CN116926142A - Production method of beta-alanine - Google Patents
Production method of beta-alanine Download PDFInfo
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- CN116926142A CN116926142A CN202311096339.1A CN202311096339A CN116926142A CN 116926142 A CN116926142 A CN 116926142A CN 202311096339 A CN202311096339 A CN 202311096339A CN 116926142 A CN116926142 A CN 116926142A
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- maleic acid
- alanine
- escherichia coli
- recombinant
- fermentation
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- UCMIRNVEIXFBKS-UHFFFAOYSA-N beta-alanine Chemical compound NCCC(O)=O UCMIRNVEIXFBKS-UHFFFAOYSA-N 0.000 title claims abstract description 148
- 229940000635 beta-alanine Drugs 0.000 title claims abstract description 72
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 49
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 claims abstract description 143
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 claims abstract description 117
- 239000011976 maleic acid Substances 0.000 claims abstract description 117
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 claims abstract description 116
- 238000000855 fermentation Methods 0.000 claims abstract description 65
- 230000004151 fermentation Effects 0.000 claims abstract description 65
- 238000000034 method Methods 0.000 claims abstract description 25
- 241000894006 Bacteria Species 0.000 claims abstract description 12
- 241000588724 Escherichia coli Species 0.000 claims description 46
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 39
- 239000011259 mixed solution Substances 0.000 claims description 32
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 27
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 27
- 108700016171 Aspartate ammonia-lyases Proteins 0.000 claims description 17
- 239000001963 growth medium Substances 0.000 claims description 16
- 108090000175 Cis-trans-isomerases Proteins 0.000 claims description 15
- 102000003813 Cis-trans-isomerases Human genes 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 12
- 230000006698 induction Effects 0.000 claims description 11
- 239000013612 plasmid Substances 0.000 claims description 9
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims description 9
- 239000001888 Peptone Substances 0.000 claims description 8
- 108010080698 Peptones Proteins 0.000 claims description 8
- 235000019319 peptone Nutrition 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 150000001413 amino acids Chemical group 0.000 claims description 7
- 239000002609 medium Substances 0.000 claims description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 5
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 4
- 230000001939 inductive effect Effects 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 241000607715 Serratia marcescens Species 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 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 claims description 2
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 claims description 2
- 238000004659 sterilization and disinfection Methods 0.000 claims description 2
- 239000013604 expression vector Substances 0.000 claims 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims 1
- 235000019797 dipotassium phosphate Nutrition 0.000 claims 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims 1
- 238000003259 recombinant expression Methods 0.000 claims 1
- 239000000758 substrate Substances 0.000 abstract description 56
- 102000004190 Enzymes Human genes 0.000 abstract description 14
- 108090000790 Enzymes Proteins 0.000 abstract description 14
- 230000002779 inactivation Effects 0.000 abstract description 5
- 238000009776 industrial production Methods 0.000 abstract description 3
- 230000010307 cell transformation Effects 0.000 abstract description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 52
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 30
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 29
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 29
- 229960005261 aspartic acid Drugs 0.000 description 29
- 239000001530 fumaric acid Substances 0.000 description 26
- 230000000052 comparative effect Effects 0.000 description 24
- 238000004128 high performance liquid chromatography Methods 0.000 description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 235000010633 broth Nutrition 0.000 description 9
- 230000000694 effects Effects 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 4
- 229960000723 ampicillin Drugs 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 4
- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical class O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 3
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 241001052560 Thallis Species 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 239000013067 intermediate product Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- WEEMDRWIKYCTQM-UHFFFAOYSA-N 2,6-dimethoxybenzenecarbothioamide Chemical compound COC1=CC=CC(OC)=C1C(N)=S WEEMDRWIKYCTQM-UHFFFAOYSA-N 0.000 description 2
- QRYRORQUOLYVBU-VBKZILBWSA-N Carnosic acid Natural products CC([C@@H]1CC2)(C)CCC[C@]1(C(O)=O)C1=C2C=C(C(C)C)C(O)=C1O QRYRORQUOLYVBU-VBKZILBWSA-N 0.000 description 2
- 108010087806 Carnosine Proteins 0.000 description 2
- 101100242684 Mesorhizobium japonicum (strain LMG 29417 / CECT 9101 / MAFF 303099) panD1 gene Proteins 0.000 description 2
- CQOVPNPJLQNMDC-UHFFFAOYSA-N N-beta-alanyl-L-histidine Natural products NCCC(=O)NC(C(O)=O)CC1=CN=CN1 CQOVPNPJLQNMDC-UHFFFAOYSA-N 0.000 description 2
- 241000254113 Tribolium castaneum Species 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- AGSPXMVUFBBBMO-UHFFFAOYSA-N beta-aminopropionitrile Chemical compound NCCC#N AGSPXMVUFBBBMO-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 229940044199 carnosine Drugs 0.000 description 2
