CN106978368B - Raoultella ornithinolytica and application thereof - Google Patents
Raoultella ornithinolytica and application thereof Download PDFInfo
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- CN106978368B CN106978368B CN201710207970.2A CN201710207970A CN106978368B CN 106978368 B CN106978368 B CN 106978368B CN 201710207970 A CN201710207970 A CN 201710207970A CN 106978368 B CN106978368 B CN 106978368B
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- hydroxyethyl methyl
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- methyl phosphoryl
- butyric acid
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- 241000531124 Raoultella ornithinolytica Species 0.000 title claims abstract description 18
- MSOSIHPIGQOOQX-YLTHGKPTSA-N (2S)-2-amino-4-[2-hydroxyethyl(methyl)phosphoryl]butanoic acid Chemical compound N[C@H](C(=O)O)CCP(=O)(C)CCO MSOSIHPIGQOOQX-YLTHGKPTSA-N 0.000 claims abstract description 25
- RBHVPEOMWFHDON-UHFFFAOYSA-N 2-amino-4-[2-hydroxyethyl(methyl)phosphoryl]butanenitrile Chemical compound NC(C#N)CCP(=O)(C)CCO RBHVPEOMWFHDON-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000000758 substrate Substances 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 36
- 239000000243 solution Substances 0.000 claims description 32
- JBKVHLHDHHXQEQ-UHFFFAOYSA-N epsilon-caprolactam Chemical compound O=C1CCCCCN1 JBKVHLHDHHXQEQ-UHFFFAOYSA-N 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 27
- 239000008367 deionised water Substances 0.000 claims description 25
- 229910021641 deionized water Inorganic materials 0.000 claims description 25
- 238000006243 chemical reaction Methods 0.000 claims description 24
- 239000000203 mixture Substances 0.000 claims description 22
- 239000001963 growth medium Substances 0.000 claims description 19
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 18
- 239000002904 solvent Substances 0.000 claims description 18
- 239000007836 KH2PO4 Substances 0.000 claims description 16
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 16
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 16
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 16
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 14
- 229930195725 Mannitol Natural products 0.000 claims description 14
- 229940041514 candida albicans extract Drugs 0.000 claims description 14
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims description 14
- 239000000594 mannitol Substances 0.000 claims description 14
- 235000010355 mannitol Nutrition 0.000 claims description 14
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 14
- 229940073490 sodium glutamate Drugs 0.000 claims description 14
- 239000012138 yeast extract Substances 0.000 claims description 14
- 238000012258 culturing Methods 0.000 claims description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
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- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 3
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- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 3
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- 238000004321 preservation Methods 0.000 claims description 2
- 239000013076 target substance Substances 0.000 claims description 2
- 241001671311 Laurus Species 0.000 claims 1
- 108010033272 Nitrilase Proteins 0.000 abstract description 14
- 239000002243 precursor Substances 0.000 abstract description 6
- 241000321184 Raoultella Species 0.000 abstract description 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
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- 238000012216 screening Methods 0.000 description 8
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- 108020004465 16S ribosomal RNA Proteins 0.000 description 6
- IAJOBQBIJHVGMQ-UHFFFAOYSA-N Phosphinothricin Natural products CP(O)(=O)CCC(N)C(O)=O IAJOBQBIJHVGMQ-UHFFFAOYSA-N 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
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- 230000009466 transformation Effects 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000005342 ion exchange Methods 0.000 description 4
- 238000002791 soaking Methods 0.000 description 4
- 239000002689 soil Substances 0.000 description 4
- KKVFPVZIZAJCBZ-UHFFFAOYSA-N (2-hydroxyphenyl) hypochlorite Chemical compound OC1=CC=CC=C1OCl KKVFPVZIZAJCBZ-UHFFFAOYSA-N 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 3
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- MSOSIHPIGQOOQX-JNKNCIAKSA-N (2R)-2-amino-4-[2-hydroxyethyl(methyl)phosphoryl]butanoic acid Chemical compound N[C@@H](C(=O)O)CCP(=O)(C)CCO MSOSIHPIGQOOQX-JNKNCIAKSA-N 0.000 description 2
- OVFWFOODFANRJL-UHFFFAOYSA-N 2-[2-hydroxyethyl(methyl)phosphoryl]butanenitrile Chemical compound OCCP(=O)(C)C(C#N)CC OVFWFOODFANRJL-UHFFFAOYSA-N 0.000 description 2
- MSOSIHPIGQOOQX-UHFFFAOYSA-N 2-amino-4-[2-hydroxyethyl(methyl)phosphoryl]butanoic acid Chemical compound NC(C(=O)O)CCP(=O)(C)CCO MSOSIHPIGQOOQX-UHFFFAOYSA-N 0.000 description 2
- 239000005552 B01AC04 - Clopidogrel Substances 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 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 2
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 2
