CN105543292A - Microbial transformation method - Google Patents
Microbial transformation method Download PDFInfo
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- CN105543292A CN105543292A CN201610085608.8A CN201610085608A CN105543292A CN 105543292 A CN105543292 A CN 105543292A CN 201610085608 A CN201610085608 A CN 201610085608A CN 105543292 A CN105543292 A CN 105543292A
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- Prior art keywords
- levodopa
- proteus
- thalline
- liquid
- salvianic acida
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- 230000000813 microbial effect Effects 0.000 title claims description 5
- 238000011426 transformation method Methods 0.000 title 1
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 claims abstract description 40
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 claims abstract description 40
- 229960004502 levodopa Drugs 0.000 claims abstract description 39
- 238000000034 method Methods 0.000 claims abstract description 17
- 241000588769 Proteus <enterobacteria> Species 0.000 claims abstract description 15
- PAFLSMZLRSPALU-MRVPVSSYSA-N (2R)-3-(3,4-dihydroxyphenyl)lactic acid Chemical compound OC(=O)[C@H](O)CC1=CC=C(O)C(O)=C1 PAFLSMZLRSPALU-MRVPVSSYSA-N 0.000 claims abstract description 7
- 238000004519 manufacturing process Methods 0.000 claims abstract 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 30
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 11
- 230000001580 bacterial effect Effects 0.000 claims description 4
- 230000009466 transformation Effects 0.000 claims description 4
- 241000588770 Proteus mirabilis Species 0.000 claims description 3
- 241000588767 Proteus vulgaris Species 0.000 claims description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 3
- 239000004310 lactic acid Substances 0.000 claims description 3
- 244000005700 microbiome Species 0.000 claims description 3
- 239000001301 oxygen Substances 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- 229940007042 proteus vulgaris Drugs 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 abstract description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 28
- 238000006243 chemical reaction Methods 0.000 description 28
- 239000007788 liquid Substances 0.000 description 26
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 20
- 239000012530 fluid Substances 0.000 description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 17
- 235000011187 glycerol Nutrition 0.000 description 14
- 239000007791 liquid phase Substances 0.000 description 12
- 230000001954 sterilising effect Effects 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 229910019142 PO4 Inorganic materials 0.000 description 10
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 10
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 10
- 239000011790 ferrous sulphate Substances 0.000 description 10
- 235000003891 ferrous sulphate Nutrition 0.000 description 10
- 238000001914 filtration Methods 0.000 description 10
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 10
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 10
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 10
- 235000019341 magnesium sulphate Nutrition 0.000 description 10
- 239000000203 mixture Substances 0.000 description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 10
- 239000010452 phosphate Substances 0.000 description 10
- 239000011591 potassium Substances 0.000 description 10
- 229910052700 potassium Inorganic materials 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 239000008223 sterile water Substances 0.000 description 10
- 238000004587 chromatography analysis Methods 0.000 description 9
- 239000012982 microporous membrane Substances 0.000 description 9
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 8
- 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 7
- 239000008103 glucose Substances 0.000 description 7
- 238000000855 fermentation Methods 0.000 description 6
- 230000004151 fermentation Effects 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 239000001888 Peptone Substances 0.000 description 5
- 108010080698 Peptones Proteins 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 235000019319 peptone Nutrition 0.000 description 5
- -1 phenolic acid compound Chemical class 0.000 description 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 4
