CN116590374A - Method for asymmetrically preparing glufosinate-ammonium by multi-enzyme coupling one-pot method - Google Patents
Method for asymmetrically preparing glufosinate-ammonium by multi-enzyme coupling one-pot method Download PDFInfo
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- CN116590374A CN116590374A CN202310427942.7A CN202310427942A CN116590374A CN 116590374 A CN116590374 A CN 116590374A CN 202310427942 A CN202310427942 A CN 202310427942A CN 116590374 A CN116590374 A CN 116590374A
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- Prior art keywords
- glufosinate
- ammonium
- amino acid
- reaction
- dehydrogenase
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- 238000000034 method Methods 0.000 title claims abstract description 61
- 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 title claims abstract description 24
- 238000005580 one pot reaction Methods 0.000 title claims abstract description 15
- 230000008878 coupling Effects 0.000 title claims abstract description 12
- 238000010168 coupling process Methods 0.000 title claims abstract description 12
- 238000005859 coupling reaction Methods 0.000 title claims abstract description 12
- 238000006243 chemical reaction Methods 0.000 claims abstract description 51
- IAJOBQBIJHVGMQ-BYPYZUCNSA-N glufosinate-P Chemical compound CP(O)(=O)CC[C@H](N)C(O)=O IAJOBQBIJHVGMQ-BYPYZUCNSA-N 0.000 claims abstract description 46
- 102000004674 D-amino-acid oxidase Human genes 0.000 claims abstract description 40
- 108010003989 D-amino-acid oxidase Proteins 0.000 claims abstract description 40
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims abstract description 38
- 101710088194 Dehydrogenase Proteins 0.000 claims abstract description 37
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- 239000013067 intermediate product Substances 0.000 claims description 8
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 8
- IAJOBQBIJHVGMQ-SCSAIBSYSA-N (2R)-glufosinate Chemical compound C[P@@](O)(=O)CC[C@@H](N)C(O)=O IAJOBQBIJHVGMQ-SCSAIBSYSA-N 0.000 claims description 7
- 244000063299 Bacillus subtilis Species 0.000 claims description 6
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 4
- 241000235058 Komagataella pastoris Species 0.000 claims description 4
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 4
- 239000012295 chemical reaction liquid Substances 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 4
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- 125000004122 cyclic group Chemical group 0.000 claims description 3
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- 108010093096 Immobilized Enzymes Proteins 0.000 claims description 2
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 claims description 2
- 229910021529 ammonia Inorganic materials 0.000 claims description 2
- 238000004176 ammonification Methods 0.000 claims description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 2
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- 238000006555 catalytic reaction Methods 0.000 claims description 2
- 244000005700 microbiome Species 0.000 claims description 2
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims description 2
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 description 2
- 150000008574 D-amino acids Chemical class 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
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- 241000221523 Rhodotorula toruloides Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
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- VDZOOKBUILJEDG-UHFFFAOYSA-M tetrabutylammonium hydroxide Chemical compound [OH-].CCCC[N+](CCCC)(CCCC)CCCC VDZOOKBUILJEDG-UHFFFAOYSA-M 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- WUWSZMDHSCNRHP-UHFFFAOYSA-N 2-amino-4-(hydroxymethylphosphanyl)butanoic acid Chemical compound NC(C(=O)O)CCPCO WUWSZMDHSCNRHP-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
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- 101001123995 Bacillus subtilis (strain 168) FMN reductase [NAD(P)H] Proteins 0.000 description 1
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- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
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- YPZRHBJKEMOYQH-UYBVJOGSSA-N FADH2 Chemical compound C1=NC2=C(N)N=CN=C2N1[C@@H]([C@H](O)[C@@H]1O)O[C@@H]1COP(O)(=O)OP(O)(=O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C(NC(=O)NC2=O)=C2NC2=C1C=C(C)C(C)=C2 YPZRHBJKEMOYQH-UYBVJOGSSA-N 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-N Gluconic acid Natural products OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 1
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- 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 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- C12P41/002—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by oxidation/reduction reactions
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
- C12N9/0024—D-Amino acid oxidase (1.4.3.3)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/001—Amines; Imines
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Abstract
The application relates to a method for asymmetrically preparing glufosinate-ammonium by a multi-enzyme coupling one-pot method. The asymmetric preparation reaction of the arginate-ammonium comprises the following steps: in the same container, D-amino acid oxidase, L-glutamate dehydrogenase and formate dehydrogenase are utilized to convert racemic D, L-glufosinate into L-glufosinate, and meanwhile, the regeneration of reduced coenzyme is realized, and the influence of byproducts on the separation and purification of subsequent products is eliminated. The method can realize the green and efficient resolution of the D, L-glufosinate-ammonium and the large-scale industrialized preparation of the glufosinate-ammonium.
Description
Technical Field
The application belongs to the technical field of biology, and particularly relates to a method for asymmetrically preparing glufosinate-ammonium by using a biological multienzyme coupling one-pot method.
Background
The ammonium glufosinate (PPT) is 2-amino-4- (hydroxymethyl phosphino) butyric acid, is a spectrum nonselective amino acid herbicide developed by Herst company of Germany in 1986, and has the advantages of high herbicidal activity, rapid weed killing, wide weed spectrum, small effective application amount, easy degradation in soil, small influence on surrounding environment and the like. The second world transgenic crop with the current dosage only inferior to glyphosate is tolerant to herbicide, and the market demand of the glufosinate is rapidly increased due to the spread of glyphosate-resistant weeds and the limit of herbicide such as paraquat and diquat, and is expected to exceed the glyphosate market in the future 5 years. However, since commercially applied glufosinate herbicides are usually composed of a racemic mixture of D, L-glufosinate, only L-glufosinate (about 40-60% by mass) has herbicidal activity, the other half of D-glufosinate reduces the atomic utilization of the pesticide and causes soil hardening, which is not environmentally friendly. Therefore, by converting D-glufosinate into L-glufosinate, the dosage of half of the glufosinate raw medicine can be reduced, which has important significance for reducing the cost, improving the atom economy and relieving the environmental pressure.
