CN117384882A - Fermentation method of recombinant DNase I - Google Patents
Fermentation method of recombinant DNase I Download PDFInfo
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- CN117384882A CN117384882A CN202311244432.2A CN202311244432A CN117384882A CN 117384882 A CN117384882 A CN 117384882A CN 202311244432 A CN202311244432 A CN 202311244432A CN 117384882 A CN117384882 A CN 117384882A
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- fermentation
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- dnase
- methanol
- calcium
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- 238000000855 fermentation Methods 0.000 title claims abstract description 272
- 230000004151 fermentation Effects 0.000 title claims abstract description 272
- 108010008532 Deoxyribonuclease I Proteins 0.000 title claims abstract description 124
- 238000000034 method Methods 0.000 title claims abstract description 107
- 102000007260 Deoxyribonuclease I Human genes 0.000 title claims abstract description 103
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 285
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 93
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 89
- 230000006698 induction Effects 0.000 claims abstract description 81
- 230000014509 gene expression Effects 0.000 claims abstract description 77
- 239000001963 growth medium Substances 0.000 claims abstract description 52
- LZCZIHQBSCVGRD-UHFFFAOYSA-N benzenecarboximidamide;hydron;chloride Chemical compound [Cl-].NC(=[NH2+])C1=CC=CC=C1 LZCZIHQBSCVGRD-UHFFFAOYSA-N 0.000 claims abstract description 41
- 159000000007 calcium salts Chemical class 0.000 claims abstract description 38
- 230000008569 process Effects 0.000 claims abstract description 38
- 241000235058 Komagataella pastoris Species 0.000 claims abstract description 37
- 239000001888 Peptone Substances 0.000 claims abstract description 35
- 108010080698 Peptones Proteins 0.000 claims abstract description 35
- 235000019319 peptone Nutrition 0.000 claims abstract description 35
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 180
- 235000011187 glycerol Nutrition 0.000 claims description 54
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 49
- 239000001301 oxygen Substances 0.000 claims description 49
- 229910052760 oxygen Inorganic materials 0.000 claims description 49
- 239000002609 medium Substances 0.000 claims description 44
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 42
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 36
- 239000007788 liquid Substances 0.000 claims description 34
- 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 claims description 28
- 239000001110 calcium chloride Substances 0.000 claims description 27
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 27
- 235000011148 calcium chloride Nutrition 0.000 claims description 27
- 238000012258 culturing Methods 0.000 claims description 26
- 101150051118 PTM1 gene Proteins 0.000 claims description 25
- 239000002904 solvent Substances 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 235000003642 hunger Nutrition 0.000 claims description 16
- 238000011218 seed culture Methods 0.000 claims description 15
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 claims description 14
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 claims description 14
- 235000020958 biotin Nutrition 0.000 claims description 14
- 239000011616 biotin Substances 0.000 claims description 14
- 229960002685 biotin Drugs 0.000 claims description 14
- 235000019837 monoammonium phosphate Nutrition 0.000 claims description 14
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 14
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 14
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims description 14
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 14
- 235000011151 potassium sulphates Nutrition 0.000 claims description 14
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 13
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 13
- 239000006012 monoammonium phosphate Substances 0.000 claims description 13
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 13
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 13
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- 238000011081 inoculation Methods 0.000 claims description 12
- ZCCIPPOKBCJFDN-UHFFFAOYSA-N calcium nitrate Chemical compound [Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ZCCIPPOKBCJFDN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 239000007789 gas Substances 0.000 claims description 5
- WHQOKFZWSDOTQP-UHFFFAOYSA-N 2,3-dihydroxypropyl 4-aminobenzoate Chemical compound NC1=CC=C(C(=O)OCC(O)CO)C=C1 WHQOKFZWSDOTQP-UHFFFAOYSA-N 0.000 claims description 3
- SIWNEELMSUHJGO-UHFFFAOYSA-N 2-(4-bromophenyl)-4,5,6,7-tetrahydro-[1,3]oxazolo[4,5-c]pyridine Chemical compound C1=CC(Br)=CC=C1C(O1)=NC2=C1CCNC2 SIWNEELMSUHJGO-UHFFFAOYSA-N 0.000 claims description 3
- ZKQDCIXGCQPQNV-UHFFFAOYSA-N Calcium hypochlorite Chemical compound [Ca+2].Cl[O-].Cl[O-] ZKQDCIXGCQPQNV-UHFFFAOYSA-N 0.000 claims description 3
- UNMYWSMUMWPJLR-UHFFFAOYSA-L Calcium iodide Chemical compound [Ca+2].[I-].[I-] UNMYWSMUMWPJLR-UHFFFAOYSA-L 0.000 claims description 3
- NKWPZUCBCARRDP-UHFFFAOYSA-L calcium bicarbonate Chemical compound [Ca+2].OC([O-])=O.OC([O-])=O NKWPZUCBCARRDP-UHFFFAOYSA-L 0.000 claims description 3
- 229910000020 calcium bicarbonate Inorganic materials 0.000 claims description 3
- YYRMJZQKEFZXMX-UHFFFAOYSA-L calcium bis(dihydrogenphosphate) Chemical compound [Ca+2].OP(O)([O-])=O.OP(O)([O-])=O YYRMJZQKEFZXMX-UHFFFAOYSA-L 0.000 claims description 3
- 229910001622 calcium bromide Inorganic materials 0.000 claims description 3
- 229940059251 calcium bromide Drugs 0.000 claims description 3
- YALMXYPQBUJUME-UHFFFAOYSA-L calcium chlorate Chemical compound [Ca+2].[O-]Cl(=O)=O.[O-]Cl(=O)=O YALMXYPQBUJUME-UHFFFAOYSA-L 0.000 claims description 3
- 229960002713 calcium chloride Drugs 0.000 claims description 3
- WGEFECGEFUFIQW-UHFFFAOYSA-L calcium dibromide Chemical compound [Ca+2].[Br-].[Br-] WGEFECGEFUFIQW-UHFFFAOYSA-L 0.000 claims description 3
- 239000004227 calcium gluconate Substances 0.000 claims description 3
- 235000013927 calcium gluconate Nutrition 0.000 claims description 3
- 229960004494 calcium gluconate Drugs 0.000 claims description 3
- 229940046413 calcium iodide Drugs 0.000 claims description 3
- 229910001640 calcium iodide Inorganic materials 0.000 claims description 3
- 229910000389 calcium phosphate Inorganic materials 0.000 claims description 3
- NEEHYRZPVYRGPP-UHFFFAOYSA-L calcium;2,3,4,5,6-pentahydroxyhexanoate Chemical compound [Ca+2].OCC(O)C(O)C(O)C(O)C([O-])=O.OCC(O)C(O)C(O)C(O)C([O-])=O NEEHYRZPVYRGPP-UHFFFAOYSA-L 0.000 claims description 3
- JXRVKYBCWUJJBP-UHFFFAOYSA-L calcium;hydrogen sulfate Chemical compound [Ca+2].OS([O-])(=O)=O.OS([O-])(=O)=O JXRVKYBCWUJJBP-UHFFFAOYSA-L 0.000 claims description 3
- 235000019691 monocalcium phosphate Nutrition 0.000 claims description 3
- 235000011118 potassium hydroxide Nutrition 0.000 claims description 3
- YAECNLICDQSIKA-UHFFFAOYSA-L calcium;sulfanide Chemical compound [SH-].[SH-].[Ca+2] YAECNLICDQSIKA-UHFFFAOYSA-L 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 29
- 230000015556 catabolic process Effects 0.000 abstract description 21
- 238000006731 degradation reaction Methods 0.000 abstract description 21
- 238000004519 manufacturing process Methods 0.000 abstract description 19
- 108091005804 Peptidases Proteins 0.000 abstract description 9
- 239000004365 Protease Substances 0.000 abstract description 9
- 239000011575 calcium Substances 0.000 abstract description 9
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract description 7
- 230000017854 proteolysis Effects 0.000 abstract description 7
- 150000001413 amino acids Chemical class 0.000 abstract description 5
- 239000000758 substrate Substances 0.000 abstract description 4
- 102000004142 Trypsin Human genes 0.000 abstract description 3
- 108090000631 Trypsin Proteins 0.000 abstract description 3
- 102000001400 Tryptase Human genes 0.000 abstract description 3
- 108060005989 Tryptase Proteins 0.000 abstract description 3
- 230000010261 cell growth Effects 0.000 abstract description 3
- 230000019522 cellular metabolic process Effects 0.000 abstract description 3
- 230000001419 dependent effect Effects 0.000 abstract description 3
- 230000007062 hydrolysis Effects 0.000 abstract description 3
- 238000006460 hydrolysis reaction Methods 0.000 abstract description 3
- 235000016709 nutrition Nutrition 0.000 abstract description 3
- 230000035764 nutrition Effects 0.000 abstract description 3
- 229920001184 polypeptide Polymers 0.000 abstract description 3
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 3
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 3
- 102000035195 Peptidases Human genes 0.000 abstract description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 abstract 1
- 229940125532 enzyme inhibitor Drugs 0.000 abstract 1
- 239000002532 enzyme inhibitor Substances 0.000 abstract 1
- 239000012588 trypsin Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 52
- 238000001262 western blot Methods 0.000 description 26
- 230000000052 comparative effect Effects 0.000 description 24
- 238000001514 detection method Methods 0.000 description 22
- 102000004190 Enzymes Human genes 0.000 description 20
- 108090000790 Enzymes Proteins 0.000 description 20
- 230000001105 regulatory effect Effects 0.000 description 18
- 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 description 14
- 239000007864 aqueous solution Substances 0.000 description 13
- 229940041514 candida albicans extract Drugs 0.000 description 13
- 239000008103 glucose Substances 0.000 description 13
- 239000012138 yeast extract Substances 0.000 description 13
- 239000003550 marker Substances 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 9
- 239000008367 deionised water Substances 0.000 description 9
- 229910021641 deionized water Inorganic materials 0.000 description 9
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 9
- 238000009423 ventilation Methods 0.000 description 9
- 239000007222 ypd medium Substances 0.000 description 9
- 239000002054 inoculum Substances 0.000 description 8
- 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 8
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 8
- CMDGQTVYVAKDNA-UHFFFAOYSA-N propane-1,2,3-triol;hydrate Chemical compound O.OCC(O)CO CMDGQTVYVAKDNA-UHFFFAOYSA-N 0.000 description 8
- 238000005070 sampling Methods 0.000 description 8
- 230000001502 supplementing effect Effects 0.000 description 8
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- 239000001965 potato dextrose agar Substances 0.000 description 4
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- 238000002474 experimental method Methods 0.000 description 3
- 239000013613 expression plasmid Substances 0.000 description 3
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- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 241000206602 Eukaryota Species 0.000 description 2