- 239000004202 carbamide Substances 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 230000003068 static effect Effects 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 240000007164 Salvia officinalis Species 0.000 description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 3
- 235000011130 ammonium sulphate Nutrition 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000004237 preparative chromatography Methods 0.000 description 3
- 235000005412 red sage Nutrition 0.000 description 3
- 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 description 2
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 2
- SNKFFCBZYFGCQN-UHFFFAOYSA-N 2-[3-[3-[1-carboxy-2-(3,4-dihydroxyphenyl)ethoxy]carbonyl-2-(3,4-dihydroxyphenyl)-7-hydroxy-2,3-dihydro-1-benzofuran-4-yl]prop-2-enoyloxy]-3-(3,4-dihydroxyphenyl)propanoic acid Chemical compound C=1C=C(O)C=2OC(C=3C=C(O)C(O)=CC=3)C(C(=O)OC(CC=3C=C(O)C(O)=CC=3)C(O)=O)C=2C=1C=CC(=O)OC(C(=O)O)CC1=CC=C(O)C(O)=C1 SNKFFCBZYFGCQN-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 229930091371 Fructose Natural products 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- SNKFFCBZYFGCQN-VWUOOIFGSA-N Lithospermic acid B Natural products C([C@H](C(=O)O)OC(=O)\C=C\C=1C=2[C@H](C(=O)O[C@H](CC=3C=C(O)C(O)=CC=3)C(O)=O)[C@H](OC=2C(O)=CC=1)C=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 SNKFFCBZYFGCQN-VWUOOIFGSA-N 0.000 description 2
- 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 description 2
- 241000806018 Proteus hauseri ATCC 700826 Species 0.000 description 2
- 241001069992 Proteus mirabilis ATCC 29906 Species 0.000 description 2
- 235000011135 Salvia miltiorrhiza Nutrition 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- SRBFZHDQGSBBOR-LECHCGJUSA-N alpha-D-xylose Chemical compound O[C@@H]1CO[C@H](O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-LECHCGJUSA-N 0.000 description 2
- 235000019270 ammonium chloride Nutrition 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 229960000448 lactic acid Drugs 0.000 description 2
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 2
- 238000007833 oxidative deamination reaction Methods 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000000419 plant extract Substances 0.000 description 2
- 244000132619 red sage Species 0.000 description 2
- STCJJTBMWHMRCD-UHFFFAOYSA-N salvianolic acid B Natural products OC(=O)C(Cc1ccc(O)c(O)c1)OC(=O)C=Cc2cc(O)c(O)c3OC(C(C(=O)OC(Cc4ccc(O)c(O)c4)C(=O)O)c23)c5ccc(O)c(O)c5 STCJJTBMWHMRCD-UHFFFAOYSA-N 0.000 description 2
- 229960003487 xylose Drugs 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 241001087672 Cosenzaea myxofaciens Species 0.000 description 1
- PAFLSMZLRSPALU-QMMMGPOBSA-N Danshensu Natural products OC(=O)[C@@H](O)CC1=CC=C(O)C(O)=C1 PAFLSMZLRSPALU-QMMMGPOBSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 102000008109 Mixed Function Oxygenases Human genes 0.000 description 1
- 108010074633 Mixed Function Oxygenases Proteins 0.000 description 1
- 241001076189 Proteus hauseri Species 0.000 description 1
- 241001472782 Proteus penneri Species 0.000 description 1
- PAFLSMZLRSPALU-UHFFFAOYSA-N Salvianic acid A Natural products OC(=O)C(O)CC1=CC=C(O)C(O)=C1 PAFLSMZLRSPALU-UHFFFAOYSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 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 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 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 description 1
- 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 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 230000007073 chemical hydrolysis Effects 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000010813 municipal solid waste Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 229940055033 proteus mirabilis Drugs 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000005204 segregation Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000012138 yeast extract Substances 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/42—Hydroxy-carboxylic acids
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for producing R-(+)-3-(3,4-dihydroxyphenyl)-2-hydroxypropanoic acid by transforming levodopa by adopting proteus microbes. The method is simple in process and has important industrial application value.
Description
Technical field
The present invention adopts Bacillus proteus to transform levodopa and produces Salvianic acidA, belongs to industrial microorganism field.
