A method for the continuous bioconversion production of L-glufosinate ammonium salt

Abstract

The present application relates to a kind of methods for preparing L-glufosinate ammonium salt by continuous bioconversion.The method comprises: (1) L-amino acid dehydrogenase wet bacteria and coenzyme regeneration enzyme wet bacteria are mixed, are treated by cell breaking, and solid bacterial residue is separated and removed, to obtain the aqueous solution containing L-amino acid dehydrogenase and coenzyme regeneration enzyme;(2) NAD + And raw material for NAD + / NADH cycle, to obtain biological catalyst solution;(3) PPO aqueous solution is reacted with ammonia solution, then the neutralization reaction liquid is reacted with the biological catalyst solution into microchannel reactor, to obtain the conversion liquid containing L-glufosinate ammonium salt.The present application realizes the continuous efficient conversion of PPO by cell breaking and impurity removing pretreatment and microchannel reaction, shortens reaction time, improves the space-time yield and conversion yield of L-glufosinate ammonium salt, and reduces the production of byproduct.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological chemical industry, in particular to a preparation method of L-glufosinate ammonium, and particularly to a method for preparing L-glufosinate ammonium through continuous biological conversion. BACKGROUND

[0002] Glufosinate ammonium, chemical name 2-amino 4-[hydroxy (methyl) phosphinyl] butyric acid, is developed and produced by Hoescht Company (now Bayer Company in Germany). Glufosinate ammonium is a broad-spectrum phosphonic acid herbicide. At present, the three major herbicides in the world are glufosinate ammonium, glyphosate and paraquat. Compared with glyphosate and paraquat, glufosinate ammonium has the characteristics of low toxicity, easy degradation, safety to crops, non-drift, wide herbicidal spectrum, high activity, small dosage, small environmental pressure, safe and convenient use, and is superior to other herbicides. In general, glufosinate ammonium will have a very large market demand in the future, and the application and development prospect is also very large.

[0003] Glufosinate ammonium has two optical isomers, in which the L-form plays a major role, and the D-form only accounts for 1 / 8 of the L-form. L-glufosinate ammonium can be degraded by microorganisms in the soil, showing a very significant environmental advantage. D-glufosinate ammonium is difficult to degrade and easy to cause soil compaction. If glufosinate ammonium is used only in the form of pure optical isomer of L-form, the usage amount will be reduced by 50%, which is of great significance to improve the atom economy, reduce the use cost and alleviate the environmental pressure.

[0004] Glufosinate ammonium salts are agronomically acceptable salts of L-glufosinate, and are commercially preferred due to their high solubility in water. In recent years, numerous methods for preparing L-glufosinate ammonium salts have been reported. In most cases, obtaining the addition salt of L-glufosinate at the end of the reaction requires its conversion to an ammonium or alkali metal salt of L-glufosinate. This can be done in two ways: either by converting the acid addition salt to a free acid, and then to the desired salt; or by converting the acid addition salt to the desired salt. Traditional chemical methods for preparing L-glufosinate or L-glufosinate ammonium salts typically involve multiple steps, complex synthetic routes, low yields, and high production costs. Compared to the disadvantages of chemical methods, such as high energy consumption, heavy pollution, and high costs, biocatalytic methods offer advantages such as mild reaction conditions, high stereoselectivity, high yields, and low pollution, making them an important trend for the industrial-scale preparation of L-glufosinate or L-glufosinate ammonium salts. However, the current biocatalytic synthesis process for L-glufosinate or L-glufosinate ammonium salt uses D,L-glufosinate as a substrate for catalytic conversion. This process involves multiple enzymes to first convert D-glufosinate to 2-oxo-4-(hydroxymethylphosphoryl)butyric acid, and then convert 2-oxo-4-(hydroxymethylphosphoryl)butyric acid to L-glufosinate or L-glufosinate ammonium salt. This method has a complex conversion system, making it difficult to achieve 100% conversion. The enzymes used are also relatively complex, requiring continuous oxygen introduction during the conversion process. The conversion system contains organic matter and produces organic byproducts. Furthermore, since the D,L-glufosinate substrate already contains some difficult-to-separate inorganic salts such as ammonium chloride, the resulting products, whether L-glufosinate ammonium salt technical grade or L-glufosinate parent compound, will contain some inorganic salts or organic matter, reducing product quality, as seen in patents CN117120625A and CN112626142A.

[0005] Another promising industrial-scale method in bioconversion is to directly use 2-oxo-4-(hydroxymethylphosphoryl)butyric acid as a substrate, and through enzymatic catalysis, obtain L-glufosinate with 100% optical purity. The separated product is free of inorganic salts and byproduct organic matter, exhibiting high purity and good product quality. However, wild-type L-glutamate dehydrogenase currently suffers from inactivity or low activity, resulting in low substrate conversion and yield. Furthermore, current bioconversion processes for L-glufosinate primarily rely on batch reactors, leading to low space-time yields. Increasing L-glufosinate production capacity necessitates the construction of numerous large stirred tanks, requiring substantial space. While increasing the size of the stirred tanks can improve L-glufosinate production, a decrease in the yield of refined glufosinate during enzymatic conversion has been observed, primarily due to the properties of 2-oxo-4-(hydroxymethylphosphoryl)butyric acid.

