CN108690854B

CN108690854B - Method for producing L-glufosinate-ammonium by using chemical-enzymatic method

Info

Publication number: CN108690854B
Application number: CN201810338960.7A
Authority: CN
Inventors: 薛亚平; 郑裕国; 曹成浩; 徐建妙; 吴哲明
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2018-04-16
Filing date: 2018-04-16
Publication date: 2022-03-18
Anticipated expiration: 2038-04-16
Also published as: CN108690854A

Abstract

The invention discloses a method for producing L-glufosinate-ammonium by a chemical-enzymatic method, which comprises the following steps: using racemic N-glufosinate-ammonium as a substrate, using wet thalli obtained by fermenting and culturing engineering bacteria containing carboxypeptidase genes or pure enzyme extracted after the wet thalli is subjected to ultrasonic disruption as a catalyst, using a buffer solution with the pH value of 5-10 as a reaction medium, reacting under the condition of 200rpm of a water bath shaker at 45 ℃, obtaining a reaction solution containing D-N-glufosinate-ammonium and L-glufosinate-ammonium after the reaction is completed, separating and purifying the reaction solution, collecting racemized D-N-glufosinate-ammonium for cyclic resolution, and obtaining the L-glufosinate-ammonium at the same time. The method does not need a coenzyme circulating system and an amino donor with a structure similar to that of a product, has simple reaction steps, easy separation, high optical selectivity (ee value of 99 percent), obvious ion exchange column separation effect and easier obtaining of the high-purity L-glufosinate-ammonium (the purity is 98 percent).

Description

Method for producing L-glufosinate-ammonium by using chemical-enzymatic method

(I) technical field

The invention relates to the field of biochemical engineering, in particular to a method for producing L-glufosinate-ammonium by a chemical-enzymatic method.

(II) background of the invention

Glufosinate, also known as glufosinate, is known by the english name: phosphonothricin (PPT for short) has the chemical name 2-amino-4- [ hydroxy (methyl) phosphono ] -butyric acid. Glufosinate is a systemic conductive herbicide and has broad-spectrum herbicidal activity. The herbicide has wide application range and huge market at home and abroad, the glufosinate-ammonium is one of three herbicides, and the market share is expected to further break through in recent years due to the action mechanism and the transgenic technology.

Glufosinate-ammonium is now on the market mainly as racemate. Glufosinate has two optical isomers: l-glufosinate-ammonium and D-glufosinate-ammonium. However, only L-glufosinate-ammonium has herbicidal activity which is twice as high as that of racemic glufosinate-ammonium, and has low toxicity to human and animals and little influence on the environment. However, commercial glufosinate-ammonium is now produced on a large scale in the form of a racemic mixture. The use of racemic glufosinate-ammonium has huge waste and serious influence on the environment. In order to reduce the environmental protection pressure and the production cost, the exploration of a production line for splitting the racemic glufosinate-ammonium with industrial application prospect has important market prospect and social significance.

The current methods for preparing L-glufosinate-ammonium are mainly divided into chemical methods and biological enzyme methods.

Wherein the chemical method mainly comprises a chemical stereo synthesis method and a chiral resolution method. The chemical stereo synthesis method needs expensive asymmetric synthesis reagents, is mainly on a laboratory research scale, and is not favorable for large-scale preparation. The chemical chiral resolution method also consumes a large amount of expensive chiral resolution reagents, and has complex process and low yield.

Compared with a chemical method, the biological enzyme method has the advantages of mild reaction conditions, strict stereoselectivity and the like. The preparation of L-glufosinate-ammonium biological enzyme method is divided into enzyme method asymmetric synthesis and enzyme method resolution. The theoretical conversion rate of the enzyme method asymmetric synthesis is high, and the enzyme method mainly relates to transaminase and amino acid dehydrogenase.