The current preparation method of L-glufosinate mainly comprises a chemical method and a biological method. However, the chemical method has the defects of complicated steps, low optical purity of the product, high production cost, poor safety, serious environmental pollution and the like. At present, manufacturers of the smart glufosinate (L-glufosinate) generally tend to adopt a biocatalytic conversion method. The biocatalytic method for producing the glufosinate-ammonium has the advantages of simple steps, mild reaction conditions, high stereoselectivity, high product yield and the like, is an important trend for industrially preparing the L-glufosinate-ammonium, and is greatly supported by national policies. The biological preparation of the arginate-ammonium phosphine mainly comprises two types of enzymatic synthesis methods and enzymatic resolution methods:
(1) The enzymatic synthesis method mainly takes derivatives of L-glufosinate (such as N-phenylacetyl-D, L-glufosinate or glufosinate-ammonium) as substrates, and the L-glufosinate is obtained by direct hydrolysis of nitrilase and the like.
(2) The enzymatic resolution method utilizes racemic D, L-glufosinate (D, L-PPT) as a substrate, performs selective resolution through a two-step enzymatic method, converts the D-glufosinate (D-PPT) into 2-carbonyl-4- [ hydroxy (methyl) phosphono ] butanoic acid (PPO) through D-amino acid oxidase (DAAO), and further reduces the D-glufosinate into L-glufosinate (L-PPT) through L-glutamate dehydrogenase, and simultaneously retains the L-glufosinate with the original mass fraction of 40-60%. The method has the advantages of simple and easily obtained raw materials, simple process, lower cost, environmental protection and the like, and has higher industrialized application value. However, the catalytic activity of the D-amino acid oxidase on the unnatural substrate D-glufosinate is low, and the reduction reaction catalyzed by the L-glutamate dehydrogenase requires the addition of stoichiometric reduced coenzyme NAD (P) H, so that the cost is wasted and the industrial application of the D-amino acid oxidase is hindered.
Disclosure of Invention
The technical problem to be solved by the application is to overcome the defects and the shortcomings in the background technology, and provide a method for asymmetrically preparing the glufosinate-ammonium by a multi-enzyme coupling one-pot method.
In order to achieve the above purpose, the specific technical scheme provided by the application is as follows:
a method for preparing arginate-ammonium phosphine by multi-enzyme coupling one-pot method is characterized in that D-amino acid oxidase, L-glutamate dehydrogenase and formate dehydrogenase are added into the same system at the same time, and the D-glufosinate-ammonium in racemic D, L-glufosinate-ammonium is completely converted into L-glufosinate-ammonium by utilizing the catalytic action of the D-amino acid oxidase, the L-glutamate dehydrogenase and the formate dehydrogenase, and meanwhile, the L-glufosinate-ammonium in the original medicine is reserved.
In the method, D-amino acid oxidase catalyzes a substrate D-PPT to be converted into an intermediate product PPO; l-glutamate dehydrogenase is used for ammonification and reduction of PPO to L-glufosinate; formate dehydrogenase is used for the cyclic regeneration of reduced coenzymes NADH or NADPH.
The D-amino acid oxidase, L-glutamate dehydrogenase and formate dehydrogenase are added in at least one form selected from crude cell extract, purified enzyme solution, immobilized enzyme, lyophilized cell containing enzyme or fermentation broth.
In the method, the host cells expressing D-amino acid oxidase, L-glutamate dehydrogenase and formate dehydrogenase are respectively selected from at least one of Saccharomyces cerevisiae (Saccharomyces cerevisiae), pichia pastoris (Pichia pastoris), streptomyces, bacillus subtilis (Bacillus subtilis) or Escherichia coli (Escherichia coli).
In the method, the addition amount of the recombinant microorganism is 1-200g of wet thallus/L reaction solution based on the weight of the wet thallus.
The method comprises the steps that the enzyme catalytic conversion reaction is carried out in a reaction liquid system with the pH value of 7-10; the reaction temperature is 25-45 ℃ and the reaction time is not less than 6 hours.
In the method, catalase and an inorganic amino donor, preferably at least one of ammonium sulfate or ammonium formate, are added into a reaction system.
The method comprises the following steps of: d, L-PPT 300-500 mM, inorganic amino donor 300-500 mM, catalase 3000-8000U/ml.
According to the method, ammonia water is added into a reaction system to control pH and then the reaction is carried out.
In the method, the mass fraction of D-glufosinate in racemic D, L-glufosinate is 40-60%.
The application relates to a method for asymmetrically preparing glufosinate-ammonium by a multi-enzyme coupling one-pot method, which comprises the following detailed steps: (1) Oxidizing and converting D-PPT with the mass fraction of about 40-60% in racemic D, L-glufosinate to an intermediate PPO in the presence of D-amino acid oxidase, and decomposing by-product hydrogen peroxide by using catalase to prevent toxic effects of accumulation of by-products on enzyme proteins; (2) In the presence of L-glutamate dehydrogenase and inorganic amino donor, the intermediate PPO is ammonified and reduced to L-PPT, and meanwhile, a coenzyme circulation system is constructed, and in the presence of formate dehydrogenase, the reduced coenzyme NAD (P) H consumed in the reaction is regenerated, so that the cost is reduced, and the reaction progress is shortened. In the whole reaction process, D-PPT in the glufosinate-ammonium original drug can be completely converted into L-PPT, meanwhile, the L-PPT with the mass fraction of 40-60% in the raw materials is reserved, the amount of the added inorganic amino donor and the reduced coenzyme is kept at a lower level, and the reaction cost is greatly reduced.