[0006] Currently, the biocatalytic conversion of 2-oxo-4-(hydroxymethylphosphono)butyric acid into L-glufosinate using a batch enzymatic process requires substrate neutralization before biocatalytic conversion, making the process complex and time-consuming. Furthermore, the neutralization process is highly exothermic, posing safety risks and making it difficult to control local hot spots, resulting in numerous byproducts and a low yield of L-glufosinate from 2-oxo-4-(hydroxymethylphosphono)butyric acid. In addition, product purification from the bioconversion system for L-glufosinate synthesis also suffers from low purity and low yield. Summary of the Invention

[0007] The existence of the above problems seriously hinders the industrial production of L-glufosinate-ammonium salt using 2-oxo-4-(hydroxymethylphosphoryl)butyric acid as a substrate.

[0008] The technical problem to be solved by this invention is: how to improve the conversion yield, space-time yield, and recovery rate of L-glufosinate-ammonium salt prepared by biotransformation, and how to improve the purity to obtain high-quality L-glufosinate-ammonium salt.

[0009] To address the problem that the L-glutamate dehydrogenase used in the current biocatalytic method for preparing L-glufosinate has no activity or low activity, resulting in low substrate conversion and yield, this invention provides an L-glutamate dehydrogenase mutant and its application in L-glufosinate biocatalysis.

[0010] Specifically, this application proposes the following technical solutions.

[0011] A type of halophilic archaea ( Halobacterium salinarum The L-glutamate dehydrogenase mutant is a three-point mutant of L-glutamate dehydrogenase. The three-point mutant of L-glutamate dehydrogenase is a combination mutation of three amino acid sites at positions 164, 286, and 365 of the amino acid sequence of L-glutamate dehydrogenase shown in SEQ ID NO.1. In the three-point mutant of L-glutamate dehydrogenase, position 164 of the amino acid sequence of L-glutamate dehydrogenase is mutated from alanine to glycine, position 286 from glutamate to arginine, and position 365 from valine to alanine. The amino acid sequence of the mutant L-glutamate dehydrogenase is shown in SEQ ID NO.2.

[0012] Furthermore, the inventors of this invention discovered through research that in the batch synthesis of L-glufosinate, the conversion system experiences an accumulation of 2-oxo-4-(hydroxymethylphosphoryl)butyric acid, leading to side reactions and a decrease in product yield. To improve the conversion yield of 2-oxo-4-(hydroxymethylphosphoryl)butyric acid, the enzyme-catalyzed conversion process must have an efficient mixing process and conversion rate, and the substrate must be continuously neutralized for better control.

[0013] Microchannel reactors possess excellent mass and heat transfer properties due to their small channel size and large surface area to volume ratio. This helps improve resource and energy utilization efficiency during the reaction process while reducing side reactions. Applying microchannel reactors to the biocatalytic conversion of 2-oxo-4-(hydroxymethylphosphono)butyric acid to synthesize L-glufosinate could potentially increase reaction rate and yield while shortening reaction time, thereby significantly improving the space-time yield of L-glufosinate. This would better facilitate industrial-scale production, reduce equipment investment, and improve operational efficiency. However, current bioconversion methods for glufosinate typically employ wet cell reactors. Since wet cells can cause feeding difficulties and reactor blockage, they are unsuitable for microchannel conversion technology, making it difficult to conduct the entire reaction within microchannels.

[0014] The inventors of this invention also discovered that after the biotransformation of 2-oxo-4-(hydroxymethylphosphoryl)butyric acid using wet bacterial cells, it is difficult to obtain high yield and high purity of glufosinate from the biotransformation solution containing glufosinate. The main reason is that during the biotransformation process, the bacterial cells gradually break down, and some components inside the cells pass through the cell wall and become free outside the cells, which causes certain difficulties for the crystallization process of L-glufosinate.

[0015] To address the shortcomings of existing technologies, one of the objectives of this invention is to provide a continuous biotransformation method for preparing L-glufosinate-ammonium salt.

[0016] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0017] This application provides a method for preparing L-glufosinate-ammonium salt, which includes the following steps:

[0018] (1) Mix L-amino acid dehydrogenase wet cells and coenzyme regeneration enzyme wet cells, and after cell disruption treatment, separate and remove solid bacterial residue to obtain an aqueous solution containing L-amino acid dehydrogenase and coenzyme regeneration enzyme.

[0019] (2) Add NAD to the aqueous solution containing L-amino acid dehydrogenase and coenzyme regeneration enzyme in step (1). + and for NAD +The raw materials from the NADH cycle are used to obtain a biocatalyst solution;

[0020] (3) Neutralize the aqueous solution of 2-oxo-4-(hydroxymethylphosphoryl)butyric acid with the aqueous solution of ammonia to obtain a neutralized reaction solution. Then, continuously pass the neutralized reaction solution and the biocatalyst solution obtained in step (2) into a microchannel reactor to carry out a bioconversion reaction to obtain a conversion solution containing L-glufosinate.

