CN107012180A - A kind of method that beta Alanine is prepared as raw material multienzyme coupling using maleic acid - Google Patents
A kind of method that beta Alanine is prepared as raw material multienzyme coupling using maleic acid Download PDFInfo
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- CN107012180A CN107012180A CN201710404758.5A CN201710404758A CN107012180A CN 107012180 A CN107012180 A CN 107012180A CN 201710404758 A CN201710404758 A CN 201710404758A CN 107012180 A CN107012180 A CN 107012180A
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- alanine
- beta
- maleic acid
- decarboxylase
- aspartase
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- UCMIRNVEIXFBKS-UHFFFAOYSA-N beta-alanine Chemical compound NCCC(O)=O UCMIRNVEIXFBKS-UHFFFAOYSA-N 0.000 title claims abstract description 165
- 229940000635 beta-alanine Drugs 0.000 title claims abstract description 82
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 title claims abstract description 36
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 title claims abstract description 35
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 title claims abstract description 35
- 239000011976 maleic acid Substances 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 25
- 239000002994 raw material Substances 0.000 title claims abstract description 13
- 230000008878 coupling Effects 0.000 title claims abstract description 12
- 238000010168 coupling process Methods 0.000 title claims abstract description 12
- 238000005859 coupling reaction Methods 0.000 title claims abstract description 12
- 102000004190 Enzymes Human genes 0.000 claims abstract description 29
- 108090000790 Enzymes Proteins 0.000 claims abstract description 28
- 108700016171 Aspartate ammonia-lyases Proteins 0.000 claims abstract description 18
- 108010030019 maleate isomerase Proteins 0.000 claims abstract description 17
- 238000006911 enzymatic reaction Methods 0.000 claims abstract description 13
- 235000011114 ammonium hydroxide Nutrition 0.000 claims abstract description 12
- 239000007788 liquid Substances 0.000 claims abstract description 7
- 238000006243 chemical reaction Methods 0.000 claims description 61
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 38
- 230000000694 effects Effects 0.000 claims description 24
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 14
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 13
- 241000588813 Alcaligenes faecalis Species 0.000 claims description 12
- 241000588724 Escherichia coli Species 0.000 claims description 12
- HZUKSQHMCTUZJL-UHFFFAOYSA-N P(=O)(O)(O)OCC=1C(=C(C(=NC1)C)O)C=O.P(=O)(O)(O)OC=1C(=NC=C(C1C=O)CO)C Chemical compound P(=O)(O)(O)OCC=1C(=C(C(=NC1)C)O)C=O.P(=O)(O)(O)OC=1C(=NC=C(C1C=O)CO)C HZUKSQHMCTUZJL-UHFFFAOYSA-N 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 10
- 238000000926 separation method Methods 0.000 claims description 8
- 229910052757 nitrogen Inorganic materials 0.000 claims description 7
- 229920000136 polysorbate Polymers 0.000 claims description 7
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 6
- 229910052799 carbon Inorganic materials 0.000 claims description 6
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 3
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 244000068988 Glycine max Species 0.000 claims description 3
- 235000010469 Glycine max Nutrition 0.000 claims description 3
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 3
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- 240000008042 Zea mays Species 0.000 claims description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 3
- 235000015278 beef Nutrition 0.000 claims description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 3
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 235000005822 corn Nutrition 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 238000005342 ion exchange Methods 0.000 claims description 3
- 239000008101 lactose Substances 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 239000011347 resin Substances 0.000 claims description 3
- 229920005989 resin Polymers 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 229940005347 alcaligenes faecalis Drugs 0.000 claims description 2
- 239000002609 medium Substances 0.000 claims description 2
- 108090000769 Isomerases Proteins 0.000 claims 1
- 102000004195 Isomerases Human genes 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 19
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 abstract description 11
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 abstract description 7
- 239000000758 substrate Substances 0.000 abstract description 7
- 230000008901 benefit Effects 0.000 abstract description 5
- 108090000489 Carboxy-Lyases Proteins 0.000 abstract description 3
- 239000003456 ion exchange resin Substances 0.000 abstract description 3
- 229920003303 ion-exchange polymer Polymers 0.000 abstract description 3
- 102000004031 Carboxy-Lyases Human genes 0.000 abstract description 2
- 235000003704 aspartic acid Nutrition 0.000 abstract 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 abstract 1
- 238000009776 industrial production Methods 0.000 abstract 1
- 210000001082 somatic cell Anatomy 0.000 abstract 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 52
- 239000012530 fluid Substances 0.000 description 33
- 238000002425 crystallisation Methods 0.000 description 24