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 2
- 229940122055 Serine protease inhibitor Drugs 0.000 description 2
- 101710102218 Serine protease inhibitor Proteins 0.000 description 2
- 229940122618 Trypsin inhibitor Drugs 0.000 description 2
- 229940122598 Tryptase inhibitor Drugs 0.000 description 2
- 229940062672 calcium dihydrogen phosphate Drugs 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
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- 239000012634 fragment Substances 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 108700021021 mRNA Vaccine Proteins 0.000 description 2
- 229940126582 mRNA vaccine Drugs 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 230000009465 prokaryotic expression Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 239000003001 serine protease inhibitor Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 239000002753 trypsin inhibitor Substances 0.000 description 2
- 239000002750 tryptase inhibitor Substances 0.000 description 2
- XDIYNQZUNSSENW-UUBOPVPUSA-N (2R,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexanal Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O XDIYNQZUNSSENW-UUBOPVPUSA-N 0.000 description 1
- 101000596279 Bacillus subtilis Type II restriction enzyme BglII Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- PXXJHWLDUBFPOL-UHFFFAOYSA-N benzamidine Chemical compound NC(=N)C1=CC=CC=C1 PXXJHWLDUBFPOL-UHFFFAOYSA-N 0.000 description 1
- LVGQIQHJMRUCRM-UHFFFAOYSA-L calcium bisulfite Chemical compound [Ca+2].OS([O-])=O.OS([O-])=O LVGQIQHJMRUCRM-UHFFFAOYSA-L 0.000 description 1
- 235000010260 calcium hydrogen sulphite Nutrition 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
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- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 239000006481 glucose medium Substances 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- KNWQLFOXPQZGPX-UHFFFAOYSA-N methanesulfonyl fluoride Chemical compound CS(F)(=O)=O KNWQLFOXPQZGPX-UHFFFAOYSA-N 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
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- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 1
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- 229920006395 saturated elastomer Polymers 0.000 description 1
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- 239000008399 tap water Substances 0.000 description 1
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- 210000001541 thymus gland Anatomy 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
Classifications
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- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/21—Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)
- C12Y301/21001—Deoxyribonuclease I (3.1.21.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/84—Pichia
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- Bioinformatics & Cheminformatics (AREA)
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- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
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- General Engineering & Computer Science (AREA)
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- Biomedical Technology (AREA)
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- Molecular Biology (AREA)
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Abstract
The invention provides a fermentation method of recombinant DNase I. According to the fermentation method, a fermentation culture medium contains peptone, and water-soluble calcium salt is added in the methanol induced protein expression process; peptone is rich in polypeptides and amino acids, provides excessive substrate for protease, can competitively inhibit the hydrolysis of target protein, and can provide nutrition for cell growth and metabolism; providing Ca in water-soluble calcium salt 2+ DNase I activity is dependent on Ca 2+ The method comprises the steps of carrying out a first treatment on the surface of the And Ca 2+ The addition of (2) can prevent DNase1 from being hydrolyzed to a certain extent. The fermentation method can reduce protein degradation in the process of producing recombinant DNase I by fermenting pichia pastoris, and improve the production level and stability; in the fermentation method, benzamidine hydrochloride which is trypsin, tryptase and serine protein is also added in the methanol induction processAn enzyme inhibitor which is effective in preventing or reducing degradation of DNase1 by proteases.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a fermentation method of recombinant DNase I.
Background
DNase I, deoxyribonuclease I, chinese name DNase I, is an endonuclease that digests single-stranded or double-stranded DNA. It hydrolyzes phosphodiester bonds to produce mono-and oligodeoxynucleotides containing 5 '-phosphate groups and 3' -OH groups. Can be widely applied to RNA preparation without DNA; removing the template DNA after in vitro transcription; preparing RNA without DNA before RT-PCR and RT-qPCR reaction; DNA labeling by nick translocation in combination with DNA polymerase I; and (5) constructing a DNA fragmentation library.
In the production process of biological products, the accounting pollution, especially the residual DNA, needs to be strictly controlled, and DNase I can play a good role in removing. For example, in recent years, the development of mRNA vaccines plays an important role in the prevention of some infectious diseases, and DNase I is one of the key enzyme raw materials in the development and production processes of mRNA vaccines.
The earliest DNase I production method is a method for extracting from bovine pancreas and other tissues, and the method has the disadvantages of unstable raw materials, very complicated preparation process, easy inactivation and unfavorable mass production; and is an animal-derived product, and the safety cannot be ensured. The current general method is a method of recombining genetically engineered bacteria, but the DNase I has serious toxicity to host cells, and the host cells are killed along with the expression of the DNase I, so that a required amount of target products cannot be obtained.
The Pichia pastoris (Pichia pastoris) expression system is characterized by single-cell lower eukaryote, simple culture condition, high propagation speed, low culture cost, contribution to large-scale production, post-translational processing capability, capability of secreting exogenous protein out of cells, more stable property than protein expressed by prokaryote, particular suitability for expressing protein from eukaryote, and can be used as a method for producing DNase I on a large scale.
Pichia pastoris is used as an expression system of foreign proteins, has the advantages of simple operation, easy culture, high growth speed, high expression quantity, low cost and the like of a prokaryotic expression system, has the characteristics of post-translational modification of the foreign proteins, such as glycosylation, protein phosphorylation and the like, which are not possessed by the prokaryotic expression system, and simultaneously avoids the defects of poor secretion efficiency, unstable expression strain, easy loss of expression plasmids and the like of Saccharomyces cerevisiae (Saccharomyces cerevisiae).
However, the applicant finds that in the process of producing DNase I by using pichia pastoris expression, the expressed DNase I protein is partially or even completely degraded, the protein yield is low, and the like; based on this, there is a need to improve the drawbacks of pichia pastoris expression production DNase I.
Disclosure of Invention
In view of the above, the invention provides a fermentation method of recombinant DNase I to solve the technical problems in the prior art.
In a first aspect, the present invention provides a fermentation process for recombinant DNase I, comprising the steps of:
amplifying recombinant Pichia pastoris containing DNase I gene by a seed culture medium, inoculating to a fermentation culture medium for fermentation culture, adding glycerol when the glycerol in the fermentation culture medium is exhausted and dissolved oxygen rises, and continuously culturing until the fermentation liquid OD is reached 600 Stopping adding glycerol after the value reaches 120-550, starving, exhausting the glycerol in the fermentation liquid, and adding methanol to induce protein expression;
wherein, water-soluble calcium salt is added in the expression process of methanol induced protein;
the fermentation medium contains peptone.
Preferably, in the fermentation method of recombinant DNase I, the water-soluble calcium salt includes at least one of calcium chloride, calcium gluconate, calcium dihydrogen phosphate, calcium nitrate, calcium bicarbonate, calcium bisulfate, calcium bisulfide, calcium hypochlorite, calcium bromide, calcium iodide, calcium chlorate, calcium perchlorate and calcium permanganate.
Preferably, in the fermentation method of the recombinant DNase I, benzamidine hydrochloride is added in the process of inducing protein expression by methanol.
Preferably, in the fermentation method of recombinant DNase I, water-soluble calcium salt is added in the methanol-induced protein expression process so that the concentration of the water-soluble calcium salt is 10-50 mM.
Preferably, in the fermentation method of recombinant DNase I, the adding mode of adding benzamidine hydrochloride in the process of inducing protein expression is as follows: adding 2.61-6.22g/L benzamidine hydrochloride into methanol, or independently feeding the same mass flow rate of benzamidine hydrochloride into the methanol, or feeding 0.5-2.34g/L benzamidine hydrochloride into the fermentation liquor in batches for multiple times.
Preferably, the fermentation medium further comprises monoammonium phosphate, monopotassium phosphate, potassium sulfate, potassium hydroxide, magnesium sulfate, calcium chloride, biotin, glycerol, PTM1 solution and solvent.
Preferably, in the fermentation method of recombinant DNase I, the concentration of peptone in the fermentation medium is 2-10 g/L, the concentration of ammonium dihydrogen phosphate is 5-40 g/L, the concentration of potassium dihydrogen phosphate is 2-10 g/L, the concentration of potassium sulfate is 3.5-26.1 g/L, the concentration of potassium hydroxide is 1.5-2 g/L, the concentration of magnesium sulfate is 10.5-18.5 g/L, the concentration of calcium chloride is 0.2-0.5 g/L, the concentration of biotin is 0.02-0.3 g/L, and the concentration of glycerol is 10-50 g/L, PTM1 solution is 2-5 ml/L;
the solvent is water;
the PTM1 solution comprises the following components in concentration: cuSO 4 5～7g/L、KI 0.08～0.09g/L、MnSO 4 2～4g/L、Na 2 MoO 4 0.1～0.3g/L、H 3 BO 3 0.01～0.03g/L、CoCl 2 0.4～0.6g/L、ZnCl 2 19～21g/L、FeSO 4 63-67 g/L, dense H 2 SO 4 4-6 ml/L; the concentration H 2 SO 4 The mass fraction is 98-99%.