- 241000097097 Laurella Species 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- GKTWGGQPFAXNFI-HNNXBMFYSA-N clopidogrel Chemical compound C1([C@H](N2CC=3C=CSC=3CC2)C(=O)OC)=CC=CC=C1Cl GKTWGGQPFAXNFI-HNNXBMFYSA-N 0.000 description 2
- 229960003009 clopidogrel Drugs 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- IAJOBQBIJHVGMQ-BYPYZUCNSA-N glufosinate-P Chemical group CP(O)(=O)CC[C@H](N)C(O)=O IAJOBQBIJHVGMQ-BYPYZUCNSA-N 0.000 description 2
- NBZBKCUXIYYUSX-UHFFFAOYSA-N iminodiacetic acid Chemical compound OC(=O)CNCC(O)=O NBZBKCUXIYYUSX-UHFFFAOYSA-N 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 229940054441 o-phthalaldehyde Drugs 0.000 description 2
- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical compound O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 description 2
- 238000004007 reversed phase HPLC Methods 0.000 description 2
- BPRHUIZQVSMCRT-VEUZHWNKSA-N rosuvastatin Chemical compound CC(C)C1=NC(N(C)S(C)(=O)=O)=NC(C=2C=CC(F)=CC=2)=C1\C=C\[C@@H](O)C[C@@H](O)CC(O)=O BPRHUIZQVSMCRT-VEUZHWNKSA-N 0.000 description 2
- 229960000672 rosuvastatin Drugs 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- CHDRYQOHTORLAX-UHFFFAOYSA-N (3-amino-3-cyanopropylidene)-(hydroxymethyl)-oxidophosphanium Chemical compound NC(C#N)CC=P(=O)CO CHDRYQOHTORLAX-UHFFFAOYSA-N 0.000 description 1
- OEJAZOGPPRWZKM-RXMQYKEDSA-N (3r)-5-ethoxy-3-hydroxy-5-oxopentanoic acid Chemical compound CCOC(=O)C[C@H](O)CC(O)=O OEJAZOGPPRWZKM-RXMQYKEDSA-N 0.000 description 1
- RWOLDZZTBNYTMS-UHFFFAOYSA-N 2-(2-chlorophenyl)-2-hydroxyacetic acid Chemical compound OC(=O)C(O)C1=CC=CC=C1Cl RWOLDZZTBNYTMS-UHFFFAOYSA-N 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- GAWIXWVDTYZWAW-UHFFFAOYSA-N C[CH]O Chemical group C[CH]O GAWIXWVDTYZWAW-UHFFFAOYSA-N 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 239000005562 Glyphosate Substances 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 231100000674 Phytotoxicity Toxicity 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 230000000702 anti-platelet effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
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- 125000004093 cyano group Chemical group *C#N 0.000 description 1
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- XDDAORKBJWWYJS-UHFFFAOYSA-N glyphosate Chemical compound OC(=O)CNCP(O)(O)=O XDDAORKBJWWYJS-UHFFFAOYSA-N 0.000 description 1
- 229940097068 glyphosate Drugs 0.000 description 1
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- 238000002156 mixing Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- PVADDRMAFCOOPC-UHFFFAOYSA-N oxogermanium Chemical compound [Ge]=O PVADDRMAFCOOPC-UHFFFAOYSA-N 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
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- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- 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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Abstract
The invention discloses Raoultella ornithinolytica and application thereof in catalyzing α -aminonitrile substrate (2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile) to prepare L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid serving as an important chiral precursor of L-glufosinate-ammonium, and provides a strain capable of producing nitrilase, namely Raoultella ornithinola ornithinolytica ZJB-16008.
Description
(I) technical field
The invention relates to a bacterial strain for producing nitrilase and application of the bacterial strain in preparing L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid serving as an important chiral precursor of L-glufosinate-ammonium by biocatalysis of α -aminonitrile substrate (2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile).
(II) background of the invention
Nitrilase (Nitrilase, EC 3.5.5.1) can directly convert cyano in nitrile compounds into carboxyl to prepare carboxylic acid, has unique chemoselectivity, stereoselectivity and regioselectivity for nitrile compounds, and can be used for synthesizing carboxylic acid with low synthesis efficiency or difficult synthesis by various chemical methods. The method has the advantages of good selectivity and high conversion rate, meanwhile, compared with the traditional process, the method has the advantages of mild reaction conditions, no need of high temperature and high pressure, strong acid and strong base, less pollution, incomparable superiority of a chemical method, accordance with the development direction of atom economy and green chemistry, great application potential in chemical synthesis, great development in application quantity and variety, and wide application in the fields of medicines and intermediates thereof, pesticides and intermediates thereof, foods and feed additives and the like: preparing key intermediate (R) -3-hydroxy glutaric acid ethyl ester of Rosuvastatin (Rosuvastatin) by nitrilase catalysis; preparing a key intermediate R-type o-chloromandelic acid of an anti-platelet aggregation medicament Clopidogrel (Clopidogrel); the key intermediate iminodiacetic acid for preparing herbicide glyphosate in the IDA route, and the like.