- 238000001816 cooling Methods 0.000 description 16
- 239000010413 mother solution Substances 0.000 description 16
- 239000007787 solid Substances 0.000 description 16
- 238000000967 suction filtration Methods 0.000 description 16
- 239000000243 solution Substances 0.000 description 14
- 230000002255 enzymatic effect Effects 0.000 description 9
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 8
- 239000003729 cation exchange resin Substances 0.000 description 8
- 238000005119 centrifugation Methods 0.000 description 8
- 230000008025 crystallization Effects 0.000 description 8
- 239000003480 eluent Substances 0.000 description 8
- 238000001728 nano-filtration Methods 0.000 description 8
- 230000002000 scavenging effect Effects 0.000 description 8
- 238000001179 sorption measurement Methods 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 238000000108 ultra-filtration Methods 0.000 description 8
- 238000010792 warming Methods 0.000 description 8
- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 6
- CKLJMWTZIZZHCS-UWTATZPHSA-N L-Aspartic acid Natural products OC(=O)[C@H](N)CC(O)=O CKLJMWTZIZZHCS-UWTATZPHSA-N 0.000 description 6
- 229960005261 aspartic acid Drugs 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 4
- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical compound O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 4
- NLHHRLWOUZZQLW-UHFFFAOYSA-N Acrylonitrile Chemical compound C=CC#N NLHHRLWOUZZQLW-UHFFFAOYSA-N 0.000 description 3
- 229910021529 ammonia Inorganic materials 0.000 description 3
- AGSPXMVUFBBBMO-UHFFFAOYSA-N beta-aminopropionitrile Chemical compound NCCC#N AGSPXMVUFBBBMO-UHFFFAOYSA-N 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
- 238000005352 clarification Methods 0.000 description 3
- -1 cyanoethyls Amine Chemical class 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 2
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000006114 decarboxylation reaction Methods 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 229940055726 pantothenic acid Drugs 0.000 description 2
- 235000019161 pantothenic acid Nutrition 0.000 description 2
- 239000011713 pantothenic acid Substances 0.000 description 2
- 229960001327 pyridoxal phosphate Drugs 0.000 description 2
- 229960002317 succinimide Drugs 0.000 description 2
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- BZSXEZOLBIJVQK-UHFFFAOYSA-N 2-methylsulfonylbenzoic acid Chemical compound CS(=O)(=O)C1=CC=CC=C1C(O)=O BZSXEZOLBIJVQK-UHFFFAOYSA-N 0.000 description 1
- 241000432824 Asparagus densiflorus Species 0.000 description 1
- 241000432767 Asparagus setaceus Species 0.000 description 1
- 102000040350 B family Human genes 0.000 description 1
- 108091072128 B family Proteins 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 238000005904 alkaline hydrolysis reaction Methods 0.000 description 1
- 238000005576 amination reaction Methods 0.000 description 1
- RQPZNWPYLFFXCP-UHFFFAOYSA-L barium dihydroxide Chemical compound [OH-].[OH-].[Ba+2] RQPZNWPYLFFXCP-UHFFFAOYSA-L 0.000 description 1
- 229910001863 barium hydroxide Inorganic materials 0.000 description 1
- 229910001422 barium ion Inorganic materials 0.000 description 1
- 159000000009 barium salts Chemical class 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000009615 deamination Effects 0.000 description 1
- 238000006481 deamination reaction Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- DMBHHRLKUKUOEG-UHFFFAOYSA-N diphenylamine Chemical class C=1C=CC=CC=1NC1=CC=CC=C1 DMBHHRLKUKUOEG-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- FVSKHRXBFJPNKK-UHFFFAOYSA-N propionitrile Chemical compound CCC#N FVSKHRXBFJPNKK-UHFFFAOYSA-N 0.000 description 1
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 1
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 description 1
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-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
- 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
- C12N9/88—Lyases (4.)
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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
- C12N9/90—Isomerases (5.)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- 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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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
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- Biotechnology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to biological technical field, and in particular to a kind of method for preparing beta Alanine as raw material multienzyme coupling using maleic acid.Certain density aqueous maleic acid is adjusted pH 6~9 by this method with ammoniacal liquor, the crude enzyme liquid containing maleate isomerase, the somatic cells of Aspartase and aspartic acid α decarboxylases or three kinds of enzymes is separately added into again, enzymatic reaction is carried out under the conditions of 30~50 DEG C, converted product is separated with the method that ion exchange resin is combined using isoelectric point crystallizing or isoelectric point crystallizing, the beta Alanine of high-purity is obtained.This method is that substrate multienzyme coupling prepares beta Alanine using maleic acid, has the advantages that raw material sources are wide, cheap, easy to operate, production cost is low, is adapted to large-scale industrial production.