D-amino acid oxidases (DAAO, EC 1.4.3.3) belong to the class of oxidoreductases, which are capable of oxidizing the amino group on the D-amino acid C.alpha.to a carbonyl group with absolute stereospecificity, while producing hydrogen peroxide as a by-product. The catalytic mechanism mainly comprises a reduction half reaction and an oxidation half reaction: the hydride equivalent of the D-amino acid is first transferred to the iso-tetraoxypyrimidine moiety of FAD to form the intermediate iminoacid, which is then spontaneously hydrolyzed to the corresponding alpha-keto acid and NH 4 + Subsequently, reduced FADH 2 Is usually cleaved by O prior to cleavage of the product iminoacid 2 Reoxidation to Hydrogen peroxide (H) 2 O 2 )。
The D-amino acid oxidase refers to an enzyme with the activity of catalyzing the conversion of a substrate D-PPT to generate an intermediate product PPO. The D-amino acid oxidase may be any enzyme known in the art having D-amino acid oxidase activity. In some embodiments, the D-amino acid oxidase is derived from a D-amino acid oxidase of Trigonella variabilis (Trigonopsis variabilis), rhodotorula gracilis (Rhodotorula agralis), candida (Candida parapsilosis), fusarium oxysporum (Fusarium oxysporum) and porcine kidney (Pig kidney), having NCBI accession numbers CAA90322.1, AAB51107.1, KAI5911410.1, EXK46480 and P00371.2, in order. Preferably, the D-amino acid oxidase is 1C0K from Rhodotorula gracilis (Rhodotorula glabrata). In some embodiments, the amino acid sequence of the D-amino acid oxidase has at least 70%, 80%, 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence set forth in SEQ ID No.1. In some embodiments, the nucleotide sequence of the D-amino acid oxidase has at least 70%, 80%, 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the nucleotide sequence set forth in SEQ ID No.2. In some embodiments, the amino acid sequence of the D-amino acid oxidase is SEQ ID NO.1. In some embodiments, the nucleotide sequence of the D-amino acid oxidase is SEQ ID NO.2.
L-glutamate dehydrogenase (GluDH, EC 1.4.1.2) belongs to the class of coenzyme NAD (P) H-dependent oxidoreductases, which are capable of catalyzing the keto acid of a precursor substance in the presence of the coenzyme NAD (P) H and an inorganic amino donor to form the corresponding L-amino acid. At the same time can also be used in NAD (P) + Catalytic reverse reaction occurs in the presence of (a).
The L-glutamate dehydrogenase disclosed by the application refers to an enzyme with the activity of catalyzing the conversion of a arginate-ammonium phosphine intermediate product PPO to generate a final product L-PPT. The L-glutamate dehydrogenase may be any enzyme known in the art having L-glutamate dehydrogenase activity. In some embodiments, the L-glutamate dehydrogenase is derived from Clostridium symbiotic (Clostridium symbiosum), bacillus subtilis (Bacillus subtilis), pseudomonas putida (Pseudomonas putida), L-glutamate dehydrogenase of Bacillus sphaericus (Lysinibacillus sphaericus) and Escherichia coli (Escherichia coli), having NCBI accession numbers CAA77805.1, WP_221249962.1, KW16710.1, KEK10552.1 and AAA87979.1, respectively. Preferably, the L-glutamate dehydrogenase is 2YFH from E.coli (Escherichia coli). In some embodiments, the amino acid sequence of the L-glutamate dehydrogenase has at least 70%, 80%, 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No.3. In some embodiments, the nucleotide sequence of the L-glutamate dehydrogenase has at least 70%, 80%, 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the nucleotide sequence set forth in SEQ ID No.4. In some embodiments, the amino acid sequence of the L-glutamate dehydrogenase is SEQ ID NO.3. In some embodiments, the nucleotide sequence of the L-glutamate dehydrogenase is SEQ ID NO.4.
The coenzyme circulation system in the application refers to the cyclic regeneration of reduced coenzyme NAD (P) H in the PPO reduction process by formate dehydrogenase. The method does not produce gluconic acid, and is favorable for separating and purifying downstream products. In some embodiments, the formate dehydrogenase is derived from Escherichia coli (Escherichia coli), escherichia coli (Enterobacter ludwigii), candida albicans (Candida boidinii), saccharomyces cerevisiae (Saccharomyces cerevisiae), and bacillus sphaericus (lysinibacillus sphaericus), having NCBI accession numbers AAA23754.2, AKM87309.1, CAB54834.1, KAF1902530.1, and KEK12543.1, respectively. Preferably, the L-glutamate dehydrogenase is 1AA6 from Escherichia coli. In some embodiments, the amino acid sequence of the L-glutamate dehydrogenase has at least 70%, 80%, 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID No.5. In some embodiments, the nucleotide sequence of the L-glutamate dehydrogenase has at least 70%, 80%, 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the nucleotide sequence set forth in SEQ ID No.6. In some embodiments, the amino acid sequence of the formate dehydrogenase is SEQ ID No.5. In some embodiments, the nucleotide sequence of the formate dehydrogenase is SEQ ID No.6.
The D-amino acid oxidase, L-glutamate dehydrogenase and formate dehydrogenase of the present application may be in the form of crude cell extracts of enzymes; purifying the enzyme solution; immobilizing an enzyme; lyophilized cells containing the enzyme, fermentation broth, or any other suitable form.