Preferably, in the fermentation method of recombinant DNase I, recombinant pichia pastoris containing DNase I gene is subjected to expansion culture by a seed culture medium and then inoculated into a fermentation culture medium for fermentation culture, wherein the fermentation culture specifically comprises the following steps:
inoculating recombinant Pichia pastoris containing DNase I gene into seed culture medium, and culturing to OD 600 The value is 20-50, and seed liquid is obtained;
inoculating the seed solution into a fermentation medium for fermentation culture;
wherein the seed culture medium comprises at least one of YPD culture medium, PDA culture medium and BMGY culture medium;
inoculating recombinant pichia pastoris containing DNase I gene into a seed culture medium, wherein the culture temperature is 28-30 ℃;
inoculating the seed solution into a fermentation culture medium for fermentation culture, wherein the inoculation amount is 5-10%.
Preferably, in the fermentation method of recombinant DNase I, seed solution is inoculated into a fermentation culture medium for fermentation culture, pH is controlled to be 4.9-5.1 by ammonia water, 1.0-1.5 VVM oxygen-containing gas is introduced, rotating speed is adjusted to control dissolved oxygen to be more than or equal to 20%, glycerol is used up in the fermentation culture medium, the dissolved oxygen is increased to be more than or equal to 60%, glycerol is added, and the culture is continued until the OD of the fermentation liquid is reached 600 Stopping adding glycerol after the value reaches 120-550, and starving for 20-40 min; wherein the fermentation culture temperature is 28-30 ℃.
Preferably, in the fermentation method of recombinant DNase I, in the step of adding methanol to induce protein expression, the methanol flow rate is regulated to ensure that the concentration of methanol is 1-10 g/L, the induction time is 70-90 h, the temperature is 20-27 ℃, and the pH is 3.5-6.5.
Compared with the prior art, the fermentation method of the recombinant DNase I has the following beneficial effects:
1. according to the fermentation method of recombinant DNase I, a fermentation culture medium contains peptone, and water-soluble calcium salt is added in the methanol induced protein expression process; wherein, the peptone is rich in polypeptide and amino acid, provides excessive substrate for protease, can competitively inhibit the hydrolysis of target protein, and can provide nutrition for cell growth and metabolism; providing Ca in water-soluble calcium salt 2+ DNase I activity is dependent on Ca 2+ The method comprises the steps of carrying out a first treatment on the surface of the And Ca 2+ The addition of (2) can prevent DNase1 from being hydrolyzed to a certain extent. Recombinant DNase of the inventionThe fermentation method of I can reduce the protein degradation in the process of producing recombinant DNase I by fermenting Pichia pastoris, and improve the production level and stability; experiments prove that the method can effectively reduce protein degradation, effectively improve the production level, ensure that DNase I enzyme activity can reach 8612U/ml level and has good stability;
2. according to the fermentation method of recombinant DNase I, benzamidine hydrochloride which is a trypsin, tryptase and serine protease inhibitor and can effectively prevent or reduce degradation of DNase1 by protease is also added in the methanol induction process.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It is evident that the drawings in the following description are only some embodiments of the present invention and that other drawings may be obtained from these drawings without inventive effort for a person of ordinary skill in the art.
FIG. 1 is a Western Blot detection chart during induction of the fermentation process of example 3;
FIG. 2 is a Western Blot detection chart during induction of the fermentation process of example 4;
FIG. 3 is a Western Blot detection plot during induction of the fermentation process of example 5;
FIG. 4 is a Western Blot detection chart during induction of the fermentation process of example 6;
FIG. 5 is a Western Blot detection plot during induction of the fermentation process in example 7;
FIG. 6 is a Western Blot detection of the induction of the fermentation process of comparative example 1;
FIG. 7 is a Western Blot detection of the induction of the fermentation process of comparative example 2;
FIG. 8 is a Western Blot detection of the induction of the fermentation process of comparative example 3;
FIG. 9 is a graph showing the enzymatic activity of fermentation supernatants during induction in the fermentation methods of examples 3-7 and comparative examples 1-3;
FIG. 10 is an SDS-PAGE diagram of the fermentation broth obtained by the fermentation method in example 5, and purified DNase I protein product.
Detailed Description
The following description of the embodiments of the present invention will be made in detail and with reference to the embodiments of the present invention, but it should be apparent that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, are intended to fall within the scope of the present invention.
For a better understanding of the present invention, and not to limit its scope, all numbers expressing quantities, percentages, and other values used in the present application are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
The following description of the embodiments is not intended to limit the preferred embodiments. In addition, in the description of the present application, the term "comprising" means "including but not limited to". Various embodiments of the invention may exist in a range of forms; it should be understood that the description in a range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention; it is therefore to be understood that the range description has specifically disclosed all possible sub-ranges and individual values within that range. For example, it should be considered that a description of a range from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the ranges, such as 1, 2, 3, 4, 5, and 6, wherever applicable. In addition, whenever a numerical range is referred to herein, it is meant to include any reference number (fractional or integer) within the indicated range.
The invention provides a fermentation method of recombinant DNase I, which comprises the following steps:
amplifying recombinant Pichia pastoris containing DNase I gene by a seed culture medium, inoculating to a fermentation culture medium for fermentation culture, adding glycerol when the glycerol in the fermentation culture medium is exhausted and dissolved oxygen rises, and continuously culturing until the fermentation liquid OD is reached 600 Stopping adding glycerol after the value reaches 120-550, starving, exhausting the glycerol in the fermentation liquid, and adding methanol to induce protein expression;
wherein, water-soluble calcium salt is added in the expression process of methanol induced protein;
the fermentation medium contains peptone.
It should be noted that, the recombinant protein expressed by pichia pastoris has certain defects, such as that in the process of expressing exogenous protein, a certain amount of protease is generated in the extracellular of the pichia pastoris cell, so that the exogenous protein has a certain risk of being degraded by the host protease, the expression quantity of the exogenous protein is affected, the production level and stability are reduced, and the purification of the subsequent protein is also not facilitated; and as DNase I has serious toxicity to host cells, the host cells are killed along with the expression of DNase I, and compared with other proteins without high toxicity, the host cells are easier to die and degrade, so that a large amount of intracellular protease is released into fermentation broth, and the expressed DNase I protein is partially or even completely degraded, so that the problems of incomplete DNase I, low protein yield, unstable yield and the like are finally caused. In the fermentation method of the recombinant DNase I, the fermentation medium contains peptone, and water-soluble calcium salt is added in the process of methanol induced protein expression; wherein, the peptone is rich in polypeptide and amino acid, provides excessive substrate for protease, can competitively inhibit the hydrolysis of target protein, and can provide nutrition for cell growth and metabolism; providing Ca in water-soluble calcium salt 2+ DNase I activity is dependent on Ca 2+ The method comprises the steps of carrying out a first treatment on the surface of the And Ca 2+ The addition of (2) can prevent DNase1 from being hydrolyzed to a certain extent. The fermentation method of the recombinant DNase I can reduce the protein degradation in the process of producing the recombinant DNase I by fermenting pichia pastoris and improve the productionLevel and stability; experiments prove that the method can effectively reduce protein degradation, effectively improve the production level, ensure that DNase I enzyme activity can reach the level of 8612U/ml, and has good stability.
In the above embodiment, the water-soluble calcium salt is added during the expression of the methanol-inducible protein, and since the expression of the methanol-inducible protein is a protein expression process which lasts for a period of time, for example, the induction time of the expression of the methanol-inducible protein is 70 to 90 hours, the expression of the methanol-inducible protein includes an initial stage, an intermediate stage and a final stage; specifically, the water-soluble calcium salt can be added into the fermentation liquor at one time, can be added into the fermentation liquor for multiple times in batches, and can also be added into the fermentation liquor in a fed-batch mode; if the water-soluble calcium salt can be added into the fermentation broth at one time, the water-soluble calcium salt is added at the beginning of the expression of the methanol-induced protein (namely, at the initial stage), and the addition of the methanol and the water-soluble calcium salt is not sequential, either the methanol is added into the fermentation broth before the water-soluble calcium salt is added, or the water-soluble calcium salt is added into the fermentation broth before the methanol is added; if the water-soluble calcium salt is added to the fermentation broth in batches, the water-soluble calcium salt is added in batches throughout the whole induction process from the beginning to the end of methanol-induced protein expression (i.e., from the initial stage, the intermediate stage, to the final stage). If the water-soluble calcium salt is added to the fermenter in a fed-batch manner, the same final concentration of the water-soluble calcium salt is added continuously during the whole induction process from the beginning to the end of the methanol-induced protein expression (i.e., from the initial stage, the intermediate stage to the final stage).
Preferably, the water-soluble calcium salt is added at the beginning of the methanol-induced protein expression, specifically, the methanol and the water-soluble calcium salt are added in no order, and the methanol and the water-soluble calcium salt can be added to the fermentation broth first, then the water-soluble calcium salt can be added to the fermentation broth first, and then the methanol can be added to the fermentation broth first.
In some embodiments, the water-soluble calcium salt comprises at least one of calcium chloride, calcium gluconate, calcium dihydrogen phosphate, calcium nitrate, calcium bicarbonate, calcium hydrogen sulfate, calcium hydrogen sulfite, calcium hypochlorite, calcium bromide, calcium iodide, calcium chlorate, calcium perchlorate, and calcium permanganate.
Preferably, the water-soluble calcium salt is calcium chloride.
In some embodiments, benzamidine hydrochloride is added during the addition of methanol to induce protein expression.
Specifically, the chemical formula of benzamidine hydrochloride is C 7 H 9 ClN 2 Benzamidine hydrochloride is a trypsin, tryptase and serine protease inhibitor that is effective in preventing or reducing degradation of DNase1 by proteases.
In some embodiments, the water-soluble calcium salt is added at the beginning of methanol-induced protein expression to provide a concentration of water-soluble calcium salt of 10 to 50mM.
In some embodiments, the added water-soluble calcium salt is a sterilized calcium chloride solution.
Specifically, the water-soluble calcium salt is added once at the beginning of the expression of the methanol-inducible protein or added in batches or streams in multiple times from the beginning to the end of the expression of the methanol-inducible protein so that the concentration of the water-soluble calcium salt in the fermentation broth is 10 to 50mM, for example, calcium chloride is added at the beginning of the expression of the methanol-inducible protein so that the concentration of calcium chloride in the fermentation broth is 10 to 50mM.