- CQOVPNPJLQNMDC-ZETCQYMHSA-N carnosine Chemical compound [NH3+]CCC(=O)N[C@H](C([O-])=O)CC1=CNC=N1 CQOVPNPJLQNMDC-ZETCQYMHSA-N 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- DMBHHRLKUKUOEG-UHFFFAOYSA-N diphenylamine Chemical compound C=1C=CC=CC=1NC1=CC=CC=C1 DMBHHRLKUKUOEG-UHFFFAOYSA-N 0.000 description 2
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 2
- 229960002598 fumaric acid Drugs 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 229940098895 maleic acid Drugs 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- FWFGVMYFCODZRD-UHFFFAOYSA-N oxidanium;hydrogen sulfate Chemical compound O.OS(O)(=O)=O FWFGVMYFCODZRD-UHFFFAOYSA-N 0.000 description 2
- 101150076071 panD gene Proteins 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 229960002385 streptomycin sulfate Drugs 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- NLHHRLWOUZZQLW-UHFFFAOYSA-N Acrylonitrile Chemical compound C=CC#N NLHHRLWOUZZQLW-UHFFFAOYSA-N 0.000 description 1
- 241000588813 Alcaligenes faecalis Species 0.000 description 1
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 1
- RGJOEKWQDUBAIZ-IBOSZNHHSA-N CoASH Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCS)O[C@H]1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-IBOSZNHHSA-N 0.000 description 1
- 241000186226 Corynebacterium glutamicum Species 0.000 description 1
- 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 1
- 102000004195 Isomerases Human genes 0.000 description 1
- 108090000769 Isomerases Proteins 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 description 1
- 229930003571 Vitamin B5 Natural products 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000005904 alkaline hydrolysis reaction Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 description 1
- 229960002079 calcium pantothenate Drugs 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- RGJOEKWQDUBAIZ-UHFFFAOYSA-N coenzime A Natural products OC1C(OP(O)(O)=O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-UHFFFAOYSA-N 0.000 description 1
- 239000005516 coenzyme A Substances 0.000 description 1
- 229940093530 coenzyme a Drugs 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000006114 decarboxylation reaction Methods 0.000 description 1
- KDTSHFARGAKYJN-UHFFFAOYSA-N dephosphocoenzyme A Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 KDTSHFARGAKYJN-UHFFFAOYSA-N 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000002085 irritant Substances 0.000 description 1
- 231100000021 irritant Toxicity 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 235000011121 sodium hydroxide Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 235000009492 vitamin B5 Nutrition 0.000 description 1
- 239000011675 vitamin B5 Substances 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
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- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
- C12Y401/01011—Aspartate 1-decarboxylase (4.1.1.11)
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- C12Y403/00—Carbon-nitrogen lyases (4.3)
- C12Y403/01—Ammonia-lyases (4.3.1)
- C12Y403/01001—Aspartate ammonia-lyase (4.3.1.1), i.e. aspartase
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- C12Y502/00—Cis-trans-isomerases (5.2)
- C12Y502/01—Cis-trans-Isomerases (5.2.1)
- C12Y502/01001—Maleate isomerase (5.2.1.1)
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
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Abstract
The invention discloses a method for producing beta-alanine. The method takes maleic acid as a substrate, and the constructed new engineering bacteria are used for whole cell transformation to generate beta-alanine, and the factors such as the maleic acid flow rate, the fermentation temperature and the like are controlled, so that the substrate utilization rate is improved, the related enzyme inactivation is reduced, the maleic acid utilization rate reaches more than 99%, and the final yield of the beta-alanine reaches more than 250 g/L. The production method can reduce the production cost, can be applied to large-scale industrial production, and meets the market demand.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a production method of beta-alanine.
Background
Beta-alanine, also known as 3-aminopropionic acid, of formula C 3 H 7 NO 2 It is one of unnatural amino acids. Beta-alanine is a precursor substance for vitamin B5 synthesis and is an important constituent of coenzyme A. It is studied that beta-alanine can increase the concentration of carnosine in human muscle, while carnosine can scavenge active oxygen as an effective buffer againstFatigue toxins are produced in the stop muscle. Therefore, the beta-alanine is paid attention to food health care, biological medicine, feed and the like, and the market demand of the beta-alanine is greatly increased.
Currently, the preparation method of beta-alanine mainly comprises a chemical synthesis method, a biological fermentation method and a biological enzyme synthesis method.
The chemical synthesis process of beta-alanine is mainly that acrylonitrile reacts with ammonia in diphenylamine and tertiary butanol solution to produce beta-aminopropionitrile, and then alkaline hydrolysis is carried out to obtain the beta-aminopropionitrile. In the process, the temperature is controlled to be about 100, the pressure is 1Mpa, a large amount of liquid caustic soda is needed for hydrolysis, and the final recovery rate is less than 90%. The method not only needs to use organic reagent, but also needs high temperature and high pressure, and has high production cost and certain danger.
The beta-alanine biological fermentation method mainly comprises the steps of genetically modifying a metabolic pathway of escherichia coli, introducing panC and panD genes into a chromosome of the escherichia coli, and fermenting and synthesizing the beta-alanine by taking glucose as a substrate, for example, patent CN111411130A. The method has long fermentation period, a large amount of byproducts are contained in fermentation liquor, the yield of beta-alanine is low, and the purification process is complicated.