[0021] In some embodiments of this application, step (1) further includes a step of purifying the aqueous solution containing L-amino acid dehydrogenase and coenzyme regenerating enzyme.

[0022] Preferably, the purification method is centrifugation and membrane filtration. Wet cells of L-amino acid dehydrogenase and wet cells of coenzyme regeneration are mixed, subjected to cell disruption, and centrifuged at 4000-5000 rpm for 10-20 minutes to separate and remove solid bacterial residue, yielding an aqueous solution containing L-amino acid dehydrogenase and coenzyme regeneration. This aqueous solution is then concentrated via membrane filtration to obtain a concentrated enzyme protein aqueous solution; the membrane has a molecular weight cutoff of 5000D.

[0023] In some embodiments of this application, the cell disruption treatment method in step (1) is homogenization; the pressure of the homogenization is 600-950 bar.

[0024] In some embodiments of this application, the L-amino acid dehydrogenase in step (1) is an L-glutamate dehydrogenase mutant with the amino acid sequence number SEQ ID NO.2. The L-glutamate dehydrogenase mutant is derived from the wild-type L-glutamate dehydrogenase with the amino acid sequence number SEQ ID NO.1. The coenzyme regenerating enzyme is formate dehydrogenase with the amino acid sequence number SEQ ID NO.3.

[0025] In some embodiments of this application, the amount of wet L-amino acid dehydrogenase cells added in step (1) is 1-20% based on the total weight of the biocatalyst solution, preferably 8-20%, more preferably 8-16%; and / or,

[0026] Based on the total weight of the biocatalyst solution, the amount of wet coenzyme regeneration enzyme cells added in step (1) is 1-10%, preferably 1-4%; and / or,

[0027] Based on the total weight of the biocatalyst solution, the NAD content in step (2) is... + The amount added is 0.04-0.15%, preferably 0.1-0.15%; and / or,

[0028] Based on the total weight of the biocatalyst solution, the amount used for NAD in step (2) +The amount of raw material added to the NADH cycle is 15-60%, preferably 30-50%.

[0029] In some embodiments of this application, the weight ratio of the L-amino acid dehydrogenase wet cells to the coenzyme regeneration enzyme wet cells is 0.25-4:1, preferably 2-4:1.

[0030] In some embodiments of this application, step (2) for NAD + The feedstock for the NADH cycle is glucose or ammonium formate, preferably ammonium formate;

[0031] Preferably, the method for NAD + The molar ratio of the feedstock to 2-oxo-4-(hydroxymethylphosphoryl)butyric acid in the NADH cycle is 0.5-3:1, preferably 1-3:1, and more preferably 1-1.2:1.

[0032] In some embodiments of this application, step (2) of the NAD + The mass ratio of the amount of [the substance] added to 2-oxo-4-(hydroxymethylphosphoryl)butyric acid is 0.0006-0.0015, preferably 0.001-0.0015.

[0033] In some embodiments of this application, the weight ratio of 2-oxo-4-(hydroxymethylphosphoryl)butyric acid and the biocatalyst solution in step (3) is 1-2:1.

[0034] In some embodiments of this application, the neutralization reaction in step (3) is carried out by continuously passing an aqueous solution of 2-oxo-4-(hydroxymethylphosphoryl)butyric acid and an aqueous solution of ammonia into a mixer.

[0035] In some embodiments of this application, in step (3), the molar ratio of NH3 to 2-oxo-4-(hydroxymethylphosphoryl)butyric acid is 1-2:1, preferably 1.5-2:1, and preferably, the temperature of the neutralization reaction is below 60°C, preferably 35-45°C.

[0036] In some embodiments of this application, the temperature of the biotransformation reaction in step (3) is 25°C-40°C;

[0037] Preferably, for every 500g of substrate 2-oxo-4-(hydroxymethylphosphoryl)butyric acid catalyzed biotransformation, the amount of L-amino acid dehydrogenase wet cells added in step (1) is 30-40g, the amount of coenzyme regeneration enzyme wet cells added is 8-10g, and the amount of coenzyme NAD in step (2) is... + The addition amount is 0.2-0.3g, used for NAD. +The raw material for the NADH cycle is ammonium formate, and the amount added is 75-100g; in step (3), the concentration of the aqueous solution of 2-oxo-4-(hydroxymethylphosphoryl)butyric acid is 30-35wt%, and the concentration of the aqueous solution of ammonia is 23-26wt%.

[0038] Preferably, the residence time of the biotransformation reaction in the microchannel reactor is 6 min to 20 min.

[0039] In some embodiments of this application, the method further includes step (4): the conversion liquid containing L-glufosinate obtained in step (3) is filtered through a membrane, concentrated and crystallized to obtain L-glufosinate.

[0040] Preferably, the membrane filtration in step (4) includes first treating the conversion liquid containing L-glufosinate with an organic membrane of 3000D-8000D to obtain a permeate containing L-glufosinate, and then treating the permeate containing L-glufosinate with an organic membrane of 100D-200D to obtain a clear aqueous solution containing L-glufosinate.