The genetically engineered bacterium for producing the D-amino acid oxidase takes E.coli BL21 (DE 3) as a host to express the D-amino acid oxidase shown in SEQ ID NO.1. The expression vector of the genetically engineered bacterium is selected from pET series plasmid vectors, and preferably, the construction of the genetically engineered bacterium adopts the expression vector pET-30a (+). The construction method comprises the steps of connecting a D-amino acid oxidase coding gene shown in SEQ ID NO.2 to an expression vector pET-30a (+) through double enzyme digestion and T4 ligase, then directly transforming the plasmid vector into E.coli BL21 (DE 3) through an electric transformation method, and obtaining a correct transformant E.coli BL21 (DE 3) -pET-30a (+) -DAAO after screening, namely the genetically engineered bacterium for expressing the valley D-amino acid oxidase.
The glutamate dehydrogenase and the formate dehydrogenase are co-expressed by a single recombinant genetic engineering bacterium, and the genetic engineering bacterium takes E.coli BL21 (DE 3) as a host to express the L-glutamate dehydrogenase shown in SEQ ID NO.3 and the formate dehydrogenase shown in SEQ ID NO.5.
The plasmid expression vector of the genetically engineered bacterium is selected from any one of pET-Duet-1, pRSF-Duet-1 and pACYC-Duet-1, and preferably, the genetically engineered bacterium is constructed by adopting the expression vector pET-Duet-1. The construction method of the genetically engineered bacterium comprises the following steps: the L-glutamate dehydrogenase encoding gene shown in SEQ ID NO.4 is connected to MCS I of the expression vector pET-Duet-1, and the formate dehydrogenase encoding gene sequence shown in SEQ ID NO.6 is connected to MCSII of the expression vector pET-Duet-1. Then directly transforming the plasmid vector connected with the genes encoding the L-glutamate dehydrogenase and the formate dehydrogenase into E.coli BL21 (DE 3) through an electrotransformation method, and obtaining a correct transformant E.coli BL21 (DE 3) -pET-Duet-1-GluDH-FDH after screening, namely the genetically engineered bacterium co-expressing the glutamate dehydrogenase and the formate dehydrogenase.
In some embodiments, the reaction method for asymmetrically preparing the glufosinate-ammonium by the multienzyme coupling one-pot method comprises the following steps: adding the enzyme solution or wet bacterial cells obtained by fermenting the genetically engineered bacteria expressing D-amino acid oxidase and co-expressing L-glutamate dehydrogenase and formate dehydrogenase into a mixed reaction liquid system with pH of 7-10 and containing racemic D, L-PPT, ammonium formate and catalase, and reacting for 6-16h at the temperature of 25-45 ℃ to obtain the L-glufosinate-ammonium.
The catalase is used for removing the byproduct hydrogen peroxide to prevent the toxic effect of accumulation of the hydrogen peroxide on an enzyme catalyst. The catalase may be any enzyme known in the art having catalase activity, such as catalase available under the trade designation CAS 9001-05-2 from Shanghai Seiyaku Biotechnology Co., ltd.
In some embodiments, the genetically engineered bacteria added into the reaction system of the application are added in an amount of 1-200g of wet bacterial cells/L reaction solution based on the weight of the wet bacterial cells.
Preferably, the initial concentration of each substance in the reaction liquid system in the reaction method of the present application may be: d, L-PPT 300-500 mM, ammonium formate 300-500 mM, catalase 3000-8000U/ml.
The yield of the product L-PPT in the production process of the present application can be measured by any method known in the art. For example, the two conformational contents of the resulting glufosinate-ammonium product can be measured by chiral HPLC. In some embodiments, the resulting glufosinate product has an enantiomeric excess (e.e.) of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.9%.
The beneficial effects of the application are mainly as follows:
(1) Directly taking racemic D, L-PPT as a substrate for resolution, completely oxidizing and converting the D-PPT in the substrate into an intermediate product PPO by D-amino acid oxidase, and further reducing the intermediate product PPO into L-PPT by L-glutamate dehydrogenase, wherein the substrate conversion rate is more than or equal to 49.95 percent. Meanwhile, the L-PPT in the raw materials is reserved, so that the waste of the raw materials is prevented.
(2) The preparation of the arginate-ammonium phosphine is carried out by adopting a one-pot method, and the intermediate product of the oxidation reaction can be directly reduced into the final product L-PPT in situ, so that the oxidation of the substrate D-PPT and the reduction of PPO are not needed to be carried out in two steps respectively, the reduction of the reaction rate caused by accumulation of the intermediate product is prevented, and the reaction process is quickened. Compared with the preparation of the L-PPT by a multi-enzyme cascade method reported in the prior art (patent number CN 112410383A), the whole reaction of the method can be completed within 16 hours at the highest speed, and meanwhile, the used inorganic amino donor has lower price and can be easily removed in the subsequent purification and separation of the product, so that the production cost is greatly reduced.
(3) The multienzyme cascade reaction only needs to add a small amount of catalase and coenzyme NAD (P) H, and has the advantages of simple reaction process, mild reaction condition, high reaction speed and byproduct CO 2 The subsequent product separation is not affected, and the optical purity of the product is high. Compared with the traditional chemical method for preparing the L-PPT, the method is more green, environment-friendly and low-carbon, and is suitable for large-scale industrial production and application.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the mechanism of asymmetric preparation of glufosinate-ammonium by multi-enzyme cascade one-pot method employed in the method of the present application.
FIG. 2 is a reaction sequence for preparing the smart glufosinate-ammonium by a 100mL system multienzyme cascade one-pot method for racemization resolution of D, L-glufosinate-ammonium in example 2.
FIG. 3 is a reaction process of preparing the smart glufosinate-ammonium by racemizing and splitting D, L-glufosinate-ammonium with different concentrations by the multi-enzyme cascade one-pot method of the amplifying system in the example 2.