The beta-alanine bioenzyme synthesis mainly comprises the following steps: one is to use an aspartase mutant, acrylic acid as a substrate, and add ammonia water to react and synthesize beta-alanine. The method uses the acrylic acid which is irritant and has corrosiveness as a substrate, byproducts are generated in the catalysis process, and the utilization rate of the acrylic acid is low, such as patent CN108546698A; the other is to synthesize beta-alanine by using L-aspartic acid-alpha-decarboxylase and L-aspartic acid as a substrate. The method has mild catalytic reaction conditions and high conversion efficiency, but uses the expensive L-aspartic acid as a substrate, so that the method is not suitable for large-scale production. And there is a serious mechanical inactivation of L-aspartic acid alpha-decarboxylase (panD) leading to lower beta-alanine yields, as in patent CN107988194B.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a method for producing beta-alanine, which improves the substrate utilization rate and reduces the related enzyme inactivation by controlling the reaction temperature, the maleic acid feeding and the like in an engineering bacteria reaction system, so as to obtain high-purity and high-yield beta-alanine fermentation liquor, and can be used for large-scale industrial production.
The route for synthesizing the beta-alanine in the invention is as follows: maleic acid is used as a substrate, maleic acid is converted into fumaric acid through cis-trans isomerism of the maleic acid, fumaric acid is then ammoniated into L-aspartic acid through aspartase, and finally L-aspartic acid is converted into beta-alanine under the action of L-aspartic acid-alpha-decarboxylase.
In order to be able to obtain high yields of beta-alanine, the present invention first optimizes the enzyme itself and the inventors screened the enzyme combination with the highest yield among the enzymes from numerous sources.
Based on the above optimization mode, the inventors further optimize the production process. The production process is realized by whole cell transformation of engineering bacteria, in the process, the pH of the whole reaction system is raised due to decarboxylation reaction, but the too high pH can reduce the activity of L-aspartic acid-alpha-decarboxylase, and in order to avoid introducing other acidic substances and reduce the fermentation cost, the invention adopts substrate maleic acid to maintain the pH. During the experiment, the inventor found that if the maleic acid flow rate is too fast, the fermentation pH is lower, the enzyme activity of maleic acid cis-trans isomerase and aspartase is reduced, the activity of L-aspartic acid-alpha-decarboxylase is further inhibited by high concentration of maleic acid or intermediate product fumaric acid, the substrate utilization rate is reduced, and the yield of beta-alanine is reduced; if the maleic acid flow rate is too slow, the fermentation pH is higher, the activity of L-aspartic acid-alpha-decarboxylase is reduced, the catalytic reaction time is prolonged, the related enzyme activity is reduced, the substrate utilization rate is reduced, and the yield of beta-alanine is reduced. Meanwhile, during the fermentation process, the temperature of the system has an important influence on the activities of maleic acid cis-trans isomerase, aspartase and L-aspartic acid-alpha-decarboxylase. For the above reasons, the present invention provides a method for producing beta-alanine, comprising:
adding a mixed solution of maleic acid and ammonia water into a culture solution of engineering bacteria, and fermenting to generate beta-alanine; wherein the total addition amount of the maleic acid is 250-375g/L, and the flow rate of the maleic acid is 15-30 g/(L.times.h); the mass volume ratio of the maleic acid to the ammonia water is 1-2:1 (g: mL);
the engineering bacteria are strains capable of expressing maleic acid cis-trans isomerase, aspartase and L-aspartic acid-alpha-decarboxylase.
In some embodiments, the total amount of maleic acid added is 325-350g/L.
In some embodiments, the maleic acid has a flow rate of 20-25 g/(L h).
In some embodiments, the temperature during fermentation is 40-45 ℃.
In some embodiments, the engineered bacterium is a recombinant E.coli comprising genes encoding maleic acid cis-trans isomerase, aspartase, and L-aspartic acid-alpha-decarboxylase.
In the present invention, L-aspartic acid alpha-decarboxylase is PLP dependent, does not have mechanical inactivation, has a certain tolerance to a substrate or an intermediate product, and still has a certain enzyme activity under higher temperature and alkaline conditions.
In some embodiments, the host of recombinant E.coli includes Escherichia coli BL (DE 3) and Escherichia coli TOP10.
In some embodiments, the amino acid sequence of the maleic acid cis-trans isomerase is set forth in SEQ ID No. 1; the amino acid sequence of the aspartase is shown as SEQ ID NO. 2; the amino acid sequence of the L-aspartic acid-alpha-decarboxylase is shown as SEQ ID NO. 3.
Specifically, the maleic acid cis-trans isomerase used in the present invention is derived from S.marcescens, the aspartase is derived from Escherichia coli, and the L-aspartic acid-alpha-decarboxylase is derived from Tribolium castaneum.
In some embodiments, the method of constructing a recombinant escherichia coli comprises: the recombinant escherichia coli is obtained by adopting pETDuet-1 and pCDFduet-1 to co-express three genes, loading the maleic acid cis-trans isomerase gene and the aspartase gene into pETduet-1, loading the L-aspartic acid-alpha-decarboxylase gene into pCDFduet-1, transforming the two plasmids into escherichia coli Escherichia coli BL (DE 3), and screening positive clones by using Ampicillin (Ampicillin) and Streptomycin sulfate (Streptomycin) plates.