Background technology
Salvianic acidA, formal name used at school R-(+)-3-(3,4-dihydroxy phenyl)-2 hydroxy propanoic acid, D-(+)-β-(3,4-dihydroxy phenyl) lactic acid, English by name: Danshensu, (R)-(+)-3-(3,4-Dihydroxyphenyl)-lacticacid, (R)-(+)-3-(3,4-Dihydroxyphenyl)-2-hydroxypropanoicacid is a kind of dextrorotation phenolic acid compound.
Salvianic acidA is that important in Salvia miltiorrhiza Bge water extract wants effective ingredient, domesticly obtained from Salvia miltiorrhiza Bge water extract in 1980 and identify the structure (research of Determination of water-soluble active constituents of radix, II .D (+) β (3,4-dihydroxy phenyl) structure of lactic acid, Shanghai first medical college journal, 1980,05 (7), 384-385), various research shows that Salvianic acidA has important pharmacological effect effect, in the treatment etc. of cardiovascular and cerebrovascular diseases, have unique treatment effect.
Current Salvianic acidA mainly extracts and obtains (patent CN200810038853.9) from the red sage root.The content of Salvianic acidA in the red sage root is lower, and the high and limits throughput of Salvianic acidA planting cost, and therefore not only price is high but also cannot meet the demand in market far away for current Salvianic acidA.Patent CN201310559498.0 proposes a kind of bacillus coli gene schedule drawing that builds and utilizes glucose fermentation to produce the method for Salvianic acidA, hydroxylase is utilized because metabolic pathway of synthesizing relates to, this enzyme is easy to metabolic process product is oxidized and affects the productive rate of Salvianic acidA, simultaneously because Escherichia coli fermentation is high oxygen process, also Salvianic acidA can be oxidized, therefore current the method productive rate is lower, and cost will higher than plant extract process.Patent CN201210190171.6 proposes the method that hydrolysis salvianolic acid B produces Salvianic acidA, and salvianolic acid B need extract from the red sage root, and chemical hydrolysis process has a large amount of side reaction, is not suitable for large-scale production equally.The catalyzer of chiral synthesize Salvianic acidA (patent CN201210420488.4) is very expensive, currently also only rests on laboratory level.
Based on the significant application value of Salvianic acidA, this patent proposes the scheme adopting proteus microbial transformation levodopa to produce Salvianic acidA.Levodopa is also named: L-3-(3,4-dihydroxy phenyl) L-Ala (L-DOPA, L-3,4-dihydroxyphenylalanine).Current levodopa also can produce (OverviewonthebiotechnologicalproductionofL-DOPA, 2015, ApplMicrobiolBiotechnol, 99:575 – 584) by fermentable from plant extract, and abundant raw material is easy to get.
Bacillus proteus (Proteus) is extensively distributed in occurring in nature, as the microorganism in the enteron aisle of soil, water, rubbish, spoilage organism and human or animal.Existing 5 kinds: proteus vulgaris (Proteusvulgaris), Proteus mirabilis (Proteusmirabilis), the glutinous Bacillus proteus (Proteusmyxofaciens) of product, Pan Shi Bacillus proteus (Proteuspenneri) and Hao Shi Bacillus proteus (Proteushauseri).Bacillus proteus has powerful oxidative deamination ability (SherrisMedicalMicrobiology, 2004,4thed.), levodopa can be deformed bacillus oxidative deamination and become 3,4-dihydroxyphenyl pyruvic acid (3-(3,4-dihydroxyphenyl)-2-oxopropanoicacid), and then be reduced into Salvianic acidA.3, although 4-dihydroxyphenyl pyruvic acid is more direct substrate, expensive and unstable, therefore levodopa is best substrate.Bacillus proteus is a kind of facultative anaerobe, can transform and produce Salvianic acidA, have high conversion and highly purified feature under anaerobic condition or aerobic condition.
