CN105543292B - A kind of method of microorganism conversion - Google Patents
A kind of method of microorganism conversion Download PDFInfo
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- CN105543292B CN105543292B CN201610085608.8A CN201610085608A CN105543292B CN 105543292 B CN105543292 B CN 105543292B CN 201610085608 A CN201610085608 A CN 201610085608A CN 105543292 B CN105543292 B CN 105543292B
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- proteus
- thallus
- levodopa
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- 238000006243 chemical reaction Methods 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 20
- 244000005700 microbiome Species 0.000 title claims abstract description 9
- 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 42
- 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
- 229960004502 levodopa Drugs 0.000 claims abstract description 38
- 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 36
- 241000588769 Proteus <enterobacteria> Species 0.000 claims abstract description 24
- 241000588770 Proteus mirabilis Species 0.000 claims description 10
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 238000011534 incubation Methods 0.000 claims description 3
- 241000193830 Bacillus <bacterium> Species 0.000 claims 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 42
- 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 34
- PAFLSMZLRSPALU-QMMMGPOBSA-N Danshensu Natural products OC(=O)[C@@H](O)CC1=CC=C(O)C(O)=C1 PAFLSMZLRSPALU-QMMMGPOBSA-N 0.000 description 30
- 238000000855 fermentation Methods 0.000 description 29
- 230000004151 fermentation Effects 0.000 description 29
- 239000007788 liquid Substances 0.000 description 28
- 239000012530 fluid Substances 0.000 description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 239000001963 growth medium Substances 0.000 description 14
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 239000007791 liquid phase Substances 0.000 description 11
- 239000000203 mixture Substances 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 10
- 229910000388 diammonium phosphate Inorganic materials 0.000 description 10
- 235000019838 diammonium phosphate Nutrition 0.000 description 10
- 238000001914 filtration Methods 0.000 description 10
- 238000004587 chromatography analysis Methods 0.000 description 9
- 239000012982 microporous membrane Substances 0.000 description 9
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 9
- 235000019796 monopotassium phosphate Nutrition 0.000 description 9
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 9
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 8
- 235000003891 ferrous sulphate Nutrition 0.000 description 8
- 239000011790 ferrous sulphate Substances 0.000 description 8
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 8
- 229910000359 iron(II) sulfate Inorganic materials 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
- 238000004458 analytical method Methods 0.000 description 7
- 239000008103 glucose Substances 0.000 description 7
- 238000011081 inoculation Methods 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 241000588767 Proteus vulgaris Species 0.000 description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 6
- 235000019341 magnesium sulphate Nutrition 0.000 description 6
- 229940007042 proteus vulgaris Drugs 0.000 description 6
- 239000008223 sterile water Substances 0.000 description 6
- 239000001888 Peptone Substances 0.000 description 5
- 108010080698 Peptones Proteins 0.000 description 5
- 235000019319 peptone Nutrition 0.000 description 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 4
- 239000004202 carbamide Substances 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000003068 static effect Effects 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- LQQFFJFGLSKYIR-UHFFFAOYSA-N 3,4-dihydroxyphenylpyruvic acid Chemical class OC(=O)C(=O)CC1=CC=C(O)C(O)=C1 LQQFFJFGLSKYIR-UHFFFAOYSA-N 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- 241001087672 Cosenzaea myxofaciens Species 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- ZGBSOTLWHZQNLH-UHFFFAOYSA-N [Mg].S(O)(O)(=O)=O Chemical compound [Mg].S(O)(O)(=O)=O ZGBSOTLWHZQNLH-UHFFFAOYSA-N 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
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 238000002360 preparation method Methods 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
- 241000208340 Araliaceae Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 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
- 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
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 2
- 235000003140 Panax quinquefolius Nutrition 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 241001472782 Proteus penneri Species 0.000 description 2
- 241000304195 Salvia miltiorrhiza Species 0.000 description 2
- 235000011135 Salvia miltiorrhiza Nutrition 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000005864 Sulphur Substances 0.000 description 2
- 235000019270 ammonium chloride Nutrition 0.000 description 2