Detailed Description
The present application will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments are shown, for the purpose of illustrating the application, but the scope of the application is not limited to the specific embodiments shown.
Unless defined otherwise, all technical and scientific terms used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the present application.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present application are commercially available or may be prepared by existing methods.
The reaction intermediate product PPO is analyzed by High Performance Liquid Chromatography (HPLC), the progress of the reaction is detected, and the HPLC analysis method is: mobile phase: 50mM (NH) 4 ) 2 HPO 4 10% acetonitrile and 1% tetrabutylammonium hydroxide are added, the flow rate is 1.0mL/min, and the detection wavelength is 210nm; the column temperature was 30℃for a Accucore Vanquish C column.
The amount of the produced L-PPT and the e.e. value of the product were detected by chiral HPLC analysis, and the chiral HPLC analysis method was as follows: mobile phase: 0.5% copper sulfate pentahydrate, flow rate is set to 0.7mL/min; the detection wavelength is 254nm; column temperature: 35 ℃.
Example 1: construction and culture of genetically engineered bacteria
The gene sequence of D-amino acid oxidase (DAAO, the amino acid sequence of which is shown as SEQ ID NO:1, and the nucleotide sequence of which is shown as SEQ ID NO: 2) derived from Rhodotorulagacilis is sent to Beijing engine biotechnology Co., ltd for total gene synthesis, cloned onto expression plasmid pET-30a (+) to obtain pET-30a-DAAO, and inserted into restriction enzyme sites NdeI and Xho I. After sequencing verification, pET-30a-daao is transferred into cloning host E.coli DH5 alpha and expression host E.coli BL21 (DE 3) for subsequent expression of D-amino acid oxidase.
The gene sequence of L-glutamate dehydrogenase (GluDH, amino acid sequence shown as SEQ ID NO:3, nucleotide sequence shown as SEQ ID NO: 4) derived from Escherichia coli was sent to Beijing Optimu Biotechnology Co., ltd for total gene synthesis and inserted into cleavage sites NcoI and Hinder III, and then ligated to MCS I of coexpression plasmid pET-Duet-1 by double cleavage and T4 DNA Ligase to obtain pET-Duet-1-GluDH. The gene sequence of formate dehydrogenase (FDH, amino acid sequence shown as SEQ ID NO:5, nucleotide sequence shown as SEQ ID NO: 6) derived from Escherichia coli was sent to Beijing engine biotechnology Co., ltd for total gene synthesis and inserted into cleavage sites NdeI and XhoI, and then ligated to MCS II of coexpression plasmid pET-Duet-1-GluDH by double cleavage and T4 DNA Ligase to obtain pET-Duet-1-GluDH-FDH. After the sequencing verification, pET-30a-DAAO is transferred into cloning host E.coli DH5 alpha and expression host E.coli BL21 (DE 3) for subsequent expression of L-glutamate dehydrogenase and formate dehydrogenase.
LB liquid medium composition: 10g/L peptone, 5g/L yeast powder and 10g/L NaCl, and is dissolved in deionized water, then the volume is fixed, and the solution is sterilized at 115 ℃ for 30min for later use. LB liquid solid medium composition (plate): adding 2% w/v agar powder based on LB liquid medium, sterilizing at 115deg.C for 30min, cooling to 50-60deg.C, adding 100mg/mL kanamycin (Kan) or ampicillin (Amp) to final concentration of 50 μg/mL, pouring into a culture dish, cooling to solidify, and sealingAnd (5) using a refrigerator at the temperature of 4 ℃ for standby. TB liquid medium composition: 12g/L tryptone, 24g/L yeast powder, 4g/L glycerol and KH 2 PO 4 2.31g/L，K 2 HPO 4 12.54g/L, dissolved in deionized water, fixed in volume, sterilized at 115 ℃ for 30min for later use.
The recombinant genetically engineered bacteria E.coli BL21 (DE 3)/pET-30 a-DAAO and E.coli BL21 (DE 3)/pET-Duet-1-GluDH-FDH are subjected to plate streak activation, and single colony is selected and inoculated into 10mL LB liquid medium containing 50 mug/mL Kan or Amp, and shake-cultured at 37 ℃ and 230rpm for overnight. Transferring into 10mL TB liquid culture medium according to 2% inoculum size, shake culturing at 37deg.C and 230rpm to OD 600 When the concentration reaches about 0.8, lactose with the final concentration of 1% w/v is added, and shake culture is carried out for 12 hours at 25 ℃. After the culture is finished, the culture solution is centrifuged at 10000rpm for 10min, the supernatant is discarded, and the thalli are collected and stored in an ultralow temperature refrigerator at-80 ℃ for standby.
Example 2: asymmetric preparation of smart glufosinate-ammonium by multi-enzyme coupling one-pot method
Genetically engineered strains E.coli BL21 (DE 3)/pET-30 a-DAAO and E.coli BL21 (DE 3)/pET-Duet-1-GluDH-FDH capable of expressing D-amino acid oxidase, L-glutamate dehydrogenase and formate dehydrogenase were cultured as in example 1, and cells were collected by centrifugation and lyophilized.
100mL of the reaction system comprises 300mM D, L-PPT,300U/mg catalase, 0.025% (v/v) defoamer, pH8.0, 50mM Tris-HCl buffer, 300mM NH 4 COOH. 20g/L E.coli BL21 (DE 3)/pET-30 a-DAAO and E.coli BL21 (DE 3)/pET-Duet-1-GluDH-FDH freeze-dried cells are added into the system to start the reaction, pH8.0 is controlled by adding ammonia water, the reaction is carried out for 6-12h at the temperature of 30 ℃, 100 mu L is sampled every 1h, 25% (w/v) trichloroacetic acid with the same volume is added to stop the reaction, the supernatant is taken to dilute 10 times, the conversion condition of D-PPT and the generation condition of L-PPT are detected by HPLC, and the reaction progress curve is shown in figure 2.