Summary of the invention
The present invention is by cultivating proteus microbial cells, and transform levodopa and produce Salvianic acidA, technical scheme is as follows:
1, bacterial strain
The present invention's bacterial strain used has the ProteusmirabilisATCC29906 of purchased from American ATCC strain library, ProteusmyxofaciensATCC19692, ProteushauseriATCC700826, ProteuspenneriATCC33519, ProteusmirabilisATCC25933, ProteusmirabilisATCC33946, ProteusvulgarisATCC19181, ProteusvulgarisATCC27972 and the ProteusvulgarisCICC10401 purchased from Chinese industrial Microbiological Culture Collection administrative center, ProteusmirabilisCICC22928.
2, yeast culture
Liquid state fermentation substratum consists of: carbon source 0-50g/L, nitrogenous source 0-50g/L, Secondary ammonium phosphate 0-2g/L, potassium primary phosphate 0-1g/L, magnesium sulfate 0-0.5g/L, ferrous sulfate 0-0.5g/L, sodium chloride 0-5g/L, adjustable pH to 2-8, can also pH nature; Inoculum size is 5-20%, and leavening temperature is 20-40 DEG C, and fermentation period is 24-72 hour.Seed and fermentation all adopt this substratum.
Can be glucose, fructose, maltose, wood sugar, sucrose, semi-lactosi, glycerine for cultivating the carbon source of bacterial classification.Cultivation nitrogenous source can be the organic nitrogen sources such as ammonium sulfate, ammonium chloride, ammonium nitrate, peptone, yeast extract paste, urea, corn steep liquor.Carbon and nitrogen sources can be one or more combination.
Liquid process of cultivating thalline can adopt anaerobism or aerobic mode.
3, product Salvianic acidA is transformed
The scheme that conversion process adopts has 2 kinds:
(1) the liquid culturing process of cell, adds levodopa substrate in above-mentioned culture system; Levodopa addition is 0-3g/L.
(2) liquid state fermentation culture centrifugal segregation fermented liquid is obtained thalline, the reactant aqueous solution system put into by thalline containing levodopa substrate is carried out; Conversion process temperature 20-40 DEG C, pH2-8, transformation time 1-24 hour.In conversion fluid, levodopa initial concentration is 0.1-3g/L.
4, the detection analysis of sample
Conversion fluid adopts PerkinElmerSeries200 high performance liquid chromatograph to detect and analyzes, chromatographic condition is: moving phase is methyl alcohol-0.1% formic acid water (40:60), adopts Chinese nation MegresC18 chromatographic column (4.6 × 250mm, 5 μm), flow velocity 0.6ml/min, column temperature 30 DEG C, sample size 20 μ l, detector wavelength 280nm.
Adopt the DAC-HB50 preparative chromatography Column preparation of Hanbon Sci. & Tech. Co., Ltd. to transform sample, preparative chromatography condition is: moving phase 50% methyl alcohol, column temperature nature, flow velocity 3ml/min, sample size 5ml.Sample reaches chromatographically pure 99.9%, and sample introduction is separated the product vacuum rotating evaporate to dryness at 50 DEG C obtained repeatedly.Take the sample 0.5g prepared, to be dissolved in deionized water and to be settled to 50ml, adopt Japan's full-automatic polarimeter of AP-300 of liking to delay to measure degree of giving out light.Adopt the nuclear magnetic data of VarianGemini2000 (VnmrS600MHz, 600/54/ASP) analytic sample further.Sample adopts UPLC-QTOF-MS method analyzing molecules amount further, and instrument is WatersMALDISYNAPTQTOFMS liquid chromatography tandem quadrupole time-of-flight mass spectrometer.
Embodiment
Embodiment 1
The analysis of converted product measures.
