Method for preparing L-glufosinate-ammonium by splitting racemic glufosinate-ammonium through electrochemical multienzyme molecular machine

Abstract

The invention relates to a method for preparing L-glufosinate-ammonium by splitting racemic glufosinate-ammonium (D, L-glufosinate-ammonium) through an electrochemical multi-enzyme molecular machine, which comprises the following steps: catalyzing D-glufosinate-ammonium by D-amino acid oxidase to obtain 2-carbonyl-4-[hydroxy (methyl) phosphonyl] butyric acid, and catalyzing by amino acid dehydrogenase to obtain L-glufosinate-ammonium. And an electrochemical multi-enzyme molecular machine is utilized to realize dynamic monitoring of the preparation process. By detecting the current change, the reaction kinetics and process are known, and the enzyme catalysis efficiency is improved; nADP < + > green efficient cyclic regeneration is achieved, separation of an intermediate product is not needed, L-glufosinate-ammonium is directly obtained, the conversion efficiency reaches up to 90% or above, and the enantiomer excess rate is gt; 99%; the method is simple to operate, green, efficient, high in selectivity and suitable for large-scale popularization and demonstration of L-glufosinate-ammonium.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for preparing L-glufosinate ammonium by electrochemical multi-enzyme molecular machine resolution of racemic glufosinate ammonium. BACKGROUND

[0002] Glufosinate ammonium (2-amino-4-[hydroxy(methyl)phosphono]butyric acid ammonium) is a high-efficiency broad-spectrum trigger-type herbicide, belonging to the phosphoric acid herbicide, which was developed by the former German Agrochemie GmbH (later belonging to the German Bayer Company) in 1986. The activity of glufosinate ammonium is between that of glyphosate and paraquat, but in recent years, it has become the second largest herbicide-resistant transgenic crop in the world due to its broad herbicidal spectrum, low toxicity, high activity and good environmental compatibility.

[0003] Glufosinate ammonium has two different conformations due to the presence of a chiral carbon atom (C2) in the molecule, and only L-glufosinate ammonium has herbicidal activity, is easy to degrade, has low toxicity, and has a small environmental impact. The commercially available glufosinate ammonium is usually a racemic mixture of L-type and D-type glufosinate ammonium. How to efficiently resolve the racemic glufosinate ammonium to prepare high-chiral-purity L-glufosinate ammonium has important practical significance in significantly reducing the use amount of glufosinate ammonium, improving the atom economy, and reducing the environmental pressure.

[0004] The mainstream preparation method of spectrally pure L-glufosinate ammonium can be divided into chemical synthesis method and biological synthesis method. The chemical synthesis method has the problems of complicated steps, high cost, low optical purity of the product, and large use of organic reagents or toxic reagents, which is poor in safety and environmental friendliness. At present, the industrial production of glufosinate ammonium mainly adopts the biological synthesis method. Compared with the chemical synthesis method, the biological synthesis method has the advantages of high stereoselectivity, mild reaction conditions, and high product yield, so it has extremely high industrial value and social benefits to explore the biological synthesis method for preparing high-chiral-purity L-glufosinate ammonium.

[0005] The biological synthesis method for preparing glufosinate ammonium can be divided into the following two types according to different synthesis raw materials: (1) using L-glufosinate ammonium derivative as a substrate to obtain by enzyme method, the main advantage is high conversion rate and high enantiomeric excess value of the product, but it needs expensive and difficult-to-obtain chiral raw materials as precursors; (2) using D,L-glufosinate ammonium as raw material, D-glufosinate ammonium is catalyzed by D-amino acid oxidase to obtain L-glufosinate ammonium precursor 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid, and then L-glufosinate ammonium is obtained by catalysis of amino acid dehydrogenase or transaminase. This method can use commercially available D,L-glufosinate ammonium as raw material to obtain high-purity L-glufosinate ammonium, and has strong operability and application prospect.

[0006] However, the current biological enzyme method for resolving D, L-glufosinate lacks a suitable detection method, which cannot clearly detect the reaction process, and cannot regulate the key factors. SUMMARY

[0007] To solve the above technical problems, the application provides a method for resolving racemic glufosinate to prepare L-glufosinate by using an electrochemical multi-enzyme molecular machine.