DL-glufosinate-ammonium, the active ingredient is phosphinothricin (PPT for short), the chemical name is 4- [ hydroxymethyl phosphonyl ] -DL-homoalanine (alias: D, L-glufosinate amine salt), the herbicide is a broad-spectrum, contact type, sterilant and non-residue herbicide, which is developed in the 20 th century 80 th generation by German Herster company (currently, Bayer company), besides the herbicidal activity, the herbicide has the characteristics of high efficiency, low toxicity, easy degradation and the like, the glufosinate-ammonium is a racemic mixture, only the L-type has phytotoxicity, the herbicidal activity is 2 times of that of the racemic mixture, the commercially available glufosinate-ammonium is a racemic mixture, and if the L-type glufosinate-ammonium can replace the L-type glufosinate-ammonium, the L-amino-4 (hydroxyethyl methylphosphonyl) -butyric acid can be used for preparing L-glufosinate-ammonium, the synthesis method can reduce the cost by using a chiral amino acid-phospho-ethyl-transferase, the L-glufosinate-amino-4 (L-glufosinate-ethyl-amino-4) is an important chiral precursor for preparing L-glufosinate, the L-glufosinate-L-homoamino acid, the L-homoamino acid is a novel sterilant herbicide developed by a broad-ketolase, the novel sterilant herbicide developed in the 20 th 80 th ages of 20 th ages of the German-Herschel, the German-Herber, the German-one, the German-Hertz, the German, the modern Hertz, the L-Carpine, the same as the same.
The method for industrially producing glufosinate-ammonium can be summarized as the following three routes, namely, Bayer company prepares D, L-glufosinate-ammonium by using a Michael free radical addition method, the D, L-glufosinate-ammonium is synthesized by using a Srecker method, and L-glufosinate-ammonium is synthesized by using an asymmetric catalytic hydrogenation method in a bright control fruit preparation process, wherein the D synthesized by the Srecker method and the L-glufosinate-ammonium route have mild reaction conditions and are a route adopted by the current industrial large production.
Disclosure of the invention
The invention aims to provide a nitrilase-producing strain-Raoultella ornithinolytica ZJB-16008 and application thereof in preparing L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid by biocatalysis of α -aminonitrile substrate (2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile).
The technical scheme adopted by the invention is as follows:
the invention provides a new strain with nitrilase production performance, namely Raoultella ornithinolytica ZJB-16008, which is preserved in China center for type culture collection with the preservation number of CCTCC No: m2017035, date of deposit 2017, month 01, day 13, address: wuhan, Wuhan university, zip code 430072.
The invention also relates to an application of the Raoultella ornithinolytica ZJB-16008 in catalyzing α -aminonitrile (2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile) to prepare L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid serving as an important chiral precursor of L-glufosinate-ammonium, wherein the application is that wet thallus obtained by fermenting and culturing the Raoultella ornithinolytica ZJB-16008 is taken as a catalyst, 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile is taken as a substrate, distilled water or 100mM phosphate buffer solution with the pH of 7.0 is taken as a reaction medium, conversion reaction is carried out under the conditions of 30-50 ℃ and 100-200rpm, the reaction liquid is separated and purified after the reaction is finished, and the L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid is obtained.
The nitrilase biocatalysis of α -aminonitrile (2-amino-4 (hydroxyethylmethylphosphoryl) -butyronitrile) to L-2-amino-4 (hydroxyethylmethylphosphoryl) -butyric acid was as follows:
furthermore, the dosage of the catalyst is 10-100g/L of reaction medium, preferably 35g/L, calculated by the weight of wet bacteria, and the final concentration of the substrate initially added is 2-100g/L of reaction medium, preferably 5 g/L.
Further, the catalyst is prepared by the following method: (1) slant culture: inoculating Raoultella ornithinolytica ZJB-16008 to slant culture medium, and culturing at 30 deg.C for 48 hr to obtain slant thallus; the final concentration of the slant culture medium is as follows: 1-20.0 g/L of mannitol, 1-10.0 g/L of sodium glutamate, 1-5.0 g/L of yeast extract and K2HPO40.1～1.0g/L，KH2PO40.1～1.0g/L，MgSO40.1-1.0 g/L, 0.1-2.0 g/L caprolactam, 20.0g/L agar, deionized water as solvent and pH 7.0-7.5; preferably, the final concentration of the slant culture medium is as follows: 10.0g/L of mannitol, 7.0g/L of sodium glutamate, 3.0g/L of yeast extract and K2HPO40.75g/L，KH2PO40.75g/L，MgSO40.5g/L, caprolactam 1.0g/L, agar 20.0g/L, deionized water as solvent, and pH 7.0-7.5;
(2) seed culture: selecting slant thallus, inoculating to seed culture medium, and culturing at 30 deg.C for 24 hr to obtain seed solution; the final concentration of the seed culture medium is as follows: 1-20.0 g/L of mannitol, 1-10.0 g/L of sodium glutamate, 1-5.0 g/L of yeast extract and K2HPO40.1～1.0g/L，KH2PO40.1～1.0g/L，MgSO40.1-1.0 g/L, 0.1-2.0 g/L caprolactam, deionized water as solvent and pH 7.0-7.5; preferably, the final concentration composition of the seed culture medium is as follows: 10.0g/L of mannitol, 7.0g/L of sodium glutamate, 3.0g/L of yeast extract and K2HPO40.75g/L，KH2PO40.75g/L，MgSO40.5g/L of caprolactam, 1.0g/L of caprolactam and deionized water as a solvent, wherein the pH value is 7.0-7.5;
(3) fermentation culture: inoculating the seed solution into a fermentation culture medium in an inoculation amount with the volume concentration of 1-10%, carrying out shaking culture at 30 ℃ and 150rpm for 48h, centrifuging at 12000g for 10min, and collecting wet thalli; the final concentration of the fermentation medium is as follows: 1-20.0 g/L of mannitol, 1-10.0 g/L of sodium glutamate, 1-5.0 g/L of yeast extract and K2HPO40.1～1.0g/L，KH2PO40.1～1.0g/L，MgSO40.1-1.0 g/L, 0.1-2.0 g/L caprolactam, deionized water as solvent, and pH 7.0-7.5. Preferably, the final concentration of the fermentation medium consists of: 10.0g/L of mannitol, 7.0g/L of sodium glutamate, 3.0g/L of yeast extract and K2HPO40.75g/L，KH2PO40.75g/L，MgSO40.5g/L, caprolactam 1.0g/L and deionized water as solvent, and the pH value is 7.0-7.5.