Preparation substratum is as follows: yeast saccharomyces cream 5g/L, peptone 10g/L, sodium-chlor 5g/L.In 500ml triangular flask, liquid amount is 100ml, totally 50 bottles, 120 DEG C, sterilizing in 20 minutes.Get ProteusmirabilisATCC29906 glycerine pipe seed liquor 1mL to inoculate, leavening temperature 30 DEG C, shaking speed 200rpm.After cultivating 24, by centrifugal for fermented liquid (rotating speed 10000rpm, time 10min), remove supernatant liquor and obtain thalline, the thalline sterile water wash of centrifugal acquisition twice.Get 20g wet thallus and put into the 1000ml solution that levodopa concentration is 2g/L, mix.In 37 DEG C of shaking tables vibration (100rpm) after 24 hours, 100 DEG C of heating in water bath kill thalline in 3 minutes.Centrifugal (rotating speed 10000rpm, time 10min) removes thalline again, obtains the supernatant liquor after transforming.Liquid phase analysis Salvianic acidA concentration is 1.3g/L, and residue levodopa concentration is 0.5g/L.Preparative chromatography is adopted to obtain 1.03g sterling.
Its specific rotation analyzed by polarimeter
nuclear magnetic data is: 1HNMR (DMSO-d6,600MHz): δ 8.76 (s, 1H), 6.63 (dd, J=24Hz, 1H), 6.64 (dd, J=12Hz, 1H), 6.45 (dd, J=24Hz, 1H), 5.48 (s, 1H), 4.06-4.17 (m, 1H), 2.61-2.81 (m, 1H); 13CNMR (DMSO-d6,600MHz): δ 175.4,174.2,144.9,144.7,143.8,143.6,128.9,128.5,120.4,120.2,117.0,116.8,115.4,115.2,71.6,71.6,51.4.The mass-spectrometric data of converted product is: (-ESI, negativemode) m/z:197.0 [M-1].
According to above data and pertinent literature (Stereochemicalconfigurationof β-(3,4-dihydroxyphenyl) lacticacidfromRosmarinusofficinalis.RicereaSci.80:255-25 9. [Chem.Abs.54:24520.1960], the research of Determination of water-soluble active constituents of radix, II .D (+) β (3,4-dihydroxy phenyl) structure of lactic acid, Shanghai first medical college journal, 1980,05 (7), 384-385), determine that its molecule and optical texture and natural Salvianic acidA are completely the same.
Embodiment 2
Aerobic cultivation with detest comparing of cultivation.Preparation substratum: glucose 30g/L, peptone 20g/L, Secondary ammonium phosphate 1g/L, potassium primary phosphate 1g/L, magnesium sulfate 0.3g/L, ferrous sulfate 0.3g/L; It is 100ml that initial pH and fermenting process pH is liquid amount in nature 500ml triangular flask, totally 2 bottles, 120 DEG C, sterilizing in 20 minutes.
Get ProteusmirabilisCICC22928 glycerine pipe seed liquor 1mL and inoculate 1 bottle, cultivate 72 hours for 35 DEG C in anaerobic culture box, the thalline sterile water wash of centrifugal acquisition twice.Get 0.1g wet thallus and put into the 4ml solution that levodopa concentration is 2g/L, mix, in anaerobic culture box, 35 DEG C of standing conversions are after 24 hours, and 100 DEG C of heating in water bath kill thalline in 3 minutes.Centrifugal (rotating speed 10000rpm, time 10min) removes thalline again, obtains the supernatant liquor after transforming.Liquid phase analysis Salvianic acidA concentration is 1.1g/L, and residue levodopa concentration is 0.6g/L.
Get ProteusmirabilisCICC22928 glycerine pipe seed liquor 1mL and inoculate 1 bottle, cultivate 24 hours for 35 DEG C in shaking table (200rpm).By centrifugal for fermented liquid (rotating speed 10000rpm, time 10min), remove supernatant liquor and obtain thalline, the thalline sterile water wash of centrifugal acquisition twice.Get 0.1g wet thallus and put into the 4ml solution that levodopa concentration is 2g/L, mix.In 35 DEG C of shaking tables vibration (100rpm) after 12 hours, 100 DEG C of heating in water bath kill thalline in 3 minutes.Centrifugal (rotating speed 10000rpm, time 10min) removes thalline again, obtains the supernatant liquor after transforming.Liquid phase analysis Salvianic acidA concentration is 1.0g/L, and residue levodopa concentration is 0.5g/L.