Schulz A (Stereospermic production of the recombinant phosphinothricin (glufosinate) by transaminase: isolation and catalysis of a phosphinothricin-specific transaminase from Escherichia coli [ J ]. Applied and Environmental Microbiology,1990,56 (1): 1-6) and the like utilize a transaminase cloned from Escherichia coli, with 2-carbonyl-4- (hydroxymethylphosphono) butyric acid as a substrate and L-glutamic acid as an amino donor, to prepare L-glufosinate. However, reversible reactions often result in reduced yields and require the addition of excess amino donor, which places a large burden on the subsequent isolation of the product.

Generally, the resolution by the biological enzyme method is to synthesize racemic D, L-glufosinate-ammonium or derivatives thereof chemically, then to catalyze a certain configuration reaction selectively by using specific enzyme to obtain one of the optical isomers, and to perform the enzyme catalysis reaction after separating, racemizing and carrying out the enzyme catalysis reaction on the other unreacted isomer derivatives, wherein the theoretical yield can reach 100%.

Disclosure of the invention

The invention aims to provide a brand-new method for producing L-glufosinate-ammonium by a chemical-enzymatic method, which has the advantages of high raw material conversion rate, low production cost and high yield.

The technical scheme adopted by the invention is as follows:

the invention provides a method for producing glufosinate-ammonium by a chemical-enzymatic method, which comprises the following steps: using racemic N-acetylglufosinate-ammonium as a substrate, using wet thalli obtained by fermenting and culturing engineering bacteria containing carboxypeptidase genes or pure enzyme extracted after the wet thalli is subjected to ultrasonic disruption as a catalyst, using a buffer solution (preferably Tris-HCl buffer solution with the pH value of 8.9 and 50mM) with the pH value of 5-10 as a reaction medium, reacting in a water bath shaker at the temperature of 45 ℃ under the condition of 200rpm, obtaining a reaction solution containing D-N-acetylglufosinate-ammonium and L-glufosinate-ammonium after the reaction is completed, separating and purifying the reaction solution, collecting racemization of the D-N-acetylglufosinate-ammonium for cyclic resolution, and simultaneously obtaining the L-glufosinate-ammonium.

Further, the carboxypeptidase is derived from Stenotrophomonas (Stenotrophomonas sp.) carboxypeptidase Ss 1. And (3) carrying out codon optimization, and carrying out recombinant expression in the engineering bacteria of escherichia coli to obtain the L-glufosinate-ammonium production catalyzing capability of the L-N-acetyl-glufosinate-ammonium. The amino acid sequence of the carboxypeptidase Ss1 is shown in SEQ ID NO.1, and the nucleotide sequence of the coding gene is shown in SEQ ID NO. 2.

Further, the final concentration of the substrate is 10-100 mM (preferably 100mM) by volume of the buffer solution, and the catalyst is 5-50 g/L (preferably 10g/L) by weight of wet cells.

Further, the racemic N-acetylglufosinate-ammonium is prepared as follows: adding acetic acid into racemic glufosinate-ammonium serving as a raw material, keeping the temperature at 10-60 ℃ (preferably 30-40 ℃), stirring, adding acetic anhydride, reacting until the solution is clear and transparent, carrying out rotary evaporation at 80 ℃ and under the condition of 0.075-0.085 MPa to remove the acetic acid, dissolving a concentrate with water, adjusting the solution to be neutral (the pH is 8.5) by using a sodium hydroxide aqueous solution, adding ethanol, standing to precipitate crystals, and drying to obtain racemic N-acetylglufosinate-ammonium; the weight ratio of the acetic acid to the racemic glufosinate-ammonium is 20-30: 1 (preferably 20:1), wherein the weight ratio of the acetic anhydride to the racemic glufosinate-ammonium is 0.7-1.5: 1 (preferably 1: 1).