[0008] The technical scheme adopted by the application is as follows: a method for resolving racemic glufosinate to prepare L-glufosinate by using an electrochemical multi-enzyme molecular machine, wherein the reactants are placed in the electrochemical multi-enzyme molecular machine, and the preparation process includes two stages (a stage for preparing 2-carbonyl-4-[hydroxy(methyl) phosphinyl] butyric acid and a stage for preparing L-glufosinate); and each stage can be observed and regulated by electrochemistry, so that a truly adjustable, controllable and visual process monitoring is realized.

[0009] In the stage for preparing 2-carbonyl-4-[hydroxy(methyl) phosphinyl] butyric acid, D, L-glufosinate is used as a substrate, and catalase and D-amino acid oxidase are added to the electrochemical multi-enzyme molecular machine of a three-electrode system, so as to monitor the reaction process by monitoring the current change.

[0010] In the stage for preparing L-glufosinate, the working electrode used in the stage for preparing 2-carbonyl-4-[hydroxy(methyl) phosphinyl] butyric acid is replaced by an electrode loaded with ferredoxin-NADP + reductase and glutamate dehydrogenase, and L-glufosinate is obtained after the reaction; and the reaction process is monitored by monitoring the current change.

[0011] Preferably, in the stage for preparing 2-carbonyl-4-[hydroxy(methyl) phosphinyl] butyric acid, a platinum mesh is used as a counter electrode, a silver-silver chloride electrode is used as a reference electrode, and an indium tin oxide-titanium sheet is used as a working electrode to construct a three-electrode system.

[0012] Preferably, in the stage for preparing L-glufosinate, an indium tin oxide-titanium sheet electrode loaded with ferredoxin-NADP + reductase and glutamate dehydrogenase is prepared in advance, and is used to replace the original indium tin oxide-titanium sheet electrode; or a mixture of ferredoxin-NADP + reductase and glutamate dehydrogenase is drop-coated on the indium tin oxide-titanium sheet electrode in the reaction system.

[0013] Preferably, ferredoxin-NADP + reductase and glutamate dehydrogenase are mixed at a molar ratio of 1:1-10.

[0014] Preferably, ferredoxin-NADP + reductase and glutamate dehydrogenase are mixed at a molar ratio of 1:5.

[0015] Preferably, in the preparation of 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid, the reaction system includes pH 8.0 TAPS buffer, D-amino acid oxidase and 20 U / mL catalase;

[0016] In the preparation of L-phosphinothricin, the reaction system is the reaction system after the preparation of 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid, and 20-30 μM NADP is added. + .

[0017] Preferably, a voltage of -0.85 V vs. silver-silver chloride reference electrode is applied for reaction kinetic monitoring.

[0018] Preferably, 0.45 mg of D-amino acid oxidase is immobilized on 2 ml of nickel-nitrilotriacetic acid resin to obtain D-amino acid oxidase-nickel-nitrilotriacetic acid microspheres, and the D-amino acid oxidase-nickel-nitrilotriacetic acid microspheres are used to prepare 2-carbonyl-4-[hydroxy(methyl)phosphono]butyric acid.

[0019] Preferably, the expression of D-amino acid oxidase and iron oxide protein-NADP is respectively constructed. + Genetically engineered bacteria that produce reductase or glutamate dehydrogenase are fermented and cultured, and D-amino acid oxidase and iron oxide protein-NADP are extracted respectively. + reductase or glutamate dehydrogenase.

[0020] The advantages and positive effects of the present invention are: placing the preparation process in an electrochemical multi-enzyme molecular machine, reflecting the synthesis process through current changes, improving the utilization efficiency of enzyme catalysis; and realizing green and efficient recycling of NADP + / NADPH, without the need to separate the intermediate product, directly prepare L-phosphinothricin; in the second step, the iron oxide protein-NADP + Reductase and glutamate dehydrogenase are co-immobilized in the micro-nanopores of the electrode, creating a high-concentration enzyme reaction compartment and achieving efficient substrate transfer, utilization and conversion;

[0021] The electrochemical multi-enzyme molecular machine is used to resolve racemic glufosinate to prepare L-glufosinate. The operation is simple and achieves a green, rapid, efficient and highly selective preparation of L-glufosinate. The method has stable performance and is suitable for large-scale demonstration and promotion of L-glufosinate. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Schematic diagram of the in vitro synthesis pathway of L-glufosinate;

[0023] Figure 2 : 12% SDS-PAGE analysis of D-amino acid oxidase expression;