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- MHUWZNTUIIFHAS-CLFAGFIQSA-N dioleoyl phosphatidic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC(COP(O)(O)=O)OC(=O)CCCCCCC\C=C/CCCCCCCC MHUWZNTUIIFHAS-CLFAGFIQSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 235000008434 ginseng Nutrition 0.000 description 2
- 239000008236 heating water Substances 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 238000007833 oxidative deamination reaction Methods 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- -1 phenolic acid compound Chemical class 0.000 description 2
- 239000000419 plant extract Substances 0.000 description 2
- 235000002639 sodium chloride Nutrition 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 230000001954 sterilising effect Effects 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
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 241000219470 Mirabilis Species 0.000 description 1
- 102000008109 Mixed Function Oxygenases Human genes 0.000 description 1
- 108010074633 Mixed Function Oxygenases Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 241001076189 Proteus hauseri Species 0.000 description 1
- 241000806018 Proteus hauseri ATCC 700826 Species 0.000 description 1
- 241001069992 Proteus mirabilis ATCC 29906 Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241001530490 Salvia rosmarinus Species 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
- 239000002253 acid Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 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
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000013452 biotechnological production Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical class OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 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
- SEGLCEQVOFDUPX-UHFFFAOYSA-N di-(2-ethylhexyl)phosphoric acid Chemical compound CCCCC(CC)COP(O)(=O)OCC(CC)CCCC SEGLCEQVOFDUPX-UHFFFAOYSA-N 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 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
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000010813 municipal solid waste Substances 0.000 description 1
- 125000001477 organic nitrogen group Chemical group 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 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
- 235000015639 rosmarinus officinalis Nutrition 0.000 description 1
- 238000007086 side reaction Methods 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
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 1
- 239000003643 water by type Substances 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 present invention relates to produce R- (+) -3- (3,4- dihydroxy phenyl) -2 hydroxy propanoic acid using Proteus microorganism conversion levodopa.This method process is simple, has important industrial application value.
Description
Technical field
The present invention produces danshensu using proteus conversion levodopa, belongs to industrial microorganism field.
Background technique
Danshensu, scientific name R- (+) -3- (3,4- dihydroxy phenyl) -2 hydroxy propanoic acid, D- (+)-β-(3,4- dihydroxy benzenes
Base) lactic acid, English name are as follows: Danshensu, (R)-(+) -3- (3,4-Dihydroxyphenyl)-lactic acid, (R) -
(+) -3- (3,4-Dihydroxyphenyl) -2-hydroxypropanoic acid is a kind of dextrorotation phenolic acid compound.
Danshensu is that important in Salvia miltiorrhiza Bge water extract wants effective ingredient, and the country obtained simultaneously from Salvia miltiorrhiza Bge water extract in 1980
Identify structure (research of Determination of water-soluble active constituents of radix, the structure of II .D (+) β (3,4- dihydroxy phenyl) lactic acid, Shanghai
One medical college's journal, 1980,05 (7), 384-385), it is various research shows that danshensu have important pharmacological effect effect,
Treatment of cardiovascular and cerebrovascular disease etc. has unique treatment effect.
Current danshensu, which is mainly extracted from Radix Salviae Miltiorrhizae, obtains (patent CN200810038853.9).Danshensu is in Radix Salviae Miltiorrhizae
Content is lower, and danshensu planting cost height and limits throughput, therefore not only price is high but also much can not for current danshensu
Meets the needs of market.Patent CN201310559498.0 proposes a kind of building bacillus coli gene engineering drawing and utilizes glucose
The method that fermentation produces danshensu, since metabolic pathway of synthesizing relates to the use of hydroxylase, which is easy to make metabolic process product
The yield of danshensu is aoxidized and influenced, simultaneously because Escherichia coli fermentation is high oxygen process, can also aoxidize danshensu, therefore work as
Preceding this method yield is lower, and cost will be higher than plant extract process.Patent CN201210190171.6 proposes the red phenol of hydrolysis
The method of sour B production danshensu, tanshin polyphenolic acid B need to be extracted from Radix Salviae Miltiorrhizae, and chemical hydrolysis process has a large amount of side reactions, same uncomfortable
For large-scale production.The catalyst of chirality synthesis danshensu (patent CN201210420488.4) is prohibitively expensive, currently also only
Rest on laboratory level.