In some embodiments, the method of preparing a culture broth of recombinant E.coli comprises: inoculating the seed solution of the recombinant bacterium into a culture medium for fermentation, and inducing the recombinant bacterium until the concentration of the recombinant escherichia coli in the culture medium is OD600 nm=20-30, so as to obtain the culture solution of the recombinant escherichia coli after the induction culture.
In some embodiments, the culture medium comprises a modified TB medium formulated by adjusting the pH to 7.0-7.5 with ammonia after high temperature sterilization, 7-17g/L peptone, 20-30g/L yeast powder, 25-35g/L glycerol, 1-3g/L potassium dihydrogen phosphate, 7-17g/L dipotassium hydrogen phosphate, 0.5-2g/L magnesium sulfate heptahydrate.
In some embodiments, the inducing culturing comprises: adding glycerol to the culture medium until the concentration is 20-50g/L, cooling to 25-30 ℃, regulating the pH to be 6.5-7.5, adding IPTG with the final concentration of 0.2-1.0mM for induction, adding TrionX-100 and PLP after induction for 10-18 hours, and continuously culturing for 0.5-3 hours; wherein, after adding TrionX-100 and PLP, the concentrations of TrionX-100 and PLP are respectively 0.1-0.3% (V/V) and 0.1-0.5g/L.
The invention has the following beneficial effects:
the invention optimizes the sources of enzymes required in the fermentation process and the production process, takes maleic acid as a substrate, utilizes recombinant escherichia coli capable of expressing maleic acid cis-trans isomerase, aspartase and L-aspartic acid-alpha-decarboxylase with specific sources to ferment and synthesize beta-alanine, and obtains beta-alanine fermentation liquor with high purity and high yield by controlling the maleic acid flow rate. The substrate maleic acid is cheap and easy to obtain, and the expensive L-aspartic acid is not used as the substrate, so that the fermentation cost is reduced, and the industrial production is facilitated. Meanwhile, the invention controls the feeding speed of the maleic acid, the fermentation temperature, the final beta-alanine yield reaches more than 250g/L, the utilization rate of the maleic acid reaches more than 99%, and the purification cost of the rear end is reduced.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
1. The strain and the plasmid are purchased from Novagen company, such as pETdur-1 plasmid, pCDFdur-1 plasmid, escherichia coli BL (DE 3) and Escherichia coli TOP.
2. Construction of recombinant escherichia coli genetic engineering bacteria: the invention adopts pETDuet-1 and pCDFduet-1 to co-express three genes, pETduet-1 loads maleic acid cis-trans isomerase gene (S.marcens) and aspartase gene (Escherichia coli), and pCDFduet-1 loads L-aspartic acid-alpha-decarboxylase gene (Tribolium castaneum). Two plasmids were transformed into E.coli Escherichia coli BL (DE 3), and positive clones were selected using Ampicillin (Ampicillin) and Streptomycin sulfate (Streptomyces) plates to obtain recombinant E.coli.
3. The culturing process of the recombinant escherichia coli comprises the following steps:
(1) The recombinant escherichia coli is placed in an LBG culture medium, the LBG culture medium comprises 10g of peptone, 5g of yeast extract, 10g of NaCl and 5g of glycerol, pure water is fixed to a volume of 1.0L, and the recombinant escherichia coli seed liquid is obtained through overnight culture under the conditions of 37 and pH 7.0.
(2) The recombinant E.coli seed solution was transferred to an improved TB medium (12 g/L peptone, 24g/L yeast powder, 30g/L glycerol, 2.31g/L potassium dihydrogen phosphate, 12.54g/L dipotassium hydrogen phosphate, 121 sterilized, pH was adjusted to 7.0 with ammonia water) and cultured under conditions of 37 and pH=7.0, and pH was adjusted with ammonia water. When the thalli in the fermentation liquid is cultivated to OD 600nm 30g/L glycerol was added additionally, the temperature was lowered to 28℃and pH=7.0, induction was performed by adding IPTG at a final concentration of 0.5mM, after 12h of induction, 0.1% TrionX-100 and 0.25g/L PLP were added, and the culture was continued for 1h to obtain a fermentation broth. The fermentation broth was used in examples 1-10 below.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
(1) The fermentation temperature is controlled to be 45 ℃, the mixed solution of maleic acid and industrial ammonia water is fed in, the total addition amount of the maleic acid in the mixed solution is 200g/L, and the feeding rate of the maleic acid is controlled to be 20 g/(L.times.h).
(2) After the substrate fed-batch was completed, fermentation was continued until the β -alanine production was no longer changed, and the substrate maleic acid remaining amount, fumaric acid remaining amount, L-aspartic acid remaining amount, and β -alanine production were detected by HPLC.
Example 2
(1) The fermentation temperature is controlled to be 45 ℃, the mixed solution of maleic acid and industrial ammonia water is fed in, the total addition amount of the maleic acid in the mixed solution is 325g/L, and the feeding rate of the maleic acid is controlled to be 20 g/(L.times.h).
(2) After the substrate fed-batch was completed, fermentation was continued until the β -alanine production was no longer changed, and the substrate maleic acid remaining amount, fumaric acid remaining amount, L-aspartic acid remaining amount, and β -alanine production were detected by HPLC.