[0024] Figure 3 SDS-PAGE analysis of glutamate dehydrogenase expression;

[0025] Figure 4 SDS-PAGE analysis of ferredoxin-NADP + reductase expression;

[0026] Figure 5 Chronoamperometry results of molar ratio optimization of ferredoxin-NADP + reductase and glutamate dehydrogenase;

[0027] Figure 6 Chronoamperometry results of molar ratio optimization of ferredoxin-NADP + reductase and glutamate dehydrogenase catalytic system; + reductase and glutamate dehydrogenase catalytic system;

[0028] Figure 7 Chronoamperometry results of ferredoxin-NADP + reductase and glutamate dehydrogenase catalytic system; + reductase and glutamate dehydrogenase catalytic system; m reductase and glutamate dehydrogenase catalytic system;

[0029] Figure 8 Electrochemical results of D-amino acid oxidase catalyzed oxidation and deamination of D-glufosinate;

[0030] Figure 9 Time course of D-amino acid oxidase catalyzed oxidation and deamination of D-glufosinate;

[0031] Figure 10 Electrochemical multi-enzyme molecular machine design device schematic diagram;

[0032] Figure 11 Chronoamperometry results of ferredoxin-NADP + reductase and glutamate dehydrogenase catalytic system for L-glufosinate production under electrical energy driving. DETAILED DESCRIPTION

[0033] The embodiments of the present application will be described below in conjunction with the accompanying drawings.

[0034] The present invention relates to a method for preparing L-phosphinothricin (L-PPT) by resolving racemic glufosinate (D,L PPT) using an electrochemical multi-enzyme molecular machine. The method is based on the catalysis of D-phosphinothricin (D-PPT) by D-amino acid oxidase (DAAO) to obtain L-PPT precursor - 2-carbonyl-4-[hydroxy(methyl)phosphonyl]butyric acid (PPO), and then catalyzing L-PPT by amino acid dehydrogenase or transaminase. Dynamic detection of the preparation process is achieved by the electrochemical multi-enzyme molecular machine.

[0035] The electrochemical multi-enzyme molecular machine uses D,L-PPT as a substrate. After adding catalase (CAT) to a three-electrode electrochemical bioreactor, D-PPT is converted to PPO under the action of DAAO. A voltage of -0.7V vs. silver / silver chloride (vs. Ag / AgCl) is applied to the working electrode. Oxygen can obtain electrons on the electrode surface. Because oxygen is a substrate for DAAO, DAAO competes with the electrode for oxygen. The extent of the reaction can be determined by observing changes in the oxygen reduction current. After the reaction is completed, the working electrode is adjusted so that the working electrode of the reaction system is loaded with wild-type iron oxide protein-NADP. + reductase (FNR) and glutamate dehydrogenase (GluDH), with the addition of a catalytic amount of NADP + A voltage of -0.85V vs. Ag / AgCl was applied to monitor the reaction and the extent of the reaction was determined by the change in the catalytic current signal. Figure 1 shown.

[0036] The above method can be used to separate the external D-PPT and L-PPT in two steps to prepare L-PPT in the same system, and the reaction progress can be monitored by using the change in the current signal. The specific preparation method is as follows:

[0037] Phase 1:

[0038] Step 1: Ultrasonicate 6 ml of an engineered bacterial suspension containing 100 g / L DAAO to prepare a DAAO lysate. The DAAO lysate was then fully combined with 2 mL of nickel-nitrilotriacetic acid (Ni-NTA) resin to elute impurities while purifying and immobilizing DAAO to obtain a DAAO-Ni-NTA catalyst. The amount of immobilized protein was 0.45 mg as calculated by Bradford. The catalyst was stored in a 4°C refrigerator until ready for use.

[0039] Step two: a three-electrode system was constructed with a platinum mesh as a counter electrode, an Ag / AgCl electrode as a reference electrode, and an indium tin oxide-titanium sheet (ITO-Ti) as a working electrode; 20 mL of pH 8.0 TAPS buffer was added to the electrolytic cell of the three-electrode system, 2 ml of DAAO-Ni-NTA prepared in step one was added to the TAPS buffer, and 20 U / mL of CAT was added; under a potential of-0.7 V vs Ag / AgCl, 10 mM D,L-PPT was added to initiate the reaction, and the reaction progress was observed by changes in current.