The method for separating and purifying the reaction liquid comprises the following steps: adjusting the pH value of the reaction liquid to 2.5, performing column chromatography by adopting anion exchange resin 201 multiplied by 7, loading the sample at the flow rate of the column of 1-6.0 BV/h (preferably 4BV/h), washing with deionized water, eluting with 0.5-2.0M (preferably 1M) of ammonia water at the elution speed of 1.0-4.0 BV/h (preferably 2BV/h), and collecting the eluent containing the target substance; distilling the eluent under reduced pressure until no solvent flows out, adding methanol for dissolving, stirring in ice bath, recrystallizing, filtering, and drying the filter cake to obtain the product L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid.
In the invention, 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile is selectively catalyzed and hydrolyzed by nitrilase to prepare L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid serving as an important chiral precursor of L-glufosinate-ammonium, and a basis is provided for synthesizing L-glufosinate-ammonium by a Srecker method with a mature industrial route. Meanwhile, the acidolysis reaction in the domestic industrial Strecker route is replaced, and the acidic wastewater pollution generated by the acidolysis reaction and the corrosion damage of the acidolysis reaction to equipment are reduced.
The invention has the following beneficial effects: the invention provides a strain capable of producing nitrilase, namely Raoultella ornithinolytica ZJB-16008, and also provides a method for preparing L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid serving as an important chiral precursor of L-glufosinate-ammonium by catalyzing 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile by using wet thallus obtained by fermenting and culturing the Raoultella ornithinolytica ZJB-16008 as a biocatalyst, and the strain has an important application prospect.
(IV) description of the drawings
FIG. 1 is a liquid phase result spectrum of D-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid and L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid under the condition of pre-column derivatization reversed phase high performance liquid chromatography.
FIG. 2 is a standard curve of ammonium ion concentration.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1: screening and identification of nitrilase-producing strains
1. Screening of nitrilase-producing strains
(1) Taking soil samples from all over the country, weighing 1g of the soil samples, suspending the soil samples by using 0.85% physiological saline, standing the soil samples, adding 1mL of supernatant into 50mL of enrichment medium added with 1g/L α -aminonitrile substrate (2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile) as a unique carbon source, culturing the mixture for 3 days on a shaking table at the temperature of 30 ℃ and the speed of 150rpm, taking 1mL of culture solution after the culture medium is turbid, transferring the culture solution into a fresh enrichment medium added with 1 g/L2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile, and culturing the mixture for 3 days again, so circularly enriching the mixture for 3-4 cycles;
(2) diluting the final enrichment culture solution with sterile water for 10 gradients step by step, and selecting 10-4、10-5、10-6、10-7、10-8Uniformly coating 0.1mL of each of five gradient diluents on a plate culture medium added with 1g/L of 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile, culturing for 2-3 days in a 30 ℃ constant temperature incubator, picking single colony on the plate, inoculating the single colony on a slant culture medium, culturing for 2-3 days in a 30 ℃ constant temperature incubator, and preserving at 4 ℃;
(3) the strain preserved on the slant was inoculated into a seed medium and cultured at 30 ℃ for 24 hours. The seed solution was inoculated into the fermentation medium at an inoculum size of 1% by volume concentration and cultured at 30 ℃ for 48 hours with shaking at 150 rpm. Centrifuging at 12000g for 10min, collecting wet thallus, suspending certain amount of thallus and substrate (2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile) in distilled water, making into reaction system with bacterium concentration of 0.2g/10mL and substrate concentration of 10g/L for conversion, and placing in a water bath shaker at 30 ℃ for conversion for 24 h. Sampling, carrying out primary screening by a phenol-hypochlorite method, detecting the concentration and optical purity of a product L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid by high performance liquid chromatography, and finally screening to obtain the strain ZJB-16008, wherein the nitrilase activity is relatively good.
The final concentration composition of the enrichment medium is (g/L): glucose 5.0, sodium chloride 1.0, K2HPO4·3H2O 0.8，KH2PO43.3，MgSO4·7H2O0.2, deionized water as solvent, pH7.0, and 1 g/L2-amino-4 (hydroxyethyl methylphosphoryl) -butyronitrile as the sole carbon source.