Embodiment 3
Substratum consists of: glucose 1g/L, urea 1g/L, Secondary ammonium phosphate 1g/L, potassium primary phosphate 0.1g/L, magnesium sulfate 0.1g/L, ferrous sulfate 0.1g/L.In 250ml triangular flask, liquid amount is 50ml, 120 DEG C, sterilizing in 20 minutes.Get ProteusvulgarisATCC19181 glycerine pipe seed liquor 0.5mL to inoculate, leavening temperature 35 DEG C, shaking speed 200rpm.After cultivating 24, by centrifugal for fermented liquid (rotating speed 10000rpm, time 10min), remove supernatant liquor and obtain thalline, the thalline sterile water wash of centrifugal acquisition twice.Get 0.05g wet thallus and put into the 2ml solution that levodopa concentration is 0.1g/L, and regulate pH to 6, mix.Static conversion 24 hours in anaerobic culture box, filtering with microporous membrane conversion fluid, in liquid-phase chromatographic analysis conversion fluid, Salvianic acidA concentration is 0.08g/L and levodopa residual quantity is 0g/L.
Embodiment 4
Substratum consists of: fructose 50g/L, ammonium sulfate 5g/L, Secondary ammonium phosphate 1g/L, potassium primary phosphate 1g/L, magnesium sulfate 0.5g/L, ferrous sulfate 0.5g/L.In 250ml triangular flask, liquid amount is 50ml, 120 DEG C, sterilizing in 20 minutes.Get ProteushauseriATCC700826 glycerine pipe seed liquor 0.5mL to inoculate, leavening temperature 20 DEG C, shaking speed 200rpm.After cultivating 72, by centrifugal for fermented liquid (rotating speed 10000rpm, time 10min), remove supernatant liquor and obtain thalline, the thalline sterile water wash of centrifugal acquisition twice.Get 0.1g wet thallus and put into the 2ml solution that levodopa concentration is 2g/L, mix.In 20 DEG C of shaking tables vibration (100rpm) after 24 hours, filtering with microporous membrane conversion fluid, in liquid-phase chromatographic analysis conversion fluid, Salvianic acidA concentration is 0.8g/L and levodopa residual quantity is 0.6g/L.
Embodiment 5
Substratum consists of: wood sugar 50g/L, ammonium sulfate 1g/L, Secondary ammonium phosphate 1g/L, potassium primary phosphate 1g/L, magnesium sulfate 0.2g/L, ferrous sulfate 0.1g/L.In 250ml triangular flask, liquid amount is 50ml, 120 DEG C, sterilizing in 20 minutes.Get ProteusvulgarisCICC10401 glycerine pipe seed liquor 0.5mL to inoculate, leavening temperature 40 DEG C, shaking speed 200rpm.After cultivating 48, by centrifugal for fermented liquid (rotating speed 10000rpm, time 10min), remove supernatant liquor and obtain thalline, the thalline sterile water wash of centrifugal acquisition twice.Get 0.1g wet thallus and put into the 2ml solution that levodopa concentration is 2g/L, mix.In 40 DEG C of shaking tables vibration (100rpm) after 24 hours, filtering with microporous membrane conversion fluid, in liquid-phase chromatographic analysis conversion fluid, Salvianic acidA concentration is 0.5g/L and levodopa residual quantity is 1.3g/L.