Specifically, the water-soluble calcium salt is fed in the form of an aqueous solution.
In some embodiments, benzamidine hydrochloride is added during the addition of methanol to induce protein expression.
Specifically, benzamidine hydrochloride is added in batches or fed-batch mode for several times from the beginning to the end of methanol-induced protein expression.
In some embodiments, benzamidine hydrochloride is added to methanol at the beginning of methanol-induced protein expression, specifically, benzamidine hydrochloride is added to methanol as it is fed with methanol at a concentration of 2.61 to 6.22g/L by volume of methanol.
In some embodiments, benzamidine hydrochloride is added during methanol-induced protein expression in the following manner: directly feeding the benzamidine hydrochloride aqueous solution into the fermentation liquor at the same mass flow rate of benzamidine hydrochloride, wherein the concentration of benzamidine hydrochloride is 2.61-6.22 g/L, and the concentration of benzamidine hydrochloride aqueous solution is 2.61-6.22 g/L based on water volume.
In some embodiments, benzamidine hydrochloride is added during the addition of methanol to induce protein expression in the following manner: from the beginning to the end of induction, benzamidine hydrochloride is added into the fermentation broth for a plurality of times, specifically, the concentration of benzamidine hydrochloride is 0.5-2.34g/L for each addition, and the concentration is calculated by the volume of the fermentation broth.
In some embodiments, the fermentation medium further comprises monoammonium phosphate, monopotassium phosphate, potassium sulfate, potassium hydroxide, magnesium sulfate, calcium chloride, biotin, glycerol, PTM1 solution, and a solvent.
In some embodiments, the concentration of peptone in the fermentation medium is 2-10 g/L, the concentration of monoammonium phosphate is 5-40 g/L, the concentration of monopotassium phosphate is 2-10 g/L, the concentration of potassium sulfate is 3.5-26.1 g/L, the concentration of potassium hydroxide is 1.5-2 g/L, the concentration of magnesium sulfate is 10.5-18.5 g/L, the concentration of calcium chloride is 0.2-0.5 g/L, the concentration of biotin is 0.02-0.3 g/L, the concentration of glycerol is 10-50 g/L, PTM1, and the concentration of solution is 2-5 ml/L;
the solvent is water, and the specific water comprises at least one of deionized water, distilled water or tap water.
In some embodiments, the PTM1 solution is a trace element solution, specifically comprising the following concentrations of components: cuSO 4 5～7g/L、KI 0.08～0.09g/L、MnSO 4 2～4g/L、Na 2 MoO 4 0.1～0.3g/L、H 3 BO 3 0.01～0.03g/L、CoCl 2 0.4～0.6g/L、ZnCl 2 19～21g/L、FeSO 4 63-67 g/L, dense H 2 SO 4 4-6 ml/L; concentrated H 2 SO 4 The mass fraction is 98-99%.
Specifically, cuSO 4 、KI、MnSO 4 、Na 2 MoO 4 、H 3 BO 3 、CoCl 2 、ZnCl 2 、FeSO 4 Concentrated H 2 SO 4 Adding into water to 1L to obtain the final productPTM1 solution.
Preferably, the PTM1 solution comprises the following concentration components: cuSO 4 ·5H 2 O 6g/L、KI 0.088g/L、MnSO 4 ·H 2 O3.0g/L、Na 2 MoO 4 ·2H 2 O 0.2g/L、H 3 BO 3 0.02g/L、CoCl 2 ·6H 2 O 0.5g/L、ZnCl 2 20.0g/L、FeSO 4 ·7H 2 O65.0g/L, dense H 2 SO 4 5.0ml/L (concentrated sulfuric acid mass concentration 98%).
In some embodiments, the recombinant pichia pastoris containing DNase I is expanded by a seed medium and then inoculated into a fermentation medium for fermentation culture specifically comprising:
inoculating recombinant Pichia pastoris containing DNase I gene into seed culture medium, and culturing to OD 600 The value is 20-50, and seed liquid is obtained;
inoculating the seed solution into a fermentation medium for fermentation culture;
wherein the seed culture medium comprises at least one of YPD culture medium, PDA culture medium and BMGY culture medium;
inoculating recombinant pichia pastoris containing DNase I gene into a seed culture medium, wherein the culture temperature is 28-30 ℃;
inoculating the seed solution into a fermentation culture medium for fermentation culture, wherein the inoculation amount is 5-10%.
Specifically, in the above examples, the inoculation amount is 5 to 10%, which means that: the volume of the seed liquid is 5-10% of the volume of the fermentation medium.
Specifically, the YPD medium is YPD liquid medium, also called yeast Extract Peptone glucose medium, which includes 1% Yeast Extract, 2% Peptone, 2% Dextrose (glucose) (glucose); the preparation method comprises the following steps:
s1, dissolving 10g Yeast Extract (Yeast Extract), and 20g Peptone (Peptone) in 900ml water;
s2, sterilizing at 115 ℃ for 15min;
s3, adding 100ml 20g Dextrose (glucose), (after sterilizing the glucose solution); glucose, yeast extract, peptone solution may react chemically at high temperature after mixing, resulting in a change in the composition of the medium, so that they are separately sterilized and then mixed. The glucose can be filtered and sterilized, or can be sterilized at 115 ℃ for 15 min.
In some embodiments, the PDA medium is potato dextrose agar medium.
In some embodiments, BMGY Medium (Buffered Glycerol-complex Medium) is a buffered complete Medium containing glycerol for the cultivation of recombinant strains of Pichia pastoris prior to secretory expression to obtain high density bacterial production.
In some embodiments, seed liquid is inoculated into a fermentation culture medium for fermentation culture, pH is controlled to be 4.9-5.1 by ammonia water, VVM oxygen-containing gas of 1.0-1.5 is introduced, rotating speed is adjusted to control dissolved oxygen to be more than or equal to 20 percent, glycerol is used up in the fermentation culture medium, the dissolved oxygen is rapidly increased to be more than or equal to 60 percent, glycerol is added, and the culture is continued until the OD of the fermentation liquid is reached 600 Stopping adding glycerol after the value reaches 120-550, and starving for 20-40 min; wherein the fermentation culture temperature is 28-30 ℃.
Specifically, in the above examples, the Dissolved Oxygen (DO) concentration is 20% or more, which is the relative Dissolved Oxygen (DO) concentration, and the maximum Dissolved Oxygen (DO) concentration (i.e., 100% concentration) in the fermentation system of the present invention is defined as: aeration (specifically air) allowed the medium to reach a saturated dissolved oxygen level before the fermentation medium was inoculated, at which time the DO concentration was calibrated to 100%.
Specifically, in the above examples, glycerin was fed in a DO-STAT manner.
Specifically, in the above embodiment, glycerin is fed in a DO-STAT manner, and the glycerin is an aqueous glycerin solution with a volume concentration of 20 to 60%, preferably, the glycerin is an aqueous glycerin solution with a volume concentration of 30%.
In the embodiment, seed liquid is inoculated into a fermentation culture medium for fermentation culture, pH is controlled to be 4.9-5.1 by ammonia water, 1.0-1.5 VVM oxygen-containing gas is introduced, the rotating speed is adjusted to control dissolved oxygen to be more than or equal to 20%, glycerol in the fermentation culture medium is exhausted, the dissolved oxygen can be rapidly increased, and the dissolved oxygen can be increased to be more than 60% within 2 min.
In some embodiments, the oxygen-containing gas may be air, oxygen, or the like. Preferably air.
In some embodiments, the methanol concentration in the fermentation broth is 1-10 g/L during the step of adding methanol to induce protein expression.
In some embodiments, the step of adding methanol to induce protein expression is performed for a period of time ranging from 70 to 90 hours.
In some embodiments, the temperature is controlled to be 20-27 ℃ during the step of adding methanol to induce protein expression.
In some embodiments, the step of adding methanol to induce protein expression is performed at a pH of 3.5 to 6.5, and specifically, at a pH of 3.5 to 6.5 by feeding ammonia.
In some embodiments, the fermentation process of recombinant DNase I comprises the steps of:
s1, inoculating recombinant Pichia pastoris strain containing DNase I gene into a seed culture medium, and culturing until OD 600 The value reaches 20 to 50, and seed liquid is obtained; wherein in the culture process, the culture temperature is 28-30 ℃ and the rotating speed is 200-250 rpm;
s2, inoculating the seed solution into a fermentation medium for fermentation culture; in the culture process, the culture temperature is 28-30 ℃, the pH is automatically controlled to be 4.9-5.1 by ammonia water, the ventilation (specifically air) is 1.0-1.5 VVM, and the dissolved oxygen is controlled to be more than or equal to 20% by adjusting the rotating speed;
s3, stirring until dissolved oxygen rises to more than 60% when the dissolved oxygen rises rapidly, feeding a glycerol aqueous solution with the volume concentration of 20-60% in a DO-STAT mode, and continuously culturing until the fermentation liquor OD 600 Stopping adding glycerol after the value reaches 120-550, and starving for 20-40 min;
s4, starting to supplement methanol to induce protein expression; adding sterile calcium chloride in batches or in a fed-batch manner for one time or multiple times at the beginning of induction to ensure that the concentration is 10-50 mM;
meanwhile, in the induction process, benzamidine hydrochloride is added into methanol to ensure that the concentration is 2.61-6.22 g/L; or, in the induction process, adding benzamidine hydrochloride into the fermentation broth every 20-30 h, and enabling the concentration of the benzamidine hydrochloride to be 0.5-2.34 g/L after adding the benzamidine hydrochloride each time;
in the induction process, the temperature is 20-27 ℃ and the pH is 3.5-6.5;
in the induction process, regulating the methanol flow rate to ensure that the methanol concentration is 1-10g/L;
the induction time is 70-90 h;
s5, after fermentation is finished, performing solid-liquid separation in a centrifugal mode, and collecting supernatant for subsequent purification to obtain DNase I.
Preferably, in step S3, an aqueous solution of glycerol having a volume concentration of 30% is fed in the DO-STAT system.