Description
First, technical field
The invention belongs to biological technical field, and in particular to one kind prepares Beta-alanine by raw material multienzyme coupling of maleic acid
Method.
2nd, background technology
Beta-alanine is the precursor of synthetic pantothenic acid, and pantothenic acid is one kind of B family vitamin, constitutes a part for coacetylase, for
Endogenous metabolism energy exchange in various tissues is extremely important.With the gradually intensification recognized Beta-alanine, Beta-alanine exists
The fields such as medicine, food, cosmetics and feed apply increasingly extensive, and the market demand of Beta-alanine is also constantly increasing, therefore
Beta-alanine is with a wide range of applications and DEVELOPMENT PROSPECT.
According to the literature, the preparation method of current Beta-alanine mainly has chemical synthesis and biological enzyme.
1st, chemical synthesis
Chemical synthesis has acrylonitrile method, propylene acid system, β-aminopropionitrile method, succinimide edman degradation Edman and double cyanoethyls
Amine method.Wherein acrylonitrile method is that acrylonitrile reacts with ammonia in diphenylamines and t-butanol solution, produces β-aminopropionitrile, then carry out
Alkaline hydrolysis is obtained, the shortcoming of this method be there are in product more than 40% NaCl and impurity, it is necessary to which purifying yield is low repeatedly；β-ammonia
Base propionitrile method is β-aminopropionitrile and barium hydroxide reaction generation Beta-alanine barium and nitrogen, is passed through CO2, barium salt precipitation, production
Raw Beta-alanine, barium ions is removed with resin, and the shortcoming of this method is that production cost is high；Succinimide edman degradation Edman is succinyl
Imines generates Beta-alanine in alkaline sodium chlorate solution, and reaction solution adjusts pH with hydrochloric acid, is made with 95% ethanol of 3 times of amounts inorganic
Salt out, filtrate is decolourized with 4 times of distilled water dilutings measured, ion exchange resin separation, condensing crystallizing, because this method need to be substantial amounts of
Ethanol, production cost is high, and production site security requirement is high.
2nd, biological enzyme
(1) amination enzyme
Qiu Juanping (CN1626665A) is using microorganism wet thallus or crude enzyme liquid as enzyme source, using acrylic acid as substrate, in 20~
2~40h of conversion reaction is stirred under the conditions of 80 DEG C of enzymatics, then conversion fluid through deamination, ion exchange, that purifying obtains Beta-alanine is pure
Product.
(2) aspartic acid-α-decarboxylase
Aspartic acid-α-decarboxylase enzymatic production process be by raw material of L-Aspartic acid, decarboxylase be the one of catalyst
Biochemical reaction is walked, reaction condition gentle, enzyme activity high, reaction speed fast, selectivity strong the characteristics of simple with equipment.Decarboxylation
Enzyme Production by Enzymes Beta-alanine has turned into the direction of people's concern in recent years and research.
Wiliamson(J.Biol.Chem.1979,254:8074-8082) using microorganism wet thallus or crude enzyme liquid as enzyme
Source, using L-Aspartic acid as substrate, 0.1mol/L pH 7.5 kaliumphosphate buffer, in 42 DEG C of enzymatic conversions, reacts 20min,
The reaction solution containing Beta-alanine is obtained, Beta-alanine is made through separation and Extraction, aspartic acid-α-decarboxylase Km values are about
160umol/L。
Cronan(J.Biol.Chem.1980,141(3):1291-1297) using microorganism wet thallus or crude enzyme liquid as enzyme
Source, using L-Aspartic acid as substrate, 0.1mol/L pH 6.8 kaliumphosphate buffer, in 37 DEG C of enzymatic conversions, reacts 1-2h,
The reaction solution containing Beta-alanine is obtained, Beta-alanine is made through separation and Extraction, aspartic acid-α-decarboxylase Km values are about
80umol/L。
(Zhejiang Polytechnical University's journal, 2011,39 (3) such as Hong Min:252-256) using L-Aspartic acid as substrate, with asparagus fern
Propylhomoserin-α-decarboxylase gene engineering bacteria pET-28c (+)-panD enzymatic clarifications Beta-alanine, enzyme activity reaches 186U.
GAO Lijuan (Zhejiang Polytechnical University's master thesis, 2007) using L-Aspartic acid as substrate, with aspartic acid-α-
Decarboxylase gene engineering bacteria pET-21c (+)-panD enzymatic clarifications Beta-alanine, enzyme activity reaches 224.96U.