Further, the catalyst is prepared by the following method: (1) wet thalli: inoculating engineering bacteria containing carboxypeptidase gene into LB liquid culture medium containing 50 ug/mL kanamycin, culturing at 37 deg.C overnight, inoculating into LB liquid culture medium containing 50 ug/mL kanamycin at an inoculum size of 1% volume concentration, and culturing at 37 deg.C and 150rpm until the thallus concentration OD₆₀₀Adding IPTG with final concentration of 0.1mM to 0.4-0.8, performing induced culture at 28 deg.C for 12 hr, centrifuging at 4 deg.C and 8000rpm for 10min, and collecting wet thallus; (2) pure enzyme: suspending the wet thalli in the step (1) in a Tris-HCl buffer solution with the concentration of 20mM and the pH value of 8.0, shaking uniformly, then carrying out ultrasonic disruption (400W, 1s disruption for 1s pause) for 15min, centrifuging the disruption solution at 12,000rpm for 10min to remove cell debris, and collecting a supernatant; carrying out Ni-NTA column chromatography on the supernatant, balancing the Ni-NTA column by using a loading balance buffer solution, loading the supernatant at the speed of 1.5mL/min, eluting by using the loading balance buffer solution to remove unadsorbed protein, eluting by using an elution buffer solution to collect target protein, dialyzing the target protein in Tris-HCl with the concentration of 20mM and the pH value of 8.5, and taking trapped fluid as pure enzyme; the sample loading balance buffer solution is 20mM Tris-HCl containing 500mM NaCl and 20mM imidazole, and the pH value is 8.5; the elution buffer was 20mM Tris-HCl, pH 8.5, containing 500mM NaCl and 500mM imidazole.

Further, the method for separating and purifying the reaction liquid comprises the following steps: after the reaction is completed, centrifuging the reaction solution, taking supernatant to adjust the pH value to 2, carrying out suction filtration, taking a filtrate to load the filtrate to a hydrogen type 001x7 cation resin, wherein the height ratio of an ion exchange column is 15:1, the loading flow rate is 1.0BV/h, washing 4BV with ultrapure water after loading, collecting effluent liquid containing unreacted substrates D-N-acetylglufosinate-ammonium, racemizing the D-N-acetylglufosinate-ammonium for cyclic resolution; then eluting with 2mol/L ammonia water at the flow rate of 0.5BV/h, collecting the eluent containing the L-glufosinate-ammonium, and obtaining the L-glufosinate-ammonium by decompression, concentration and crystallization.

Further, the racemization method of the D-N-glufosinate-ammonium comprises the following steps: evaporating the effluent containing D-N-acetylglufosinate-ammonium to dryness under reduced pressure, adding acetic acid for mixing, adding acetic anhydride, stirring for racemization reaction at the temperature of 100 ℃ and 125 ℃ (preferably at the temperature of 120 ℃), evaporating the reaction solution to dryness under reduced pressure after the reaction is finished to obtain racemized N-acetylglufosinate-ammonium for cyclic resolution; the mass ratio of the acetic anhydride to the D-N-glufosinate-ammonium is 0.1-3: 1 (preferably 1.7:1), wherein the mass ratio of acetic acid to D-N-glufosinate-ammonium is 10-20: 1 (preferably 10: 1).

The invention has the beneficial effects that: the invention provides a method for producing L-glufosinate-ammonium by a chemical-enzymatic method, which does not need a coenzyme circulating system and an amino donor with a structure similar to that of a product, and has simple reaction steps and easy separation. The catalyst carboxypeptidase has high optical selectivity (ee value of 99%), obvious ion exchange column separation effect and capacity of obtaining high purity L-glufosinate-ammonium in purity of 98%.

(IV) description of the drawings

FIG. 1 shows the reaction scheme for the carboxypeptidase-based chemo-enzymatic preparation of L-glufosinate-ammonium.

FIG. 2 is a liquid phase spectrum of N-acetylglufosinate-ammonium in example 1, with a retention time of 9.0 min.

FIG. 3 is a mass spectrum of N-acetylglufosinate-ammonium in example 1.

FIG. 4 is a liquid phase spectrum of L-glufosinate-D-glufosinate in example 5, with a retention time of 4.6min for L-glufosinate-D-glufosinate and 5.4min for D-glufosinate.