[0041] The concentration temperature in step (4) is 70℃-90℃, and the absolute pressure is 5KPa-10KPa;

[0042] And / or, the crystallization operation temperature in step (4) is 10℃-60℃, preferably 30℃-50℃.

[0043] The beneficial effects of this invention are:

[0044] (1) The present invention obtained an enzyme mutant with significantly improved catalytic activity by mutating wild-type L-glutamate dehydrogenase derived from halophilic archaea. Compared with wild-type glutamate dehydrogenase, the catalytic activity of the mutant glutamate dehydrogenase obtained in the present invention for 2-carbonyl-4-(hydroxymethylphosphono)butyric acid is 252 times that of wild-type. Using this mutant as a catalyst significantly improves the substrate conversion rate and L-glufosinate yield of L-glufosinate catalyzed by 2-carbonyl-4-(hydroxymethylphosphono)butyric acid as substrate.

[0045] (2) This invention achieves continuous and efficient microchannel conversion of 2-oxo-4-(hydroxymethylphosphoryl)butyric acid by pre-treating wet bacterial cells with bio-enzymes to break down cells and remove impurities. By enhancing mass transfer in the reactor, the reaction time is shortened, and the space-time yield of L-glufosinate is greatly improved. Its space-time yield is more than 100 times that of L-glufosinate synthesized by batch reaction.

[0046] (3) This invention realizes the continuous neutralization and continuous conversion reaction of 2-oxo-4-(hydroxymethylphosphoryl)butyric acid, reduces the generation of by-products, and improves the conversion yield of L-glufosinate.

[0047] (4) The continuous microchannel synthesis technology of L-glufosinate-ammonium salt implemented in this invention can significantly reduce the production of L-amino acid dehydrogenase, coenzyme regeneration enzyme, and coenzyme NAD. + Reduce the amount of substrate used for coenzyme regeneration and decrease production costs. Attached Figure Description

[0048] Figure 1 This is a schematic diagram of the process flow of the present invention. Detailed Implementation

[0049] To make the objectives, technical solutions, and technical effects of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. The embodiments described below are some embodiments of the present invention, but not all embodiments. All other embodiments obtained by those skilled in the art in conjunction with the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0050] The term "glufosinate-ammonium salt" in this application is also known as L-glufosinate-ammonium salt.

[0051] The term "2-oxo-4-(hydroxymethylphosphoryl)butyric acid" in this application is also called 2-carbonyl-4-(hydroxymethylphosphoryl)butyric acid, or simply "keto acid" or PPO.

[0052] When using 2-oxo-4-(hydroxymethylphosphoryl)butyric acid as a substrate for bioconversion to produce L-glufosinate, the following main problems exist:

[0053] (1) After neutralization of 2-oxo-4-(hydroxymethylphosphoryl)butyric acid, uneven mixing or untimely conversion in the conversion tank can lead to the occurrence of byproducts;

[0054] (2) The bioconversion of L-glufosinate using wet cells in a stirred tank has a long reaction time and a low space-time yield, making it difficult to carry out large-scale production.

[0055] (3) When L-glufosinate is synthesized by batch reaction with wet cells, the conversion rate is slow due to the limited mass transfer capacity of the reaction system and the diffusion penetration capacity of the substrate and product on both sides of the cell wall.

[0056] (4) During the process of synthesizing L-glufosinate by batch reaction with wet bacterial cells, the bacterial cells will gradually break down, and the substances inside the bacterial cells will mix with L-glufosinate in the same system, making it difficult to purify L-glufosinate.

[0057] To address the aforementioned issues, this application provides a method for the continuous and efficient production of L-glufosinate. This method achieves high stereoselectivity and high yield, reduces coenzyme input, and significantly shortens reaction time. It not only improves the space-time yield of L-glufosinate ammonium salt, simplifies the production process, and reduces production costs, but also purifies L-glufosinate ammonium salt in high yield.

[0058] like Figure 1 As shown in one specific embodiment of this application, this application provides a method for preparing L-glufosinate-ammonium salt, which includes the following steps:

[0059] (1) Mix the wet cells of coenzyme regeneration enzyme and the wet cells of L-amino acid dehydrogenase, break the cells, centrifuge to remove solid bacterial residue, and then concentrate through a membrane to obtain an aqueous solution containing L-amino acid dehydrogenase and coenzyme regeneration enzyme.

[0060] (2) Add NAD to the aqueous solution containing L-amino acid dehydrogenase and coenzyme regeneration enzyme in step (1). + and for NAD + The raw materials from the NADH cycle are used to obtain a biocatalyst solution;

[0061] (3) Neutralize the aqueous solution of 2-oxo-4-(hydroxymethylphosphoryl)butyric acid with the aqueous solution of ammonia to obtain a neutralized reaction solution. Then, the neutralized reaction solution and the biocatalyst solution obtained in step (2) are simultaneously and continuously introduced into a microchannel reactor to carry out a bioconversion reaction to obtain a conversion solution containing L-glufosinate.