Example 3
(1) The fermentation temperature is controlled to be 45 ℃, the mixed solution of maleic acid and industrial ammonia water is fed in, the total addition amount of the maleic acid in the mixed solution is 350g/L, and the feeding rate of the maleic acid is controlled to be 20 g/(L.times.h).
(2) After the substrate fed-batch was completed, fermentation was continued until the β -alanine production was no longer changed, and the substrate maleic acid remaining amount, fumaric acid remaining amount, L-aspartic acid remaining amount, and β -alanine production were detected by HPLC.
Example 4
(1) The fermentation temperature is controlled to be 45 ℃, the mixed solution of maleic acid and industrial ammonia water is fed in, the total addition amount of the maleic acid in the mixed solution is 325g/L, and the feeding rate of the maleic acid is controlled to be 25 g/(L.times.h).
(2) After the substrate fed-batch was completed, fermentation was continued until the β -alanine production was no longer changed, and the substrate maleic acid remaining amount, fumaric acid remaining amount, L-aspartic acid remaining amount, and β -alanine production were detected by HPLC.
Example 5
(1) The fermentation temperature is controlled to be 40 ℃, the mixed solution of maleic acid and industrial ammonia water is fed in, the total addition amount of the maleic acid in the mixed solution is 325g/L, and the feeding rate of the maleic acid is controlled to be 25 g/(L.times.h).
(2) After the substrate fed-batch was completed, fermentation was continued until the β -alanine production was no longer changed, and the substrate maleic acid remaining amount, fumaric acid remaining amount, L-aspartic acid remaining amount, and β -alanine production were detected by HPLC.
Example 6
(1) The fermentation temperature is controlled to be 40 ℃, the mixed solution of maleic acid and industrial ammonia water is fed in, the total addition amount of the maleic acid in the mixed solution is 350g/L, and the feeding rate of the maleic acid is controlled to be 25 g/(L.times.h).
(2) After the substrate fed-batch was completed, fermentation was continued until the β -alanine production was no longer changed, and the substrate maleic acid remaining amount, fumaric acid remaining amount, L-aspartic acid remaining amount, and β -alanine production were detected by HPLC.
Example 7
(1) The fermentation temperature is controlled to be 40 ℃, the mixed solution of maleic acid and industrial ammonia water is fed in, the total addition amount of the maleic acid in the mixed solution is 350g/L, and the feeding rate of the maleic acid is controlled to be 20 g/(L.times.h).
(2) After the substrate fed-batch was completed, fermentation was continued until the β -alanine production was no longer changed, and the substrate maleic acid remaining amount, fumaric acid remaining amount, L-aspartic acid remaining amount, and β -alanine production were detected by HPLC.
Example 8
(1) The fermentation temperature is controlled to be 42.5 ℃, the mixed solution of maleic acid and industrial ammonia water is started to be fed, the total addition amount of the maleic acid in the mixed solution is 325g/L, and the feeding rate of the maleic acid is controlled to be 22.5 g/(L.times.h).
(2) After the substrate fed-batch was completed, fermentation was continued until the β -alanine production was no longer changed, and the substrate maleic acid remaining amount, fumaric acid remaining amount, L-aspartic acid remaining amount, and β -alanine production were detected by HPLC.
Example 9
(1) The fermentation temperature is controlled to be 42.5 ℃, the mixed solution of maleic acid and industrial ammonia water is fed, the total addition amount of the maleic acid in the mixed solution is 350g/L, and the feeding rate of the maleic acid is controlled to be 22.5 g/(L.times.h).
(2) After the substrate fed-batch was completed, fermentation was continued until the β -alanine production was no longer changed, and the substrate maleic acid remaining amount, fumaric acid remaining amount, L-aspartic acid remaining amount, and β -alanine production were detected by HPLC.
Example 10
(1) The fermentation temperature is controlled to be 45 ℃, the mixed solution of maleic acid and industrial ammonia water is started to be fed, the total addition amount of the maleic acid in the mixed solution is 325g/L, and the feeding rate of the maleic acid is controlled to be 22.5 g/(L.times.h).
(2) After the substrate fed-batch was completed, fermentation was continued until the β -alanine production was no longer changed, and the substrate maleic acid remaining amount, fumaric acid remaining amount, L-aspartic acid remaining amount, and β -alanine production were detected by HPLC.
Example 11
This example differs from example 1 in that the cultivation process of recombinant E.coli is different:
(1) The recombinant escherichia coli is placed in an LBG culture medium, the LBG culture medium comprises 10g of peptone, 5g of yeast extract, 10g of NaCl and 5g of glycerol, pure water is fixed to 1.0L, and the recombinant escherichia coli seed liquid is obtained by overnight culture at 37 ℃ and pH 7.0.
(2) The recombinant E.coli seed solution was transferred to an improved TB medium (12 g/L peptone, 24g/L yeast powder, 30g/L glycerol, 2.31g/L potassium dihydrogen phosphate, 12.54g/L dipotassium hydrogen phosphate, sterilized at 121 ℃, pH=7.0 with ammonia water), and the fermentation conditions were 37℃and pH=7.0, and pH was adjusted with ammonia water.