Embodiment 6
Substratum consists of: glycerine 15g/L, ammonium nitrate 1g/L, Secondary ammonium phosphate 1g/L, potassium primary phosphate 0.1g/L, magnesium sulfate 0.1g/L, ferrous sulfate 0.1g/L.In 250ml triangular flask, liquid amount is 50ml, 120 DEG C, sterilizing in 20 minutes.Get ProteusmyxofaciensATCC19692 glycerine pipe seed liquor 0.5mL to inoculate, leavening temperature 30 DEG C, shaking speed 200rpm.After cultivating 24, by centrifugal for fermented liquid (rotating speed 10000rpm, time 10min), remove supernatant liquor and obtain thalline, the thalline sterile water wash of centrifugal acquisition twice.Get 0.1g wet thallus and put into the 2ml solution that levodopa concentration is 2g/L, mix.Static conversion 24 hours in 20 DEG C of anaerobic culture boxes, filtering with microporous membrane conversion fluid, in liquid-phase chromatographic analysis conversion fluid, Salvianic acidA concentration is 0.7g/L and levodopa residual quantity is 1.0g/L.
Embodiment 7
Substratum consists of: maltose 15g/L, ammonium chloride 1g/L, Secondary ammonium phosphate 1g/L, potassium primary phosphate 0.1g/L, magnesium sulfate 0.1g/L, ferrous sulfate 0.1g/L, regulates pH to 5.In 250ml triangular flask, liquid amount is 50ml, 120 DEG C, sterilizing in 20 minutes.Get ProteusvulgarisATCC27972 glycerine pipe seed liquor 0.5mL to inoculate, leavening temperature 35 DEG C, shaking speed 200rpm.After cultivating 24, by centrifugal for fermented liquid (rotating speed 10000rpm, time 10min), remove supernatant liquor and obtain thalline, the thalline sterile water wash of centrifugal acquisition twice.Get 0.1g wet thallus and put into the 2ml solution that levodopa concentration is 3g/L, mix.Static conversion 24 hours in 40 DEG C of anaerobic culture boxes, filtering with microporous membrane conversion fluid, in liquid-phase chromatographic analysis conversion fluid, Salvianic acidA concentration is 1.1g/L and levodopa residual quantity is 1.6g/L.
Embodiment 8
Substratum consists of: semi-lactosi 1g/L, urea 1g/L, Secondary ammonium phosphate 1g/L, magnesium sulfate 0.1g/L, ferrous sulfate 0.1g/L, regulates pH to 2.In 250ml triangular flask, liquid amount is 50ml, 120 DEG C, sterilizing in 20 minutes.Get ProteusvulgarisATCC25933 glycerine pipe seed liquor 0.5mL to inoculate, leavening temperature 35 DEG C, shaking speed 200rpm.After cultivating 24, by centrifugal for fermented liquid (rotating speed 10000rpm, time 10min), remove supernatant liquor and obtain thalline, the thalline sterile water wash of centrifugal acquisition twice.Get 0.1g wet thallus and put into the 2ml solution that levodopa concentration is 2g/L, and regulate pH to 2, mix.Static conversion 24 hours in 37 DEG C of anaerobic culture boxes, filtering with microporous membrane conversion fluid, in liquid-phase chromatographic analysis conversion fluid, Salvianic acidA concentration is 0.3g/L and levodopa residual quantity is 1.4g/L.
Embodiment 9
Substratum consists of: glucose 5g/L, peptone 1g/L, potassium primary phosphate 0.1g/L, magnesium sulfate 0.1g/L, ferrous sulfate 0.1g/L, regulates pH to 8.In 250ml triangular flask, liquid amount is 50ml, 120 DEG C, sterilizing in 20 minutes.The levodopa adding filtration sterilization makes substratum degree at end reach 2g/L, and ProteusmirabilisATCC33946 glycerine pipe seed liquor 0.5mL inoculates, leavening temperature 35 DEG C, shaking speed 200rpm.After cultivating 24, filtering with microporous membrane conversion fluid, in liquid-phase chromatographic analysis conversion fluid, Salvianic acidA concentration is 1g/L and levodopa residual quantity is 0.2g/L.