[0062] It should be noted that the wet bacterial cells in this application are the precipitate obtained after solid-liquid separation of the culture medium of microbial cells (e.g., centrifugation followed by discarding the supernatant and collecting the precipitate). L-amino acid dehydrogenase cells are bacterial cells containing the L-amino acid dehydrogenase gene, and may be, for example, recombinant engineered bacteria. Coenzyme regeneration enzyme cells are bacterial cells containing the coenzyme regeneration enzyme gene, and may be recombinant engineered bacteria. The coenzyme regeneration enzyme can perform the main reaction through its activity of regenerating coenzyme NADH; the coenzyme regeneration enzyme can be formate dehydrogenase, glucose dehydrogenase, alcohol dehydrogenase, etc.

[0063] In some embodiments of this application, step (1) further includes a step of concentrating and purifying the aqueous solution containing L-amino acid dehydrogenase and coenzyme regeneration enzyme. Preferably, the concentration and purification method is membrane filtration, preferably organic membrane filtration; more preferably, the molecular weight cutoff of the organic membrane is 5000D.

[0064] It should be noted that the unit of molecular weight cutoff in this application is Dalton (D).

[0065] In some embodiments of this application, the coenzyme regeneration enzyme wet cells in step (1) are formate dehydrogenase wet cells.

[0066] In some embodiments of this application, the amount of wet L-amino acid dehydrogenase cells added in step (1) is 1-20%, preferably 8-20%, and more preferably 8-16%, based on the total weight of the biocatalyst solution obtained in step (2).

[0067] In some embodiments of this application, the amount of coenzyme regeneration enzyme wet cells added in step (1) is 1-10%, preferably 1-4%, based on the total weight of the biocatalyst solution obtained in step (2).

[0068] In some embodiments of this application, based on the total weight of the biocatalyst solution obtained in step (2), the NAD in step (2) + The amount added is 0.06-0.15%, preferably 0.1-0.15%.

[0069] In some embodiments of this application, the total weight of the biocatalyst solution obtained in step (2) is used for NAD in step (2). + The amount of raw material added to the NADH cycle is 15-60%, preferably 30-50%.

[0070] In this application, NAD+ refers to oxidized NAD (nicotinamide adenine dinucleotide), and NADP refers to reduced NAD (nicotinamide adenine dinucleotide).

[0071] In some embodiments of this application, the weight ratio of the L-amino acid dehydrogenase wet cells to the coenzyme regeneration enzyme wet cells is 0.2-4:1, preferably 2-4:1. L-amino acid dehydrogenase and coenzyme regeneration enzyme work in coordination; an excess of one enzyme inevitably leads to a deficiency of the other. Consequently, the reaction catalyzed by the less abundant enzyme becomes the rate-controlling step. Therefore, by adjusting the weight ratio of coenzyme regeneration enzyme to L-amino acid dehydrogenase wet cells, the conversion efficiency of keto acids can be improved.

[0072] In some embodiments of this application, wet cells of L-amino acid dehydrogenase and wet cells of coenzyme regeneration enzyme can be prepared by the following method: a recombinant strain containing the gene encoding the target enzyme (L-amino acid dehydrogenase or formate dehydrogenase) is inoculated into LB liquid medium containing kanamycin sulfate at a final concentration of 10-100 μg / mL and cultured at 35-40℃ for 10-20 h. Then, it is inoculated into fresh LB liquid medium containing 10-100 μg / mL kanamycin at a volume concentration of 1-3% and cultured at 35-40℃ and 150-200 rpm for 1-5 h. Next, IPTG (isopropyl-β-D-thiogalactoside) at a final concentration of 0.05-0.2 mM is added to the culture medium for induction, and the culture is carried out at 20-30℃ and 150-200 rpm for 10-20 h. After centrifugation, the collected precipitate is the wet cell containing the target enzyme. The LB liquid medium consists of 8-12 g / L peptone, 9-11 g / L sodium chloride, 4-6 g / L yeast extract, and the remainder is water.

[0073] In some embodiments of this application, step (2) for NAD + The feedstock for the NADH cycle is glucose or ammonium formate, preferably ammonium formate. In some embodiments, the feedstock for NAD... + The molar ratio of the feedstock to 2-oxo-4-(hydroxymethylphosphoryl)butyric acid in the NADH cycle is 0.5-3:1, preferably 1-3:1, and more preferably 1-1.2:1.

[0074] In some embodiments of this application, the cell disruption treatment method in step (1) is homogenization treatment. Preferably, the pressure of the homogenization treatment is 300 bar to 950 bar, more preferably 600 bar to 950 bar, and even more preferably 800 to 950 bar.

[0075] In some embodiments of this application, step (2) of the NAD + The mass ratio of the amount of [the substance] to 2-oxo-4-(hydroxymethylphosphoryl)butyric acid is 0.0006-0.0015, preferably 0.001-0.0015, and more preferably 0.0012-0.0015.