(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:

the ultrapure water is treated by an ultrapure water instrument, and water molecules (H) are removed from the ultrapure water₂O), almost no impurities, bacteria, viruses, organic matters containing green dioxin and the like, and mineral trace elements.

Example 1: synthesis of racemic N-acetylglufosinate-ammonium

Taking 5g of racemic glufosinate-ammonium, putting the racemic glufosinate-ammonium into a 250mL three-neck flask, adding 100g of acetic acid, keeping the temperature and stirring at 40 ℃, adding 5g of acetic anhydride, and reacting until the solution is clear and transparent. And (3) evaporating acetic acid at 80 ℃ by using a rotary evaporator and under the vacuum degree of 0.075-0.085 MPa. Dissolving the concentrate with water, adjusting pH to 8.5 with sodium hydroxide aqueous solution, adding ethanol, standing to precipitate crystal, and drying to obtain racemic N-acetylglufosinate-ammonium. The liquid chromatogram is shown in FIG. 2 and the mass spectrum is confirmed to be N-acetylglufosinate-ammonium by liquid chromatography (Shimadzu LC-16) (FIG. 3). The chromatographic column is

C-18column (250 mM. times.4.6 mM, 5 μm), 10mM phosphoric acid solution (pH 2.1) as mobile phase, 0.75mL/min flow rate, ultraviolet detection wavelength 210nm, and column temperature 35 ℃.

Example 2: construction of engineering bacteria

A strain derived from Stenotrophomonas sp (Stenotrophoromonas sp) and annotated as the sequence of carboxypeptidase Ss1 was subjected to codon optimization and then subjected to total gene synthesis, and the expression plasmid was pET-28 b. The stop codon was site-directed to allow the tail (C-terminus) of the expressed recombinase to contain the histidine tag in pET28 b. The insertion sequence (the amino acid sequence is shown as SEQ ID NO.1, and the nucleotide sequence is shown as SEQ ID NO. 2) is transferred into an expression host escherichia coli E.coli BL21(DE3) after being sequenced and verified to be correct for subsequent expression of recombinase.

Example 3: inducible expression of recombinase

The engineered bacterium constructed in example 2 was inoculated into LB liquid medium containing 50. mu.g/mL kanamycin, cultured overnight at 37 ℃, further inoculated into LB liquid medium containing 50. mu.g/mL kanamycin at 1% inoculum size (v/v), cultured at 37 ℃ at 150rpm until the cell density OD₆₀₀Adding IPTG with final concentration of 0.1mM to 0.6, performing induced culture at 28 deg.C for 12 hr, centrifuging at 8000rpm for 10min at 4 deg.C, and collecting wet thallus cells.

Example 4: separation and purification of enzyme

The wet cells collected in example 3 were suspended in 50mL of Tris-HCl buffer (20mM, pH 8.0), shaken, and then disrupted by ultrasonication (400W, 15min, 1s disruption, 1s pause, effective time 15 min). The disruption solution was centrifuged at 12,000rpm for 10min to remove cell debris, and the supernatant (crude enzyme solution) was collected for subsequent separation and purification. The purification column was Ni-NTA, the column volume was 20mL, the Ni-NTA column was equilibrated with a loading equilibration buffer (20mM Tris-HCl,500mM NaCl and 20mM imidazole, pH 8.5), the crude enzyme solution was loaded at a rate of 1.5mL/min, the crude enzyme solution was eluted with the loading equilibration buffer to remove non-adsorbed proteins, and finally the target protein was collected by elution with an elution buffer (20mM Tris-HCl,500mM NaCl, and 500mM imidazole, pH 8.5). The enzyme solution was dialyzed overnight (2 dialysis solutions were changed) against 20mM Tris-HCl pH 8.5, and the retentate from the last dialysis was taken as the enzyme solution.