Embodiment 10
Substratum consists of: glucose 10g/L, urea 1g/L, Secondary ammonium phosphate 1g/L, potassium primary phosphate 0.1g/L, ferrous sulfate 0.1g/L, regulates pH to 8.In 500ml triangular flask, liquid amount is 100ml, 120 DEG C, sterilizing in 20 minutes, totally 10 bottles.Respectively get ProteuspenneriATCC33519 glycerine pipe seed liquor 0.5mL to inoculate, leavening temperature 35 DEG C, shaking speed 200rpm.After cultivating 24, by centrifugal for fermented liquid (rotating speed 10000rpm, time 10min), remove supernatant liquor and obtain thalline, the thalline sterile water wash of centrifugal acquisition twice.Get 5g wet thallus and put into the 10ml solution that levodopa concentration is 3g/L, and regulate pH to 5, mix.In 35 DEG C of shaking tables vibration (100rpm) after 1 hour, filtering with microporous membrane conversion fluid, in liquid-phase chromatographic analysis conversion fluid, Salvianic acidA concentration is 2.3g/L and levodopa residual quantity is 0.5g/L.
Embodiment 11
Substratum consists of: glucose 10g/L, peptone 1g/L, Secondary ammonium phosphate 1g/L, potassium primary phosphate 0.1g/L, magnesium sulfate 0.1g/L, pH to 5.In 250ml triangular flask, liquid amount is 50ml, 120 DEG C, sterilizing in 20 minutes.Get ProteusmirabilisATCC33946 glycerine pipe seed liquor 0.5mL to inoculate, leavening temperature 35 DEG C, shaking speed 200rpm.After cultivating 24, filtering with microporous membrane conversion fluid, in liquid-phase chromatographic analysis conversion fluid, Salvianic acidA concentration is 0.01g/L and levodopa content is 0.05g/L.
Claims (4)
1. proteus microbial transformation levodopa produces R-(+)-3-(3,4-dihydroxy phenyl) the concrete microorganism of-Lactic acid has: proteus vulgaris, Proteus mirabilis, product stick Bacillus proteus, Pan Shi Bacillus proteus and Hao Shi Bacillus proteus.
2. the bacterial classification according to right 1, its culturing process also can be carried out at good oxygen condition at anaerobic state.
3. the thalline that the cultivation according to right 2 obtains, it transforms levodopa and also can carry out at good oxygen condition at anaerobic state.
4. directly can add levodopa length of side thalline limit in the yeast culture process according to right 2 and transform production R-(+)-3-(3,4-dihydroxy phenyl)-2 hydroxy propanoic acid.
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| CN108424937A (en) * | 2018-04-27 | 2018-08-21 | 鲁东大学 | A kind of method of enzymatic clarification danshensu |
| ES2825205R1 (en) * | 2018-04-19 | 2021-06-11 | Univ Jiangnan | Strain modified by genetic engineering and its application in the production of danshensu |
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| ANNE-MARIE LABOURÉ等: "Regulation of Phenylalanine Oxidase Synthesis in Proteus mirabilis", 《JOURNAL OF BACTERIOLOGY》 * |
| HANS E. MÜLLER: "Production and Degradation of Indole by Gram-negative Bacteria", 《ZBL. BAKT. HYG. A》 * |
| SVERRE DICK HENRIKSEN: "A comparison of the phenylpyruvic acid reaction and the urease test in the differentiation of Proteus from other enteric organisms", 《J BACTERIOL.》 * |
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| CN106191139A (en) * | 2016-08-17 | 2016-12-07 | 汕头市佳禾生物科技有限公司 | Lai Bituo moves about the method that danshensu is prepared in coccus catalysis |
| ES2825205R1 (en) * | 2018-04-19 | 2021-06-11 | Univ Jiangnan | Strain modified by genetic engineering and its application in the production of danshensu |
| CN108424937A (en) * | 2018-04-27 | 2018-08-21 | 鲁东大学 | A kind of method of enzymatic clarification danshensu |
| CN108424937B (en) * | 2018-04-27 | 2022-06-21 | 鲁东大学 | Method for synthesizing tanshinol by enzyme method |
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