[0076] In some embodiments of this application, in step (3), the molar ratio of NH3 to 2-oxo-4-(hydroxymethylphosphoryl)butyric acid is 1:1-2:1, preferably 1.5-2:1; preferably, the temperature of the neutralization reaction is below 60°C, preferably 40-60°C, and more preferably 55-60°C.

[0077] In some embodiments of this application, the neutralization reaction in step (3) is carried out using a continuous neutralization reaction process, which includes continuously passing an aqueous solution of 2-oxo-4-(hydroxymethylphosphoryl)butyric acid and an aqueous solution of ammonia into a mixer to carry out the neutralization reaction; preferably, the product of the continuous neutralization reaction is simultaneously and continuously passed into a microchannel reactor with the aqueous solution of the biological enzyme obtained in step (2) to carry out the biotransformation reaction, thereby obtaining a conversion solution containing L-glufosinate.

[0078] In some embodiments of this application, the temperature of the biotransformation reaction in step (3) is 20°C-40°C; preferably, the residence time of the biotransformation reaction in the microchannel reactor is 6 min-20 min.

[0079] In some embodiments of this application, the above method further includes step (4): the bioconversion liquid containing L-glufosinate obtained in step (3) is filtered through a membrane, concentrated and crystallized to obtain L-glufosinate.

[0080] In some embodiments of this application, the membrane filtration in step (4) involves first treating the solution with a 3000D-8000D organic membrane to obtain a permeate containing L-glufosinate, and then treating it with a 100D-200D organic membrane to obtain a clear aqueous solution containing L-glufosinate. This application uses a specific type of membrane to treat the conversion solution, which can remove some impurities and improve the crystallization purity and yield of L-glufosinate.

[0081] In some embodiments of this application, the concentration temperature in step (4) is 70℃-90℃ and the absolute pressure is 5KPa-10KPa; preferably, the crystallization operation temperature is 10℃-60℃, and more preferably 30℃-50℃.

[0082] In some embodiments of this application, the crystallization operation described herein is as follows: the concentrated bioconversion solution is added to a methanol solvent, then cooled to the crystallization temperature and kept at that temperature for 5-20 hours.

[0083] The method for continuous biotransformation preparation of L-glufosinate ammonium salt according to the present invention will be specifically described below through specific embodiments. In the embodiments, all original reagent materials are commercially available, and experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0084] The present invention will now be described in more detail with reference to examples and comparative examples, but the scope of the present invention is not limited to these examples. It should be noted that, unless otherwise specified, all percentages, parts, and ratios used in the present invention are based on mass.

[0085] The sources of the reagents and instruments used in the following examples and comparative examples are shown in Table 1.

[0086] Table 1. Raw material information used in the embodiments.

[0087]

[0088] The model numbers of the components of the microchannel reaction device are as follows:

[0089] The micro mixer model is Cascade Mixer 06, purchased from EhrfeldMikrotechnik GmbH.

[0090] The microreactor model is: Miprowa ® Lab Reactor (ca. 30mL, 45° / strut 1.0mm / spacing 2.0mm) HC, purchased from EhrfeldMikrotechnik GmbH.

[0091] The three-point mutant of L-glutamate dehydrogenase is a combination mutation of three amino acid sites at positions 164, 286, and 365 of the amino acid sequence of L-glutamate dehydrogenase shown in SEQ ID NO.1. In the three-point mutant of L-glutamate dehydrogenase, position 164 of the amino acid sequence of L-glutamate dehydrogenase is mutated from alanine to glycine, position 286 is mutated from glutamate to arginine, and position 365 is mutated from valine to alanine.

[0092] The L-amino acid dehydrogenase wet cells and formic acid dehydrogenase wet cells used in the examples and comparative examples were prepared according to the following method:

[0093] The encoding genes of wild-type L-glutamate dehydrogenase and L-glutamate dehydrogenase mutant, with amino acid sequences as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively, were sent to a gene synthesis company for whole-gene synthesis and cloned into the pET28a vector. The recombinant vector containing the target gene was expressed in recombinant strain E. coli BL21(DE3) (purchased from Novagen). After verification by PCR and sequencing, positive colonies were selected, cultured, and induced to prepare wet cells expressing wild-type L-glutamate dehydrogenase with amino acid sequences as shown in SEQ ID NO.1 and L-glutamate dehydrogenase mutant as shown in SEQ ID NO.2.

[0094] The encoding gene of formate dehydrogenase, with an amino acid sequence as shown in SEQ ID NO.3, was sent to a gene synthesis company for full gene synthesis and cloned into the pET28a vector. The recombinant vector containing the target gene was expressed in the recombinant strain E. coli BL21(DE3). After verification by PCR and sequencing, positive colonies were selected, cultured, and induced to prepare wet cells expressing formate dehydrogenase with an amino acid sequence as shown in SEQ ID NO.3.