The result shows that the reaction is carried out for 10 hours, the content of PPO is 2mM, the content of L-PPT is 299mM, the conversion rate of the substrate D and the L-PPT is more than 49.9%, and the e.e. value of the product glufosinate-ammonium is 99.9%.
In a 3L reactor, respectively, to 1600mL of Tris-HCl buffer (pH 8.0, 50 mM) system containing 300mM,400mM and 500mM of D, L-PPT,8000U/L catalase, 1% (v/v) defoamer, 500mM NH 4 50g/L of E.coli BL21 (DE 3)/pET-30 a-DAAO and E.coli BL21 (DE 3)/pET-Duet-1-GluDH-FDH cells were added to COOH, pH8.0 was controlled by adding ammonia, and the reaction was carried out at 30℃for 10-40 hours. 1mL of the sample was sampled every 2 hours, an equal volume of 25% (w/v) trichloroacetic acid was added to terminate the reaction, the content of L-PPT in the sample was measured by high performance liquid chromatography, and the substrate conversion was calculated, and the result was shown in FIG. 3.
As can be seen from FIG. 3, the concentration of L-glufosinate gradually increased over time when the catalytic reaction was carried out using the multienzyme coupling system described in example 2. At a substrate concentration of 300mM, after the reaction is finished, the concentration of the liquid phase detection L-PPT is more than 298mM, the existence of the D-PPT can not be detected almost, the conversion rate of the substrate D, L-PPT can reach 49.9%, and the reaction can be completed within 16 hours at the highest. Under the condition of higher substrate concentration (500 mM), the method can still show higher catalytic efficiency, can generate L-PPT with the conversion rate of more than 446mM in 36h, has the conversion rate of more than 49.5 percent and the e.e. value of 99.9 percent, and shows the large-scale application prospect of asymmetrically preparing the glufosinate-ammonium by the multienzyme coupling one-pot method.
SEQ ID NO.1
LMMHSQKRVVVLGSGVIGLSSALILARKGYSVHILARDLPEDVSSQTFASPW
AGANWTPFMTLTDGPRQAKWEESTFKKWVELVPTGHAMWLKGTRRFAQNE
DGLLGHWYKDITPNYRPLPSSECPPGAIGVTYDTLSVHAPKYCQYLARELQK
LGATFERRTVTSLEQAFDGADLVVNATGLGAKSIAGIDDQAAEPIRGQTVLV
KSPCKRCTMDSSDPASPAYIIPRPGGEVICGGTYGVGDWDLSVNPETVQRILK
HCLRLDPTISSDGTIEGIEVLRHNVGLRPARRGGPRVEAERIVLPLDRTKSPLSL
GRGSARAAKEKEVTLVHAYGFSSAGYQQSWGAAEDVAQLVDEAFQRYHGSEQ ID NO.2
atgcactctcagaagcgcgtcgttgtcctcggatcaggcggtgcgtcttttccctctcctccccacacccgacagtcctcgac
gaggtgtaggacggcgagcaaagctgccgagggcgatctgggctgactgagcgctcgagtgtacagttatcggtctgag
cagcgccctcatcctcgctcggaagggctacagcgtgcatattctcgcgcgcgacttgccggaggacgtctcgagccaga
ctttcgcttcaccatgggctgtgcgtcgtctcactgtagttggaggatgtcagcgagagctgagcaatctcgtcatccccgca
gggcgcgaattggacgcctttcatgacgcttacagacggtcctcgacaagcaaaatgggaagaatcgactttgtgcgtctc
cttctacctcattcttggcctcgagctgacgagtgtatgatacacagcaagaagtgggtcgagttggtcccgacgggccatg
ccatgtggctcaaggggacgaggcggttcgcgcagaacgaagacggcttgctcgggcactggtacaaggacatcacgc
caaatgtgcgcccacattcactcttcccttcgcatgtctccgtttactgacccgccctctttcgccgtgcgcagtaccgccccc
tcccatcttccgaatgtccacctggcgctatcggcgtaacctacgacaccctctccgtccacgcaccaaagtactgccagta
ccttgcaagagagctgcagaagctcggcgcgacgtttgagagacggaccgttacgtcgcttgagcaggcgttcgacggt
gcggatttggtggtcaacgctacgggacttggtatgtcccgaactgcccctctctacctgcaattttgctgattgatatgctcg
caggcgccaagtcgattgcgggcatcgacgaccaagccgccgagccaatccgcggccaaaccgtcctcgtcaagtccc
catgcaagcgatgcacgatggactcgtccgaccccgcttctcccgcctacatcattccccgaccaggtggcgaagtcatct
gcggcgggacgtacggcgtgggagactgggacttgtctgtcaacccagagacggtccagcggatcctcaagcactgctt
gcgcctcgacccgaccatctcgagcgacggaacgatcgaaggcatcgaggtcctccgccacaacgtcggcttgcgacct
gcacgacgaggcggaccccgcgtcgaggcagaacggatcgtcctgcctctcgaccggacaaagtcgcccctctcgctc
ggcaggggcagcgcacgagcggcgaaggagaaggaggtcacgcttgtgcatgcgtatggcttctcgagtgcgggatac
cagcagagttggggcgcggcggaggatgtcgcgcagctcgtcgacgaggcgttccagcggtaccacggcgcggcgcg
ggagtcgaagttgtag
SEQ ID NO.3
MSKYVDRVIAEVEKKYADEPEFVQTVEEVLSSLGPVVDAHPEYEEVALLERM