Example 5: enzyme activity assay

The reaction system was 10mL, contained Tris-HCl buffer (20mM, pH 8.9), and the enzyme prepared in example 4 was added to a final concentration of 7.8. mu.g/mL, racemic N-acetylglufosinate-ammonium prepared in example 1 was added to a final concentration of 20g/L, and the enzyme activity was measured by reacting at 45 ℃ for 10min under 200rpm conditions in a water bath shaker. After the reaction was completed, 100. mu.L of the reaction mixture was added with 5. mu.L of HCl (4M) to terminate the reaction, and then the conversion and the ee value of the product were analyzed by derivatization. The chromatographic column is

C-18column (150 mm. times.4.6 mm, 5 μm), mobile phase methanol: 0.05mol/L ammonium acetate solution ═ 10: the flow rate of 90(v/v) is 1mL/min, the ultraviolet detection wavelength is 215nm, and the column temperature is 35 ℃. Preparing a derivatization reagent: equal amounts of derivatization reagent OPA and NAC (0.015M) were weighed out separately and mixed, dissolved in absolute ethanol, added with prepared boric acid buffer (20mM pH 9.8) to a constant volume and stored at 4 ℃. And (3) a derivatization reaction system: 200. mu.l of sample solution: (<2.0g/L) and 400 mul of derivatization reagent (excessive derivatization reagent) at normal temperature, and oscillating for 6min, wherein a liquid phase spectrogram of the L-glufosinate-ammonium is shown in figure 4.

Definition of enzyme activity (U): the amount of enzyme required to produce 1. mu. mol L-glufosinate-ammonium per minute at 45 ℃ and pH 8.9. The specific enzyme activity refers to the enzyme activity per milligram of enzyme protein, U/mg. The specific enzyme activity is 23.1U/mg by determination.

Example 6: effect of Metal ions on the Activity of recombinant enzymes

The enzyme collected in example 4 was used as a catalyst in Co²⁺,Cu²⁺,Fe²⁺,Ba²⁺,Mn²⁺,Mg²⁺,Fe³⁺,Ni²⁺,Al²⁺,Zn²⁺And in the presence of EDTA, catalyzing the stereoselective hydrolysis of the substrate N-acetylglufosinate-ammonium to produce L-glufosinate-ammonium. The biocatalysis system comprises the following components and is operated as follows: 10mL of the reaction system, the buffer solution was Tris-HCl (pH 8.9, 50mM), the final concentration of metal ions and EDTA was 1mM, the amount of recombinase added was 8.1. mu.g/mL, and the final concentration of substrate was 20 g/L. The reaction was carried out at 45 ℃ in a water bath shaker at 200rpm, and the enzyme activity was measured by the method of example 5, and the results are shown in Table 1. Enzyme activity determination shows that Co²⁺The addition of (2) has obvious influence on the improvement of enzyme activity, the enzyme activity is improved by 1.57 times, and Zn²⁺The addition of (A) has a remarkable inhibitory effect on the enzyme.

TABLE 1 Effect of Metal ions on recombinant enzymes

Example 7: recombinant escherichia coli catalysis generation L-glufosinate-ammonium containing recombinase

The wet thalli of the recombinant engineering bacteria collected in the example 3 is used as a biocatalyst to catalyze the stereoselective hydrolysis of the substrate N-acetylglufosinate-ammonium to generate L-glufosinate-ammonium. The biocatalysis system comprises the following components and is operated as follows: 2.5g of wet cells were suspended in 250mL of Tris-HCl buffer (pH 8.9, 50mM), N-acetylglufosinate-ammonium was added to the suspension to a final concentration of 100mM, and the reaction was carried out in a shaker at 45 ℃ at 200rpm in a water bath for 30 minutes, after which the conversion was 49% and the ee value was 99% in the liquid phase by the method of example 5.

Example 8: separation of L-glufosinate-ammonium and D-N-acetylglufosinate-ammonium

Pretreatment of hydrogen 001x7 cation resin: (1) washing the column with deionized water at a flow rate of 1.0BV/h for 2 BV; (2) washing the column with 2M sodium hydroxide aqueous solution at a flow rate of 0.5BV/h for 2 BV; (3) washing the column with deionized water at a flow rate of 1.0BV/h for 2 BV; (4) washing the column with 2M hydrochloric acid aqueous solution at a flow rate of 0.5BV/h for 2 BV; (5) the column was washed with deionized water at a flow rate of 1.0BV/h and 2 BV.