According to the recombinant DNase I fermentation method, peptone is added into a fermentation culture medium, calcium chloride is added in the methanol-induced protein expression process, benzamidine hydrochloride is added in the methanol-induced protein expression process, and the induction time and the induction duration are regulated, so that the method for reducing protein degradation and improving the production level and stability in the pichia pastoris fermentation production process of recombinant DNase I is realized. Experiments prove that the method can effectively reduce protein degradation, effectively improve the production level, ensure that DNase I enzyme activity can reach 8612U/ml level and has good stability; the recombinant DNase I fermentation method can greatly reduce the degradation of DNase I in the process of expressing DNase I by pichia pastoris, remarkably improve the production level, almost can not collect complete target protein from the initial degradation of a large amount of protein, greatly reduce the degradation degree of the protein, greatly improve the production level, shorten the fermentation period and improve the production stability, and is very suitable for the industrialized mass production of the recombinant DNase I expressed by pichia pastoris, thereby having good industrialized practical value.
The fermentation process of recombinant DNase I of the present application is further illustrated in the following specific examples. This section further illustrates the summary of the invention in connection with specific embodiments, but should not be construed as limiting the invention. The technical means employed in the examples are conventional means well known to those skilled in the art, unless specifically stated. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Example 1
Construction of recombinant Pichia pastoris containing DNase I gene
Synthesizing a gene sequence for expressing DNase I, wherein the sequence of the gene sequence is shown as SEQ ID NO. 1, the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2, and inserting a gene fragment of SEQ ID NO. 1 between Eco RI and Not I of a pPICZ alpha A vector to obtain an expression plasmid pPICZ alpha ADNase I; after linearizing the obtained expression plasmid by using endonuclease BglII, respectively converting the linearization fragments into pichia pastoris X33 competent cells in an electrotransformation mode, selecting positive clones, culturing in YPD culture medium, and detecting the enzyme activity of DNase I to obtain an expression strain of recombinant DNase I;
the gene sequence shown in SEQ ID NO. 1 is:
gggtagagct gtagcagctc ccgcttgtgt ttcgctttgt attaatttaa gcagccaaag gagaaaattg gtactgaagaataccagttc ttggcagaat tctttgtgaa aggtttcaggatgaggggca ccaggctgat ggggctgctg ctcgccctcg ctggtctgctgcagctgggc ttgtccctga agatagcagc cttcaacatc cgcacctttg gggagaccaa gatgtccaat gctacgctcg ccagctacattgttcggatc gtgcgtcgtt acgacatcgt cctcatccag gaggtcagag acagccacct ggtggctgtg gggaagctcc tggactatctcaaccaggat gacccaaaca cctaccacta tgtggtcagt gagccgctgg gccgcaacag ctacaaggag cgctacctctttctgttcag acccaacaag gtgtccgtgc tggacaccta ccagtacgac gacggctgcg agtcctgcgg gaacgacagcttcagccggg agcccgctgt ggtcaagttc tcatcccact ccaccaaggt caaggaattt gccattgttg ccctgcactc ggccccatcggacgcagtgg ctgagattaa ttctctctac gatgtctacc tggatgtcca gcagaagtgg cacttgaacg atgtcatgtt gatgggcgatttcaatgctg actgcagcta cgtgacctcc tcgcagtggt catccatccg cctgcgtacg agctccacct tccagtggct gattcctgacagtgccgaca ccacggctac gtccacgaac tgcgcctatg acaggatcgt ggtcgcaggg tctctgctcc agagttctgt ggttcctggctcggccgctc cctttgactt ccaagctgca tacggactga gcaatgagat ggccctggcc atcagtgacc attacccggtggaggtgacg ctgacataag tcccttctgg ggtccagaac ctctgcccca cgctgggagc tgaagacaac aactgacccgtcacagttaa gagcggaggg cggagcttaa ggaacccagt gccgttcgtc cccgtgtgcc ctcaactacc ccacagcagctggaatttga ggagagggat ggaaggtagg cttttcctca cctggcccaa cttccattgt ccgtgtggcg gtcgtgtcac cagagcaccccactttcatc agtctggaaa gaaagtctaa aactaagaaa tgccttttaa tttaaataaa gctcaaaagg gggtgttgtc attcacgcaaaaaaa。
the amino acid sequence shown in SEQ ID NO. 2 is:
MRGTRLMGLLLALAGLLQLGLSLKIAAFNIRTFGETKMSNATLASYIVRIVRRYDIVLIQEVRDSHLVAVGKLLDYLNQDDPNTYHYVVSEPLGRNSYKERYLFLFRPNKVSVLDTYQYDDGCESCGNDSFSREPAVVKFSSHSTKVKEFAIVALHSAPSDAVAEINSLYDVYLDVQQKWHLNDVMLMGDFNADCSYVTSSQWSSIRLRTSSTFQWLIPDSADTTATSTNCAYDRIVVAGSLLQSSVVPGSAAPFDFQAAYGLSNEMALAISDHYPVEVTLT。
Example 2
DNase I enzyme activity detection method
The specific detection method of DNase I enzyme activity is described in patent CN114250208B, and the definition of DNase I enzyme activity is as follows: the amount of enzyme required to increase the absorbance at 260nm per ml of the reaction system by 0.001 in 1 minute at 37℃and pH5.0 was defined as 1 enzyme activity unit (U) using calf thymus DNA as a substrate at a final concentration of 33.33. Mu.g/ml.
Example 3
The embodiment of the application provides a fermentation method of recombinant DNase I, which comprises the following steps:
s1, inoculating the recombinant Pichia pastoris strain containing DNase I gene constructed in the embodiment 1 into YPD medium (comprising 1% yeast extract, 2% peptone and 2% glucose), and culturing at 30deg.C and 200rpm to OD 600 The value reaches 20 to 50, and seed liquid is obtained;
s2, inoculating the seed solution into a fermentation tank filled with a fermentation medium, wherein the inoculum size is 10%; after inoculation, the initial culture conditions were: the ventilation (specifically air) amount is 1.2VVM, the temperature is 30 ℃, the pH is automatically controlled to be 5.0 by ammonia water, and the dissolved oxygen is controlled to be more than or equal to 20% by regulating the rotating speed;
when glycerol in the fermentation culture medium is exhausted and dissolved oxygen rises to more than 60% when the dissolved oxygen rises rapidly, adding 30% glycerol aqueous solution in a DO-STAT mode, and continuously culturing until the fermentation broth OD is obtained 600 Stopping adding the glycerol water solution after the value reaches 120-150, and starving for 30min to obtain fermentation liquor;
wherein the fermentation medium comprises the following concentration components:
32.2g/L of monoammonium phosphate, 3.85g/L of monopotassium phosphate, 10.2g/L of potassium sulfate, 1.73g/L of potassium hydroxide, 14.9g/L of magnesium sulfate heptahydrate, 0.2g/L of anhydrous calcium chloride, 0.1g/L of biotin, 3ml/L of glycerol 40g/L, PTM1 solution, 3.3g/L of peptone and the balance of solvent, wherein the solvent is deionized water;
the PTM1 solution included the following concentration components:
CuSO 4 ·5H 2 O 6g/L、KI 0.088g/L、MnSO 4 ·H 2 O 3.0g/L、Na 2 MoO 4 ·2H 2 O 0.2g/L、H 3 BO 3 0.02g/L、CoCl 2 ·6H 2 O 0.5g/L、ZnCl 2 20.0g/L、FeSO 4 ·7H 2 o65.0 g/L, dense H 2 SO 4 5.0ml/L (concentrated sulfuric acid mass concentration 98%);
s3, adding sterile calcium chloride into the fermentation liquor to enable the concentration of the calcium chloride to be 30mM (based on the volume of the fermentation liquor), then starting to supplement methanol to induce protein expression, and simultaneously adding benzamidine hydrochloride into methanol to enable the concentration of the benzamidine hydrochloride to be 5.22g/L (based on the volume of the methanol); the temperature is 25 ℃ and the pH value is 6.0 in the induction process, the methanol concentration is detected at certain intervals, and the methanol supplementing rate is regulated according to the detection result, so that the methanol concentration is kept in the range of 1.0-10.0 g/L (calculated by the volume of fermentation liquor), and the induction time is 70-90 h;
sampling every certain time in the fermentation process, and detecting protein expression condition and enzyme activity of fermentation liquid by using Western Blot; the results are shown in fig. 1 and 9.
Example 4
The embodiment of the application provides a fermentation method of recombinant DNase I, which comprises the following steps:
s1, inoculating the recombinant Pichia pastoris strain containing DNase I gene constructed in the embodiment 1 into YPD medium (comprising 1% yeast extract, 2% peptone and 2% glucose), and culturing at 30deg.C and 200rpm to OD 600 The value reaches 20 to 50, and seed liquid is obtained;
s2, inoculating the seed solution into a fermentation tank filled with a fermentation medium, wherein the inoculum size is 10%; after inoculation, the initial culture conditions were: the ventilation (specifically air) amount is 1.2VVM, the temperature is 30 ℃, the pH is automatically controlled to be 5.0 by ammonia water, and the dissolved oxygen is controlled to be more than or equal to 20% by regulating the rotating speed;
when glycerol in the fermentation culture medium is exhausted and dissolved oxygen rises to more than 60% when the dissolved oxygen rises rapidly, adding 30% glycerol aqueous solution in a DO-STAT mode, and continuously culturing until the fermentation broth OD is obtained 600 Stopping adding the glycerol water solution after the value reaches 250-300, and starving for 30min to obtain fermentation liquor;
wherein the fermentation medium comprises the following concentration components:
32.2g/L of monoammonium phosphate, 3.85g/L of monopotassium phosphate, 10.2g/L of potassium sulfate, 1.73g/L of potassium hydroxide, 14.9g/L of magnesium sulfate heptahydrate, 0.2g/L of anhydrous calcium chloride, 0.1g/L of biotin, 3ml/L of glycerol 40g/L, PTM1 solution, 3.3g/L of peptone and the balance of solvent, wherein the solvent is deionized water;
The PTM1 solution included the following concentration components:
CuSO 4 ·5H 2 O 6g/L、KI 0.088g/L、MnSO 4 ·H 2 O 3.0g/L、Na 2 MoO 4 ·2H 2 O 0.2g/L、H 3 BO 3 0.02g/L、CoCl 2 ·6H 2 O 0.5g/L、ZnCl 2 20.0g/L、FeSO 4 ·7H 2 o65.0 g/L, dense H 2 SO 4 5.0ml/L (concentrated sulfuric acid mass concentration 98%);
s3, after starvation, starting to supplement methanol to induce protein expression, simultaneously adding benzamidine hydrochloride into methanol to enable the concentration of benzamidine hydrochloride to be 5.22g/L (calculated by the volume of methanol), and simultaneously adding sterile calcium chloride into fermentation broth to enable the concentration of benzamidine to be 30mM (calculated by the volume of fermentation broth) when induction is started; the temperature is 25 ℃ and the pH value is 6.0 in the induction process, the methanol concentration is detected at certain intervals, and the methanol supplementing rate is regulated according to the detection result, so that the methanol concentration is kept in the range of 1.0-10.0 g/L (calculated by the volume of fermentation liquor), and the induction time is 70-90 h;
sampling every certain time in the fermentation process, detecting protein expression condition by using Western Blot and detecting enzyme activity of fermentation liquor. The results are shown in fig. 2 and 9.