[0040] Second stage:

[0041] Step three: the reaction solution in step two was used as an electrolyte, and 20-30 μM NADP + In the electrolytic cell of the three-electrode system, the working electrode was replaced with an ITO-Ti electrode sheet loaded with FNR and GluDH; a voltage of-0.85 V vs Ag / AgCl was applied, and the reaction progress was observed by changes in current; and L-PPT was prepared.

[0042] Wherein, according to the disclosed sequence information, a DAAO expression vector can be constructed, which is transferred into a host cell to construct an engineering strain, and after expression, a DAAO crude enzyme solution is obtained by cell disruption, the DAAO enzyme amino acid sequence is shown as SEQ ID No. 1, and the nucleic acid sequence is shown as SEQ ID No. 2; in some embodiments of the present application, the DAAO can be prepared according to the method mentioned in patent CN116064446A. According to the disclosed sequence information, a GluDH expression vector can be constructed, which is transferred into a host cell to construct an engineering strain, and after expression, GluDH is extracted, the amino acid sequence is shown as SEQ ID No. 3, and the nucleic acid sequence is shown as SEQ ID No. 4; in some embodiments of the present application, the GluDH can be prepared according to the method mentioned in patent CN113817694 A. According to the disclosed sequence information, a FNR expression vector can be constructed, which is transferred into a host cell to construct an engineering strain, and after expression, FNR is extracted, the amino acid sequence is shown as SEQ ID No. 5, the nucleic acid sequence is shown as SEQ ID No. 6, and the NCBI accession number is WP_011430120.1.

[0043] SEQ ID No. 1:

[0044] MHSQKRVVVLGSGVIGLSSALILARKGYSVHILARDLPEDVSSQTFASPWAGATWTPFMTLTDGPRQAKWEESTFKKWVELVPTGHAMWLKGTRRFAQNEDGLLGHWYKDITPNYRPLPSSECPPGAIGVTYDTLSVHAPKYCQYLARELQKLGATFERRTVTSLEQAFDGADLVVNATGLGAKSIAGIDDQAAEPIRGQTVLVKSPCKRCTTDSSDPASPAYIIPRPGGEVICGGTYGVGDWDLSVNPETVQRILKHCLRLDPTISSDGTIEGIEVLRHNVGLRPARRGGPRVEAERIVLPLDRTKSPLSLGRGSARAAKEKEVTLVHAYGFSQAGYQQSWGAAEDVAQLVDEAFQRYHGAARESKL

[0045] SEQ ID No. 2:

[0046]

[0047] SEQ ID No. 3:

[0048] MIESVDSFLARLQQRDPGQPEFHQAVEEVLRTLWPFLEANPRYLQSGILERMVEPERAVLFRVSWVDDQGKVQVNRGYRIQMSSAIGPYKGGLRFHPSVNLSVLKFLAFEQVFKNSLTSLPMGGGKGGSDFDPKGKSDAEVMRFCQAFMSELYRHIGADCDVPAGDIGVGAREIGFMFGQYKRLANQFTSVLTGKGMTYGGSLIRPEATGYGCVYFAEEMLKRQGLRVDGRRVAISGSGNVAQYAARKVMDLGGKVISLSDSEGTLYAEGGLTEAQWEAVMQLKNVARGRISELAEAFGLEFRKGQTPWSLPCDIALPCATQNELGIEDARTLLRNGCICVAEGANMPTTLAAVDLFIDAGILYAPGKASNAGGSAVSGLEMSQNAMRLLWTAGEVDSKLHNIMQSIHHACVHYGEEADGKVNYVKGANIAGFVKVADAMLAQGVV

[0049] SEQ ID No. 4:

[0050]

[0051] SEQ ID No. 5:

[0052] MVASAQKREVVVNLYRPNAPLIGQCVETYSLVGEGAPGLTKHIVLSLDPNYRYLEGQSVGIIPPGVDDKGKPHKPRLYSIASTRYGDDGEGRTVSLSVKRAEYVDKETGQPGVGVCSGFLTDLKPGDEVMITGPSGKTFLLPEDENANLILIATGTGIAPFRAFIKHLFEEDPNYQGKIWLFFGVPTTSTLLYHGDLEAWKAQYGDRFRVDYAISREQQTPDGKKMYVQNRMAEYGPELWEMLQQPNTYTYICGLKGMEDGINSVMAPLAEQAGQDWSKFQKELKKANRWHEETY