VIPERVIEFRVPWEDDNGKVHVNTGYRVQFNGAIGPYKGGLRFAPSVNLSIM
KFLGFEQAFKDSLTTLPMGGAKGGSDFDPNGKSDREVMRFCQAFMTELYRHI
GPDIDVPAGDLGVGAREIGYMYGQYRKIVGGFYNGVLTGKARSFGGSLIRPE
ATGYGLVYFTEAMLKRHGMGFEGMRVSVSGSGNVAQYAIEKAMEFGARVIT
ASDSSGTVVDESGFTKEKLARLIEIKASRDGRVADYAKEFGLVYLEGQQPWS
LPVDIALPCATQNELDVDAAHQLIANGVKAVAEGANMPTTIEATELFQQAGV
LFAPGKAANAGGVATSGLEMAQNAARLGWKAEKVDARLHHIMTDIHDGSA
AAAERYGLGYNLVAGANIVGFQKIADAMMAQGIAW
SEQ ID NO.4
atggatcagacatattctctggagtcattcctcaaccatgtccaaaagcgcgacccgaatcaaaccgagttcgcgcaagcc
gttcgtgaagtaatgaccacactctggccttttcttgaacaaaatccaaaatatcgccagatgtcattactggagcgtctggttg
aaccggagcgcgtgatccagtttcgcgtggtatgggttgatgatcgcaaccagatacaggtcaaccgtgcatggcgtgtgc
agttcagctctgccatcggcccgtacaaaggcggtatgcgcttccatccgtcagttaacctttccattctcaaattcctcggctt
tgaacaaaccttcaaaaatgccctgactactctgccgatgggcggtggtaaaggcggcagcgatttcgatccgaaaggaa
aaagcgaaggtgaagtgatgcgtttttgccaggcgctgatgactgaactgtatcgccacctgggcgcggataccgacgttc
cggcaggtgatatcggggttggtggtcgtgaagtcggctttatggcggggatgatgaaaaagctctccaacaataccgcct
gcgtcttcaccggtaagggcctttcatttggcggcagtcttattcgcccggaagctaccggctacggtctggtttatttcacag
aagcaatgctaaaacgccacggtatgggttttgaagggatgcgcgtttccgtttctggctccggcaacgtcgcccagtacgc
tatcgaaaaagcgatggaatttggtgctcgtgtgatcactgcgtcagactccagcggcactgtagttgatgaaagcggattc
acgaaagagaaactggcacgtcttatcgaaatcaaagccagccgcgatggtcgagtggcagattacgccaaagaatttgg
tctggtctatctcgaaggccaacagccgtggtctctaccggttgatatcgccctgccttgcgccacccagaatgaactggat
gttgacgccgcgcatcagcttatcgctaatggcgttaaagccgtcgccgaaggggcaaatatgccgaccaccatcgaagc
gactgaactgttccagcaggcaggcgtactatttgcaccgggtaaagcggctaatgctggtggcgtcgctacatcgggcct
ggaaatggcacaaaacgctgcgcgcctgggctggaaagccgagaaagttgacgcacgtttgcatcacatcatgctggata
tccaccatgcctgtgttgagcatggtggtgaaggtgagcaaaccaactacgtgcagggcgcgaacattgccggttttgtga
aggttgccgatgcgatgctggcgcagggtgtgatttaa
SEQ ID NO 5
MKKVVTVCPYCASGCKINLVVDNGKIVRAEAAQGKTNQGTLCLKGYYGWD
FINDTQILTPRLKTPMIRRQRGGKLEPVSWDEALNYVAERLSAIKEKYGPDAI
QTTGSSRGTGNETNYVMQKFARAVIGTNNVDCCARVUHGPSVAGLHQSVGN
GAMSNAINEIDNTDLVFVFGYNPADSHPIVANHVINAKRNGAKIIVCDPRKIET
ARIADMHIALKNGSNIALLNAMGHVIIEENLYDKAFVASRTEGFEEYRKIVEG
YTPESVEDITGVSASEIRQAARMYAQAKSAAILWGMGVTQFYQGVETVRSLT
SLAMLTGNLGKPHAGVNPVRGQNNVQGACDMGALPDTYPGYQYVKDPAN
REKFAKAWGVESLPAHTGYRISELPHRAAHGEVRAAYIMGEDPLQTDAELSA
VRKAFEDLELVIVQDIFMTKTASAADVILPSTSWGEHEGVFTAADRGFQRFFK
AVEPKWDLKTDWQIISEIATRMGYPMHYNNTQEIWDELRHLCPDFYGATYE
KMGELGFIQWPCRDTSDADQGTSYLFKEKFDTPNGLAQFFTCDWVAPIDKLT
DEYPMVLSTVREVGHYSCRSMTGNCAALAALADEPGYAQINTEDAKRLGIE
DEALVWVHSRKGKIITRAQVSDRPNKGAIYMTYQWWIGACNELVTENLSPIT
KTPEYKYCAVRVEPIADQRAAEQYVIDEYNKLKTRLREAALASEQ ID NO 6
atgaaaaaagtcgtcacggtttgcccctattgcgcatcaggttgcaaaatcaacctggtcgtcgataacggcaaaatcgtcc
gggcggaggcagcgcaggggaaaaccaaccagggtaccctgtgtctgaagggttattatggctgggacttcattaacgat
acccagatcctgaccccgcgcctgaaaacccccatgatccgtcgccagcgtggcggcaaactcgaacctgtttcctggga
tgaggcactgaattacgttgccgagcgcctgagcgccatcaaagagaagtacggtccggatgccatccagacgaccggc
tcctcgcgtggtacgggtaacgaaaccaactatgtaatgcaaaaatttgcgcgcgccgttattggtaccaataacgttgactg
ctgcgctcgtgtctgacacggcccatcggttgcaggtctgcaccaatcggtcggtaatggcgcaatgagcaatgctattaac
gaaattgataataccgatttagtgttcgttttcgggtacaacccggcggattcccacccaatcgtggcgaatcacgtaattaac
gctaaacgtaacggggcgaaaattatcgtctgcgatccgcgcaaaattgaaaccgcgcgcattgctgacatgcacattgca
ctgaaaaacggctcgaacatcgcgctgttgaatgcgatgggccatgtcattattgaagaaaatctgtacgacaaagcgttcg
tcgcttcacgtacagaaggctttgaagagtatcgtaaaatcgttgaaggctacacgccggagtcggttgaagatatcaccgg
cgtcagcgccagtgagattcgtcaggcggcacggatgtatgcccaggcgaaaagcgccgccatcctgtggggcatgggt
gtaacccagttctaccagggcgtggaaaccgtgcgttctctgaccagcctcgcgatgctgaccggtaacctcggtaagccg
catgcgggtgttaacccggttcgtggtcagaacaacgttcagggtgcctgcgatatgggcgcgctgccggatacgtatccg
ggataccagtacgtgaaagatccggctaaccgcgagaaattcgccaaagcctggggcgtggaaagcctgccagcgcata
ccggctatcgcatcagcgagctgccgcaccgcgcagcgcatggcgaagtgcgtgccgcgtacattatgggcgaagatcc
gctacaaactgacgcggagctgtcggcagtacgtaaagcctttgaagatctggaactggttatcgttcaggacatctttatga