Example 5
The embodiment of the application provides a fermentation method of recombinant DNase I, which comprises the following steps:
s1, inoculating the recombinant Pichia pastoris strain containing DNase I gene constructed in the embodiment 1 into YPD medium (comprising 1% yeast extract, 2% peptone and 2% glucose), and culturing at 30deg.C and 200rpm to OD 600 The value reaches 20 to 50, and seed liquid is obtained;
S2, inoculating the seed solution into a fermentation tank filled with a fermentation medium, wherein the inoculum size is 10%; after inoculation, the initial culture conditions were: the ventilation (specifically air) amount is 1.2VVM, the temperature is 30 ℃, the pH is automatically controlled to be 5.0 by ammonia water, and the dissolved oxygen is controlled to be more than or equal to 20% by regulating the rotating speed;
when glycerol in the fermentation culture medium is exhausted and dissolved oxygen rises to more than 60% when the dissolved oxygen rises rapidly, adding 30% glycerol aqueous solution in a DO-STAT mode, and continuously culturing until the fermentation broth OD is obtained 600 Stopping adding the glycerol water solution after the value reaches 400-450, and starving for 30min to obtain fermentation liquor;
wherein the fermentation medium comprises the following concentration components:
32.2g/L of monoammonium phosphate, 3.85g/L of monopotassium phosphate, 10.2g/L of potassium sulfate, 1.73g/L of potassium hydroxide, 14.9g/L of magnesium sulfate heptahydrate, 0.2g/L of anhydrous calcium chloride, 0.1g/L of biotin, 3ml/L of glycerol 40g/L, PTM1 solution, 3.3g/L of peptone and the balance of solvent, wherein the solvent is deionized water;
the PTM1 solution included the following concentration components:
CuSO 4 ·5H 2 O 6g/L、KI 0.088g/L、MnSO 4 ·H 2 O 3.0g/L、Na 2 MoO 4 ·2H 2 O 0.2g/L、H 3 BO 3 0.02g/L、CoCl 2 ·6H 2 O 0.5g/L、ZnCl 2 20.0g/L、FeSO 4 ·7H 2 o65.0 g/L, dense H 2 SO 4 5.0ml/L (concentrated sulfuric acid mass concentration 98%);
s3, adding methanol to the fermentation broth to induce protein expression, and simultaneously adding sterile calcium chloride to the fermentation broth to ensure that the calcium chloride concentration is 30mM (calculated by volume of the fermentation broth), and adding benzamidine hydrochloride (0.78 g/L of benzamidine hydrochloride is added each time according to volume of the fermentation broth) to the fermentation broth at intervals of 24 hours from the beginning of induction; the temperature is 25 ℃ and the pH value is 6.0 in the induction process, the methanol concentration is detected at certain intervals, and the methanol supplementing rate is regulated according to the detection result, so that the methanol concentration is kept in the range of 1.0-10.0 g/L (calculated by the volume of fermentation liquor), and the induction time is 70-90 h;
Sampling every certain time in the fermentation process, and detecting protein expression condition and enzyme activity of fermentation liquid by using Western Blot; the results are shown in fig. 3 and 9.
Example 6
The embodiment of the application provides a fermentation method of recombinant DNase I, which comprises the following steps:
s1, inoculating the recombinant Pichia pastoris strain containing DNase I gene constructed in the embodiment 1 into YPD medium (comprising 1% yeast extract, 2% peptone and 2% glucose), and culturing at 30deg.C and 200rpm to OD 600 The value reaches 20 to 50, and seed liquid is obtained;
s2, inoculating the seed solution into a fermentation tank filled with a fermentation medium, wherein the inoculum size is 10%; after inoculation, the initial culture conditions were: the ventilation (specifically air) amount is 1.2VVM, the temperature is 30 ℃, the pH is automatically controlled to be 5.0 by ammonia water, and the dissolved oxygen is controlled to be more than or equal to 20% by regulating the rotating speed;
when glycerol in the fermentation culture medium is exhausted and dissolved oxygen rises to more than 60% when the dissolved oxygen rises rapidly, adding 30% glycerol aqueous solution in a DO-STAT mode, and continuously culturing until the fermentation broth OD is obtained 600 Stopping adding the glycerol water solution after the value reaches 500-550, and starving for 30min to obtain fermentation liquor;
wherein the fermentation medium comprises the following concentration components:
32.2g/L of monoammonium phosphate, 3.85g/L of monopotassium phosphate, 10.2g/L of potassium sulfate, 1.73g/L of potassium hydroxide, 14.9g/L of magnesium sulfate heptahydrate, 0.2g/L of anhydrous calcium chloride, 0.1g/L of biotin, 3ml/L of glycerol 40g/L, PTM1 solution, 3.3g/L of peptone and the balance of solvent, wherein the solvent is deionized water;
the PTM1 solution included the following concentration components:
CuSO 4 ·5H 2 O 6g/L、KI 0.088g/L、MnSO 4 ·H 2 O 3.0g/L、Na 2 MoO 4 ·2H 2 O 0.2g/L、H 3 BO 3 0.02g/L、CoCl 2 ·6H 2 O 0.5g/L、ZnCl 2 20.0g/L、FeSO 4 ·7H 2 o65.0 g/L, dense H 2 SO 4 5.0ml/L (concentrated sulfuric acid mass concentration 98%);
s3, adding methanol to the fermentation broth to induce protein expression, and simultaneously adding sterile calcium chloride to the fermentation broth to ensure that the calcium chloride concentration is 30mM (calculated by volume of the fermentation broth), and adding benzamidine hydrochloride (0.78 g/L of benzamidine hydrochloride is added each time according to volume of the fermentation broth) to the fermentation broth at intervals of 24 hours from the beginning of induction; the temperature is 25 ℃ and the pH value is 6.0 in the induction process, the methanol concentration is detected at certain intervals, and the methanol supplementing rate is regulated according to the detection result, so that the methanol concentration is kept in the range of 1.0-10.0 g/L (calculated by the volume of fermentation liquor), and the induction time is 70-90 h;
sampling every certain time in the fermentation process, and detecting protein expression condition and enzyme activity of fermentation liquid by using Western Blot; the results are shown in fig. 4 and 9.
Example 7
The embodiment of the application provides a fermentation method of recombinant DNase I, which comprises the following steps:
S1, inoculating the recombinant Pichia pastoris strain containing DNase I gene constructed in the embodiment 1 into YPD medium (comprising 1% yeast extract, 2% peptone and 2% glucose), and culturing at 30deg.C and 200rpm to OD 600 The value reaches 20 to 50, and seed liquid is obtained;
s2, inoculating the seed solution into a fermentation tank filled with a fermentation medium, wherein the inoculum size is 10%; after inoculation, the initial culture conditions were: the ventilation (specifically air) amount is 1.2VVM, the temperature is 30 ℃, the pH is automatically controlled to be 5.0 by ammonia water, and the dissolved oxygen is controlled to be more than or equal to 20% by regulating the rotating speed;
when the glycerol in the fermentation culture medium is exhausted and the dissolved oxygen rises rapidly, the dissolved oxygen rises toAdding 30% glycerol aqueous solution by DO-STAT method, and continuously culturing until fermentation broth OD 600 Stopping adding the glycerol water solution after the value reaches 120-150, and starving for 30min to obtain fermentation liquor;
wherein the fermentation medium comprises the following concentration components:
32.2g/L of monoammonium phosphate, 3.85g/L of monopotassium phosphate, 10.2g/L of potassium sulfate, 1.73g/L of potassium hydroxide, 14.9g/L of magnesium sulfate heptahydrate, 0.2g/L of anhydrous calcium chloride, 0.1g/L of biotin, 3ml/L of glycerol 40g/L, PTM1 solution, 3.3g/L of peptone and the balance of solvent, wherein the solvent is deionized water;
The PTM1 solution included the following concentration components:
CuSO 4 ·5H 2 O 6g/L、KI 0.088g/L、MnSO 4 ·H 2 O 3.0g/L、Na 2 MoO 4 ·2H 2 O 0.2g/L、H 3 BO 3 0.02g/L、CoCl 2 ·6H 2 O 0.5g/L、ZnCl 2 20.0g/L、FeSO 4 ·7H 2 o65.0 g/L, dense H 2 SO 4 5.0ml/L (concentrated sulfuric acid mass concentration 98%);
s3, adding sterile calcium chloride into the fermentation broth to ensure that the concentration of the calcium chloride is 30mM (calculated by the volume of the fermentation broth), and then starting to supplement methanol to induce protein expression; the temperature is 25 ℃ and the pH value is 6.0 in the induction process, the methanol concentration is detected at certain intervals, and the methanol supplementing rate is regulated according to the detection result, so that the methanol concentration is kept in the range of 1.0-10.0 g/L (calculated by the volume of fermentation liquor), and the induction time is 120-144 hours;
sampling every certain time in the fermentation process, and detecting protein expression condition and enzyme activity of fermentation liquid by using Western Blot; the results are shown in fig. 5 and 9.