[0053] SEQ ID No. 6:

[0054] ATGGTTGCCAGTGCACAGAAACGCGAAGTGGTGGTTAATCTGTATCGCCCGAATGCCCCGCTGATTGGCCAGTGTGTTGAAACCTATAGTCTGGTTGGCGAAGGTGCCCCGGGCCTGACCAAACATATTGTGCTGAGCCTGGACCCTAATTATCGTTATCTGGAAGGTCAGAGCGTTGGCATTATTCCGCCGGGCGTTGATGATAAAGGTAAACCGCATAAACCGCGTCTGTATAGTATTGCAAGTACCCGCTATGGCGATGATGGCGAAGGCCGTACCGTTAGTCTGAGTGTGAAACGTGCCGAATATGTGGATAAAGAAACCGGCCAGCCGGGCGTGGGCGTGTGTAGTGGTTTTCTGACCGATCTGAAACCGGGCGATGAAGTGATGATTACCGGCCCGAGTGGTAAAACCTTTCTGCTGCCGGAAGATGAAAATGCCAATCTGATTCTGATTGCAACCGGTACCGGCATTGCCCCGTTTCGTGCATTCATTAAGCATCTGTTTGAAGAAGATCCGAATTATCAGGGCAAAATTTGGCTGTTTTTCGGCGTTCCGACCACCAGCACCCTGCTGTATCATGGTGACCTGGAAGCATGGAAAGCACAGTATGGCGATCGCTTTCGTGTGGATTATGCAATTAGTCGCGAACAGCAGACCCCGGATGGTAAAAAGATGTATGTTCAGAATCGCATGGCCGAATATGGTCCGGAACTGTGGGAAATGCTGCAGCAGCCGAATACCTATACCTATATTTGCGGTCTGAAAGGCATGGAAGATGGTATTAATAGCGTGATGGCACCGCTGGCAGAACAGGCAGGTCAGGATTGGAGTAAATTTCAGAAAGAACTGAAAAAGGCAAACCGCTGGCATGAAGAAACCTATTAA

[0055] In some embodiments of the present application, the working electrode ITO-Ti or indium tin oxide-pyrolytic edge graphite electrode (ITO-PGE) is a porous electrode. The preparation process is as follows: 10 mg of iodine and 40 mg of indium tin oxide (ITO) nanoparticles are added into 40 mL of acetone, and ultrasonic treatment is performed for 30 minutes to make the mixture uniformly dispersed. Then, the uniformly dispersed suspension is poured into a small beaker, and the anode and cathode are placed in parallel with a distance of 1 cm. A voltage of 10 V is applied by a direct current power supply, and electrophoretic deposition is performed for 5 min to obtain the ITO porous electrode.

[0056] In the second stage reaction, the FNR and GluDH are mixed and diluted appropriately, and the molar ratio of FNR to GluDH is preferably 1:5. The protein mixture solution is drop-coated on the surface of the ITO-Ti electrode, and incubated in a refrigerator at 4°C for 30 min. Then, the electrode is washed with ultrapure water to remove the excess unbound proteins, and the ITO-Ti electrode loaded with FNR and GluDH is obtained. The FNR and GluDH are co-immobilized in the pores of the porous electrode, which can realize efficient transmission of substrates. In some embodiments of the present application, the pre-mixed FNR and GluDH protein mixture can also be added dropwise to the ITO-Ti electrode in the electrode system after the completion of the first stage reaction, to form the ITO-Ti electrode loaded with FNR and GluDH.

[0057] The above method can efficiently prepare L-PPT, and realize a conversion efficiency of more than 90%. The multi-enzyme synergistic immobilization method improves the utilization efficiency of enzyme catalysts, and realizes NADPH regeneration in situ. + Green and efficient recycling without separation of intermediate products for L-PPT synthesis.

[0058] The embodiments of the present application will be described below with reference to the accompanying drawings, wherein the experimental methods not specifically described in the operation steps are performed according to the corresponding product instructions. The instruments, reagents and consumables used in the embodiments can be purchased from commercial companies, unless otherwise specified.