The plate screening culture medium is (g/L): glucose 5.0, sodium chloride 1.0, K2HPO4·3H2O 0.8，KH2PO43.3，MgSO4·7H2O0.2, agar 20.0, solvent deionized water, pH 7.0. Sterilizing at 115 deg.C for 30 min. After cooling to room temperature, 1.0g/L of 2-amino-4- (hydroxymethyl-phosphoryl) -butyronitrile was added.
The slant cultureThe final concentration composition was (g/L): 10.0 percent of mannitol, 7.0 percent of sodium glutamate, 3.0 percent of yeast extract and K2HPO40.75，KH2PO40.75，MgSO40.5, caprolactam 1.0, agar 20.0 and deionized water as solvent, and the pH value is 7.0-7.5.
The final concentration composition of the fermentation medium is (g/L): 10.0 percent of mannitol, 7.0 percent of sodium glutamate, 3.0 percent of yeast extract and K2HPO40.75，KH2PO40.75，MgSO40.5, caprolactam 1.0 and deionized water as solvent, and the pH value is 7.0-7.5.
(4) Primary screening by phenol-hypochlorite method
2ml of the solution A is taken by a pipette and placed in a 10ml centrifuge tube, 4. mu.L of the solution to be tested is added, a cover is covered, and the mixture is vigorously mixed. Then 2ml of solution B was added by pipette, capped and mixed again. And (3) heating in a water bath at 37 ℃ for 5min until the color in the centrifugal tube does not change any more, placing 200 mu L of the solution in a 96-well plate, measuring the absorbance at 620nm, and comparing the absorbance with a standard curve to obtain the concentration of ammonium in the solution to be measured, wherein the concentration of the ammonium is equal to the concentration of the converted 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid, so that the yield of the 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid in the conversion process can be detected.
The solution A comprises: 5g of phenol and 25mg of sodium nitrosoferricyanide were weighed, placed in a 500mL volumetric flask, added with ultrapure water to a constant volume, and then stored in a brown bottle in a refrigerator at 4 ℃.
And the liquid B: 2.5g of NaOH was weighed into a 500mL volumetric flask, and 4.4mL of an aqueous sodium hypochlorite solution containing 5.2% available chlorine was added by a pipette, and then the volume was fixed with ultrapure water, and the mixture was stored in a brown bottle and stored in a refrigerator at 4 ℃.
Preparation of a standard curve: the phenol-hypochlorite method can measure the concentration of ammonium ions at room temperature by using a cuvette and an ultraviolet-visible spectrophotometer or a 96-well plate and a universal microplate spectrophotometer, and the OD620 value has a good linear relation with the concentration of ammonium ions (shown in figure 2) at the wavelength of 620nm, R is20.9982, which shows that the method has high feasibility and accuracy.
(5) Pre-column derivatization reversed-phase high performance liquid chromatography rescreening
And (3) taking 500 mu L of reaction liquid with a better result obtained after primary screening, adding an equal volume of derivatization reagent, uniformly mixing, carrying out derivatization reaction for 5min, then injecting 15 mu L of sample, and carrying out HPLC analysis.
The derivatization reagent is as follows: 10mg of o-phthalaldehyde (OPA) and 12mg of N-acetyl-L-cysteine (NAC) were weighed out, dissolved in lmL anhydrous ethanol, and diluted to 5mL with 0.2mol/L boric acid buffer (pH 9.8) and stored in a refrigerator at-4 ℃ for later use (the storage time should not exceed 3 days).
The analysis conditions of the high performance liquid chromatography are as follows: the adopted gas chromatograph is a U3000 liquid chromatograph (provided with a fluorescence detector) of Daian in America; a chromatographic column: daran C18(4.6 mm. times.250 mm, silicon hydroxyl filler); mobile phase: methanol: 0.05mol/L ammonium acetate solution (volume ratio 10:90, pH value 5.7); flow rate: 1 mL/min; column temperature: 35 ℃; sample introduction amount: 15 mu L of the solution; fluorescence detection wavelength: excitation wavelength Ex 350nm, emission wavelength Em 450 nm.
Under the condition, the peak-off time of L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid and D-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid is 9.2min and 11.1min respectively, as shown in figure 1.
2. Molecular characterization of Strain ZJB-16008
Extracting the total DNA of the strain ZJB-16008 obtained by screening in the step 1 by using a molecular kit, amplifying the 16S rDNA gene (shown in SEQ ID NO: 1) of the strain by using the total DNA of the strain ZJB-16008 as a template and using primers P1:5'-AGAGTTTGATCCTGGCTCAG-3' and P2:5'-AAGGAGGTGATCCAGCCGCA-3', connecting the gene product with a T vector, entrusting Shanghai worker to amplify and sequence the 16S rDNA of the strain to obtain the 16S rDNA sequence of the strain, searching the 16S rDNA gene sequence of related strains in GenBank by using BLAST on an NCBI website, and performing homology comparison. The microorganism has the highest homology (homology, 100 percent, basedon 16S rDNA) with the Raoultella ornitholinolytica strain MG, and the identified bacterium basically belongs to a control bacterium based on the homology of a 16S rDNA sequence higher than 95 percent according to the identification principle of microbial molecular genetics. Therefore, the strain ZJB-16008 identified in this experiment is Laurella ornithinolytica (Raoultella ornithinolytica), and is intended to be named Laurella ornithinolytica (Raoultella ornithinolytica) ZJB-16008.
