/h after 6h, maintaining feeding at the speed until the fermentation is finished, maintaining the dissolved oxygen value at 20% fluctuation, and introducing liquid oxygen after the feeding speed is improved, wherein the whole fermentation process is finished when the wet bacterial weight or the protein expression quantity of the pichia pastoris is no longer increased; the methanol feed medium comprises the following components in concentration: methanol 988mL/L, PTM1 12mL/L.
Experimental example 1
Detecting recombinant humanized type I collagen expression of recombinant humanized type I collagen Pichia genetic engineering bacteria fermented in example 1 and comparative example, respectively taking fermentation liquids before induction and expression feeding and after induction and expression feeding are cultured for 30h, 36h, 42h, 48h, 54h and 60h in the fermentation process of example 1, centrifuging to obtain supernatant, and detecting protein expression by using polyacrylamide gel electrophoresis (SDS-PAGE), wherein the results are shown in figure 1; respectively taking fermentation liquids obtained by culturing for 20h, 30h, 40h, 50h, 60h, 70h, 80h and 90h after induction expression feeding in the fermentation process of the comparative example, centrifuging to obtain supernatant, and detecting protein expression by using polyacrylamide gel electrophoresis (SDS-PAGE), wherein the result is shown in figure 2; and the SDS-PAGE result map was analyzed by gray level analysis, and the analysis results are shown in Table 1.
Determination criteria: the theoretical molecular weight of the recombinant humanized type I collagen is about 66.2kD, and whether the target protein is expressed or not is judged according to the molecular weight marked by a Marker. As can be seen from fig. 1 and fig. 2, the pichia pastoris genetically engineered bacteria fermented by the conventional fermentation method of the comparative example have obvious degradation in the whole induction process after the induction and expression of the fed-batch; the Pichia pastoris engineering bacteria fermented by the fermentation method only slightly degrade the recombinant humanized I-type collagen in the whole induction process after the induction and expression of the feed supplement.
TABLE 1 SDS-PAGE result profile analysis
As can be seen from Table 1, in the whole induction process of the Pichia pastoris engineering bacteria fermented by the fermentation method of the application after the induction and the feed supplement, the expression level of the recombinant humanized type I collagen is far higher than that of the recombinant humanized type I collagen obtained by the traditional fermentation method of the comparative example, the ratio of the recombinant humanized type I collagen is over 90 percent, only slight degradation occurs, and the ratio of the recombinant humanized type I collagen obtained by the comparative example is only 37 percent, so that serious degradation occurs.
Experimental example 2
Recording related parameters of the recombinant humanized collagen pichia pastoris gene engineering bacteria fermented in the examples 1-3 and the comparative example; the results are shown in Table 2.
TABLE 2 fermentation related parameters
As can be seen from Table 2, under the condition that the wet bacterial weights of the tank-releasing bacteria are basically the same, the consumption of methanol in examples 1 to 3 is reduced by more than half compared with that of comparative examples, meanwhile, liquid oxygen is not introduced in the fermentation process of examples 1 to 3, liquid oxygen is required to be introduced in comparative examples to maintain a higher methanol feed rate, and although the higher methanol feed rate is maintained in comparative examples, higher yield is not obtained.
In summary, the fermentation method of the recombinant humanized collagen provided by the application has the advantages that in the process of inducing protein expression, the recombinant humanized collagen is only slightly degraded, the expression quantity is improved, the consumption of methanol in the fermentation process is reduced, the fermentation period is shortened, and stable industrial production is facilitated.