3rd, the content of the invention
The present invention needs to be to provide a kind of method that efficient, low cost prepares Beta-alanine the problem of solution.The present invention with
Maleic acid is raw material, and β-the third ammonia is prepared with maleate isomerase, Aspartase and aspartic acid-α-decarboxylase multienzyme coupling
Acid.
The present invention can reach by the following technical programs：
A kind of method for preparing Beta-alanine as raw material multienzyme coupling using maleic acid, its step is：
(1) by the bacterial strain respectively with maleate isomerase, Aspartase and aspartic acid-α-decarboxylase in training
Support in base and cultivate, produce maleate isomerase, Aspartase and the aspartic acid-α-decarboxylase of high activity；
(2) by certain density aqueous maleic acid with ammoniacal liquor adjust pH 6~9, be separately added into maleate isomerase,
The wet thallus or crude enzyme liquid of Aspartase and aspartic acid-α-decarboxylase, add appropriate phosphopyridoxal pyridoxal phosphate and surface
Activating agent, carries out enzymatic reaction under the conditions of 30~50 DEG C, utilizes isoelectric point crystallizing or isoelectric point crystallizing and ion exchange resin
The Beta-alanine for the method separation reaction generation being combined.
Maleate isomerase described in above-mentioned steps (1) derives from Alcaligenes faecalis Alcaligenes faecalis, day
Winter propylhomoserin enzyme source derives from Corynebacterium glutamicum in Escherichia coli Escherichia coli, aspartic acid-α-decarboxylase
Corynebacterium glutamicum, three of the above bacterial strain is purchased from China General Microbiological culture presevation administrative center；
Culture medium carbon source described in above-mentioned steps (1) is used in glucose or maltose or sucrose or lactose, culture medium
Total carbon source mass concentration is 1~30g/L；Nitrogen source uses beef extract or yeast extract or corn steep liquor or peptone or soya-bean cake hydrolyzate,
Total nitrogen source mass concentration is 1~30g/L in culture medium；
Aqueous maleic acid mass concentration is 50~200g/L in above-mentioned steps (2)；Phosphopyridoxal pyridoxal phosphate mass concentration is
0.01~1.0g/L；Described surfactant be tween or cetyl trimethylammonium bromide (CTAB) or TritonX 100,
Mass concentration is 0.01~1.0g/L.
At present, the production of Beta-alanine mainly by chemical synthesis or uses aspartic acid-α-decarboxylase with L- asparagus ferns
Propylhomoserin is that raw material decarboxylation is obtained.One step enzyme method production cost and chemical synthesis production cost are relatively；By contrast, with Malaysia
Acid prepares the separation and Extraction mistake that Beta-alanine eliminates intermediate product fumaric acid and L-Aspartic acid for the enzyme of raw material three is coupling catalysed
Journey, coordinates enzyme-catalyzed reaction condition, many enzymic catalytic reactions is carried out simultaneously, simplify production procedure, reduce production cost, carry
High production efficiency, meets environmental protection and the requirement of clean manufacturing, implementation result is protruded, with good application value.This
Invention reaction principle is as follows：
The present invention has the following advantages that compared with prior art：
(1) Alcaligenes faecalis Alcaligenes faecalis, the Escherichia coli Escherichia coli that the present invention is used
With Corynebacterium glutamicum Corynebacterium glutamicum, can be different with high efficient expression maleic acid in preferred culture medium
Structure enzyme, Aspartase and aspartic acid-α-decarboxylase, make three enzymes coupling synthesis Beta-alanine have higher catalytic rate and turn
Rate, wherein maleic acid molar yield reaches more than 95%.
(2) present invention is raw material using maleic acid with low cost, and three enzyme coupling production Beta-alanines simplify production stream
Journey, reduces production cost, improves production efficiency, with good economic benefit and social benefit.
(3) present invention prepares Beta-alanine using enzyme law catalysis, and substrate conversion is complete, and product separation is simple.
(4) enzymatic clarification Beta-alanine has that reaction condition is gentle, enzyme stereoselectivity is strong, high catalytic efficiency, technique stream
The advantages of journey is simple, is adapted to industrialized production.
4th, embodiment
Following examples are only used for that the present invention is specifically described, but protection scope of the present invention be not limited in it is following
Embodiment.