ccaaaaccgcgtcggcggcggatgttattttaccgtcaacgtcgtggggcgagcatgaaggcgtgtttactgcggctgacc
gtggcttccagcgtttcttcaaggcggttgaaccgaaatgggatctgaaaacggactggcaaatcatcagtgaaatcgcca
cccgtatgggttatccgatgcactacaacaacacccaggagatctgggatgagttgcgtcatctgtgcccggatttctacggt
gcgacttacgagaaaatgggcgaactgggcttcattcagtggccttgccgcgatacttcagatgccgatcaggggacttctt
atctgtttaaagagaagtttgataccccgaacggtctggcgcagttcttcacctgcgactgggtagcgccaatcgacaaact
caccgacgagtacccgatggtactgtcaacggtgcgtgaagttggtcactactcttgccgttcgatgaccggtaactgtgcg
gcactggcggcgctggctgatgaacctggctacgcacaaatcaataccgaagacgccaaacgtctgggtattgaagatga
ggcattggtttgggtgcactcgcgtaaaggcaaaattatcacccgtgcgcaggtcagcgatcgtccgaacaaaggggcga
tttacatgacctaccagtggtggattggtgcctgtaacgagctggttaccgaaaacttaagcccgattacgaaaacgccgga
gtacaaatactgcgccgttcgcgtcgagccgatcgccgatcagcgcgccgccgagcagtacgtgattgacgagtacaac
aagttgaaaactcgcctgcgcgaagcggcactggcgtaa
Claims (10)
1. A method for preparing refined glufosinate-ammonium by a multi-enzyme coupling one-pot method is characterized in that D-amino acid oxidase, L-glutamate dehydrogenase and formate dehydrogenase are added simultaneously in the same system, and the D-glufosinate-ammonium in racemic D, L-glufosinate-ammonium is completely converted into L-glufosinate-ammonium by utilizing the catalysis of the D-amino acid oxidase, the L-glutamate dehydrogenase and the formate dehydrogenase, and meanwhile, the L-glufosinate-ammonium in the original medicine is reserved.
2. The method of claim 1, wherein the step of determining the position of the substrate comprises,
d-amino acid oxidase catalyzes the substrate D-PPT to be converted into an intermediate product PPO; l-glutamate dehydrogenase is used for ammonification and reduction of PPO to L-glufosinate; formate dehydrogenase is used for the cyclic regeneration of reduced coenzymes NADH or NADPH.
3. The method according to claim 1, wherein the D-amino acid oxidase, L-glutamic acid dehydrogenase and formate dehydrogenase are added in at least one form selected from the group consisting of crude cell extract of enzyme, purified enzyme solution, immobilized enzyme, lyophilized cell containing enzyme and fermentation broth.
4. A method according to claim 3, wherein the host cell expressing D-amino acid oxidase, L-glutamate dehydrogenase and formate dehydrogenase is selected from at least one of saccharomyces cerevisiae (Saccharomyces cerevisiae), pichia pastoris (Pichia pastoris), streptomyces (Streptomyces), bacillus subtilis (Bacillus subtilis) or Escherichia coli, respectively.
5. The method according to claim 3 or 4, wherein the recombinant microorganism is added in an amount of 1 to 200g of wet cell/L reaction solution based on the weight of the wet cell.
6. The method according to any one of claims 1 to 5, wherein the enzymatic conversion reaction is carried out in a reaction liquid system having a pH of 7 to 10; the reaction temperature is 25-45 ℃ and the reaction time is not less than 6 hours.
7. The method according to any one of claims 1 to 6, wherein the reaction system further requires the addition of catalase and an inorganic amino donor, preferably at least one of ammonium sulfate or ammonium formate.
8. The method according to any one of claims 1 to 7, wherein the initial concentration of each substance in the reaction system is: d, L-PPT 300-500 mM, inorganic amino donor 300-500 mM, catalase 3000-8000U/ml.
9. The method according to any one of claims 1 to 8, wherein the reaction system is pH controlled by adding ammonia.
10. The method according to claim 1, wherein the D-glufosinate in the racemic D, L-glufosinate is present in an amount of 40-60% by mass.
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| CN202310427942.7A CN116590374A (en) | 2023-04-20 | 2023-04-20 | Method for asymmetrically preparing glufosinate-ammonium by multi-enzyme coupling one-pot method |
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