Any pair of SEQ ID NOs: 1 by substitution, deletion or insertion of one or more nucleotides, provided that it has more than 90% homology with the nucleotide, is within the scope of the present invention.
Example 2: preparation of Wet cells
(1) Slant culture
Inoculating Raoultella ornithinolytica ZJB-16008 to slant culture medium, and culturing at 30 deg.C for 48 hr to obtain slant thallus.
The final concentration composition of the slant culture medium is (g/L): 10.0 percent of mannitol, 7.0 percent of sodium glutamate, 3.0 percent of yeast extract and K2HPO40.75，KH2PO40.75，MgSO40.5, caprolactam 1.0, agar 20.0 and deionized water as solvent, and the pH value is 7.0-7.5.
(2) Seed culture
Selecting bacteria from the inclined plane bacteria, inoculating the bacteria to a seed culture medium, and culturing at 30 ℃ for 24h to obtain a seed solution; the final concentration composition of the seed culture medium is (g/L): 10.0 percent of mannitol, 7.0 percent of sodium glutamate, 3.0 percent of yeast extract and K2HPO40.75，KH2PO40.75，MgSO40.5, 1.0 caprolactam and deionized water as solvent, wherein the pH value is 7.0-7.5;
(3) fermentation culture
Inoculating the seed solution into a fermentation medium at a volume concentration of 1%, performing shaking culture at 30 deg.C and 150rpm for 48h, centrifuging at 12000g for 10min, and collecting wet thallus.
The final concentration composition of the fermentation medium is (g/L): 10.0 percent of mannitol, 7.0 percent of sodium glutamate, 3.0 percent of yeast extract and K2HPO40.75，KH2PO40.75，MgSO40.5, caprolactam 1.0 and deionized water as solvent, and the pH value is 7.0-7.5.
EXAMPLE 3 bioconversion reaction Using α -aminonitrile (2-amino-4 (hydroxyethylmethylphosphoryl) -butyronitrile) as substrate
(1) 0.2g of wet cells prepared in example 2 and a final concentration of 2-amino-4 (hydroxyethylmethylphosphoryl) -butyronitrile of 2g/L as a substrate were added to 10mL of phosphate buffer (100mM, pH 7.0), and the mixture was transferred for 24 hours at 150rpm in a shaker 30 ℃.1ml of the transformation solution is put into an EP tube and centrifuged at 12000r/min for 2min, and the supernatant is taken for chiral liquid phase detection. The result shows that the optical purity of the L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid obtained by the conversion of the strain ZJB-16005 on 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile is 99.9%.
(2) The conversion solution containing the product L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid obtained by the enzyme reaction is separated on an ion exchange column by adopting anion exchange resin 201 x 7.
1) Resin pretreatment
Soaking in warm water at 50 deg.C to fully expand and remove fine particles (inclined or flotation method); soaking in l.0M NaOH aqueous solution for 3 hr, washing with deionized water to neutrality, soaking in l.0M HCl aqueous solution for 3 hr, washing with deionized water to neutrality, soaking in l.0M NaOH aqueous solution for 3 hr, and converting into OH-And finally washing the mixture to be neutral by deionized water for later use.
2) Column mounting
The method comprises the steps of filling a column (with the inner diameter of 1.5cm and the height of 40cm) by a wet method, firstly adding deionized water (generally 1/3 with the length of the column) with a certain height into an ion exchange column, filling 30mL of wet resin 201 multiplied by 7 into a glass cup, adding 50mL of deionized water, slowly stirring, pouring suspended resin into the ion exchange column, and naturally settling the resin, wherein the resin must be uniformly distributed in the column, no obvious boundary line is required, and no air bubbles are generated.
3) Loading and eluting
Adjusting the pH value of the conversion solution obtained in the step (1) to 2.5, sampling at the column flow rate of 4.0BV/h, taking the effluent liquid at intervals to perform liquid phase detection, when the adsorption reaches the maximum value, washing the column with deionized water, then eluting with 1.0M ammonia water at the elution speed of 2.0BV/h, collecting the eluent, and detecting the L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid contained in the eluent at intervals. After the elution is finished, the ion exchange column is washed by deionized water, and the resin 201 multiplied by 7 is converted into OH-ForAnd (5) next separation and extraction.
4) Purification of
Distilling the eluate under reduced pressure to obtain yellow viscous substance, dissolving in methanol, stirring in ice bath, recrystallizing to obtain white solid, and filtering to obtain L-2-amino-4 (hydroxyethyl methylphosphoryl) -butyric acid.
Preparing 1L of conversion solution under the reaction condition of the step (1), and finally obtaining 0.72g of L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid solid after the separation and purification.
Example 4:
0.2g of wet cells prepared in example 2 and a final concentration of 10g/L of the substrate 2-amino-4 (hydroxyethylmethylphosphoryl) -butyronitrile were added to 10mL of phosphate buffer (100mM, pH 7.0), and the mixture was transferred for 24 hours at 150rpm in a shaker at 30 ℃.1ml of the transformation solution is put into an EP tube and centrifuged at 12000r/min for 2min, and the supernatant is taken for chiral liquid phase detection. 1L of the converted solution was prepared under the same reaction conditions, and after separation and purification by the method of example 3, 3.5g of L-2-amino-4 (hydroxyethylmethylphosphoryl) -butyric acid having an optical purity of 99.9% was obtained as a product.