Example 8
Separation and purification of DNase I
The recombinant DNase I was fermented according to the method of example 5, and after the fermentation was completed, solid-liquid separation was performed by centrifugation, and the supernatant was collected and subjected to subsequent purification to obtain DNase I, and the method for separating and purifying DNase I was specifically described in patent document CN 115433726A, and the obtained recombinant DNase I was shown in fig. 10.
Comparative example 1
The comparative example provides a fermentation method of recombinant DNase I, comprising the following steps:
S1, inoculating the recombinant Pichia pastoris strain containing DNase I gene constructed in the embodiment 1 into YPD medium (comprising 1% yeast extract, 2% peptone and 2% glucose), and culturing at 30deg.C and 200rpm to OD 600 The value reaches 20 to 50, and seed liquid is obtained;
s2, inoculating the seed solution into a fermentation tank filled with a fermentation medium, wherein the inoculum size is 10%; after inoculation, the initial culture conditions were: the ventilation (specifically air) amount is 1.2VVM, the temperature is 30 ℃, the pH is automatically controlled to be 5.0 by ammonia water, and the dissolved oxygen is controlled to be more than or equal to 20% by regulating the rotating speed;
when glycerol in the fermentation culture medium is exhausted and dissolved oxygen rises to more than 60% when the dissolved oxygen rises rapidly, adding 30% glycerol aqueous solution in a DO-STAT mode, and continuously culturing until the fermentation broth OD is obtained 600 Stopping adding the glycerol water solution after the value reaches 120-150, and starving for 30min to obtain fermentation liquor;
wherein the fermentation medium comprises the following concentration components:
32.2g/L of monoammonium phosphate, 3.85g/L of monopotassium phosphate, 10.2g/L of potassium sulfate, 1.73g/L of potassium hydroxide, 14.9g/L of magnesium sulfate heptahydrate, 0.2g/L of anhydrous calcium chloride, 0.1g/L of biotin, 3ml/L of glycerol 40g/L, PTM1 solution, and the balance of solvent, wherein the solvent is deionized water;
The PTM1 solution included the following concentration components:
CuSO 4 ·5H 2 O 6g/L、KI 0.088g/L、MnSO 4 ·H 2 O 3.0g/L、Na 2 MoO 4 ·2H 2 O 0.2g/L、H 3 BO 3 0.02g/L、CoCl 2 ·6H 2 O 0.5g/L、ZnCl 2 20.0g/L、FeSO 4 ·7H 2 o65.0 g/L, dense H 2 SO 4 5.0ml/L (concentrated sulfuric acid mass concentration 98%);
s3, adding methanol into the fermentation broth to induce protein expression; the temperature is 25 ℃ and the pH value is 6.0 in the induction process, the methanol concentration is detected at certain intervals, and the methanol supplementing rate is regulated according to the detection result, so that the methanol concentration is kept in the range of 1.0-10.0 g/L (calculated by the volume of fermentation liquor), and the induction time is 120-144 hours;
sampling every certain time in the fermentation process, and detecting protein expression condition and enzyme activity of fermentation liquid by using Western Blot; the results are shown in fig. 6 and 9.
Comparative example 2
The comparative example provides a fermentation method of recombinant DNase I, comprising the following steps:
s1, inoculating the recombinant Pichia pastoris strain containing DNase I gene constructed in the embodiment 1 into YPD medium (comprising 1% yeast extract, 2% peptone and 2% glucose), and culturing at 30deg.C and 200rpm to OD 600 The value reaches 20 to 50, and seed liquid is obtained;
s2, inoculating the seed solution into a fermentation tank filled with a fermentation medium, wherein the inoculum size is 10%; after inoculation, the initial culture conditions were: the ventilation (specifically air) amount is 1.2VVM, the temperature is 30 ℃, the pH is automatically controlled to be 5.0 by ammonia water, and the dissolved oxygen is controlled to be more than or equal to 20% by regulating the rotating speed;
When glycerol in the fermentation culture medium is exhausted and dissolved oxygen rises to more than 60% when the dissolved oxygen rises rapidly, adding 30% glycerol aqueous solution in a DO-STAT mode, and continuously culturing until the fermentation broth OD is obtained 600 Stopping adding the glycerol water solution after the value reaches 120-150, and starving for 30min to obtain fermentation liquor;
wherein the fermentation medium comprises the following concentration components:
32.2g/L of monoammonium phosphate, 3.85g/L of monopotassium phosphate, 10.2g/L of potassium sulfate, 1.73g/L of potassium hydroxide, 14.9g/L of magnesium sulfate heptahydrate, 0.2g/L of anhydrous calcium chloride, 0.1g/L of biotin, 3ml/L of glycerol 40g/L, PTM1 solution, 3.3g/L of peptone and the balance of solvent, wherein the solvent is deionized water;
the PTM1 solution included the following concentration components:
CuSO 4 ·5H 2 O 6g/L、KI 0.088g/L、MnSO 4 ·H 2 O 3.0g/L、Na 2 MoO 4 ·2H 2 O 0.2g/L、H 3 BO 3 0.02g/L、CoCl 2 ·6H 2 O 0.5g/L、ZnCl 2 20.0g/L、FeSO 4 ·7H 2 o65.0 g/L, dense H 2 SO 4 5.0ml/L (concentrated sulfuric acid mass concentration 98%);
s3, adding methanol into the fermentation broth to induce protein expression; the temperature is 25 ℃ and the pH value is 6.0 in the induction process, the methanol concentration is detected at certain intervals, and the methanol supplementing rate is regulated according to the detection result, so that the methanol concentration is kept in the range of 1.0-10.0 g/L (calculated by the volume of fermentation liquor), and the induction time is 120-144 hours;
sampling every certain time in the fermentation process, and detecting protein expression condition and enzyme activity of fermentation liquid by using Western Blot; the results are shown in fig. 7 and 9.
Comparative example 3
The comparative example provides a fermentation method of recombinant DNase I, comprising the following steps:
s1, inoculating the recombinant Pichia pastoris strain containing DNase I gene constructed in the embodiment 1 into YPD medium (comprising 1% yeast extract, 2% peptone and 2% glucose), and culturing at 30deg.C and 200rpm to OD 600 The value reaches 20 to 50, and seed liquid is obtained;
s2, inoculating the seed solution into a fermentation tank filled with a fermentation medium, wherein the inoculum size is 10%; after inoculation, the initial culture conditions were: the ventilation (specifically air) amount is 1.2VVM, the temperature is 30 ℃, the pH is automatically controlled to be 5.0 by ammonia water, and the dissolved oxygen is controlled to be more than or equal to 20% by regulating the rotating speed;
when glycerol in the fermentation culture medium is exhausted and dissolved oxygen rises to more than 60% when the dissolved oxygen rises rapidly, adding 30% glycerol aqueous solution in a DO-STAT mode, and continuously culturing until the fermentation broth OD is obtained 600 Stopping adding the glycerol water solution after the value reaches 120-150, and starving for 30min to obtain fermentation liquor;
wherein the fermentation medium comprises the following concentration components:
32.2g/L of monoammonium phosphate, 3.85g/L of monopotassium phosphate, 10.2g/L of potassium sulfate, 1.73g/L of potassium hydroxide, 14.9g/L of magnesium sulfate heptahydrate, 0.2g/L of anhydrous calcium chloride, 0.1g/L of biotin, 3ml/L of glycerol 40g/L, PTM1 solution, 3.3g/L of peptone and the balance of solvent, wherein the solvent is deionized water;
The PTM1 solution included the following concentration components:
CuSO 4 ·5H 2 O 6g/L、KI 0.088g/L、MnSO 4 ·H 2 O 3.0g/L、Na 2 MoO 4 ·2H 2 O 0.2g/L、H 3 BO 3 0.02g/L、CoCl 2 ·6H 2 O 0.5g/L、ZnCl 2 20.0g/L、FeSO 4 ·7H 2 o65.0 g/L, dense H 2 SO 4 5.0ml/L (concentrated sulfuric acid mass concentration 98%);
s3, adding sterile calcium chloride into the fermentation broth to ensure that the concentration of the calcium chloride is 30mM (calculated by the volume of the fermentation broth), then starting to supplement methanol to induce protein expression, wherein the temperature is 25 ℃ in the induction process, the pH is 6.0, and simultaneously, 0.2mM (calculated by the volume of the fermentation broth) of phenylmethylsulfonyl fluoride (PMSF) is added into the fermentation broth respectively at the beginning of induction, at the beginning of induction for 23h, at the beginning of induction for 47h and at the beginning of induction for 53h, detecting the concentration of the methanol at certain intervals, and adjusting the methanol supplementing rate according to the detection result to ensure that the concentration of the methanol is kept within the range of 1.0-10.0 g/L (calculated by the volume of the fermentation broth) for 70-90 h;
sampling every certain time in the fermentation process, and detecting protein expression condition and enzyme activity of fermentation liquid by using Western Blot; the results are shown in fig. 8 and 9.
Results analysis of fermentation methods in examples 3 to 7 and comparative examples 1 to 3
FIG. 1 is a Western Blot detection plot during induction of the fermentation process of example 3; in FIG. 1, 1 represents a molecular weight Marker,2 represents a molecular weight Marker before induction, and 3 to 9 represent induction for 14h, 24h, 36h, 49h, 58h, 72h and 8h, respectively.
FIG. 2 is a Western Blot detection plot during induction of the fermentation process of example 4; in FIG. 2, 1 represents a molecular weight Marker,2 represents a molecular weight Marker before induction, and 3 to 8 represent induction for 14h, 24h, 36h, 47h, 58h and 73h, respectively.
FIG. 3 is a Western Blot detection plot during induction of the fermentation process of example 5; in FIG. 3, 1 shows the induction times before induction, 2 to 8 show induction times of 14h, 24h, 36h, 47h, 58h, 73h and 90h, and 9 shows the molecular weight Marker.