When the thalli in the fermentation broth is cultivated to OD600 nm=20, 30g/L glycerol is additionally added, the temperature is reduced to 28 ℃, the pH=7.0, the final concentration of 0.5mM IPTG is added for induction, 0.1% TrionX-100 and 0.25g/L PLP are added after 12 hours of induction, and the cultivation is continued for 1 hour, so as to obtain the fermentation broth.
Example 12
This example differs from example 1 in that the cultivation process of recombinant E.coli is different:
(1) The recombinant escherichia coli is placed in an LBG culture medium, the LBG culture medium comprises 10g of peptone, 5g of yeast extract, 10g of NaCl and 5g of glycerol, pure water is fixed to 1.0L, and the recombinant escherichia coli seed liquid is obtained by overnight culture at 37 ℃ and pH 7.0.
(2) The recombinant E.coli seed solution was transferred to an improved TB medium (12 g/L peptone, 24g/L yeast powder, 30g/L glycerol, 2.31g/L potassium dihydrogen phosphate, 12.54g/L dipotassium hydrogen phosphate, sterilized at 121 ℃, pH=7.0 with ammonia water), and the fermentation conditions were 37℃and pH=7.0, and pH was adjusted with ammonia water.
When the thalli in the fermentation broth is cultivated to OD600 nm=30, 30g/L glycerol is additionally added, the temperature is reduced to 28 ℃, the pH=7.0, the final concentration of 0.5mM IPTG is added for induction, 0.1% TrionX-100 and 0.25g/L PLP are added after 12 hours of induction, and the cultivation is continued for 1 hour, so as to obtain the fermentation broth.
Comparative example 1
(1) The fermentation temperature is controlled to be 45 ℃, the mixed solution of maleic acid and industrial ammonia water is fed in, the total addition amount of the maleic acid in the mixed solution is 400g/L, and the feeding rate of the maleic acid is controlled to be 20 g/(L.times.h).
(2) After the substrate fed-batch was completed, fermentation was continued until the β -alanine production was no longer changed, and the substrate maleic acid remaining amount, fumaric acid remaining amount, L-aspartic acid remaining amount, and β -alanine production were detected by HPLC.
Other embodiments, conditions, and the like in this comparative example are the same as in example 1.
Comparative example 2
(1) The fermentation temperature is controlled to be 45 ℃, the mixed solution of maleic acid and industrial ammonia water is fed in, the total addition amount of the maleic acid in the mixed solution is 325g/L, and the feeding rate of the maleic acid is controlled to be 10 g/(L.times.h).
(2) After the substrate fed-batch was completed, fermentation was continued until the β -alanine production was no longer changed, and the substrate maleic acid remaining amount, fumaric acid remaining amount, L-aspartic acid remaining amount, and β -alanine production were detected by HPLC.
Other embodiments, conditions, and the like in this comparative example are the same as in example 1.
Comparative example 3
(1) The fermentation temperature is controlled to be 45 ℃, the mixed solution of maleic acid and industrial ammonia water is started to be fed, the total addition amount of the maleic acid in the mixed solution is 325g/L, and the feeding rate of the maleic acid is controlled to be 35 g/(L.times.h).
(2) After the substrate fed-batch was completed, fermentation was continued until the β -alanine production was no longer changed, and the substrate maleic acid remaining amount, fumaric acid remaining amount, L-aspartic acid remaining amount, and β -alanine production were detected by HPLC.
Other embodiments, conditions, and the like in this comparative example are the same as in example 1.
Comparative example 4
(1) The fermentation temperature is controlled to be 30 ℃, the mixed solution of maleic acid and industrial ammonia water is fed in, the total addition amount of the maleic acid in the mixed solution is 325g/L, and the feeding rate of the maleic acid is controlled to be 20 g/(L.times.h).
(2) After the substrate fed-batch was completed, fermentation was continued until the β -alanine production was no longer changed, and the substrate maleic acid remaining amount, fumaric acid remaining amount, L-aspartic acid remaining amount, and β -alanine production were detected by HPLC.
Other embodiments, conditions, and the like in this comparative example are the same as in example 1.
Comparative example 5
(1) The fermentation temperature is controlled to be 55 ℃, the mixed solution of maleic acid and industrial ammonia water is fed in, the total addition amount of the maleic acid in the mixed solution is 325g/L, and the feeding rate of the maleic acid is controlled to be 20 g/(L.times.h).
(2) After the substrate fed-batch was completed, fermentation was continued until the β -alanine production was no longer changed, and the substrate maleic acid remaining amount, fumaric acid remaining amount, L-aspartic acid remaining amount, and β -alanine production were detected by HPLC.
Other embodiments, conditions, and the like in this comparative example are the same as in example 1.
Comparative example 6
This comparative example differs from example 5 only in that: the species sources of some enzymes used were different, and specifically, the maleic acid isomerase used in this comparative example was derived from Alcaligenes faecalis Alcaligenes faecalis, and the aspartic acid-. Alpha. -decarboxylase was derived from Corynebacterium glutamicum Corynebacterium glutamicum.
The rest of the experimental procedure and the implementation conditions were the same as in example 5.
Comparative example 7
This comparative example differs from example 5 only in that: the aspartic acid- α -decarboxylase used was different, specifically, the aspartic acid- α -decarboxylase used in this comparative example was PYR-dependent, and its amino acid sequence was shown in SEQ ID NO.4, for a total of 127 amino acids.