Significant application value based on danshensu, this patent are proposed using Proteus microorganism conversion levodopa
Produce the scheme of danshensu.Levodopa is also named: L-3- (3,4- dihydroxy phenyl) alanine (L-DOPA, L-3,4-
dihydroxyphenylalanine).Current levodopa can produce (Overview from plant extract also microbial fermentation
On the biotechnological production of L-DOPA, 2015, ApplMicrobiol Biotechnol,
99:575-584), abundant raw material is easy to get.
Proteus (Proteus) be it is widely distributed in nature, such as soil, water, rubbish, spoilage organism and people or
Microorganism in the enteron aisle of animal.Existing 5 kinds: proteus vulgaris (Proteus vulgaris), proteus mirabilis
(Proteus mirabilis), glutinous proteus (Proteus myxofaciens), Pan Shi proteus (Proteus are produced
) and Hao Shi proteus (Proteus hauseri) penneri.Proteus has powerful oxidative deamination ability
(Sherris Medical Microbiology, 2004,4th ed.), levodopa can by proteus oxidative deamination at
3,4- dihydroxyphenyl pyruvic acids (3- (3,4-dihydroxyphenyl) -2-oxopropanoic acid), are then then reduced into
Danshensu.Although 3,4- dihydroxyphenyl pyruvic acids are more direct substrates, but expensive and unstable, therefore levodopa
It is optimal substrate.Proteus is a kind of facultative anaerobic bacteria, and production Radix Salviae Miltiorrhizae can be converted under anaerobic condition or aerobic condition
Element has the characteristics that high conversion and high-purity.
Summary of the invention
The present invention produces danshensu by culture Proteus microbial cells, conversion levodopa, and technical solution is such as
Under:
1, bacterial strain
Bacterial strain used in the present invention have Proteus mirabilis ATCC 29906 purchased from U.S.'s ATCC strain library,
Proteus myxofaciens ATCC 19692、Proteus hauseri ATCC 700826、Proteus penneri
ATCC 33519、Proteus mirabilis ATCC 25933、Proteus mirabilis ATCC 33946、Proteus
Vulgaris ATCC 19181, Proteus vulgaris ATCC 27972 and be purchased from Chinese industrial Microbiological Culture Collection
Proteus vulgaris CICC 10401, the Proteus mirabilis CICC 22928 of administrative center.
2, thallus culture
Liquid state fermentation culture medium composition are as follows: carbon source 0-50g/L, nitrogen source 0-50g/L, diammonium hydrogen phosphate 0-2g/L, di(2-ethylhexyl)phosphate
PH to 2-8 is adjusted in hydrogen potassium 0-1g/L, magnesium sulfate 0-0.5g/L, ferrous sulfate 0-0.5g/L, common salt 0-5g/L, can also pH
It is natural；Inoculum concentration is 5-20%, and fermentation temperature is 20-40 DEG C, and fermentation period is 24-72 hours.Seed and fermentation are all made of this
Culture medium.
Carbon source for cultivating strain can be glucose, fructose, maltose, xylose, sucrose, galactolipin, glycerol.Culture
It can be the organic nitrogen sources such as ammonium sulfate, ammonium chloride, ammonium nitrate, peptone, yeast extract, urea, corn pulp with nitrogen source.Carbon source and nitrogen
Source can be one or more combinations.
The process of liquid culture thallus can use anaerobism or aerobic mode.
3, conversion produces danshensu
The scheme that conversion process uses has 2 kinds:
(1) the liquid incubation of cell, levodopa substrate is added in above-mentioned cultivating system；Levodopa addition
Amount is 0-3g/L.
(2) liquid state fermentation culture is centrifuged removal fermentation liquid and obtains thallus, thallus is put into containing levodopa substrate
Reactant aqueous solution system in carry out；20-40 DEG C of conversion process temperature, pH 2-8, transformation time 1-24 hours.It is left in conversion fluid
Rotation DOPA initial concentration is 0.1-3g/L.
4, the detection and analysis of sample
Conversion fluid is tested and analyzed using 200 high performance liquid chromatograph of PerkinElmer Series, chromatographic condition are as follows: stream
Dynamic phase is -0.1% formic acid water of methanol (40:60), using Chinese nation Megres C18 chromatographic column (4.6 × 250mm, 5 μm), flow velocity
0.6ml/min, 30 DEG C of column temperature, 20 μ l of sample volume, detector wavelength 280nm.