FIG. 4 is a Western Blot detection plot during induction of the fermentation process of example 6; in FIG. 4, 1 represents a molecular weight Marker,2 represents a molecular weight Marker before induction, and 3 to 8 represent induction for 13h, 24h, 34h, 47h, 55h, and 71h, respectively.
FIG. 5 is a Western Blot analysis of the fermentation process of example 7 during induction; in FIG. 5, 1 represents a molecular weight Marker,2 represents a molecular weight Marker before induction, and 3 to 11 represent induction for 16h, 24h, 37h, 48h, 56h, 75h, 89h, 115h, 135h, respectively.
FIG. 6 is a Western Blot analysis of the fermentation process of comparative example 1 during induction; in FIG. 6, 1 shows that before induction, 2 to 10 show induction for 15h, 24h, 36h, 48h, 59h, 71h, 90h, 115h, 138h,11 show molecular weight markers, respectively.
FIG. 7 is a Western Blot analysis of the fermentation process of comparative example 2, during induction; in FIG. 7, 1 shows that before induction, 2 to 10 show induction for 16h, 24h, 36h, 48h, 58h, 72h, 91h, 115h, 136h, and 11 shows molecular weight markers, respectively.
FIG. 8 is a Western Blot analysis of the fermentation process of comparative example 3 during induction; in FIG. 8, 1 represents the induction time, 2 to 5 represent the induction time 18h, 23h, 47h and 72h, respectively, and 6 represents the molecular weight Marker.
FIG. 9 shows the enzymatic activity curves of fermentation supernatants during induction (i.e., at different induction times) for the fermentation methods of examples 3-7 and comparative examples 1-3.
FIG. 10 is an SDS-PAGE diagram of the fermentation broth obtained by the fermentation method in example 5, and purified DNase I protein product.
Example 3 differs from the fermentation method in example 7 in that in example 3, a certain amount of benzamidine hydrochloride is added along with methanol flow in the induction process, and the induction time is 70-90 h. The results of FIGS. 1 and 9 show that the degradation degree of the protein is greatly improved in the DNase I induced expression process, the degradation band is obviously reduced, and a great amount of DNase I protein is obviously contained in the fermentation liquor at the end of fermentation; as the degradation is reduced, the enzyme activity is greatly improved to 6989U/ml, and the enzyme activity is also improved by 63% compared with the highest enzyme activity in the fermentation process in the example 7.
The results of FIGS. 2 and 9 show that the fermentation method in example 4 has significantly improved degradation degree and little protein band degradation during DNase I induced expression, and a significant amount of DNase I protein is present in the fermentation broth at the end of fermentation; the enzyme activity can reach 8021U/ml.
The results of FIGS. 3 and 9 show that the fermentation method in example 5 has significantly improved degradation degree and little protein band degradation during DNase I induced expression, and a significant amount of DNase I protein is present in the fermentation broth at the end of fermentation; the enzyme activity can reach 8612U/ml.
FIGS. 4 and 9 show that the fermentation method in example 6 has significantly improved degradation degree and little protein band degradation during DNase I induced expression, and a significant amount of DNase I protein is present in the fermentation broth at the end of fermentation; the enzyme activity can reach 6317U/ml.
Example 7 differs from the fermentation process of comparative example 2 in that, on the basis of comparative example 2, in example 7, at the start of induction, a certain amount of calcium chloride is added; the results in fig. 5 and 9 show that the degradation of DNase I is further improved, the protein band is obviously reduced, the activity is greatly improved, the degradation is still carried out to a certain extent, and after the highest 4289U/ml is reached at the time of 75h induction, the protein is greatly degraded, which indicates that the degradation of DNase I by calcium chloride is reduced to a certain extent.
The results of FIGS. 6 and 9 show that the fermentation method in comparative example 1 has low DNase I expression level during the induction of DNase I expression, and the protein is degraded in a large amount after the induction for 36-48 hours, the target protein is not detected by Western Blot, and the enzyme activity is reduced to nearly 0U/ml.
Comparative example 2 differs from the fermentation process in comparative example 1 in that a certain amount of peptone was added to comparative example 2; from the results of FIGS. 7 and 9, it was found that the protein expression amount was relatively higher and the enzyme activity was also higher in comparative example 2, indicating that addition of peptone could improve DNase I to some extent, but still there was a larger degree of degradation.
Comparative example 3 differs from the fermentation process in example 7 in that, on the basis of example 7, comparative example 3 is fed in portions during induction with a certain amount of the protease inhibitor methylsulfonyl fluoride (PMSF); the results in FIGS. 8 and 9 show that, despite the addition of the protease inhibitor PMSF, there was no further improvement in DNase I degradation, and Western Blot detected that there was still a large amount of degraded protein bands, and DNase I activity reached only 2915U/ml at the end of fermentation, indicating that PMSF addition did not significantly reduce DNase I degradation.
As can be seen from FIG. 10, the fermentation broth obtained by the fermentation method in example 5 can be separated and purified to obtain pure DNase I product, and the purity is more than 95% by SDS-PAGE detection, so that the market demand can be satisfied.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. A fermentation method of recombinant DNase I, comprising the steps of:
amplifying recombinant Pichia pastoris containing DNase I gene by a seed culture medium, inoculating to a fermentation culture medium for fermentation culture, adding glycerol when the glycerol in the fermentation culture medium is exhausted and dissolved oxygen rises, and continuously culturing until the fermentation liquid OD is reached 600 Stopping adding glycerol after the value reaches 120-550, starving, exhausting the glycerol in the fermentation liquid, and adding methanol to induce protein expression;
wherein, water-soluble calcium salt is added in the expression process of methanol induced protein;
the fermentation medium contains peptone.
2. The fermentation process of recombinant DNase I according to claim 1, wherein the water-soluble calcium salt comprises at least one of calcium chloride, calcium gluconate, calcium biphosphate, calcium nitrate, calcium bicarbonate, calcium bisulfate, calcium bisulfide, calcium hypochlorite, calcium bromide, calcium iodide, calcium chlorate, calcium perchlorate, calcium permanganate.
3. The method for fermenting recombinant DNase I according to claim 1, wherein benzamidine hydrochloride is added during methanol-induced protein expression.
4. The method of fermentation of recombinant DNase I according to claim 1, wherein the water-soluble calcium salt is added during methanol-induced protein expression such that the concentration of the water-soluble calcium salt is between 10 and 50mM.
5. The method for fermenting recombinant DNase I according to claim 3, wherein the adding of benzamidine hydrochloride during the methanol-induced protein expression process is as follows: adding 2.61-6.22g/L benzamidine hydrochloride into methanol, or independently feeding the same mass flow rate of benzamidine hydrochloride into the methanol, or feeding 0.5-2.34g/L benzamidine hydrochloride into the fermentation liquor in batches for multiple times.
6. The method of fermentation of recombinant DNase I according to any one of claims 1 to 5, wherein the fermentation medium further comprises monoammonium phosphate, monopotassium phosphate, potassium sulfate, potassium hydroxide, magnesium sulfate, calcium chloride, biotin, glycerol, PTM1 solution, and a solvent.
7. The method for fermenting recombinant DNase I according to claim 6, wherein the concentration of peptone in the fermentation medium is 2-10 g/L, the concentration of monoammonium phosphate is 5-40 g/L, the concentration of monopotassium phosphate is 2-10 g/L, the concentration of potassium sulfate is 3.5-26.1 g/L, the concentration of potassium hydroxide is 1.5-2 g/L, the concentration of magnesium sulfate is 10.5-18.5 g/L, the concentration of calcium chloride is 0.2-0.5 g/L, the concentration of biotin is 0.02-0.3 g/L, the concentration of glycerol is 10-50 g/L, PTM and the concentration of solution is 2-5 ml/L;
the solvent is water;
the PTM1 solution comprises the following components in concentration: cuSO 4 5～7g/L、KI 0.08～0.09g/L、MnSO 4 2～4g/L、Na 2 MoO 4 0.1～0.3g/L、H 3 BO 3 0.01～0.03g/L、CoCl 2 0.4～0.6g/L、ZnCl 2 19～21g/L、FeSO 4 63-67 g/L, dense H 2 SO 4 4-6 ml/L; the concentration H 2 SO 4 The mass fraction is 98-99%.
8. The method for fermenting recombinant DNase I according to claim 1, wherein the fermenting and culturing recombinant pichia pastoris containing DNase I gene in a seed culture medium comprises:
Inoculating recombinant Pichia pastoris containing DNase I gene into seed culture medium, and culturing to OD 600 The value is 20-50, and seed liquid is obtained;
inoculating the seed solution into a fermentation medium for fermentation culture;
wherein the seed culture medium comprises at least one of YPD culture medium, PDA culture medium and BMGY culture medium;
inoculating recombinant pichia pastoris containing DNase I gene into a seed culture medium, wherein the culture temperature is 28-30 ℃;
inoculating the seed solution into a fermentation culture medium for fermentation culture, wherein the inoculation amount is 5-10%.
9. The method for fermenting recombinant DNase I according to claim 8, wherein the seed solution is inoculated into a fermentation medium for fermentation culture, pH is controlled to be 4.9-5.1 by ammonia water, 1.0-1.5 VVM oxygen-containing gas is introduced, the rotating speed is adjusted to control the dissolved oxygen to be more than or equal to 20%, glycerol is used up in the fermentation medium, the dissolved oxygen is increased to be more than or equal to 60%, glycerol is added, and the culture is continued until the OD of the fermentation liquid is reached 600 Stopping adding glycerol after the value reaches 120-550, and starving for 20-40 min; wherein the fermentation culture temperature is 28-30 ℃.
10. The method of fermenting recombinant DNase I according to any one of claims 1 to 5, wherein in the step of adding methanol to induce protein expression, the methanol flow rate is adjusted so that the methanol concentration is 1 to 10g/L, the induction time is 70 to 90 hours, the temperature is 20 to 27 ℃ and the pH is 3.5 to 6.5.
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