Example 5:
1g of the wet cell prepared in example 2 was added to 10mL of phosphate buffer (100mM, pH 7.0) to give a final substrate concentration of 10g/L, and the mixture was transferred at 150rpm for 24 hours in a shaker 30 ℃.1ml of the transformation solution is put into an EP tube and centrifuged at 12000r/min for 2min, and the supernatant is taken for chiral liquid phase detection. 1L of the converted solution was prepared under the same reaction conditions, and after separation and purification by the method of example 3, 4.5g of L-2-amino-4 (hydroxyethylmethylphosphoryl) -butyric acid having an optical purity of 99.9% was obtained as a product.
Example 6:
0.2g of wet cells prepared in example 2 and a final concentration of 10g/L of the substrate 2-amino-4 (hydroxyethylmethylphosphoryl) -butyronitrile were added to 10mL of phosphate buffer (100mM, pH 7.0), and the mixture was transferred for 24 hours at 150rpm in a shaker at 40 ℃ in a water bath. 1ml of the transformation solution is put into an EP tube and centrifuged at 12000r/min for 2min, and the supernatant is taken for chiral liquid phase detection. 1L of the converted solution was prepared under the same reaction conditions, and after separation and purification by the method of example 3, 3.5g of L-2-amino-4 (hydroxyethylmethylphosphoryl) -butyric acid having an optical purity of 99.9% was obtained as a product.
Example 7:
5g of the wet cell prepared in example 2 was added to 100mL of phosphate buffer (100mM, pH 7.0) to give a final substrate concentration of 10g/L, and the mixture was transferred at 150rpm for 48 hours in a shaker 30 ℃.1ml of the transformation solution is put into an EP tube and centrifuged at 12000r/min for 2min, and the supernatant is taken for chiral liquid phase detection. 1L of the converted solution was prepared under the same reaction conditions, and after separation and purification by the method of example 3, 5.5g of L-2-amino-4 (hydroxyethylmethylphosphoryl) -butyric acid having an optical purity of 99.9% was obtained as a product.
SEQUENCE LISTING
<110> Zhejiang industrial university
<120> Raoultella ornithinolytica and application thereof
<130>
<160>1
<170>PatentIn version 3.5
<210>1
<211>1444
<212>DNA
<213>Raoultella ornithinolytica
<400>1
tgcaagtcga gcggtagcac agagagcttg ctctcgggtg acgagcggcg gacgggtgag 60
taatgtctgg gaaactgcct gatggagggg gataactact ggaaacggta gctaataccg 120
cataacgtcg caagaccaaa gtgggggacc ttcgggcctc atgccatcag atgtgcccag 180
atgggattag ctagtaggtg gggtaatggc tcacctaggc gacgatccct agctggtctg 240
agaggatgac cagccacact ggaactgaga cacggtccag actcctacgg gaggcagcag 300
tggggaatat tgcacaatgg gcgcaagcct gatgcagcca tgccgcgtgt atgaagaagg 360
ccttcgggtt gtaaagtact ttcagcgagg aggaaggcat taaggttaat aaccttagtg 420
attgacgtta ctcgcagaag aagcaccggc taactccgtg ccagcagccg cggtaatacg 480
gagggtgcaa gcgttaatcg gaattactgg gcgtaaagcg cacgcaggcg gtctgttaag 540
tcagatgtga aatccccggg ctcaacctgg gaactgcatt tgaaactggc aggcttgagt 600
cttgtagagg ggggtagaat tccaggtgta gcggtgaaat gcgtagagat ctggaggaat 660
accggtggcg aaggcggccc cctggacaaa gactgacgct caggtgcgaa agcgtgggga 720
gcaaacagga ttagataccc tggtagtcca cgctgtaaac gatgtcgact tggaggttgt 780
tcccttgagg agtggcttcc ggagctaacg cgttaagtcg accgcctggg gagtacggcc 840
gcaaggttaa aactcaaatg aattgacggg ggcccgcaca agcggtggag catgtggttt 900
aattcgatgc aacgcgaaga accttaccta ctcttgacat ccagagaact tagcagagat 960
gctttggtgc cttcgggaac tctgagacag gtgctgcatg gctgtcgtca gctcgtgttg 1020
tgaaatgttg ggttaagtcc cgcaacgagc gcaaccctta tcctttgttg ccagcgattc 1080
ggtcgggaac tcaaaggaga ctgccagtga taaactggag gaaggtgggg atgacgtcaa 1140
gtcatcatgg cccttacgag tagggctaca cacgtgctac aatggcatat acaaagagaa 1200
gcgacctcgc gagagcaagc ggacctcata aagtatgtcg tagtccggat tggagtctgc 1260
aactcgactc catgaagtcg gaatcgctag taatcgtaga tcagaatgct acggtgaata 1320
cgttcccggg ccttgtacac accgcccgtc acaccatggg agtgggttgc aaaagaagta 1380
ggtagcttaa ccttcgggag ggcgcttacc actttgtgat tcatgactgg ggtgaagtcg 1440
aaca 1444
Claims (6)
1. Raoultella ornithinolytica (Raoultella ornithinolytica) ZJB-16008, which is preserved in China center for type culture Collection with the preservation number of CCTCC No: m2017035, date of deposit 2017, month 01, day 13, address: china, wuhan university, 430072.