The method of the present invention for preparing Beta-alanine as raw material multienzyme coupling using maleic acid, is made up of following steps：
(1) by the Alcaligenes faecalis with maleate isomerase activity, Escherichia coli and tool with aspartase activity
The Corynebacterium glutamicum for having aspartic acid-α-decarboxylase is cultivated in the medium respectively, and the maleic acid for producing high activity is different
Structure enzyme, Aspartase and aspartic acid-α-decarboxylase；
(2) culture medium carbon source uses total carbon source mass concentration in glucose or maltose or sucrose or lactose, culture medium to be 1
~30g/L；Nitrogen source uses total nitrogen source quality in beef extract or yeast extract or corn steep liquor or peptone or soya-bean cake hydrolyzate, culture medium
Concentration is 1~30g/L；
(3) mass concentration is adjusted into pH 6~9 for 50~200g/L aqueous maleic acid ammoniacal liquor, be separately added into horse
Carry out the wet thallus or crude enzyme liquid of acid isomer enzyme, Aspartase and aspartic acid-α-decarboxylase, adding mass concentration is
0.01~1.0g/L phosphopyridoxal pyridoxal phosphate, mass concentration for 0.01~1.0g/L tween or cetyl trimethylammonium bromide or
TritonX 100, carries out enzymatic reaction under the conditions of 30~50 DEG C, utilizes isoelectric point crystallizing or isoelectric point crystallizing and ion exchange
The Beta-alanine for the method separation reaction generation that resin is combined.
Embodiment one
1. take the Alcaligenes faecalis Alcaligenes faecalis wet thallus 5g with maleate isomerase activity, with day
Escherichia coli Escherichia coli wet thallus 0.5g of winter propylhomoserin enzymatic activity and with aspartic acid-α-decarboxylase
Corynebacterium glutamicum Corynebacterium glutamicum wet thallus 10g, are added in 1000ml conversion fluids, in conversion fluid
Maleic acid containing 50g/L, 0.01g/L phosphopyridoxal pyridoxal phosphates and 0.01g/L Tween 80s, 6.0,30 DEG C of enzymatic reaction 15h of pH, reaction is eventually
Only.It is 36.1g/L that reaction, which terminates Beta-alanine concentration in rear conversion fluid, is 94% to maleic acid molar yield.
2. conversion fluid 4000r/min centrifugations 15min is removed into thalline, supernatant is warming up to 70~80 DEG C, adds activated carbon
Decolourize, destainer passes through ultrafiltration and nanofiltration purified treatment, scavenging solution is concentrated in vacuo to 40ml, crystallisation by cooling to room temperature, suction filtration, baking
Do to obtain solid Beta-alanine 14g.
3. being separated after crystalline mother solution is diluted by 732 type cation exchange resin columns, Beta-alanine is eluted with 3% ammoniacal liquor
Saturation adsorption column, is concentrated in vacuo to 50ml after collecting the eluent 500ml containing Beta-alanine, activated carbon decolorizing, two is added while hot
Times ethanol of volume 95%, crystallisation by cooling to room temperature, dries to obtain solid Beta-alanine 18g at suction filtration, and crystalline mother solution is followed after reclaiming ethanol
Ring set is used.Crystallization there are Beta-alanine 32g, yield 88.6% twice.
Embodiment two
1. take the Alcaligenes faecalis Alcaligenes faecalis wet thallus 5g with maleate isomerase activity, with day
Escherichia coli Escherichia coli wet thallus 0.5g of winter propylhomoserin enzymatic activity and with aspartic acid-α-decarboxylase
Corynebacterium glutamicum Corynebacterium glutamicum wet thallus 15g, are added in 1000ml conversion fluids, in conversion fluid
Maleic acid containing 100g/L, 7.0,37 DEG C of enzymatic reaction 20h of 0.1g/L phosphopyridoxal pyridoxal phosphates and 0.1g/LTritonX 100, pH, instead
It should terminate.It is 72.9g/L that reaction, which terminates Beta-alanine concentration in rear conversion fluid, is 95% to maleic acid molar yield.
2. conversion fluid 4000r/min centrifugations 15min is removed into thalline, supernatant is warming up to 70~80 DEG C, adds activated carbon
Decolourize, destainer passes through ultrafiltration and nanofiltration purified treatment, scavenging solution is concentrated in vacuo to 80ml, crystallisation by cooling to room temperature, suction filtration, baking
Do to obtain solid Beta-alanine 29g.
3. being separated after crystalline mother solution is diluted by 732 type cation exchange resin columns, Beta-alanine is eluted with 3% ammoniacal liquor
Saturation adsorption column, is concentrated in vacuo to 100ml after collecting the eluent 1100ml containing Beta-alanine, activated carbon decolorizing, adds while hot
Diploid accumulates 95% ethanol, crystallisation by cooling to room temperature, suction filtration, dries to obtain solid Beta-alanine 37g, and crystalline mother solution is reclaimed after ethanol
Recycled.Crystallization there are Beta-alanine 66g, yield 90.5% twice.