Using the DAC-HB50 preparation chromatographic column preparation conversion sample of Hanbon Sci. & Tech. Co., Ltd., chromatographic condition is prepared
Are as follows: 50% methanol of mobile phase, column temperature nature, flow velocity 3ml/min, sample volume 5ml.Sample reaches chromatographically pure 99.9%, repeatedly into
The isolated product of sample vacuum rotating at 50 DEG C is evaporated.The sample 0.5g being prepared is weighed, is dissolved in deionized water simultaneously
It is settled to 50ml, the full-automatic polarimeter measurement degree of giving out light of AP-300 of delaying is liked using Japan.Further adopt Varian Gemini
The nuclear magnetic data of 2000 (VnmrS 600MHz, 600/54/ASP) analysis sample.Sample further uses UPLC-QTOF-MS method
Analyzing molecules amount, instrument are Waters MALDI SYNAPT QTOF MS liquid chromatography tandem quadrupole rod time of-flight mass spectrometer.
Specific embodiment
Embodiment 1
The analysis of converted product measures.
It is as follows to prepare culture medium: yeast saccharomyces cream 5g/L, peptone 10g/L, sodium chloride 5g/L.It is filled in 500ml triangular flask
Liquid measure is 100ml, totally 50 bottles, 120 DEG C, is sterilized within 20 minutes.Take 29906 glycerol tube seed of Proteus mirabilis ATCC
Liquid 1mL inoculation, 30 DEG C of fermentation temperature, shaking speed 200rpm.After culture 24, fermentation liquid is centrifuged (revolving speed 10000rpm, time
10min), removal supernatant obtains thallus, is centrifuged the thallus of acquisition with twice of sterile water wash.20g wet thallus is taken to be put into left-handed
DOPA concentration is to be uniformly mixed in the 1000ml solution of 2g/L.After 37 DEG C of shaking tables vibrate (100rpm) 24 hours, 100 DEG C of water
Bath 3 minutes killing thallus of heating.It is centrifuged (revolving speed 10000rpm, time 10min) removal thallus, the supernatant after being converted again
Liquid.Liquid phase analysis danshensu concentration is 1.3g/L, and remaining levodopa concentration is 0.5g/L.1.03g is obtained using preparation chromatography
Sterling.
Polarimeter analyzes its optical activityNuclear magnetic data are as follows: 1H NMR (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)；13C NMR(DMSO-d6,600MHz):δ175.4,174.2,
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 are as follows: (- ESI, negative mode) m/z:197.0 [M-1].
According to above data and pertinent literature (Stereochemical configuration of β-(3,4-
dihydroxyphenyl)lactic acid from Rosmarinus officinalis.Ricerea Sci.80:255-
259. [Chem.Abs.54:24520.1960], the research of Determination of water-soluble active constituents of radix, II .D (+) β (3,4- dihydroxy benzenes
Base) lactic acid structure, the first medical college of Shanghai journal, 1980,05 (7), 384-385), determine its molecule and optical texture and day
Right danshensu is completely the same.
Embodiment 2
Aerobic culture is compared with detesting culture.Prepare culture medium: glucose 30g/L, peptone 20g/L, diammonium hydrogen phosphate
1g/L, potassium dihydrogen phosphate 1g/L, magnesium sulfate 0.3g/L, ferrous sulfate 0.3g/L；Originating pH and fermentation process pH is nature
Liquid amount is 100ml in 500ml triangular flask, totally 2 bottles, 120 DEG C, is sterilized within 20 minutes.
22928 glycerol tube seed liquor 1mL of Proteus mirabilis CICC is taken to be inoculated with 1 bottle, 35 in anaerobic culture box
DEG C culture 72 hours, be centrifuged the thallus of acquisition with twice of sterile water wash.Taking 0.1g wet thallus to be put into levodopa concentration is 2g/
It in the 4ml solution of L, is uniformly mixed, after 35 DEG C of standings convert 24 hours in anaerobic culture box, 100 DEG C are killed for heating water bath 3 minutes
Thallus.It is centrifuged (revolving speed 10000rpm, time 10min) removal thallus, the supernatant after being converted again.Liquid phase analysis danshensu
Concentration is 1.1g/L, and remaining levodopa concentration is 0.6g/L.