The reaction solution in example 7 was centrifuged to remove the cells, the supernatant was adjusted to pH 2 with hydrochloric acid and filtered, the filtrate was loaded onto pretreated hydrogen 001X7 cation resin in a column volume of 120mL at a column height ratio of 15:1 on an ion exchange column at a flow rate of 1.0BV/h, after loading, 4BV was rinsed with ultrapure water, and the effluent containing the unreacted substrate D-N-acetylglufosinate-ammonium was collected. Then eluting with 2mol/L ammonia water at the flow rate of 0.5BV/h, and collecting the eluent containing L-glufosinate-ammonium. And (3) concentrating and crystallizing the eluent under the vacuum degree of 0.075-0.085 MPa at 60 ℃ to obtain the L-glufosinate-ammonium with the purity of 98%.

Example 9: chemical racemization reaction of D-N-acetylglufosinate-ammonium

100mL of the effluent containing the unreacted substrate D-N-acetylglufosinate-ammonium in example 8 was concentrated under reduced pressure to 50mL, the optical rotation was measured to be-0.59, and then, the mixture was evaporated to dryness under pressure to obtain 3g of D-N-acetylglufosinate-ammonium. Adding 30g of acetic acid, mixing, adding 5g of acetic anhydride, stirring and racemizing for 12h at 120 ℃, after the reaction is finished, evaporating the reaction solution under reduced pressure to obtain racemized N-acetylglufosinate-ammonium, adding water to dissolve the racemized N-acetylglufosinate-ammonium to 50mL, measuring the optical rotation to be-0.002, and storing for cyclic resolution.

Comparative example 1: separating and extracting L-glufosinate-ammonium from enzyme conversion liquid

The reaction solution obtained in example 7 was centrifuged to remove the bacterial cells, evaporated to dryness under reduced pressure to remove part of the acetic acid, dissolved in water, adjusted to neutral (pH 8.5) with aqueous sodium hydroxide solution, added with ethanol, and allowed to stand while the temperature was lowered to 4 ℃ to find that no crystal was precipitated. And adding acetone, and detecting a liquid phase to find that the precipitate contains 30-50% of D-N-acetylglufosinate-ammonium and 10-20% of L-glufosinate-ammonium.

Sequence listing

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Claims

1. A chemical-enzymatic method for producing glufosinate-ammonium, which is characterized by comprising the following steps: using racemic N-glufosinate-ammonium as a substrate, using wet thalli obtained by fermenting and culturing engineering bacteria containing carboxypeptidase genes shown in SEQ ID NO.2 or pure enzyme extracted after the wet thalli is subjected to ultrasonic disruption as a catalyst, using a buffer solution with the pH value of 5-10 as a reaction medium, reacting under the condition of 200rpm of a water bath shaker at 45 ℃, obtaining a reaction solution containing D-N-glufosinate-ammonium and L-glufosinate-ammonium after the reaction is complete, separating and purifying the reaction solution, collecting the racemized D-N-glufosinate-ammonium for cyclic resolution, and simultaneously obtaining the L-glufosinate-ammonium; the engineering bacteria host is Escherichia coli E.coli BL21(DE 3).

2. The chemical-enzymatic method for producing glufosinate-ammonium according to claim 1, wherein the final concentration of the substrate is 10-100 mM based on the volume of the buffer solution, and the amount of the catalyst is 5-50 g/L of the buffer solution based on the weight of the wet cells.