Embodiment three
1. take the Alcaligenes faecalis Alcaligenes faecalis wet thallus 10g with maleate isomerase activity, have
Escherichia coli Escherichia coli wet thallus 1.0g of aspartase activity and with aspartic acid-α-decarboxylase
Corynebacterium glutamicum Corynebacterium glutamicum wet thallus 20g, be added in 1000ml conversion fluids, conversion fluid
In maleic acid containing 100g/L, 8.0,42 DEG C of enzymatic reaction 18h of 0.1g/L phosphopyridoxal pyridoxal phosphates and 0.1g/L CTAB, pH, reaction eventually
Only.It is 73.6g/L that reaction, which terminates Beta-alanine concentration in rear conversion fluid, is 96% to maleic acid molar yield.
2. conversion fluid 4000r/min centrifugations 15min is removed into thalline, supernatant is warming up to 70~80 DEG C, adds activated carbon
Decolourize, destainer passes through ultrafiltration and nanofiltration purified treatment, scavenging solution is concentrated in vacuo to 80ml, crystallisation by cooling to room temperature, suction filtration, baking
Do to obtain solid Beta-alanine 29.5g.
3. being separated after crystalline mother solution is diluted by 732 type cation exchange resin columns, Beta-alanine is eluted with 3% ammoniacal liquor
Saturation adsorption column, is concentrated in vacuo to 100ml after collecting the eluent 1100ml containing Beta-alanine, activated carbon decolorizing, adds while hot
Diploid accumulates 95% ethanol, crystallisation by cooling to room temperature, suction filtration, dries to obtain solid Beta-alanine 38g, and crystalline mother solution is reclaimed after ethanol
Recycled.Crystallization there are Beta-alanine 67.5g, yield 91.7% twice.
Example IV
1. take the Alcaligenes faecalis Alcaligenes faecalis wet thallus 10g with maleate isomerase activity, have
Escherichia coli Escherichia coli wet thallus 1.0g of aspartase activity and with aspartic acid-α-decarboxylase
Corynebacterium glutamicum Corynebacterium glutamicum wet thallus 25g, be added in 1000ml conversion fluids, conversion fluid
In maleic acid containing 150g/L, 0.5g/L phosphopyridoxal pyridoxal phosphates and 0.5g/L Tween 80s, 7.0,40 DEG C of enzymatic reaction 22h of pH, reaction eventually
Only.It is 110.5g/L that reaction, which terminates Beta-alanine concentration in rear conversion fluid, is 96% to maleic acid molar yield.
2. conversion fluid 4000r/min centrifugations 15min is removed into thalline, supernatant is warming up to 70~80 DEG C, adds activated carbon
Decolourize, destainer passes through ultrafiltration and nanofiltration purified treatment, scavenging solution is concentrated in vacuo to 120ml, crystallisation by cooling to room temperature, suction filtration,
Dry to obtain solid Beta-alanine 44g.
3. being separated after crystalline mother solution is diluted by 732 type cation exchange resin columns, Beta-alanine is eluted with 3% ammoniacal liquor
Saturation adsorption column, is concentrated in vacuo to 150ml after collecting the eluent 1600ml containing Beta-alanine, activated carbon decolorizing, adds while hot
Diploid accumulates 95% ethanol, crystallisation by cooling to room temperature, suction filtration, dries to obtain solid Beta-alanine 55.8g, and crystalline mother solution reclaims ethanol
Posterior circle is applied mechanically.Crystallization there are Beta-alanine 99.8g, yield 90.3% twice.
Embodiment five
1. take the Alcaligenes faecalis Alcaligenes faecalis wet thallus 15g with maleate isomerase activity, have
Escherichia coli Escherichia coli wet thallus 1.0g of aspartase activity and with aspartic acid-α-decarboxylase
Corynebacterium glutamicum Corynebacterium glutamicum wet thallus 20g, be added in 1000ml conversion fluids, conversion fluid
In maleic acid containing 150g/L, 0.3g/L phosphopyridoxal pyridoxal phosphates and 0.3g/L Tween 80s, 6.0,40 DEG C of enzymatic reaction 25h of pH, reaction eventually
Only.It is 110.6g/L that reaction, which terminates Beta-alanine concentration in rear conversion fluid, is 96% to maleic acid molar yield.