[0095] The various wet bacterial cells used in this invention can be prepared according to the following general preparation method: Recombinant strain E. coli BL21(DE3) glycerol bacteria containing the gene encoding the target enzyme (L-amino acid dehydrogenase or formate dehydrogenase) are inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin sulfate and cultured at 37°C for 15 h. Then, 1% (v / v) is inoculated into fresh LB liquid medium containing 50 μg / mL kanamycin sulfate and cultured at 37°C and 180 rpm for 3 h. Next, IPTG is added to the culture medium to induce incubation, and the culture is carried out at 25°C and 180 rpm for 16 h. The medium is then centrifuged at 4000 rpm for 15 min, and the collected precipitate is the wet bacterial cell containing the target enzyme. The LB liquid medium formulation consists of 10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast extract, and the remainder water.

[0096] L-glutamate dehydrogenase mutant is derived from halophilic archaea ( Halobacterium salinarum The amino acid sequence of the mutant wild-type glutamate dehydrogenase is shown in GenBank accession number AY840088.1. The amino acid sequence of the wild-type glutamate dehydrogenase derived from halophilic archaea is shown in SEQ ID NO.1, and the amino acid sequence of the mutant wild-type glutamate dehydrogenase is shown in SEQ ID NO.2.

[0097] The amino acid sequence of wild-type L-glutamate dehydrogenase, SEQ ID NO.1:

[0098] 1 MTMASKSDSTHDESGDEAADSTEPESALETARRQLYHAASYLDIDQNIVERLKYPKKVHE

[0099] 61 VTIPIERDDGTVEVFTGYRAQHDSVRGPYKGGLRYHPDVTDRDECVGLGMWMTWKCAVMDL

[0100] 121 PFGGAKGGVAVNPKELSPEEKERLTRRFTQEIRDVIGPNQDIPAPDMGTDPQTMAWLMDA

[0101] 181 YSMQEGETTPGVVTGKPPVVGGSEGREEAPGRSVAIITQLVCEYYDQPLDETTVAVQGYG

[0102] 241 SVGANAARLLDKWGATIVAISDVNGAMYEPDGIDTASVPSHDEEPEAVTTYADTVISNEE

[0103] 301 LLTLDVDVLIPAALGNVITKENAEAIAADLVVEGANGPTTSTADSILADRDVAVIPDILA

[0104] 361 NAGGVTVSYFEWLQDINRRAWSLERVNDELEAEMQAAWRAVKDEYENRDVTWRDAAYIVA

[0105] 421 LSRIAEAHEARGLWP。

[0106] Amino acid sequence of the L-glutamate dehydrogenase mutant SEQ ID NO.2:

[0107] 1 MTMASKSDST HDESGDEAAD STEPESALET ARRQLYHAAS YLDIDQNIVE RLKYPKKVHE

[0108] 61 VTIPIERDDG TVEVFTGYRA QHDSVRGPYK GGLRYHPDVT RDECVGLGMW MTWKCAVMDL

[0109] 121 PFGGAKGGVA VNPKELSPEE KERLTRRFTQ EIRDVIGPNQ DIPGPDMGTD PQTMAWLMDA

[0110] 181 YSMQEGETTP GVVTGKPPVV GGSEGREEAP GRSVAIITQL VCEYYDQPLD ETTVAVQGYG

[0111] 241 SVGANAARLL DKWGATIVAI SDVNGAMYEP DGIDTASVPS HDEEPRAVTT YADTVISNEE

[0112] 301 LLTLDVDVLI PAALGNVITK ENAEAIAADL VVEGANGPTT STADSILADR DVAVIPDILA

[0113] 361 NAGGATVSYF EWLQDINRRA WSLERVNDEL EAEMQAAWRA VKDEYENRDV TWRDAAYIVA

[0114] 421 LSRIAEAHEA RGLWP.

[0115] The amino acid sequence of formate dehydrogenase is shown in SEQ ID NO.3 below. The amino acid sequence of formate dehydrogenase is accessed in GenBank under accession number ACF35003.1.

[0116] The amino acid sequence of formate dehydrogenase is SEQ ID NO.3:

[0117] 1 MATVLCVLYP DPVDGYPPHY VRDTIPVITR YADGQTAPTP AGPPGFRPGE LVGSVSGALG

[0118] 61 LRGYLEAHGH TLIVTSDKDG PDSEFERRLP DADVVISQPF WPAYLTAERI ARAPKLRLAL

[0119] 121 TAGIGSDHVD LDAAARAHIT VAEVTGSNSI SVAEHVVMTT LALVRNYLPS HAIAQQGGWN

[0120] 181 IADCVSRSYD VEGMHFGTVG AGRIGLAVLR RLKPFGLHLH YTQRHRLDAA IEQELGLTYH

[0121] 241 ADPASLAAAV DIVNLQIPLY PSTEHLFDAA MIARMKRGAY LINTARAKLV DRDAVVRAVT

[0122] 301 SGHLAGYGGD VWFPQPAPAD HPWRAMPFNG MTPHISGTSL SAQARYAAGT LEILQCWFDG

[0123] 361 RPIRNEYLIV DGGTLAGTGA QSYRLT.