The rest of the experimental procedure and the implementation conditions were the same as in example 5.
Experimental example
(1) Standard curve plotting of beta-alanine
Weighing 0.1g of beta-alanine standard sample, transferring the standard sample into a 50mL volumetric flask, dissolving the standard sample with water to fix the volume, and then respectively transferring 0.5mL, 1mL, 2mL, 4mL and 5mL into 5 10mL volumetric flasks to obtain standard samples with the concentration of 0.1g/L, 0.2g/L, 0.4g/L, 0.8g/L and 1 g/L. Taking 800 mu L of standard sample, adding 200 mu L of OPA derivative, mixing for 1min at 25 ℃, detecting by HPLC, and performing chromatographic conditions: the chromatographic column is C18, the mobile phase A is 0.1% formic acid water, the mobile phase B is methanol, and the mobile phase A: mobile phase B (v: v) =30: 70, flow rate: 1mL/L, column temperature: 30 ℃, wavelength: 333nm, sample volume: 10 μl, retention time: and (3) drawing a beta-alanine standard curve by taking the concentration (g/L) as an abscissa and the peak area as an ordinate after the peak time of the beta-alanine is about 7.5min for 11 min.
OPA derivative preparation: 10g/L phthalic aldehyde+100 mL/L methanol+900 mL/L ph= 10.40.4M boric acid buffer+50 mL/L β -mercaptoethanol.
(2) Drawing standard vertebral curve of L-aspartic acid
Weighing 0.1g of L-aspartic acid standard sample, transferring the standard sample into a 50mL volumetric flask, dissolving with water to fix the volume, transferring 0.5mL, 1mL, 2mL, 4mL and 5mL into 6 10mL volumetric flasks respectively, and dissolving with water to fix the volume to obtain standard samples with the concentrations of 0.1g/L, 0.2g/L, 0.4g/L, 0.8g/L and 1g/L respectively. Taking 800 mu L of standard sample, adding 200 mu L of OPA derivative, mixing for 1min at 25 ℃, detecting by HPLC, and performing chromatographic conditions: the chromatographic column is C18, the mobile phase A is 0.1% formic acid water, the mobile phase B is methanol, and the mobile phase A: mobile phase B (v: v) =30: 70, flow rate: 1mL/L, column temperature: 30 ℃, wavelength: 333nm, sample volume: 10 μl, retention time: 11min, the peak time of L-aspartic acid is about 4.6min, and the L-aspartic acid standard curve is drawn by taking the concentration (g/L) as the abscissa and the peak area as the ordinate.
(3) Mapping of fumaric acid on vertebral curve
Weighing 0.1g of fumaric acid standard sample, transferring into 50mL volumetric flasks, dissolving with water to constant volume, transferring 0.5mL, 1mL, 2mL, 4mL and 5mL into 6 10mL volumetric flasks respectively, dissolving with water to constant volume to obtain standard samples with concentration of 0.1g/L, 0.2g/L, 0.4g/L, 0.8g/L and 1g/L respectively, detecting by HPLC respectively, and chromatographic conditions: the chromatographic column is an H column, the mobile phase A is 0.0257% sulfuric acid water, and the flow rate is as follows: 0.5mL/L, column temperature: 40 ℃, wavelength: 210nm, sample volume: 10 μl, retention time: and (3) drawing a fumaric acid standard curve by taking the concentration (g/L) as an abscissa and the peak area as an ordinate after 20min and the fumaric acid peak time of about 16.5 min.
(4) Mapping of maleic acid on vertebral curves
Weighing 0.1g of maleic acid standard sample, transferring the standard sample into a 50mL volumetric flask, dissolving with water to fix volume, transferring 0.5mL, 1mL, 2mL, 4mL and 5mL into 6 10mL volumetric flasks respectively, dissolving with water to fix volume to obtain standard samples with the concentration of 0.1g/L, 0.2g/L, 0.4g/L, 0.8g/L and 1g/L respectively, detecting by HPLC respectively, and performing chromatographic conditions: the chromatographic column is an H column, the mobile phase A is 0.0257% sulfuric acid water, and the flow rate is as follows: 0.5mL/L, column temperature: 40 ℃, wavelength: 210nm, sample volume: 10 μl, retention time: and (3) drawing a maleic acid standard curve by taking the concentration (g/L) as an abscissa and the peak area as an ordinate after 20min and the maleic acid peak time of about 14.5 min.
(5) Determination of the beta-alanine, maleic acid, fumaric acid, L-aspartic acid concentration in the fermentation broths of examples 1-12 and comparative examples 1-7
2mL of the fermentation broths obtained in examples 1 to 12 and comparative examples 1 to 7 were respectively centrifuged, 1mL of the supernatant was diluted with water to different multiples, and the beta-alanine, maleic acid, fumaric acid and L-aspartic acid concentrations in this example were obtained by HPLC detection and analysis.
The beta-alanine production amounts, the residual maleic acid amount, the residual fumaric acid amount, and the residual L-aspartic acid amount shown in Table 1 were all obtained according to the corresponding standard curves; maleic acid utilization = (β -alanine yield × maleic acid relative molecular mass)/(maleic acid addition × β -alanine relative molecular mass).