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (5)
1. A method for fermenting recombinant humanized collagen, comprising the steps of:
s1: inoculating Pichia pastoris genetically engineered bacteria into a seed tank for culture, and transferring to a production fermentation tank after dissolved oxygen is greatly risen;
s2: culturing Pichia pastoris genetically engineered bacteria in a production fermentation tank until dissolved oxygen is greatly raised, and then using glucose solution and methanol for mixed carbon source feeding;
wherein the flow rate of the glucose solution is 10-30 mL/h/L, and the flow rate of the methanol is 0.1-1.5 mL/h/L;
s3: after the mixed carbon source feeding is finished, feeding is firstly carried out by using a methanol solution in a gradient pulse mode, and then constant-speed feeding is carried out by using a glucose solution and a methanol solution until the fermentation is finished;
the pulse gradients are respectively 1%, 1.5% and 2% of the volume of the fermentation broth, each gradient is respectively subjected to pulse feeding for 2-5 times, and after each pulse feeding is finished, the next pulse feeding is started after the dissolved oxygen is returned; when constant-speed feeding is carried out, the feeding rate of the glucose solution is 0.1-1 mL/h/L, and the feeding rate of the methanol solution is 4-10 mL/h/L; PTM1 is added into methanol solutions used for pulse and constant-speed feeding, and the addition amount is 12-24 mL/L;
the mass fraction of the glucose solution is 40% -80%.
2. The fermentation method of the recombinant humanized collagen according to claim 1, wherein the pichia pastoris gene engineering bacteria are pichia pastoris engineering bacteria for expressing the recombinant humanized collagen; the recombinant humanized collagen is recombinant humanized type I collagen, recombinant humanized type II collagen, recombinant humanized type III collagen or a combination of functional region fragments of the recombinant humanized type I, type II and type III collagen.
3. The fermentation method of recombinant humanized collagen according to claim 1, wherein the specific method for culturing the genetically engineered pichia pastoris in step S1 is as follows: (1) Inoculating Pichia pastoris genetically engineered bacteria into YPD culture medium in a shake flask according to an inoculum size of 0.1% -0.3%, and culturing to obtain first-class seeds; (2) Inoculating the primary seed liquid into a bottom material culture medium in a 15L seed tank according to the inoculum size of 6% -12%, and culturing to obtain secondary seeds; (3) After the dissolved oxygen of the secondary seed liquid is greatly risen, inoculating the secondary seed liquid into a bottom material culture medium in a 150L seed tank according to the inoculum size of 6% -12%, and culturing to obtain tertiary seeds; (4) After the dissolved oxygen of the three-level seed liquid is greatly risen, inoculating the three-level seed liquid into a bottom material culture medium in a 1500L production fermentation tank according to the inoculum size of 6% -12%, and culturing to obtain four-level seeds; in the fermentation process, the pH of the fermentation liquid is regulated by adopting 1-5 mol/L KOH, so that the pH in the fermentation process is 3.0-7.0.
4. A method of fermenting recombinant humanized collagen according to claim 3, wherein the YPD medium comprises the following concentrations of components: 0.8-1.2 g/L of yeast powder, 1.8-2.2 g/L of peptone and 1.8-2.2 g/L of glucose.
5. A method of fermenting recombinant humanized collagen according to claim 3, wherein the primer medium comprises the following concentrations of components: KH2PO4 21.5-43.0 g/L, (NH 4) 2SO4.5-7.5 g/L, caSO4.2H2O 0.2-1.0 g/L, K2SO4 7-14.3 g/L, mgSO4.7H2O 6-11.7 g/L, glucose 80-120 g/L, PTM 1.2-8.4 mL/L and vitamin C0.01-0.1 g/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110999047.3A CN113667709B (en) | 2021-08-28 | 2021-08-28 | Fermentation method of recombinant humanized collagen |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110999047.3A CN113667709B (en) | 2021-08-28 | 2021-08-28 | Fermentation method of recombinant humanized collagen |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113667709A CN113667709A (en) | 2021-11-19 |
| CN113667709B true CN113667709B (en) | 2023-11-21 |
Family
ID=78547367