2. The use of Raoultella ornithinolytica ZJB-16008 in the catalysis of 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile to prepare L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid, wherein the 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile is:
the structural formula of the L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid is as follows:
3. the use according to claim 2, characterized in that said use is: taking wet thalli obtained by fermenting and culturing Laurus ornithine-resolving ZJB-16008 as a catalyst, taking 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile as a substrate, taking distilled water or 100mM phosphate buffer solution with the pH value of 7.0 as a reaction medium, carrying out conversion reaction at the temperature of 30-50 ℃ and the speed of 100 plus 200rpm, separating and purifying the reaction liquid after the reaction is finished, and obtaining the L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid.
4. The use according to claim 3, wherein the amount of catalyst is 20-100g/L of reaction medium based on the weight of wet cells, and the final concentration of substrate initially added is 2-10g/L of reaction medium.
5. The use according to claim 3, wherein the catalyst is prepared by the following process: (1) slant culture: inoculating Raoultella ornithinolytica ZJB-16008 to slant culture medium, and culturing at 30 deg.C for 48 hr to obtain slant thallus; the slant culture mediumThe concentration composition is as follows: 1-20.0 g/L of mannitol, 1-10.0 g/L of sodium glutamate, 1-5.0 g/L of yeast extract and K2HPO40.1～1.0g/L，KH2PO40.1～1.0g/L，MgSO40.1-1.0 g/L, 0.1-2.0 g/L caprolactam, 20.0g/L agar, deionized water as solvent and pH 7.0-7.5;
(2) seed culture: selecting slant thallus, inoculating to seed culture medium, and culturing at 30 deg.C for 24 hr to obtain seed solution; the final concentration of the seed culture medium is as follows: 1-20.0 g/L of mannitol, 1-10.0 g/L of sodium glutamate, 1-5.0 g/L of yeast extract and K2HPO40.1～1.0g/L，KH2PO40.1～1.0g/L，MgSO40.1-1.0 g/L, 0.1-2.0 g/L caprolactam, deionized water as solvent and pH 7.0-7.5;
(3) fermentation culture: inoculating the seed solution into a fermentation culture medium in an inoculation amount of 1% of volume concentration, carrying out shaking culture at 30 ℃ and 150rpm for 48h, centrifuging at 12000g for 10min, and collecting wet thalli; the final concentration of the fermentation medium is as follows: 1-20.0 g/L of mannitol, 1-10.0 g/L of sodium glutamate, 1-5.0 g/L of yeast extract and K2HPO40.1～1.0g/L，KH2PO40.1～1.0g/L，MgSO40.1-1.0 g/L, 0.1-2.0 g/L caprolactam, deionized water as solvent and pH 7.0-7.5.
6. The use of claim 3, wherein the reaction solution is separated and purified by the following steps: adjusting the pH value of the reaction liquid to 2.5, performing column chromatography by adopting anion exchange resin 201 multiplied by 7, loading the sample at the flow rate of 1-6.0 BV/h, washing with deionized water, then eluting with 0.5-2.0M ammonia water at the elution speed of 1.0-4.0 BV/h, and collecting the eluent containing the target substance; distilling the eluent under reduced pressure until no solvent flows out, adding methanol for dissolving, stirring in ice bath, recrystallizing, filtering, and drying the filter cake to obtain the product L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid.
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| EP3423585A1 (en) | 2016-03-02 | 2019-01-09 | Agrimetis, LLC | Methods for making l-glufosinate |
| CN110240271B (en) * | 2018-04-28 | 2022-06-17 | 华东理工大学 | Starting method of bioreactor for treating triethyl phosphate-containing wastewater |
| CN108570071B (en) * | 2018-06-27 | 2020-07-03 | 安徽国星生物化学有限公司 | Separation and purification method of 4- (methyl hydroxyl phosphoryl) -2-carbonyl butyric acid mother liquor |
| CN109182200B (en) * | 2018-09-30 | 2020-10-09 | 浙江工业大学 | Lysinibacillus fusiformis ZJB-17006 and application thereof |
| CN109321492B (en) * | 2018-09-30 | 2020-08-21 | 浙江工业大学 | Non-decarboxylation lechler bacterium ZJB-17008 and application thereof |
| CN109321493B (en) * | 2018-09-30 | 2020-10-02 | 浙江工业大学 | B-lysine-resistant bacillus ZJB-17007 and application thereof |
| CN110628679B (en) * | 2019-10-09 | 2022-05-24 | 江苏海洋大学 | Raoultella ornithinolytica G10, enzyme production method, product and application |
| CN111621482B (en) * | 2020-06-30 | 2022-04-29 | 浙江工业大学 | Glufosinate-ammonium dehydrogenase mutant, gene engineering bacteria and one-pot multi-enzyme synchronous directed evolution method |
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