[0059] Example 1: Protein expression and purification

[0060] 1. Expression of DAAO

[0061] According to the sequence of NCBI accession number YP_009724397.2 28274-29533, the DAAO protein recombinant plasmid (DAAO-pET-28a(+)) was prepared, the competent E. coli BL21(DE3) was transformed, and the induction expression was carried out: overexpression and induction for 24 hours at 1 mM IPTG, 25°C, and middle exponential phase, after the end of culture, the culture solution was centrifuged at 4000 rpm for 40 min, the supernatant was discarded, the bacterial body was collected, resuspended and diluted with 50 mM, pH 8.0 TAPS buffer, the final concentration was 100 g / L, and it was stored in a ultra-low temperature refrigerator at -80°C for standby. The prepared protein was detected by electrophoresis, and the purity was as shown in Figure 2 .

[0062] 2. Expression and purification of GluDH

[0063] According to the sequence of NCBI accession number YP_009724397.2 28274-29533, the GluDH protein recombinant plasmid (Glu-pET-28a(+)) was prepared, the competent E. coli BL21(DE3) was transformed, and the induction expression was carried out: overexpression and induction for 24 hours at 0.5 mM IPTG, 25°C, and middle exponential phase, the cells were harvested by centrifugation, broken and lysed to obtain the supernatant. Then, gradient elution was carried out by using Ni-NTA resin (5 mL, Cytiva) and AKTA protein purification instrument (Cytiva, AKTA Pure) (buffer A: 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 30 mM imidazole; buffer B: 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 500 mM imidazole). The purity of the protein was determined by 12% SDS-PAGE, as shown in Figure 3 , and the protein concentration was detected by using Bradford Kit (Sangon Biotech), diluted to 50 mg / mL, and stored at -80°C for standby.

[0064] 3. Expression and purification of FNR

[0065] The FNR protein recombinant plasmid (FNR-pET-28a(+)) was prepared according to the sequence of NCBI accession number YP_009724397.2 at positions 28274-29533, and transformed into competent Escherichia coli BL21 (DE3) for induction and expression: overexpression and induction for 7 hours at 37°C in the middle exponential phase with 1 mM IPTG, and the cells were harvested by centrifugation, broken and lysed to obtain the supernatant. Then, gradient elution was performed using Ni-NTA resin (5 mL, Cytiva) and an AKTA protein purification instrument (Cytiva, AKTA Pure) (buffer A: 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 30 mM imidazole; buffer B: 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 500 mM imidazole). The purity of the protein was determined by 12% SDS-PAGE, and the protein concentration was detected using a Bradford Kit (Sangon Biotech), diluted to 10 mg / mL, and stored at -80°C for standby. Figure 4

[0066] Example 2: Immobilization of DAAO

[0067] The DAAO-BL21 (DE3) bacterial solution prepared in step 1 of Example 1 was taken out in a beaker, and under the condition of ice water bath, ultrasonic treatment was performed at 325 W for 10 min (on for 2 s, off for 2 s), and the supernatant was collected after centrifugation at 12000 rpm for 20 min. 2 mL of the crude enzyme solution was added to 2 mL of Ni-NTA agarose beads, and the target protein DAAO was fully combined with Ni-NTA by stirring for 30 min. Then, 30 mM imidazole was used to wash away impurities, and 50 mM pH 8.0 TAPS buffer was used to wash 3 times, to obtain DAAO-Ni-NTA microspheres, which were stored in a refrigerator at 4°C for standby.

[0068] Example 3: Preparation of ITO-Ti / ITO-PGE electrode loaded with FNR and GluDH

[0069] In 40 mL of acetone, 10 mg of iodine was added, and 40 mg of ITO nanoparticles was ultrasonically dispersed for 30 minutes. The uniformly dispersed suspension was poured into a small beaker, and the anode and cathode were placed in parallel with a distance of 1 cm. A voltage of 10 V was applied by a direct current power supply, and electrophoretic deposition was performed for 5 min to obtain an ITO porous electrode.

[0070] ​After FNR and GluDH were mixed evenly, they were diluted in proper amount. FNR and GluDH were mixed in a certain ratio, and then dropped on the surface of ITO-Ti or ITO-PGE electrode, and incubated in 4°C refrigerator for 30 min. Then the electrode was washed with ultrapure water to remove the excess unbound protein, and the ITO-Ti / ITO-PGE electrode loaded with FNR and GluDH was obtained.