2. conversion fluid 4000r/min centrifugations 15min is removed into thalline, supernatant is warming up to 70~80 DEG C, adds activated carbon
Decolourize, destainer passes through ultrafiltration and nanofiltration purified treatment, scavenging solution is concentrated in vacuo to 120ml, crystallisation by cooling to room temperature, suction filtration,
Dry to obtain solid Beta-alanine 44.8g.
3. being separated after crystalline mother solution is diluted by 732 type cation exchange resin columns, Beta-alanine is eluted with 3% ammoniacal liquor
Saturation adsorption column, is concentrated in vacuo to 150ml after collecting the eluent 1600ml containing Beta-alanine, activated carbon decolorizing, adds while hot
Diploid accumulates 95% ethanol, crystallisation by cooling to room temperature, suction filtration, dries to obtain solid Beta-alanine 55.4g, and crystalline mother solution reclaims ethanol
Posterior circle is applied mechanically.Crystallization there are Beta-alanine 100.2g, yield 90.6% twice.
Embodiment six
1. take the Alcaligenes faecalis Alcaligenes faecalis wet thallus 12g with maleate isomerase activity, have
Escherichia coli Escherichia coli wet thallus 0.8g of aspartase activity and with aspartic acid-α-decarboxylase
Corynebacterium glutamicum Corynebacterium glutamicum wet thallus 15g, be added in 1000ml conversion fluids, conversion fluid
In maleic acid containing 150g/L, 0.3g/L phosphopyridoxal pyridoxal phosphates and 0.3g/L Tween 80s, 9.0,50 DEG C of enzymatic reaction 25h of pH, reaction eventually
Only.It is 107g/L that reaction, which terminates Beta-alanine concentration in rear conversion fluid, is 93% to maleic acid molar yield.
2. conversion fluid 4000r/min centrifugations 15min is removed into thalline, supernatant is warming up to 70~80 DEG C, adds activated carbon
Decolourize, destainer passes through ultrafiltration and nanofiltration purified treatment, scavenging solution is concentrated in vacuo to 120ml, crystallisation by cooling to room temperature, suction filtration,
Dry to obtain solid Beta-alanine 42.2g.
3. being separated after crystalline mother solution is diluted by 732 type cation exchange resin columns, Beta-alanine is eluted with 3% ammoniacal liquor
Saturation adsorption column, is concentrated in vacuo to 150ml after collecting the eluent 1600ml containing Beta-alanine, activated carbon decolorizing, adds while hot
Diploid accumulates 95% ethanol, crystallisation by cooling to room temperature, suction filtration, dries to obtain solid Beta-alanine 53.4g, and crystalline mother solution reclaims ethanol
Posterior circle is applied mechanically.Crystallization there are Beta-alanine 95.6g, yield 89.3% twice.
Embodiment seven
1. take the Alcaligenes faecalis Alcaligenes faecalis wet thallus 20g with maleate isomerase activity, have
Escherichia coli Escherichia coli wet thallus 1.0g of aspartase activity and with aspartic acid-α-decarboxylase
Corynebacterium glutamicum Corynebacterium glutamicum wet thallus 30g, be added in 1000ml conversion fluids, conversion fluid
In maleic acid containing 200g/L, 7.0,40 DEG C of enzymatic reaction 29h of 1.0g/L phosphopyridoxal pyridoxal phosphates and 1.0g/L TritonX 100, pH,
Reaction terminating.It is 145.8g/L that reaction, which terminates Beta-alanine concentration in rear conversion fluid, is 95% to maleic acid molar yield.
2. conversion fluid 4000r/min centrifugations 15min is removed into thalline, supernatant is warming up to 70~80 DEG C, adds activated carbon
Decolourize, destainer passes through ultrafiltration and nanofiltration purified treatment, scavenging solution is concentrated in vacuo to 160ml, crystallisation by cooling to room temperature, suction filtration,
Dry to obtain solid Beta-alanine 59.2g.
3. being separated after crystalline mother solution is diluted by 732 type cation exchange resin columns, Beta-alanine is eluted with 3% ammoniacal liquor
Saturation adsorption column, is concentrated in vacuo to 200ml after collecting the eluent 2200ml containing Beta-alanine, activated carbon decolorizing, adds while hot
Diploid accumulates 95% ethanol, crystallisation by cooling to room temperature, suction filtration, dries to obtain solid Beta-alanine 72.2g, and crystalline mother solution reclaims ethanol
Posterior circle is applied mechanically.Crystallization there are Beta-alanine 131.4g, yield 90.1% twice.