22928 glycerol tube seed liquor 1mL of Proteus mirabilis CICC is taken to be inoculated with 1 bottle, in shaking table (200rpm)
35 DEG C are cultivated 24 hours.Fermentation liquid is centrifuged (revolving speed 10000rpm, time 10min), removal supernatant obtains thallus, and centrifugation obtains
The thallus obtained is with twice of sterile water wash.0.1g wet thallus is taken to be put into the 4ml solution that levodopa concentration is 2g/L, mixing is equal
It is even.After 35 DEG C of shaking tables vibrate (100rpm) 12 hours, 100 DEG C of heating water baths, 3 minutes killing thallus.It is centrifuged (revolving speed again
10000rpm, time 10min) removal thallus, the supernatant after being converted.Liquid phase analysis danshensu concentration is 1.0g/L, is remained
Remaining levodopa concentration is 0.5g/L.
Embodiment 3
Culture medium composition are as follows: glucose 1g/L, urea 1g/L, diammonium hydrogen phosphate 1g/L, potassium dihydrogen phosphate 0.1g/L, sulfuric acid
Magnesium 0.1g/L, ferrous sulfate 0.1g/L.Liquid amount is 50ml in 250ml triangular flask, 120 DEG C, is sterilized within 20 minutes.Take Proteus
19181 glycerol tube seed liquor 0.5mL of vulgaris ATCC inoculation, 35 DEG C of fermentation temperature, shaking speed 200rpm.Culture 24
Afterwards, fermentation liquid is centrifuged (revolving speed 10000rpm, time 10min), removal supernatant obtains thallus, is centrifuged the thallus nothing of acquisition
Bacterium water cleans twice.It takes 0.05g wet thallus to be put into the 2ml solution that levodopa concentration is 0.1g/L, and adjusts pH to 6, mix
It closes uniform.Static conversion 24 hours in anaerobic culture box, filtering with microporous membrane conversion fluid are red in liquid-phase chromatographic analysis conversion fluid
The plain concentration of ginseng is 0.08g/L and levodopa residual quantity is 0g/L.
Embodiment 4
Culture medium composition are as follows: fructose 50g/L, ammonium sulfate 5g/L, diammonium hydrogen phosphate 1g/L, potassium dihydrogen phosphate 1g/L, sulfuric acid
Magnesium 0.5g/L, ferrous sulfate 0.5g/L.Liquid amount is 50ml in 250ml triangular flask, 120 DEG C, is sterilized within 20 minutes.Take Proteus
700826 glycerol tube seed liquor 0.5mL of hauseri ATCC inoculation, 20 DEG C of fermentation temperature, shaking speed 200rpm.Culture 72
Afterwards, fermentation liquid is centrifuged (revolving speed 10000rpm, time 10min), removal supernatant obtains thallus, is centrifuged the thallus nothing of acquisition
Bacterium water cleans twice.It takes 0.1g wet thallus to be put into the 2ml solution that levodopa concentration is 2g/L, is uniformly mixed.It is shaken in 20 DEG C
Bed oscillation (100rpm) is after 24 hours, filtering with microporous membrane conversion fluid, and danshensu concentration is in liquid-phase chromatographic analysis conversion fluid
0.8g/L and levodopa residual quantity are 0.6g/L.
Embodiment 5
Culture medium composition are as follows: xylose 50g/L, ammonium sulfate 1g/L, diammonium hydrogen phosphate 1g/L, potassium dihydrogen phosphate 1g/L, sulfuric acid
Magnesium 0.2g/L, ferrous sulfate 0.1g/L.Liquid amount is 50ml in 250ml triangular flask, 120 DEG C, is sterilized within 20 minutes.Take Proteus
10401 glycerol tube seed liquor 0.5mL of vulgaris CICC inoculation, 40 DEG C of fermentation temperature, shaking speed 200rpm.Culture 48
Afterwards, fermentation liquid is centrifuged (revolving speed 10000rpm, time 10min), removal supernatant obtains thallus, is centrifuged the thallus nothing of acquisition
Bacterium water cleans twice.It takes 0.1g wet thallus to be put into the 2ml solution that levodopa concentration is 2g/L, is uniformly mixed.It is shaken in 40 DEG C
Bed oscillation (100rpm) is after 24 hours, filtering with microporous membrane conversion fluid, and danshensu concentration is in liquid-phase chromatographic analysis conversion fluid
0.5g/L and levodopa residual quantity are 1.3g/L.
Embodiment 6
Culture medium composition are as follows: glycerol 15g/L, ammonium nitrate 1g/L, diammonium hydrogen phosphate 1g/L, potassium dihydrogen phosphate 0.1g/L, sulphur
Sour magnesium 0.1g/L, ferrous sulfate 0.1g/L.Liquid amount is 50ml in 250ml triangular flask, 120 DEG C, is sterilized within 20 minutes.It takes
19692 glycerol tube seed liquor 0.5mL of Proteus myxofaciens ATCC inoculation, 30 DEG C of fermentation temperature, shaking speed
200rpm.After culture 24, fermentation liquid is centrifuged (revolving speed 10000rpm, time 10min), removal supernatant obtains thallus, centrifugation
The thallus of acquisition is with twice of sterile water wash.0.1g wet thallus is taken to be put into the 2ml solution that levodopa concentration is 2g/L, mixing
Uniformly.Static conversion 24 hours, filtering with microporous membrane conversion fluid, in liquid-phase chromatographic analysis conversion fluid in 20 DEG C of anaerobic culture boxes
Danshensu concentration is 0.7g/L and levodopa residual quantity is 1.0g/L.
Embodiment 7
Culture medium composition are as follows: maltose 15g/L, ammonium chloride 1g/L, diammonium hydrogen phosphate 1g/L, potassium dihydrogen phosphate 0.1g/L,
Magnesium sulfate 0.1g/L, ferrous sulfate 0.1g/L adjust pH to 5.In 250ml triangular flask liquid amount be 50ml, 120 DEG C, 20 minutes
Sterilizing.27972 glycerol tube seed liquor 0.5mL of Proteus vulgaris ATCC is taken to be inoculated with, 35 DEG C of fermentation temperature, shaking speed
200rpm.After culture 24, fermentation liquid is centrifuged (revolving speed 10000rpm, time 10min), removal supernatant obtains thallus, centrifugation
The thallus of acquisition is with twice of sterile water wash.0.1g wet thallus is taken to be put into the 2ml solution that levodopa concentration is 3g/L, mixing
Uniformly.Static conversion 24 hours, filtering with microporous membrane conversion fluid, in liquid-phase chromatographic analysis conversion fluid in 40 DEG C of anaerobic culture boxes
Danshensu concentration is 1.1g/L and levodopa residual quantity is 1.6g/L.
Embodiment 8
Culture medium composition are as follows: galactolipin 1g/L, urea 1g/L, diammonium hydrogen phosphate 1g/L, magnesium sulfate 0.1g/L, ferrous sulfate
0.1g/L adjusts pH to 2.Liquid amount is 50ml in 250ml triangular flask, 120 DEG C, is sterilized within 20 minutes.Take Proteus
25933 glycerol tube seed liquor 0.5mL of vulgaris ATCC inoculation, 35 DEG C of fermentation temperature, shaking speed 200rpm.Culture 24
Afterwards, fermentation liquid is centrifuged (revolving speed 10000rpm, time 10min), removal supernatant obtains thallus, is centrifuged the thallus nothing of acquisition
Bacterium water cleans twice.It takes 0.1g wet thallus to be put into the 2ml solution that levodopa concentration is 2g/L, and adjusts pH to 2, mixing is equal
It is even.Static conversion 24 hours in 37 DEG C of anaerobic culture boxes, filtering with microporous membrane conversion fluid are red in liquid-phase chromatographic analysis conversion fluid
The plain concentration of ginseng is 0.3g/L and levodopa residual quantity is 1.4g/L.