[0071] Example 4: Optimization of the mixing ratio of FNR and GluDH

[0072] After FNR and GluDH were mixed evenly, they were diluted in proper amount. FNR and GluDH were mixed in a certain ratio, and then dropped on the surface of ITO-Ti or ITO-PGE electrode, and incubated in 4°C refrigerator for 30 min. Then the electrode was washed with ultrapure water to remove the excess unbound protein, and the ITO-Ti / ITO-PGE electrode loaded with FNR and GluDH was obtained.

[0073] The electrochemical results and reaction progress in the reaction process were detected, and the results are shown in Figure 5 From the results in the figure, it can be seen that when the molar ratio of FNR and GluDH is 1:5, the reaction effect is best. Further increasing the ratio will not further improve the reaction performance due to the limited amount of protein fixed on the electrode surface. Therefore, 1:5 is preferably used as the ratio of the subsequent two-enzyme reaction.

[0074] Example 5: NADP + Optimization of the amount of addition

[0075] PGE was used as a model electrode, FNR and GluDH were mixed in a molar ratio of 1:5 and loaded on the surface of ITO / PGE electrode to construct a three-electrode system. A potential of -0.7 V vs. Ag / AgCl was applied, and the NADP + concentration in the solution was gradually increased, and the change of current signal was observed. The optimal NADP + concentration of the reaction system was calculated by the NADP + concentration and current intensity. The results are shown in Figure 6 、 Figure 7 and the preferred NADP + concentration is 20-30 μM.

[0076] Example 6: Electrochemical multi-enzyme molecular machine for splitting D, L-PPT to prepare L-PPT

[0077] 6.1 Preparation of PPO

[0078] Take 0.04 g D, L-PPT into 20 ml, 50 mM pH 8.0 TAPS buffer, placed in an electrochemical reaction vessel, so that the final concentration of D, L-PPT is 10 mM, add 2 mL DAAO-Ni-NTA microspheres or the same concentration of DAAO protein solution, add 20 U / ml CAT, take a piece of ITO-Ti sheet electrode inserted into the electrochemical cell as the working electrode; with platinum mesh as the counter electrode, Ag / AgCl electrode as the reference electrode, ITO-Ti sheet as the working electrode, to build a three-electrode system.

[0079] After the construction of the electrochemical biological reactor prepared by PPO, the voltage of-0.7 V vs. Ag / AgCl was applied by the electrochemical workstation, and the change of current signal was observed as shown in Figure 8 After adding DAAO, the current signal decreased due to the consumption of oxygen in the solution by DAAO, and when the reaction substrate (D-PPT) was completely consumed, the signal basically recovered to the original intensity. Compared with immobilized DAAO, DAAO-Ni-NTA had faster reaction rate and more complete reaction. In order to verify the relationship between the change of current signal and the reaction process, the reaction system was sampled at regular time, and the decrease of D-PPT was detected by pre-column derivatization high performance liquid chromatography as shown in Figure 9

[0080] The reaction system with DAAO-Ni-NTA was taken for subsequent experiments.

[0081] 6.2 Preparation of L-PPT

[0082] The reaction solution in step 6.1 was deoxygenated by nitrogen, and was used as electrolyte. The ITO-Ti sheet electrode loaded with FNR and GluDH at a molar ratio of 1:5 prepared according to the method of Example 3 was placed in the electrochemical reactor as the working electrode; after the electrolytic cell was sealed, the reactor structure was shown as Figure 10 , and the change of current signal was monitored at-0.85 V vs. Ag / AgCl potential, as shown in Figure 11 When the current decayed to the baseline, it indicated that the reaction was completed.

[0083] The reaction system was sampled at regular time, and the increase of L-PPT and ee value were detected by pre-column derivatization high performance liquid chromatography. According to the chromatographic data and the integral of electrochemical signal, the input amount of 10 mM D, L-PPT produced 4.5 mM L-PPT after the first step of oxidative deamination and the second step of adding ammonia. The total amount of 9.5 mM L-PPT was calculated, and the yield was 90%, and the enantiomeric excess rate (ee) was > 99%.

[0084] ​The above detailed description of the embodiments of the present application is only preferred embodiments of the present application, and should not be considered as limiting the scope of the present application. Any equivalent changes and improvements made according to the scope of the present application should still belong to the patent coverage of the present application.