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110999047.3A Active CN113667709B (en) | 2021-08-28 | 2021-08-28 | Fermentation method of recombinant humanized collagen |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113667709B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114106202B (en) * | 2021-11-25 | 2023-10-27 | 西安德诺海思医疗科技有限公司 | Conus toxin and collagen fusion protein and preparation method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN102115735A (en) * | 2010-11-12 | 2011-07-06 | 广东溢多利生物科技股份有限公司 | High-density industrial fermentation method for recombinant pichia pastoris phytase |
| WO2018014453A1 (en) * | 2016-07-22 | 2018-01-25 | 江苏江山聚源生物技术有限公司 | Fermentation technique to improve production level of recombinant human collagen |
| CN107988291A (en) * | 2018-01-18 | 2018-05-04 | 江苏江山聚源生物技术有限公司 | The methanol feeding zymotechnique of Pichia anomala expression recombination human source collagen |
| CN111733200A (en) * | 2020-06-30 | 2020-10-02 | 浙江诸暨聚源生物技术有限公司 | Fermentation process for improving production level of recombinant collagen by adjusting addition mode of PTM1 |
-
2021
- 2021-08-28 CN CN202110999047.3A patent/CN113667709B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN102115735A (en) * | 2010-11-12 | 2011-07-06 | 广东溢多利生物科技股份有限公司 | High-density industrial fermentation method for recombinant pichia pastoris phytase |
| WO2018014453A1 (en) * | 2016-07-22 | 2018-01-25 | 江苏江山聚源生物技术有限公司 | Fermentation technique to improve production level of recombinant human collagen |
| CN107988291A (en) * | 2018-01-18 | 2018-05-04 | 江苏江山聚源生物技术有限公司 | The methanol feeding zymotechnique of Pichia anomala expression recombination human source collagen |
| CN111733200A (en) * | 2020-06-30 | 2020-10-02 | 浙江诸暨聚源生物技术有限公司 | Fermentation process for improving production level of recombinant collagen by adjusting addition mode of PTM1 |
Non-Patent Citations (1)
| Title |
|---|
| 以葡萄糖为碳源的毕赤酵母发酵工艺优化及诱导调控;李宇然;《中国优秀硕士学位论文全文数据库 工程科技I辑》;20190115(第01期);第B018-205页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113667709A (en) | 2021-11-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR102132132B1 (en) | Fermentation method to improve recombinant human collagen production level | |
| Chen | High cell density culture of microalgae in heterotrophic growth | |
| Chen et al. | Heterotrophic growth of Chlamydomonas reinhardtii on acetate in chemostat culture | |
| CN103224965B (en) | Method for producing pyrroloquinoline quinine through microbial fermentation and fermentation medium used in same | |
| CN102154189B (en) | A kind of fermentation culture method of rhG-CSF recombinant bacterial strain | |
| CN101570771A (en) | Method for producing S-adenosylmethionine through fermentation of recombinant pichia pastoris | |
| CN117210343A (en) | Candida tropicalis and application thereof, and production method of long-chain dibasic acid | |
| CN113667709B (en) | Fermentation method of recombinant humanized collagen | |
| CN106434830A (en) | Method for improving 2-keto-L-gluconic acid fermentation efficiency | |
| CN111748480B (en) | Candida virginiana and application thereof | |
| CN116179356A (en) | Method for high-density heterotrophic culture of chlamydomonas reinhardtii and application thereof | |
| EP0136802B1 (en) | Industrial-scale process for the production of polyols by fermentation of sugars (1111111) | |
| CN107988294A (en) | Adjust the zymotechnique that temperature improves recombination human source collagen production level | |
| CN100537775C (en) | A kind of method of coercing strategy raising hyaluronic acid volume of production of fermentation production with intermittent high pH | |
| CN114276937A (en) | Method for fermenting paecilomyces hepiali by using Chinese yam as carbon source | |
| CN111909859B (en) | Low-temperature culture medium for pichia pastoris | |
| CN108048511B (en) | Fermentation process for adjusting pH and improving production level of recombinant human collagen | |
| CN102517223A (en) | High-density fermentation process and equipment for methanol type pichia pastoris | |
| CN107988293B (en) | Fermentation process for improving production level of recombinant human-derived collagen by adjusting pressure | |
| CN101984053B (en) | Preparation method of lipase by fermentation | |
| CN202415541U (en) | Special equipment being applicable to high-concentration culture of methanol pichia methanolica | |
| CN102041285B (en) | Method for fermenting and producing Tremella polysaccharides by adopting constant pH feeding strategy | |
| CN101412991B (en) | Method for preparing mannanase by using glycerol as carbon source and fedbatch of substrate for induction | |
| CN111411131B (en) | Glucose feed supplement control method for producing glutamic acid through fermentation | |
| KR102202694B1 (en) | Novel methylotrophic bacteria variant with overexpression of ftfL gene and method for producing PHB using the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |