CN108660167A - The biological synthesis method of L-glufosinate-ammonium - Google Patents
The biological synthesis method of L-glufosinate-ammonium Download PDFInfo
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- CN108660167A CN108660167A CN201710195282.9A CN201710195282A CN108660167A CN 108660167 A CN108660167 A CN 108660167A CN 201710195282 A CN201710195282 A CN 201710195282A CN 108660167 A CN108660167 A CN 108660167A
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- glufosinate
- ammonium
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- biological synthesis
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- 238000001308 synthesis method Methods 0.000 title claims abstract description 29
- 238000006243 chemical reaction Methods 0.000 claims abstract description 88
- 239000000843 powder Substances 0.000 claims abstract description 47
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 40
- 230000009466 transformation Effects 0.000 claims abstract description 40
- 239000000654 additive Substances 0.000 claims abstract description 36
- 229940069521 aloe extract Drugs 0.000 claims abstract description 33
- 230000000996 additive effect Effects 0.000 claims abstract description 27
- 239000012530 fluid Substances 0.000 claims abstract description 23
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 21
- 239000001110 calcium chloride Substances 0.000 claims abstract description 21
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 21
- 102000004190 Enzymes Human genes 0.000 claims abstract description 20
- 108090000790 Enzymes Proteins 0.000 claims abstract description 20
- 235000019441 ethanol Nutrition 0.000 claims abstract description 20
- 239000000758 substrate Substances 0.000 claims abstract description 20
- 239000011942 biocatalyst Substances 0.000 claims abstract description 18
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000005515 coenzyme Substances 0.000 claims abstract description 7
- 239000002904 solvent Substances 0.000 claims abstract description 7
- 125000003277 amino group Chemical group 0.000 claims abstract description 5
- 108090000340 Transaminases Proteins 0.000 claims description 29
- 102000003929 Transaminases Human genes 0.000 claims description 24
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 24
- 239000000203 mixture Substances 0.000 claims description 24
- 239000002893 slag Substances 0.000 claims description 24
- 238000002360 preparation method Methods 0.000 claims description 23
- 241000894006 Bacteria Species 0.000 claims description 22
- 241001116389 Aloe Species 0.000 claims description 17
- 235000011399 aloe vera Nutrition 0.000 claims description 17
- 239000000243 solution Substances 0.000 claims description 17
- 238000000926 separation method Methods 0.000 claims description 14
- 239000007853 buffer solution Substances 0.000 claims description 13
- 241000607291 Vibrio fluvialis Species 0.000 claims description 11
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 10
- 230000003139 buffering effect Effects 0.000 claims description 10
- 239000007993 MOPS buffer Substances 0.000 claims description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 238000010353 genetic engineering Methods 0.000 claims description 7
- 239000004471 Glycine Substances 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 239000007979 citrate buffer Substances 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 4
- 239000008055 phosphate buffer solution Substances 0.000 claims description 4
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 3
- 239000002202 Polyethylene glycol Substances 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 229920001223 polyethylene glycol Polymers 0.000 claims description 3
- FNIQKHXZAHAFCZ-UHFFFAOYSA-N phosphoric acid;1h-pyrrole Chemical compound C=1C=CNC=1.OP(O)(O)=O FNIQKHXZAHAFCZ-UHFFFAOYSA-N 0.000 claims description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims 4
- ZBMRKNMTMPPMMK-UHFFFAOYSA-N 2-amino-4-[hydroxy(methyl)phosphoryl]butanoic acid;azane Chemical compound [NH4+].CP(O)(=O)CCC(N)C([O-])=O ZBMRKNMTMPPMMK-UHFFFAOYSA-N 0.000 claims 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical class OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims 1
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 claims 1
- 239000013078 crystal Substances 0.000 abstract description 24
- 238000000034 method Methods 0.000 abstract description 16
- 238000000746 purification Methods 0.000 abstract description 14
- 230000003287 optical effect Effects 0.000 abstract description 13
- 230000008569 process Effects 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 5
- 238000013329 compounding Methods 0.000 abstract description 3
- 230000003197 catalytic effect Effects 0.000 abstract description 2
- FBWXUNCARHZYFO-UHFFFAOYSA-N C(=O)=C(C(=O)O)CCP(=O)(OCO)O Chemical compound C(=O)=C(C(=O)O)CCP(=O)(OCO)O FBWXUNCARHZYFO-UHFFFAOYSA-N 0.000 abstract 1
- 238000004904 shortening Methods 0.000 abstract 1
- IAJOBQBIJHVGMQ-UHFFFAOYSA-N 2-amino-4-[hydroxy(methyl)phosphoryl]butanoic acid Chemical compound CP(O)(=O)CCC(N)C(O)=O IAJOBQBIJHVGMQ-UHFFFAOYSA-N 0.000 description 27
- 238000004128 high performance liquid chromatography Methods 0.000 description 16
- 238000012544 monitoring process Methods 0.000 description 14
- 238000004519 manufacturing process Methods 0.000 description 13
- 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 group 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 description 11
- 230000000052 comparative effect Effects 0.000 description 8
- IBHWYBRNKAMYKE-UHFFFAOYSA-N 2-[hydroxy(hydroxymethoxy)phosphoryl]butanoic acid Chemical compound OCOP(=O)(O)C(C(=O)O)CC IBHWYBRNKAMYKE-UHFFFAOYSA-N 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- 229910019142 PO4 Inorganic materials 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 230000002363 herbicidal effect Effects 0.000 description 6
- 239000011734 sodium Substances 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 239000004009 herbicide Substances 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 4
- 239000012531 culture fluid Substances 0.000 description 4
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 4
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 4
- 229930027917 kanamycin Natural products 0.000 description 4
- 238000003259 recombinant expression Methods 0.000 description 4
- 230000006798 recombination Effects 0.000 description 4
- 238000005215 recombination Methods 0.000 description 4
- 238000011218 seed culture Methods 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- 230000002194 synthesizing effect Effects 0.000 description 3
- RHQOZBPLEPGZDK-UHFFFAOYSA-N 4-[hydroxy(hydroxymethoxy)phosphoryl]butanoic acid Chemical compound OCOP(=O)(O)CCCC(=O)O RHQOZBPLEPGZDK-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 2
- 239000005562 Glyphosate Substances 0.000 description 2
- 241000607598 Vibrio Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- CDPKWOKGVUHZFR-UHFFFAOYSA-N dichloro(methyl)phosphane Chemical compound CP(Cl)Cl CDPKWOKGVUHZFR-UHFFFAOYSA-N 0.000 description 2
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- XDDAORKBJWWYJS-UHFFFAOYSA-N glyphosate Chemical compound OC(=O)CNCP(O)(O)=O XDDAORKBJWWYJS-UHFFFAOYSA-N 0.000 description 2
- 229940097068 glyphosate Drugs 0.000 description 2
- 238000000703 high-speed centrifugation Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 239000002777 nucleoside Substances 0.000 description 2
- 125000003835 nucleoside group Chemical group 0.000 description 2
- FIKAKWIAUPDISJ-UHFFFAOYSA-L paraquat dichloride Chemical compound [Cl-].[Cl-].C1=C[N+](C)=CC=C1C1=CC=[N+](C)C=C1 FIKAKWIAUPDISJ-UHFFFAOYSA-L 0.000 description 2
- 239000000575 pesticide Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 150000003457 sulfones Chemical class 0.000 description 2
- 238000007514 turning Methods 0.000 description 2
- 108700023418 Amidases Proteins 0.000 description 1
- 108010073324 Glutaminase Proteins 0.000 description 1
- 102000009127 Glutaminase Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- XYFCBTPGUUZFHI-UHFFFAOYSA-N Phosphine Chemical compound P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 description 1
- 102000004861 Phosphoric Diester Hydrolases Human genes 0.000 description 1
- 108090001050 Phosphoric Diester Hydrolases Proteins 0.000 description 1
- ABLZXFCXXLZCGV-UHFFFAOYSA-N Phosphorous acid Chemical compound OP(O)=O ABLZXFCXXLZCGV-UHFFFAOYSA-N 0.000 description 1
- 235000014676 Phragmites communis Nutrition 0.000 description 1
- 241000209504 Poaceae Species 0.000 description 1
- 206010044565 Tremor Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 108010027597 alpha-chymotrypsin Proteins 0.000 description 1
- 102000005922 amidase Human genes 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- QLULGSLAHXLKSR-UHFFFAOYSA-N azane;phosphane Chemical compound N.P QLULGSLAHXLKSR-UHFFFAOYSA-N 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000002360 explosive Substances 0.000 description 1
- 244000037671 genetically modified crops Species 0.000 description 1
- 102000005396 glutamine synthetase Human genes 0.000 description 1
- 108020002326 glutamine synthetase Proteins 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- GPRLSGONYQIRFK-UHFFFAOYSA-N hydron Chemical compound [H+] GPRLSGONYQIRFK-UHFFFAOYSA-N 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000003754 machining Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 125000001476 phosphono group Chemical group [H]OP(*)(=O)O[H] 0.000 description 1
- 229920002338 polyhydroxyethylmethacrylate Polymers 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000001429 stepping effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- OBSZRRSYVTXPNB-UHFFFAOYSA-N tetraphosphorus Chemical compound P12P3P1P32 OBSZRRSYVTXPNB-UHFFFAOYSA-N 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- CYTQBVOFDCPGCX-UHFFFAOYSA-N trimethyl phosphite Chemical compound COP(OC)OC CYTQBVOFDCPGCX-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
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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)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of biological synthesis methods of L glufosinate-ammoniums, including：It is substrate, using 2 propylamine as amino group donor using 2 carbonyl 4 (hydroxymethyl phosphono) butyric acid; reaction system is constituted with solvent; biocatalyst, coenzyme and additive are added into the reaction system again and carries out bioconversion reaction, obtains the conversion fluid containing L glufosinate-ammoniums.Its technological process is simple, to equipment without particular/special requirement, is suitable for industrialization；The yield of L glufosinate-ammoniums reaches 98% or more up to 85% or more, after purification L glufosinate-ammoniums crystal purity, and optical purity is 99% or more；Used biocatalyst excellent catalytic effect, dosage is few, single-minded efficient；Aloe extract powder, ethyl alcohol and calcium chloride compounding are used as additive, and shortening transformation time is with obvious effects, and the yield of L glufosinate-ammoniums is also obviously improved.
Description
Technical field
The present invention relates to the preparation methods of pesticide, in particular to a kind of biological synthesis method of L-glufosinate-ammonium.
Background technology
Glufosinate-ammonium, chemical name are 4- [hydroxyl (methyl) phosphono]-DL- high lactamines, by (existing Germany of Hirst company
Beyer Co., Ltd) in the last century 80's development and production, belong to phosphonic acid herbicide, is glutamine synthetase inhibitor, it is non-selection
Property contact killing type herbicide, as herbicide obtain registration use be 1984.Just there is Glufosinate-ammonium registration in China within 2004,
Just there is product registration in China within 2005.
Since extensive use, resistance glyphosate weeds are continuously increased glyphosate, and harm gradually aggravates.Paraquat is a kind of strong
People and animals are had very strong toxic effect by strong killing weeds herbicide.On July 1st, 2014, China cancel the registration of paraquat aqua
With production permit, stopping production；Stop aqua on July 1st, 2016 to sell and use at home.Glufosinate-ammonium is world's large-tonnage agriculture
Drug kind and second-biggest-in-the-world genetically modified crops herbicide-tolerant.Glufosinate-ammonium toxicity is low, safer, is easy in the soil
Degradation, to crop safety, is not easy drift, and herbicidal spectrum is wide, and activity is high, and dosage is few, and environmental pressure is small, and herbicide is rapid, can quickly kill
Dead 100 kinds or more of grass family and broad leaved weed can make base of water, safe and convenient to use, these are that the product are removed better than other
The characteristics of careless agent, so this product can still be in great demand after the product for numerous efficient ultra high efficiencies occur.
The synthetic technology route of glufosinate-ammonium is more both at home and abroad at present, tight Hydron etc. (《Pesticide》, 2002,41 (9), 46-48)
Reporting of Overview was done, but each route generally existing reaction step is more, the high problem of production cost.Bayer AG is in patent
It is proposed in US4521348 and US6359162 to synthesize dichloromethyl phosphine synthesizing methyl phosphite ester again, by series reaction
The method for synthesizing glufosinate-ammonium.It is the route high income, at low cost, but originate route dichloromethyl phosphine synthesis material, product materialization
Matter is active, inflammable and explosive.Building-up process be in 500~600 DEG C, such as control it is unstable, easy to produce easily natural yellow phosphorus and
Hydrogen phosphide is dangerous very big.In addition, material has strong corrosive, the selection of consersion unit material and its process for machining and manufacturing are wanted
Ask harsh, country's processing and manufacturing level is difficult to meet production requirement at present.
These methods are all the methods for preparing glufosinate-ammonium, and glufosinate-ammonium is L/D mixed types, wherein what is played a major role is L-type,
The effect of D types is only the 1/8 of L-type；L-glufosinate-ammonium can be in the soil through microbial degradation, and D- type glufosinate-ammoniums are more difficult to degrade, finally
It can lead to soil hardening.Therefore, the more traditional glufosinate-ammonium of L-glufosinate-ammonium is more efficient, more inexpensive, safer.
The pure L-glufosinate-ammonium of chemical synthesis synthesizing optical, processing step is more, and yield is low, and asymmetric syntheses reagent used is most
It is more expensive, cause production cost higher, be unfavorable for industrialized production, chiral separation is by chemical synthesis racemic DL-
Glufosinate-ammonium or derivatives thereof, then isomer separation is carried out, complex process, chiral resolving agent is expensive, and D- type glufosinate-ammoniums need weight
New racemization recycles；And biological synthesis process has that stereoselectivity is strong, reaction condition is mild, high income, it is at low cost etc. significantly
Advantage.
Currently, the approach of domestic external application bioanalysis synthesis L-glufosinate-ammonium probably has following several, alpha -chymotrypsin is torn open
Divide double propylamine phosphorus ethyl esters to prepare L-glufosinate-ammonium, can not recycle, increase into after the D configuration mapping derivatizations in raw material
This；Racemic glufosinate-ammonium obtains L-glufosinate-ammonium after 3 phosphodiesterase, acylase and glutaminase enzyme stepping actions,
It needs 3 enzyme synergistic effects, is difficult to apply in actual production；2- carbonyls -4- (hydroxymethyl phosphono) butyric acid is turning ammonia
Under the action of enzyme, L-glufosinate-ammonium is generated, but used amino group donor separation is difficult in the prior art, transformation time is long, efficiency
It is low.
Therefore, research and development are a kind of can improve raw material availability, improve transformation efficiency, shorten transformation time, reduce production cost
The biological synthesis method for being easy to industrialized L-glufosinate-ammonium simultaneously seems very necessary.
Invention content
Problem to be solved by this invention seeks to provide a kind of biological synthesis method of L-glufosinate-ammonium, and this method is ensureing
On the basis of highly-solid selectively, high conversion and high yield pulp1, raw material availability can be improved, shortens transformation time, reduce production
Cost, while being easy to industrialize.
In order to solve the above technical problems, the biological synthesis method of L-glufosinate-ammonium provided by the present invention, including：With 2- carbonyls
Base -4- (hydroxymethyl phosphono) butyric acid is substrate, using 2- propylamine as amino group donor, constitutes reaction system with solvent, then to institute
It states and biocatalyst, coenzyme and additive progress bioconversion reaction is added in reaction system, obtain turning containing L-glufosinate-ammonium
Change liquid.
Preferably, biocatalyst is the glufosinate-ammonium transaminase from Vibrio fluvialis, derives from Vibrio
The encoding gene of the genetic engineering bacterium of the glufosinate-ammonium transaminase of fluvialis is SEQ ID NO in sequence table：Nucleosides shown in 1
Acid sequence；Coenzyme is phosphopyridoxal pyridoxal phosphate.
Further, solvent is buffer solution, buffer solution be selected from phosphate buffer solution, carbonate buffer solution,
In Tri-HCl buffer solutions, borate buffer solution, glycine buffer, citrate buffer solution, MOPS buffer solutions
One kind.
Still further, additive is selected from aloe extract powder, Folium Pterocaryae extract powder, acetone, polyethylene glycol, dimethyl Asia
The mixture of one or more of sulfone, ethyl alcohol and calcium chloride.
Still further, it is 2-5 in mass ratio that additive, which is aloe extract powder, ethyl alcohol and calcium chloride,:3-8:1 preferably 3:
5:The mixture of 1 composition.
Again further, the preparation method of aloe extract powder is：Fresh aloe is crushed, is squeezed the juice, juice slag separation, by gained
Slag dry 6-8h at 80 DEG C, crushes, and crosses 30 mesh sieve, obtains aloe and take object powder.The preparation method of Folium Pterocaryae extract powder is：It will be fresh
Folium Pterocaryae is crushed, squeezes the juice, the separation of juice slag, and by gained slag, dry 6-8h, crushing at 80 DEG C, cross 30 mesh sieve, obtain Folium Pterocaryae and take
Object powder.
Again further, the pH value control of transformation system is 5-10, and preferably 6-7 controls the temperature of the transformation system
Degree is 20-60 DEG C, preferably 20-40 DEG C；The control of reaction system speed of agitator is 150-250r/min, preferably 180-220r/
min。
Again further, the concentration control of biocatalyst is 10-30g/L, preferably 15-20g/L；The additive
Mass ratio control with biocatalyst is 1-5%, preferably 2.5-4%.
Biocatalyst is the genetic engineering bacterium of the glufosinate-ammonium transaminase from Vibrio fluvialis, specific to prepare
Method is：Selection carries out engineer, after design from the gene order of the glufosinate-ammonium transaminase of Vibrio fluvialis
Gene order such as sequence table in SEQ ID NO：Shown in nucleotide sequence shown in 1；The sequence is synthesized by full genome, gram
It is grand enter expression vector pET28a Nde I and Xho I restriction enzyme sites, conversion host strain E.coli BL21 (DE3) competence it is thin
Born of the same parents；Picking positive transformant and after identification is sequenced, obtains recombinant expression carrier；Recombinant expression carrier is transferred to E.coli BL21
(DE3) in bacterial strain, the recombination glufosinate-ammonium aminotransferase gene engineering bacteria that glufosinate-ammonium transaminase can be recombinated with induced expression is obtained.
Recombination glufosinate-ammonium aminotransferase gene engineering bacteria is inoculated into the LB culture mediums containing kanamycins, overnight in 37 DEG C
Culture, obtains seed culture fluid；Seed culture fluid is inoculated into the TB culture mediums containing kanamycins, inoculum concentration is that is mould containing card
The 1% of the TB culture volumes of element；It is subsequently placed at 37 DEG C and cultivates 2-5h, sterile IPTG inductions are added, make IPTG final concentrations
Reach 0.1mM, is placed at 25 DEG C and continues to cultivate 20h.It is obtained from Vibrio fluvialis' finally by high speed centrifugation
The full cell of genetic engineering bacterium of glufosinate-ammonium transaminase.
The present invention is monitored in biotransformation using HPLC-MS and HPLC, until substrate is fully utilized.
Advantages of the present invention is mainly reflected in following several respects：
First, present invention process flow is simple, to equipment without particular/special requirement, it is suitable for industrialized production；
Second, the yield of L-glufosinate-ammonium of the present invention is up to 85% or more, after purification L-glufosinate-ammonium crystal purity reach 98% with
On, optical purity is 99% or more；
Third, biocatalyst of the present invention is the glufosinate-ammonium aminotransferase gene engineering bacteria from Vibrio fluvialis
Full cell, excellent catalytic effect, dosage is few, single-minded efficient；
Fourth, the present invention is compounded with aloe extract powder and calcium chloride as additive, aloe contains abundant carbohydrate, protein, dimension
Raw element C and minerals etc., ethyl alcohol, calcium chloride contribute to penetration cell, aloe extract powder, ethyl alcohol and calcium chloride compounding contracting
Short transformation time is with obvious effects, and the yield of L-glufosinate-ammonium is also obviously improved.
Specific implementation mode
Below in conjunction with specific embodiment, the present invention is described in further detail, but the embodiment should not be construed pair
The limitation of the present invention.
The biological synthesis method of L-glufosinate-ammonium, including：Using 2- carbonyls -4- (hydroxymethyl phosphono) butyric acid as substrate, with
2- propylamine is amino group donor, constitutes reaction system with solvent, then biocatalyst, coenzyme is added into the reaction system and adds
Add agent to carry out bioconversion reaction, obtains the conversion fluid containing L-glufosinate-ammonium.
Preferably, biocatalyst is the glufosinate-ammonium transaminase from Vibrio fluvialis, derives from Vibrio
The encoding gene of the genetic engineering bacterium of the glufosinate-ammonium transaminase of fluvialis is SEQ ID NO in sequence table：Nucleosides shown in 1
Acid sequence；Coenzyme is phosphopyridoxal pyridoxal phosphate.
Further, solvent is buffer solution, buffer solution be selected from phosphate buffer solution, carbonate buffer solution,
In Tri-HCl buffer solutions, borate buffer solution, glycine buffer, citrate buffer solution, MOPS buffer solutions
One kind.
Still further, additive is selected from aloe extract powder, Folium Pterocaryae extract powder, acetone, polyethylene glycol, dimethyl Asia
The mixture of one or more of sulfone, ethyl alcohol and calcium chloride.
Still further, it is 2-5 in mass ratio that additive, which is aloe extract powder, ethyl alcohol and calcium chloride,:3-8:1 preferably 3:
5:The mixture of 1 composition.
Again further, the preparation method of aloe extract powder is：Fresh aloe is crushed, is squeezed the juice, juice slag separation, by gained
Slag dry 6-8h at 80 DEG C, crushes, and crosses 30 mesh sieve, obtains aloe and take object powder.The preparation method of Folium Pterocaryae extract powder is：It will be fresh
Folium Pterocaryae is crushed, squeezes the juice, the separation of juice slag, and by gained slag, dry 6-8h, crushing at 80 DEG C, cross 30 mesh sieve, obtain Folium Pterocaryae and take
Object powder.
Again further, the pH value control of transformation system is 6-10, and preferably 5-7 controls the temperature of the transformation system
Degree is 20-60 DEG C, preferably 20-40 DEG C；The control of reaction system speed of agitator is 150-250r/min, preferably 180-220r/
min。
Again further, the concentration control of biocatalyst is 10-30g/L, preferably
15-20g/L；The control of the mass ratio of the additive and biocatalyst is 1-5%, preferably 2.5-4%.
Biocatalyst is the genetic engineering bacterium of the glufosinate-ammonium transaminase from Vibrio fluvialis, specific to prepare
Method is：Selection carries out engineer, after design from the gene order of the glufosinate-ammonium transaminase of Vibrio fluvialis
Gene order such as sequence table in SEQ ID NO：Shown in nucleotide sequence shown in 1；The sequence is synthesized by full genome, gram
It is grand enter expression vector pET28a Nde I and Xho I restriction enzyme sites, conversion host strain E.coli BL21 (DE3) competence it is thin
Born of the same parents；Picking positive transformant and after identification is sequenced, obtains recombinant expression carrier；Recombinant expression carrier is transferred to E.coli BL21
(DE3) in bacterial strain, the recombination glufosinate-ammonium aminotransferase gene engineering bacteria that glufosinate-ammonium transaminase can be recombinated with induced expression is obtained.
Recombination glufosinate-ammonium aminotransferase gene engineering bacteria is inoculated into the LB culture mediums containing kanamycins, overnight in 37 DEG C
Culture, obtains seed culture fluid；Seed culture fluid is inoculated into the TB culture mediums containing kanamycins, inoculum concentration is that is mould containing card
The 1% of the TB culture volumes of element；It is subsequently placed at 37 DEG C and cultivates 2-5h, sterile IPTG inductions are added, make IPTG final concentrations
Reach 0.1mM, is placed at 25 DEG C and continues to cultivate 20h.It is obtained from Vibrio fluvialis' finally by high speed centrifugation
The full cell of genetic engineering bacterium of glufosinate-ammonium transaminase.
Embodiment 1
The preparation process of L-glufosinate-ammonium crystal, includes the following steps：
The biological synthesis method of L-glufosinate-ammonium, reaction carries out in 1L shaking flasks, by the 2- carbonyls -4- of 30g (0.1657mol)
The MOPS buffer solutions of 300mL are added (with 3- N-morpholinyls and Na in (hydroxymethyl phosphono) butyric acid substrate2PO4For buffering pair
Saline) in, control Glufosinate-ammoniumpesticideng transaminase the full cell of engineering bacteria a concentration of 18g/L, control phosphopyridoxal pyridoxal phosphate
A concentration of 11mM, then be added into reaction system the 2- propylamine of 22.8g (0.2mol) and 0.162g additives convert it is anti-
It answers, obtains the conversion fluid containing L-glufosinate-ammonium.Wherein, additive be the aloe extract powder of 0.054g, 0.09g ethyl alcohol and
The mixture of the calcium chloride composition of 0.018g.The preparation method of aloe extract powder is：Fresh aloe is crushed, is squeezed the juice, the separation of juice slag,
By gained slag, dry 7h, crushing at 80 DEG C, cross 30 mesh sieve, obtain aloe extract powder.Control transformation system pH value be
6.5 the temperature for controlling transformation system is 30 DEG C；Conversion reaction carries out in shaking table, and the rotating speed control of shaking table is 200r/min.Instead
During answering, pass through the progress of high performance liquid chromatography (HPLC) monitoring reaction.
When reaction proceeds to 14.9h, high performance liquid chromatography monitoring, substrate converts completely, the receipts of prepared L-glufosinate-ammonium
Rate is 94.6%, is purified to the conversion fluid containing L-glufosinate-ammonium, and after purification, L-glufosinate-ammonium crystal purity reaches 99.6%,
Optical purity is 99.7%.
Embodiment 2
The preparation process of L-glufosinate-ammonium crystal, includes the following steps：
The biological synthesis method of L-glufosinate-ammonium, reaction carries out in 5L beakers, by the 2- carbonyls-of 200g (1.1050mol)
The phosphate buffer solution that 2L is added in 4- (hydroxymethyl phosphono) butyric acid substrate (is slow with disodium hydrogen phosphate and sodium dihydrogen phosphate
The saline of punching pair) in, a concentration of 18g/L of the full cell of engineering bacteria of Glufosinate-ammoniumpesticideng transaminase is controlled, phosphoric acid pyrrole is controlled
Tremble a concentration of 11.05mM of aldehyde, then the 2- propylamine of 228g (2mol) is added into reaction system and 1.08g additives are converted
Reaction, obtains the conversion fluid containing L-glufosinate-ammonium.Wherein, additive be the aloe extract powder of 0.36g, 0.6g ethyl alcohol and
The mixture of the calcium chloride composition of 0.12g.The preparation method of aloe extract powder is：Fresh aloe is crushed, is squeezed the juice, the separation of juice slag,
By gained slag, dry 7h, crushing at 80 DEG C, cross 30 mesh sieve, obtain aloe extract powder.Control transformation system pH value be
6.5, the temperature for controlling transformation system is 30 DEG C；The control of mechanical agitation rotating speed is 200r/min.In reaction process, pass through efficient liquid
The progress of phase chromatography (HPLC) monitoring reaction.
When reaction proceeds to 15.3h, high performance liquid chromatography monitoring, substrate converts completely, the receipts of prepared L-glufosinate-ammonium
Rate is 94.2%, is purified to the conversion fluid containing L-glufosinate-ammonium, and after purification, L-glufosinate-ammonium crystal purity reaches 99.5%,
Optical purity is 99.8%.
Embodiment 3
The preparation process of L-glufosinate-ammonium crystal, includes the following steps：
The biological synthesis method of L-glufosinate-ammonium, reaction carries out in 5L beakers, by the 2- carbonyls-of 180g (0.9945mol)
The MOPS buffer solutions of 2L are added (with 3- N-morpholinyls and Na in 4- (hydroxymethyl phosphono) butyric acid substrate2PO4For buffering pair
Saline) in, control Glufosinate-ammoniumpesticideng transaminase the full cell of engineering bacteria a concentration of 20g/L, control phosphopyridoxal pyridoxal phosphate
A concentration of 9.945mM, then into reaction system be added 228g (2mol) 2- propylamine and 1g additives carry out conversion reaction, obtain
To the conversion fluid containing L-glufosinate-ammonium.Wherein, additive is aloe extract powder, the ethyl alcohol of 0.4g and the calcium chloride of 0.1g of 0.5g
The mixture of composition.The preparation method of aloe extract powder is：Fresh aloe is crushed, is squeezed the juice, juice slag separation, by gained slag at 80 DEG C
Lower dry 7h is crushed, and is crossed 30 mesh sieve, is obtained aloe extract powder.The pH value for controlling transformation system is 7, controls transformation system
Temperature is 25 DEG C；The control of mechanical agitation rotating speed is 220r/min.In reaction process, monitored by high performance liquid chromatography (HPLC) anti-
The progress answered.
When reaction proceeds to 17.7h, high performance liquid chromatography monitoring, substrate converts completely, the receipts of prepared L-glufosinate-ammonium
Rate is 92.3%, is purified to the conversion fluid containing L-glufosinate-ammonium, and after purification, L-glufosinate-ammonium crystal purity reaches 99.2%,
Optical purity is 99.6%.
Embodiment 4
The preparation process of L-glufosinate-ammonium crystal, includes the following steps：
The biological synthesis method of L-glufosinate-ammonium, reaction carries out in 500mL shaking flasks, by the 2- carbonyls of 18g (0.0994mol)
Base -4- (hydroxymethyl phosphono) butyric acid substrate be added 150mL citrate buffer solution (be with citric acid and sodium citrate
The solution of buffering pair) in, a concentration of 15g/L of the full cell of engineering bacteria of Glufosinate-ammoniumpesticideng transaminase is controlled, phosphopyridoxal pyridoxal phosphate is controlled
A concentration of 13.3mM, then be added into reaction system the 2- propylamine of 22.8g (0.2mol) and 0.09g additives convert it is anti-
It answers, obtains the conversion fluid containing L-glufosinate-ammonium.Wherein, additive be the aloe extract powder of 0.03g, 0.05g ethyl alcohol and 0.01g
Calcium chloride composition mixture.The preparation method of aloe extract powder is：Fresh aloe is crushed, is squeezed the juice, juice slag separation, by gained
Slag dry 7h at 80 DEG C, crushes, and crosses 30 mesh sieve, obtains aloe extract powder.The pH value for controlling transformation system is 6, and control turns
The temperature of change system is 40 DEG C；Conversion reaction carries out in shaking table, and the rotating speed control of shaking table is 250r/min.In reaction process,
Pass through the progress of high performance liquid chromatography (HPLC) monitoring reaction.
When reaction proceeds to 16.5h, high performance liquid chromatography monitoring, substrate converts completely, the receipts of prepared L-glufosinate-ammonium
Rate is 90.4%, is purified to the conversion fluid containing L-glufosinate-ammonium, and after purification, L-glufosinate-ammonium crystal purity reaches 98.7%,
Optical purity is 99.5%.
Embodiment 5
The preparation process of L-glufosinate-ammonium crystal, includes the following steps：
The biological synthesis method of L-glufosinate-ammonium, reaction carries out in 1L shaking flasks, by the 2- carbonyls -4- of 45g (0.1657mol)
The glycine buffer that 300mL is added in (hydroxymethyl phosphono) butyric acid substrate (is buffering pair with glycine and sodium hydroxide
Solution) in, control Glufosinate-ammoniumpesticideng transaminase the full cell of engineering bacteria a concentration of 30g/L, control the concentration of phosphopyridoxal pyridoxal phosphate
For 11mM, then the 2- propylamine of addition 22.8g (0.2mol) and the progress conversion reaction of 0.45g additives into reaction system, obtain
Conversion fluid containing L-glufosinate-ammonium.Wherein, additive be the aloe extract powder of 0.0818g, 0.3273g ethyl alcohol and 0.0409g
Calcium chloride composition mixture.The preparation method of aloe extract powder is：Fresh aloe is crushed, is squeezed the juice, juice slag separation, by gained
Slag dry 7h at 80 DEG C, crushes, and crosses 30 mesh sieve, obtains aloe extract powder.The pH value for controlling transformation system is 10, and control turns
The temperature of change system is 60 DEG C；Conversion reaction carries out in shaking table, and the rotating speed control of shaking table is 180r/min.In reaction process,
Pass through the progress of high performance liquid chromatography (HPLC) monitoring reaction.
When reaction proceeds to 16.8h, high performance liquid chromatography monitoring, substrate converts completely, the receipts of prepared L-glufosinate-ammonium
Rate is 91.6%, is purified to the conversion fluid containing L-glufosinate-ammonium, and after purification, L-glufosinate-ammonium crystal purity reaches 97.8%,
Optical purity is 99.5%.
Embodiment 6
The preparation process of L-glufosinate-ammonium crystal, includes the following steps：
The biological synthesis method of L-glufosinate-ammonium, reaction carries out in 1L shaking flasks, by the 2- carbonyls -4- of 24g (0.1326mol)
The citrate buffer solution that 300mL is added in (hydroxymethyl phosphono) butyric acid substrate (is buffering with citric acid and sodium citrate
To solution) in, control Glufosinate-ammoniumpesticideng transaminase the full cell of engineering bacteria a concentration of 10g/L, control the dense of phosphopyridoxal pyridoxal phosphate
Degree is 8.8mM, then 2- propylamine and the progress conversion reaction of 0.03g additives of 22.8g (0.2mol) are added into reaction system, is obtained
To the conversion fluid containing L-glufosinate-ammonium.Wherein, additive be the aloe extract powder of 0.0115g, 0.0139g ethyl alcohol and
The mixture of the calcium chloride composition of 0.0046g.The preparation method of aloe extract powder is：Fresh aloe is crushed, is squeezed the juice, juice slag point
From by gained slag, dry 7h, crushing at 80 DEG C, cross 30 mesh sieve, obtain aloe extract powder.Control transformation system pH value be
5, the temperature for controlling transformation system is 20 DEG C；Conversion reaction carries out in shaking table, and the rotating speed control of shaking table is 150r/min.Reaction
In the process, pass through the progress of high performance liquid chromatography (HPLC) monitoring reaction.
Transformation time is 19.2h, and the yield of prepared L-glufosinate-ammonium is 89.1%, to the conversion fluid containing L-glufosinate-ammonium
It is purified, after purification, L-glufosinate-ammonium crystal purity reaches 98.0%, optical purity 99.3%.
Comparative example 1
The preparation process of L-glufosinate-ammonium crystal, includes the following steps：
The biological synthesis method of L-glufosinate-ammonium, reaction carries out in 1L shaking flasks, by the 2- carbonyls -4- of 30g (0.1657mol)
The MOPS buffer solutions of 300mL are added (with 3- N-morpholinyls and Na in (hydroxymethyl phosphono) butyric acid substrate2PO4For buffering pair
Saline) in, control Glufosinate-ammoniumpesticideng transaminase the full cell of engineering bacteria a concentration of 18g/L, control phosphopyridoxal pyridoxal phosphate
A concentration of 11mM, then the 2- propylamine of 22.8g (0.2mol) is added into reaction system and carries out conversion reaction, obtain careless containing L-
The conversion fluid of ammonium phosphine.The pH value for controlling transformation system is 6.5, and the temperature for controlling transformation system is 30 DEG C；Conversion reaction is in shaking table
The rotating speed control of middle progress, shaking table is 200r/min.In reaction process, by high performance liquid chromatography (HPLC) monitoring reaction into
Row.
Transformation time is 27.2h, and the yield of prepared L-glufosinate-ammonium is 80.4%, to the conversion fluid containing L-glufosinate-ammonium
It is purified, after purification, L-glufosinate-ammonium crystal purity reaches 96.9%, optical purity 99.1%.
Comparative example 2
The preparation process of L-glufosinate-ammonium crystal, includes the following steps：
The biological synthesis method of L-glufosinate-ammonium, reaction carries out in 1L shaking flasks, by the 2- carbonyls -4- of 30g (0.1657mol)
The MOPS buffer solutions of 300mL are added (with 3- N-morpholinyls and Na in (hydroxymethyl phosphono) butyric acid substrate2PO4For buffering pair
Saline) in, control Glufosinate-ammoniumpesticideng transaminase the full cell of engineering bacteria a concentration of 18g/L, control phosphopyridoxal pyridoxal phosphate
A concentration of 11mM, then be added into reaction system the 2- propylamine of 22.8g (0.2mol) and 0.162g additives convert it is anti-
It answers, obtains the conversion fluid containing L-glufosinate-ammonium.Wherein, additive is the aloe extract powder of 0.162g.The preparation of aloe extract powder
Method is：Fresh aloe is crushed, is squeezed the juice, the separation of juice slag, dry 7h, crushing at 80 DEG C, cross 30 mesh sieve, obtain reed by gained slag
Luxuriant growth extract powder.The pH value for controlling transformation system is 6, and the temperature for controlling transformation system is 30 DEG C；Conversion reaction in shaking table into
The rotating speed control of row, shaking table is 200r/min.In reaction process, pass through the progress of high performance liquid chromatography (HPLC) monitoring reaction.
Transformation time is 23.9h, and the yield of prepared L-glufosinate-ammonium is 86.3%, to the conversion fluid containing L-glufosinate-ammonium
It is purified, after purification, L-glufosinate-ammonium crystal purity reaches 97.2%, optical purity 99.5%.
Comparative example 3
The preparation process of L-glufosinate-ammonium crystal, includes the following steps：
The biological synthesis method of L-glufosinate-ammonium, reaction carries out in 1L shaking flasks, by the 2- carbonyls -4- of 30g (0.1657mol)
The MOPS buffer solutions of 300mL are added (with 3- N-morpholinyls and Na in (hydroxymethyl phosphono) butyric acid substrate2PO4For buffering pair
Saline) in, control Glufosinate-ammoniumpesticideng transaminase the full cell of engineering bacteria a concentration of 18g/L, control phosphopyridoxal pyridoxal phosphate
A concentration of 11mM, then be added into reaction system the 2- propylamine of 22.8g (0.2mol) and 0.162g additives convert it is anti-
It answers, obtains the conversion fluid containing L-glufosinate-ammonium.Wherein, additive is the ethyl alcohol of 0.162g.The pH value for controlling transformation system is 6,
The temperature for controlling transformation system is 30 DEG C；Conversion reaction carries out in shaking table, and the rotating speed control of shaking table is 200r/min.It reacted
Cheng Zhong passes through the progress of high performance liquid chromatography (HPLC) monitoring reaction.
Transformation time is 24.6h, and the yield of prepared L-glufosinate-ammonium is 83.9%, to the conversion fluid containing L-glufosinate-ammonium
It is purified, after purification, L-glufosinate-ammonium crystal purity reaches 96.8%, optical purity 99.2%.
Comparative example 4
The preparation process of L-glufosinate-ammonium crystal, includes the following steps：
The biological synthesis method of L-glufosinate-ammonium, reaction carries out in 1L shaking flasks, by the 2- carbonyls -4- of 30g (0.1657mol)
The MOPS buffer solutions of 300mL are added (with 3- N-morpholinyls and Na in (hydroxymethyl phosphono) butyric acid substrate2PO4For buffering pair
Saline) in, control Glufosinate-ammoniumpesticideng transaminase the full cell of engineering bacteria a concentration of 18g/L, control phosphopyridoxal pyridoxal phosphate
A concentration of 11mM, then be added into reaction system the 2- propylamine of 22.8g (0.2mol) and 0.162g additives convert it is anti-
It answers, obtains the conversion fluid containing L-glufosinate-ammonium.Wherein, additive is the calcium chloride of 0.162g.Control transformation system pH value be
6, the temperature for controlling transformation system is 30 DEG C；Conversion reaction carries out in shaking table, and the rotating speed control of shaking table is 200r/min.Reaction
In the process, pass through the progress of high performance liquid chromatography (HPLC) monitoring reaction.
Transformation time is 25.1h, and the yield of prepared L-glufosinate-ammonium is 84.7%, to the conversion fluid containing L-glufosinate-ammonium
It is purified, after purification, L-glufosinate-ammonium crystal purity reaches 97.6%, optical purity 99.3%.
Embodiment 1, embodiment 2 are compared with comparative example 1 it is found that when additive is added in transformation system, and transformation time is bright
Aobvious to shorten, the yield of L-glufosinate-ammonium is also obviously improved, and the quality of L-glufosinate-ammonium crystal after purification also significantly improves.Embodiment 1,
Embodiment 2 is with comparative example 1, comparative example 2, the comparison of comparative example 3 it is found that additive is aloe extract powder, ethyl alcohol and calcium chloride compounding
The mixture of composition is compared with single addition aloe extract powder, ethyl alcohol or calcium chloride, and transformation time is obviously shortened, L-glufosinate-ammonium
Yield is also obviously improved, and the quality of L-glufosinate-ammonium crystal after purification also significantly improves, and aloe extract powder, ethyl alcohol and calcium chloride are multiple
Unexpected technique effect is reached with addition.
The content not being described in detail in this specification belongs to the prior art well known to those skilled in the art.
<110>Wuhan Yin Maote Bioisystech Co., Ltd
<120>The biological synthesis method of L-glufosinate-ammonium
<160> 1
<211> 1359
<212> DNA
<213>Artificial sequence
<400> 1
atgaacaaac cgcagagctg ggaagcgcgt gcggaaacct atagcctgta tggctttacc 60
gatatgccga gcctgcatca gcgtggcacc gtggtggtga cccatggcga aggcccgtat 120
attgtggatg tgaacggccg tcgttatctg gatgcgaaca gcggcctgtg gaacatggtg 180
ccgggctttg atcataaagg cctgattgat gcggcgaaag cgcagtatga acgttttccg 240
ggctatcatg cgttttttgg ccgtatgagc gatcagaccg tgatgctgag cgaaaaactg 300
gtggaagtga gcccgtttga tagcggccgt gtgttttata ccaacagcgg cagcgaagcg 360
aacgatacca tggtgaaaat gctgtggttt ctgcatgcgg cggaaggcaa accgcagaaa 420
cgtaaaattc tgacccgttg gaacgcgtat catggcgtga ccgcggtgag cgcgagcatg 480
accggcaaac cgtataacag cgtgtttggc ctggcgctgc cgggctttgt gcatctgacc 540
tgcccgcatt attggcgtta tggcgaagaa ggcgaaaccg aagaacagtt tgtggcgcgt 600
ctggcgcgtg aactggaaga aaccattcag cgtgaaggcg cggataccat tgcgggcttt 660
tttgcggaac cggtgatggg cgcgggcggc gtgattccgc cggcgaaagg ctattttcag 720
gcgattctgc cgattctgcg taaatatgat attccggtga ttagcgatga agtgatttgc 780
ggctttggcc gtaccggcaa cacctggggc tgcgtgacct atgattttac cccggatgcg 840
attattagca gcaaaaacct gaccgcgggc ttttttccga tgggcgcggt gattctgggc 900
ccggaactga gcaaacgtct ggaaaccgcg attgaagcga ttgaagaatt tccgcatggc 960
tttaccgcga gcggccatcc ggtgggctgc gcgattgcgc tgaaaccgat tgatgtggtg 1020
atgaacgaag gcctggcgga aaacgtgcgt cgtctggcgc cgcgttttga agaacgtctg 1080
aaacatattg cggaacgtcc gaacattggc gaatatcgtg gcattggctt tatgtgggcg 1140
ctggaagcgg tgaaagataa agcgagcaaa accccgtttg atggcaacct gagcgtgagc 1200
gaacgtattg cgaacacctg caccgatctg ggcctgattt gccgtccgct gggccagagc 1260
gtggtgctgt gcccgccgtt tattctgacc gaagcgcaga tggatgaaat gtttgataaa 1320
ctggaaaaag cgctggataa agtgtttgcg gaagtggcg 1359
Claims (10)
1. a kind of biological synthesis method of L-glufosinate-ammonium, which is characterized in that including：With 2- carbonyls -4- (hydroxymethyl phosphono)
Butyric acid is substrate, using 2- propylamine as amino group donor, constitutes reaction system with solvent, then biology is added into the reaction system and urges
Agent, coenzyme and additive carry out bioconversion reaction, obtain the conversion fluid containing L-glufosinate-ammonium.
2. the biological synthesis method of L-glufosinate-ammonium according to claim 1, which is characterized in that the biocatalyst is next
Derived from the glufosinate-ammonium transaminase of Vibrio fluvialis, the glufosinate-ammonium transaminase from Vibrio fluvialis
The encoding gene of genetic engineering bacterium is SEQ ID NO in sequence table：Nucleotide sequence shown in 1；The coenzyme is that phosphoric acid pyrrole is trembled
Aldehyde.
3. the biological synthesis method of L-glufosinate-ammonium according to claim 2, which is characterized in that the solvent is that buffering is molten
Liquid, it is molten that the buffer solution is selected from phosphate buffer solution, carbonate buffer solution, Tri-HCl buffer solutions, boric acid salt buffer
One kind in liquid, glycine buffer, citrate buffer solution, MOPS buffer solutions.
4. the biological synthesis method of L-glufosinate-ammonium according to claim 3, which is characterized in that the additive is selected from aloe
The mixing of one or more of extract powder, Folium Pterocaryae extract powder, acetone, polyethylene glycol, dimethyl sulfoxide (DMSO), ethyl alcohol and calcium chloride
Object.
5. the biological synthesis method of L-glufosinate-ammonium according to claim 4, which is characterized in that the additive carries for aloe
It is 2-5 to take powder, ethyl alcohol and calcium chloride in mass ratio:3-8:The mixture of 1 composition.
6. the biological synthesis method of L-glufosinate-ammonium according to claim 5, which is characterized in that the additive carries for aloe
It is 3 to take powder, ethyl alcohol and calcium chloride in mass ratio:5:The mixture of 1 composition.
7. the biological synthesis method of L-glufosinate-ammonium according to claim 5 or 6, which is characterized in that the aloe extract powder
Preparation method be：Fresh aloe is crushed, is squeezed the juice, the separation of juice slag, 30 mesh are crossed in dry 6-8h, crushing at 80 DEG C by gained slag
Sieve, obtains aloe and takes object powder.
8. the biological synthesis method of L-glufosinate-ammonium according to claim 7, which is characterized in that the pH value of transformation system controls
For 5-10, the temperature for controlling the transformation system is 20-60 DEG C；The control of reaction system speed of agitator is 150-250r/min.
9. the biological synthesis method of L-glufosinate-ammonium according to claim 7, which is characterized in that the biocatalyst it is dense
Degree control is 10-30g/L；The control of the mass ratio of the additive and biocatalyst is 1-5%.
10. the biological synthesis method of L-glufosinate-ammonium according to claim 8 or claim 9, which is characterized in that the pH value of transformation system
Control is 6-7, and the temperature control of the transformation system is 25-40 DEG C；The control of reaction system speed of agitator is 180-220r/min,
The concentration control of the biocatalyst is 15-20g/L；The control of the mass ratio of the additive and biocatalyst is 2.5-
4%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710195282.9A CN108660167A (en) | 2017-03-29 | 2017-03-29 | The biological synthesis method of L-glufosinate-ammonium |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609476A (en) * | 2019-01-14 | 2019-04-12 | 浙江工业大学 | α-Transaminases and mutants and their application in asymmetric synthesis of L-glufosinate |
| US10260078B2 (en) | 2016-03-02 | 2019-04-16 | Agrimetis, Llc | Methods for making L-glufosinate |
| CN113234767A (en) * | 2021-05-13 | 2021-08-10 | 永农生物科学有限公司 | Method for producing solid L-glufosinate ammonium salt powder free of crystal water |
| CN114150026A (en) * | 2021-05-10 | 2022-03-08 | 永州恒飞生物医药有限公司 | Application of providencia in producing 1-substituted-propanesulfonic acid |
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- 2017-03-29 CN CN201710195282.9A patent/CN108660167A/en active Pending
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| US11560577B2 (en) | 2016-03-02 | 2023-01-24 | Basf Se | Methods for making L-glufosinate |
| US10260078B2 (en) | 2016-03-02 | 2019-04-16 | Agrimetis, Llc | Methods for making L-glufosinate |
| US10781465B2 (en) | 2016-03-02 | 2020-09-22 | Agrimetis, Llc | Methods for making L-glufosinate |
| US11732281B2 (en) | 2016-03-02 | 2023-08-22 | Basf Se | Methods for making L-glufosinate |
| US11905538B2 (en) | 2016-03-02 | 2024-02-20 | Basf Se | Methods for making L-glufosinate |
| US11913048B2 (en) | 2016-03-02 | 2024-02-27 | Basf Se | Methods for making L-glufosinate |
| US12305207B2 (en) | 2016-03-02 | 2025-05-20 | Basf Se | Methods for making L-glufosinate |
| US12305206B2 (en) | 2016-03-02 | 2025-05-20 | Basf Se | Methods for making L-glufosinate |
| CN109609476B (en) * | 2019-01-14 | 2020-06-19 | 浙江工业大学 | α-Transaminases and mutants and their application in asymmetric synthesis of L-glufosinate |
| CN109609476A (en) * | 2019-01-14 | 2019-04-12 | 浙江工业大学 | α-Transaminases and mutants and their application in asymmetric synthesis of L-glufosinate |
| CN114150026A (en) * | 2021-05-10 | 2022-03-08 | 永州恒飞生物医药有限公司 | Application of providencia in producing 1-substituted-propanesulfonic acid |
| CN114150026B (en) * | 2021-05-10 | 2024-05-28 | 永州恒飞生物医药有限公司 | Use of providencia in producing 1-substituted-propanesulfonic acid |
| CN113234767A (en) * | 2021-05-13 | 2021-08-10 | 永农生物科学有限公司 | Method for producing solid L-glufosinate ammonium salt powder free of crystal water |
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