Embodiment eight
1. take the Alcaligenes faecalis Alcaligenes faecalis wet thallus 20g with maleate isomerase activity, have
Escherichia coli Escherichia coli wet thallus 1.0g of aspartase activity and with aspartic acid-α-decarboxylase
Corynebacterium glutamicum Corynebacterium glutamicum wet thallus 40g, be added in 1000ml conversion fluids, conversion fluid
In maleic acid containing 200g/L, 6.0,42 DEG C of enzymatic reaction 24h of 1.0g/L phosphopyridoxal pyridoxal phosphates and 1.0g/L CTAB, pH, reaction eventually
Only.It is 147.4g/L that reaction, which terminates Beta-alanine concentration in rear conversion fluid, is 96% to maleic acid molar yield.
2. conversion fluid 4000r/min centrifugations 15min is removed into thalline, supernatant is warming up to 70~80 DEG C, adds activated carbon
Decolourize, destainer passes through ultrafiltration and nanofiltration purified treatment, scavenging solution is concentrated in vacuo to 160ml, crystallisation by cooling to room temperature, suction filtration,
Dry to obtain solid Beta-alanine 60.3g.
3. being separated after crystalline mother solution is diluted by 732 type cation exchange resin columns, Beta-alanine is eluted with 3% ammoniacal liquor
Saturation adsorption column, is concentrated in vacuo to 200ml after collecting the eluent 2200ml containing Beta-alanine, activated carbon decolorizing, adds while hot
Diploid accumulates 95% ethanol, crystallisation by cooling to room temperature, suction filtration, dries to obtain solid Beta-alanine 73.9g, and crystalline mother solution reclaims ethanol
Posterior circle is applied mechanically.Crystallization there are Beta-alanine 134.2g, yield 91% twice.
Claims (1)
1. a kind of method for preparing Beta-alanine as raw material multienzyme coupling using maleic acid, its feature is made up of following steps：
(1) by the Alcaligenes faecalis with maleate isomerase activity, Escherichia coli with aspartase activity and with day
The Corynebacterium glutamicum of winter propylhomoserin-α-decarboxylase is cultivated in the medium respectively, the maleate isomerase of generation high activity,
Aspartase and aspartic acid-α-decarboxylase；
(2) culture medium carbon source uses glucose or maltose or sucrose or lactose, in culture medium total carbon source mass concentration be 1~
30g/L；Nitrogen source uses total nitrogen source quality in beef extract or yeast extract or corn steep liquor or peptone or soya-bean cake hydrolyzate, culture medium dense
Spend for 1~30g/L；
(3) mass concentration is adjusted into pH 6~9 for 50~200g/L aqueous maleic acid ammoniacal liquor, be separately added into maleic acid
The wet thallus or crude enzyme liquid of isomerase, Aspartase and aspartic acid-α-decarboxylase, it is 0.01 to add mass concentration
~1.0g/L phosphopyridoxal pyridoxal phosphate, mass concentration for 0.01~1.0g/L tween or cetyl trimethylammonium bromide or
TritonX 100, carries out enzymatic reaction under the conditions of 30~50 DEG C, utilizes isoelectric point crystallizing or isoelectric point crystallizing and ion exchange
The Beta-alanine for the method separation reaction generation that resin is combined.
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|---|---|---|---|---|
| CN1235641A (en) * | 1996-11-01 | 1999-11-17 | 索罗蒂亚公司 | Improved preparation of L-aspartic acid |
| CN1626665A (en) * | 2003-12-10 | 2005-06-15 | 浙江工业大学 | Biology method for synthesizing beta alanine |
| CN103320480A (en) * | 2013-06-25 | 2013-09-25 | 南京大学 | Method for preparing beta-alanine by coupled enzymatic reaction |
| CN104531820A (en) * | 2015-01-05 | 2015-04-22 | 南京大学 | Method for preparing DL-alanine with fumaric acid as raw material through multi-enzyme coupling |
-
2017
- 2017-06-01 CN CN201710404758.5A patent/CN107012180A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1235641A (en) * | 1996-11-01 | 1999-11-17 | 索罗蒂亚公司 | Improved preparation of L-aspartic acid |
| CN1626665A (en) * | 2003-12-10 | 2005-06-15 | 浙江工业大学 | Biology method for synthesizing beta alanine |
| CN103320480A (en) * | 2013-06-25 | 2013-09-25 | 南京大学 | Method for preparing beta-alanine by coupled enzymatic reaction |
| CN104531820A (en) * | 2015-01-05 | 2015-04-22 | 南京大学 | Method for preparing DL-alanine with fumaric acid as raw material through multi-enzyme coupling |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110724680A (en) * | 2019-10-29 | 2020-01-24 | 天津大学 | 3-phenylmaleic acid isomerase and application thereof |
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