Claims

1. A method for preparing L-glufosinate-ammonium by electrochemically resolving racemic glufosinate-ammonium with an electrochemical multi-enzyme molecular machine, characterized in that: The preparation process is placed in an electrochemical multi-enzyme molecular machine, realizing "one pot two steps", including two stages of preparing 2-carbonyl-4-[hydroxy(methyl) phosphinyl] butyric acid and preparing L-glufosinate ammonium; In the stage of preparing 2-carbonyl-4-[hydroxy(methyl) phosphinyl] butyric acid, peroxidase and D-amino acid oxidase are added into the electrochemical multi-enzyme molecular machine of a three-electrode system with D,L-glufosinate ammonium as a substrate, and the reaction process is monitored by observing current changes; For the L-glufosinate production stage, the working electrode used in the preparation of 2-oxo-4-[hydroxy(methyl)phosphinoyl]butyric acid stage was replaced by an electrode loaded with ferredoxin-NADP + The electrodes for the reductase and glutamate dehydrogenase, after reaction, give L-glufosinate; the progress of the reaction was monitored by observing the current changes.

2. The process for preparing L-glufosinate-ammonium by electrochemical multi-enzymatic molecular machine resolution of racemic glufosinate-ammonium according to claim 1, characterized by the fact that: In the stage of preparing 2-carbonyl-4-[hydroxy(methyl) phosphinyl] butyric acid, a platinum mesh is used as a counter electrode, a silver-silver chloride electrode is used as a reference electrode, and an indium tin oxide-titanium sheet electrode is used as a working electrode to construct a three-electrode system.

3. The method for preparing L-glufosinate-ammonium by electrochemically resolving racemic glufosinate-ammonium through an electrochemical multi-enzyme molecular machine according to claim 2, characterized in that: In the preparation stage of L-glufosinate-ammonium, iron-oxiderubredoxin-NADP + The indium tin oxide-titanium sheet electrode of the reductase and glutamate dehydrogenase replaces the original indium tin oxide-titanium sheet electrode.

4. The method for preparing L-glufosinate-ammonium by electrochemically resolving racemic glufosinate-ammonium through an electrochemical multi-enzyme molecular machine according to claim 3, characterized in that: Iron-oxidizing protein-NADP + The reductase and glutamate dehydrogenase are mixed in a molar ratio of 1 : 1-10.

5. The method for preparing L-glufosinate-ammonium by electrochemically resolving racemic glufosinate-ammonium through an electrochemical multi-enzymatic molecular machine according to claim 4, characterized in that: Iron-oxidizing protein-NADP + The reductase and glutamate dehydrogenase are mixed in a molar ratio of 1 :

5.

6. The process for preparing L-glufosinate-ammonium by electrochemically resolving racemic glufosinate-ammonium using an electrochemical multi-enzymatic molecular machine according to claim 1, characterized in that: The reaction system of the stage of preparing 2-carbonyl-4-[hydroxy(methyl) phosphinoyl] butyric acid comprises pH 8.0 TAPS buffer, D-amino acid oxidase and 20 U / mL catalase; the reaction system of the stage of preparing L-glufosinate is the reaction system after the stage of preparing 2-carbonyl-4-[hydroxy(methyl) phosphinoyl] butyric acid, and 20-30 μM NADP is added + .

7. The process for preparing L-glufosinate-ammonium by electrochemical multi-enzymatic molecular machine resolution of racemic glufosinate-ammonium according to claim 1 or 6, characterized by the fact that: The working electrode is applied with a voltage of -0.85 V vs. silver / silver chloride, and a chronoamperometry method is used to monitor the reaction process.

8. The process for preparing L-glufosinate-ammonium by electrochemical multi-enzymatic molecular machine resolution of racemic glufosinate-ammonium according to claim 1 or 6, characterized by: D-amino acid oxidase-nickel-nitrilotriacetic acid resin microspheres are obtained by fixing D-amino acid oxidase on nickel-nitrilotriacetic acid resin, and the D-amino acid oxidase-nickel-nitrilotriacetic acid resin microspheres are used for preparing 2-carbonyl-4-[hydroxy(methyl) phosphinyl] butyric acid.

9. The process for preparing L-glufosinate-ammonium by electrochemical multi-enzyme molecular machine resolution of racemic glufosinate-ammonium according to any one of claims 1 to 6, characterized in that: respectively, which can be used for expressing D-amino acid oxidase, ferredoxin-NADP + D-amino acid oxidase, ferredoxin-NADP + reductase or glutamate dehydrogenase.

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