3. The chemical-enzymatic method for producing glufosinate-ammonium according to claim 1, characterized in that the racemic N-acetylglufosinate-ammonium is prepared as follows: adding acetic acid into racemic glufosinate-ammonium serving as a raw material, keeping the temperature at 10-60 ℃, stirring, adding acetic anhydride, reacting until the solution is clear and transparent, carrying out rotary evaporation at 80 ℃ and under the pressure of 0.075-0.085 MPa to remove the acetic acid, dissolving a concentrate with water, adjusting the concentrate to be neutral with sodium hydroxide aqueous solution, adding ethanol, standing to separate out crystals, and drying to obtain racemic N-acetylglufosinate-ammonium; the weight ratio of the acetic acid to the racemic glufosinate-ammonium is 20-30: 1, the weight ratio of the acetic anhydride to the racemic glufosinate-ammonium is 0.7-1.5: 1.

4. the chemical-enzymatic process for producing glufosinate-ammonium according to claim 1, characterized in that the catalyst is prepared as follows: (1) wet thalli: inoculating engineering bacteria containing carboxypeptidase gene into LB liquid culture medium containing 50 ug/mL kanamycin, culturing at 37 deg.C overnight, inoculating into LB liquid culture medium containing 50 ug/mL kanamycin at an inoculum size of 1% volume concentration, and culturing at 37 deg.C and 150rpm until the thallus concentration OD₆₀₀To 0.4-0.8, adding final concentrationIPTG with the concentration of 0.1mM, inducing and culturing for 12h at the temperature of 28 ℃, centrifuging for 10min at the temperature of 4 ℃ and the rpm of 8000, and collecting wet thalli; (2) pure enzyme: suspending the wet thalli in the step (1) in a Tris-HCl buffer solution with the concentration of 20mM and the pH value of 8.0, shaking uniformly, then carrying out ultrasonic crushing for 15min, centrifuging the crushed solution at 12,000rpm for 10min to remove cell fragments, and collecting a supernatant; carrying out Ni-NTA column chromatography on the supernatant, balancing the Ni-NTA column by using a loading balance buffer solution, loading the supernatant at the speed of 1.5mL/min, eluting by using the loading balance buffer solution to remove unadsorbed protein, eluting by using an elution buffer solution to collect target protein, dialyzing the target protein in Tris-HCl with the concentration of 20mM and the pH value of 8.5, and taking trapped fluid as pure enzyme; the sample loading balance buffer solution is 20mM Tris-HCl containing 500mM NaCl and 20mM imidazole, and the pH value is 8.5; the elution buffer was 20mM Tris-HCl, pH 8.5, containing 500mM NaCl and 500mM imidazole.

5. The chemical-enzymatic method for producing glufosinate-ammonium according to claim 1, wherein the reaction solution is separated and purified by: after the reaction is completed, centrifuging the reaction solution, taking supernatant to adjust the pH value to 2, carrying out suction filtration, taking a filtrate to load the filtrate to a hydrogen type 001x7 cation resin, wherein the height ratio of an ion exchange column is 15:1, the loading flow rate is 1.0BV/h, washing 4BV with ultrapure water after loading, collecting effluent liquid containing unreacted substrates D-N-acetylglufosinate-ammonium, racemizing the D-N-acetylglufosinate-ammonium for cyclic resolution; eluting with 2mol/L ammonia water at the flow rate of 0.5BV/h, collecting the eluent containing L-glufosinate-ammonium, concentrating the eluent under reduced pressure at 60 ℃ and the vacuum degree of 0.075-0.085 MPa, and crystallizing to obtain the L-glufosinate-ammonium.

6. The chemical-enzymatic method for producing glufosinate-ammonium according to claim 5, characterized in that the racemization method of D-N-acetylglufosinate-ammonium is: evaporating the effluent containing D-N-acetylglufosinate-ammonium to dryness under reduced pressure, adding acetic acid for mixing, adding acetic anhydride, stirring for racemization reaction at the temperature of 100-125 ℃, and evaporating the reaction solution to dryness under reduced pressure after the reaction is finished to obtain racemic N-acetylglufosinate-ammonium for cyclic resolution; the mass ratio of the acetic anhydride to the D-N-glufosinate-ammonium is 0.1-3: 1, the mass ratio of acetic acid to D-N-glufosinate-ammonium is 10-20: 1.