TABLE 1 summary of data for examples 1-12 above and comparative examples 1-7
The production method can ensure that the yield of beta-alanine is more than 250g/L and the utilization rate of maleic acid is more than 99 percent.
From the experimental data and results of comparative examples 1 and 2, it can be seen that when the addition amount of the substrate maleic acid is too high or the flow rate of the substrate during fermentation is too low, the residual amounts of the substrate and intermediate after fermentation are increased, resulting in incomplete application of the raw materials; however, under the method of the invention, better experimental effect can be obtained, and better substrate utilization rate is maintained.
From the experimental data and results of comparative example 3, it can be seen that when the substrate flow rate is too high during fermentation, incomplete contact between the strain and the substrate during fermentation can be caused, and the residual quantity of the substrate and intermediate product can be greatly improved, so that the substrate utilization rate of the experiment is greatly affected.
As can be seen from the experimental data and results of comparative examples 4 and 5, when the temperature is too high or too low, the fermentation process is affected, wherein the reason is that the strain organism is destroyed and the various functional enzymes are deteriorated and deactivated due to the too high temperature, so that the substrate utilization rate is greatly reduced.
From the experimental data and results of comparative example 6, it can be seen that when other enzyme groups of the same type are adopted, better experimental effects cannot be achieved, which indicates that each enzyme combination of the invention has a certain synergistic effect.
From the experimental data and results of comparative example 7, it can be seen that when the aspartic acid- α -decarboxylase is PLP-independent, even though the utilization ratio of the front-end substrate and the intermediate is high, the yield is not good finally because the PYR-dependent aspartic acid- α -decarboxylase is susceptible to mechanical inactivation by maleic acid and fumaric acid, thereby greatly reducing the yield of the product.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for producing β -alanine, the method comprising: adding a mixed solution of maleic acid and ammonia water into a culture solution of engineering bacteria, and fermenting to generate beta-alanine;
the total addition amount of the maleic acid is 250-375g/L, and the flow rate of the maleic acid is 15-30 g/(L.times.h); the mass volume ratio of the maleic acid to the ammonia water is 1-2:1 (g: mL);
the engineering bacteria are strains capable of expressing maleic acid cis-trans isomerase, aspartase and L-aspartic acid-alpha-decarboxylase.
2. The production method according to claim 1, wherein the total amount of maleic acid added is 325-350g/L.
3. The production method according to claim 2, wherein the maleic acid has a feeding rate of 20-25 g/(lxh).
4. A production method according to claim 3, wherein the temperature during fermentation is 37-50;
preferably, the temperature during fermentation is 40-45.
5. The method according to claim 4, wherein the engineering bacterium is recombinant E.coli comprising genes encoding maleic acid cis-trans isomerase, aspartase and L-aspartic acid-alpha-decarboxylase;
hosts for the recombinant E.coli include Escherichia coli BL (DE 3) and Escherichia coli TOP.
6. The method according to claim 5, wherein the amino acid sequences of maleic acid cis-trans isomerase, aspartase and L-aspartic acid-alpha-decarboxylase are shown in SEQ ID NO. 1-3;
preferably, the maleic acid cis-trans isomerase is derived from S.marcescens, the aspartase is derived from Escherichia coli, and the L-aspartic acid-alpha-decarboxylase is derived from Triboliumcastanum.
7. The method according to claim 6, wherein the method for constructing recombinant E.coli comprises: connecting genes of the maleic acid cis-trans isomerase, the aspartase and the L-aspartic acid-alpha-decarboxylase to an expression vector, and then introducing the obtained recombinant expression vector into the escherichia coli to obtain the recombinant escherichia coli;
preferably, the expression vector comprises pETDuet-1 plasmid and pCDFdur-1 plasmid;
preferably, the pETdur-1 plasmid is loaded with a maleic acid cis-trans isomerase gene and an aspartase gene, and the pCDFdur-1 plasmid is loaded with an L-aspartic acid-alpha-decarboxylase gene.
8. The production method according to claim 7, wherein the preparation method of the culture solution of recombinant E.coli comprises: inoculating the seed solution of the recombinant escherichia coli into a culture medium for fermentation until the concentration of the recombinant escherichia coli in the culture medium is OD 600nm When the recombinant escherichia coli is included in the culture medium, the recombinant escherichia coli is induced and cultured to obtain the culture medium of the recombinant escherichia coli.
9. The method of preparation of claim 8, wherein the medium comprises a modified TB medium;
the preparation method of the improved TB culture medium comprises the steps of regulating the pH value to 7.0-7.5 by using ammonia water after high-temperature sterilization, wherein 7-17g/L of peptone, 20-30g/L of yeast powder, 25-35g/L of glycerol, 1-3g/L of monopotassium phosphate and 7-17g/L of dipotassium phosphate, and 0.5-2g/L of magnesium sulfate heptahydrate.
10. The method of claim 9, wherein the inducing culture comprises: adding glycerol to the culture medium until the concentration is 20-50g/L, cooling to 25-30, regulating pH=6.5-7.5, adding IPTG with the final concentration of 0.2-1.0mM for induction, adding TrionX-100 and PLP after induction for 10-18h, and continuously culturing for 0.5-3h.
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