Embodiment 9
Culture medium composition are as follows: glucose 5g/L, peptone 1g/L, potassium dihydrogen phosphate 0.1g/L, magnesium sulfate 0.1g/L, sulfuric acid
Ferrous 0.1g/L adjusts pH to 8.Liquid amount is 50ml in 250ml triangular flask, 120 DEG C, is sterilized within 20 minutes.Filtration sterilization is added
Levodopa make culture medium end degree up to 2g/L, 33946 glycerol tube seed liquor 0.5mL of Proteusmirabilis ATCC connects
Kind, 35 DEG C of fermentation temperature, shaking speed 200rpm.After culture 24, filtering with microporous membrane conversion fluid, liquid-phase chromatographic analysis conversion fluid
Middle danshensu concentration is 1g/L and levodopa residual quantity is 0.2g/L.
Embodiment 10
Culture medium composition are as follows: glucose 10g/L, urea 1g/L, diammonium hydrogen phosphate 1g/L, potassium dihydrogen phosphate 0.1g/L, sulphur
Sour ferrous iron 0.1g/L, adjusts pH to 8.Liquid amount is 100ml in 500ml triangular flask, 120 DEG C, is sterilized within 20 minutes, totally 10 bottles.Respectively
33519 glycerol tube seed liquor 0.5mL of Proteus penneri ATCC is taken to be inoculated with, 35 DEG C of fermentation temperature, shaking speed
200rpm.After culture 24, fermentation liquid is centrifuged (revolving speed 10000rpm, time 10min), removal supernatant obtains thallus, centrifugation
The thallus of acquisition is with twice of sterile water wash.It takes 5g wet thallus to be put into the 10ml solution that levodopa concentration is 3g/L, and adjusts
PH to 5 is saved, is uniformly mixed.After 35 DEG C of shaking tables vibrate (100rpm) 1 hour, filtering with microporous membrane conversion fluid, liquid chromatogram point
Danshensu concentration is 2.3g/L in analysis conversion fluid and levodopa residual quantity is 0.5g/L.
Embodiment 11
Culture medium composition are as follows: glucose 10g/L, peptone 1g/L, diammonium hydrogen phosphate 1g/L, potassium dihydrogen phosphate 0.1g/L,
Magnesium sulfate 0.1g/L, pH are to 5.Liquid amount is 50ml in 250ml triangular flask, 120 DEG C, is sterilized within 20 minutes.Take Proteus
33946 glycerol tube seed liquor 0.5mL of mirabilis ATCC inoculation, 35 DEG C of fermentation temperature, shaking speed 200rpm.Culture 24
Afterwards, filtering with microporous membrane conversion fluid, danshensu concentration is 0.01g/L in liquid-phase chromatographic analysis conversion fluid and levodopa content is
0.05g/L。
Claims (4)
1. a kind of method of conversion levodopa production R- (+) -3- (3,4- dihydroxy phenyl) -2 hydroxy propanoic acid, feature exist
In the method is to carry out conversion production using proteus microorganism belonging to genus；The proteus microorganism belonging to genus has: common variation
Bacillus, proteus mirabilis produce glutinous proteus, Pan Shi proteus and Hao Shi proteus.
2. the method according to claim 1, wherein the incubation of the proteus microorganism belonging to genus can be
Anaerobic state can also be carried out in aerobic state.
3. according to the method described in claim 2, it is characterized in that, the obtained thallus of culture, conversion levodopa can be
Anaerobic state can also be carried out in aerobic state.
4. according to the method described in claim 2, it is characterized in that, levodopa side can be directly added into thallus incubation
Long thallus side conversion production R- (+) -3- (3,4- dihydroxy phenyl) -2 hydroxy propanoic acid.
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| A comparison of the phenylpyruvic acid reaction and the urease test in the differentiation of Proteus from other enteric organisms;SVERRE DICK HENRIKSEN;《J Bacteriol.》;19501231;第60卷;225-231 |
| ANNE-MARIE LABOURÉ等.Regulation of Phenylalanine Oxidase Synthesis in Proteus mirabilis.《JOURNAL OF BACTERIOLOGY》.1979,第137卷(第1期),161-168. |
| Production and Degradation of Indole by Gram-negative Bacteria;HANS E. MüLLER;《Zbl. Bakt. Hyg. A》;19861231;第261卷;1-11 |
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