[0124] The 2-oxo-4-(hydroxymethylphosphoryl)butyric acid used in the examples and comparative examples was prepared according to the following method:

[0125] (1) Take 208g of diethyl methylphosphonic acid and place it in a 500ml three-necked flask. Then add 72g of ethyl acrylate to the three-necked flask dropwise for 2 hours. Stir mechanically at 300rpm. During the dropwise addition, control the temperature of the liquid in the three-necked flask to 0℃. After the dropwise addition is complete, raise the temperature to 10℃ and keep it warm for 2 hours. The reaction is then complete.

[0126] (2) Take 68g sodium ethoxide and 146g diethyl oxalate and place them in a 1000ml three-necked flask. Mix them by mechanical stirring. Then add the product liquid obtained in step (1) dropwise. The dropwise addition time is 1h. During the dropwise addition, control the liquid temperature at about 5℃. After the dropwise addition is completed, raise the temperature to 20℃ and keep it warm for 2h. The reaction is complete.

[0127] (3) Transfer the product liquid obtained in step (2) to a 10000ml three-necked flask, add 7410g of water and 1mol of HCl, then heat to 90℃ and hydrolyze for 20h until the reaction ends.

[0128] (4) The product liquid obtained in step (3) was evaporated by a rotary evaporator at 90°C and 2 kPa absolute pressure to remove water. Then, 2500 ml of acetone was added to dissolve it, and the solution was filtered to remove salt. Then, 10000 ml of methyl isobutyl ketone was added to the filtrate, and the mixture was stirred to crystallize for 48 h. After filtration and drying, 2-oxo-4-(hydroxymethylphosphoryl)butyric acid solid powder was obtained.

[0129] The calculation methods or definitions for the yield of PPO to synthesize glufosinate-ammonium salt, the space-time yield of glufosinate-ammonium salt, and the crystallization yield in the examples and comparative examples are as follows:

[0130] Space-time yield of glufosinate-ammonium salt: the mass of glufosinate-ammonium salt generated per unit time and unit volume.

[0131]

[0132] The mass fraction of glufosinate in the samples prepared in the examples and comparative examples was determined according to the method specified in GB / T 43172-2023 glufosinate, and then the molar number of glufosinate was calculated.

[0133] Example 1 Enzyme activity assay of wild-type L-glutamate dehydrogenase and its mutants

[0134] The encoding genes of wild-type glutamate dehydrogenase and glutamate dehydrogenase mutant, as shown in SEQ ID NO.1 and SEQ ID NO.2 respectively, were sent to a gene synthesis company for whole-gene synthesis and cloned into the commercial vector pET28a provided by the gene synthesis company. Then, the recombinant vector containing the target gene was transformed into Escherichia coli BL21(DE3). After the gene sequencing company confirmed that the gene was correct, positive colonies were selected, cultured, and induced to prepare wet cells expressing wild-type glutamate dehydrogenase and glutamate dehydrogenase mutant.

[0135] The encoding gene of formate dehydrogenase with the amino acid sequence SEQ ID NO.3 was sent to a gene synthesis company for whole-gene synthesis and cloned into a commercial vector provided by the gene synthesis company. Then, the recombinant vector containing the target gene was transformed into Escherichia coli BL21(DE3). After the gene sequencing company verified that the gene was correct, positive colonies were selected, cultured, and induced to prepare wet cells expressing formate dehydrogenase containing SEQ ID NO.3.

[0136] The collected wet bacterial cells were diluted with water to prepare a bacterial suspension containing 40 g / L glutamate dehydrogenase wet bacterial cells and 10 g / L formate dehydrogenase wet bacterial cells. After cooling, the cells were ruptured. The crude enzyme solution prepared in this way was used as a catalyst. 30 g / L 2-carbonyl-4-(hydroxymethylphosphono)butyric acid was used as the substrate, 0.2 g / L NAD was used as the coenzyme, and 15 g / L ammonium formate was used as the amino donor. The reaction medium was an ammonium formate-ammonia solution with pH 7.5. The reaction was carried out at 35℃ and 180 rpm for 10 min. After the reaction was terminated, the amount of L-glufosinate produced was analyzed by high-performance liquid chromatography (HPLC).

[0137] Determination of L-glufosinate formation (strong anion exchange column) (non-derivative detection method): Accurately weigh 0.1 g (accurate to 0.0001 g) of the reaction product and place it in a 50 mL volumetric flask. Add 40 mL of mobile phase, sonicate for 5 min, cool to room temperature, dilute to the mark with the mobile phase, mix well, and filter. The mobile phase is obtained by dissolving 5.8 g of potassium dihydrogen phosphate in 850 mL of water, adding 150 mL of acetonitrile, sonicating for 20 min, filtering through a filter membrane, and degassing at a flow rate of 1.0 mL / min. The column temperature is 30 °C, the detection wavelength is 195 nm, the injection volume is 20 μL, and the retention time is 10 min.

[0138] Enzyme activity assays showed that the concentration of L-glufosinate in the product of the wild-type L-glutamate dehydrogenase conversion system was 0.10 g / L, while the concentration of L-glufosinate in the product of the L-glutamate dehydrogenase mutant conversion system was 25.2 g / L. The mutant enzyme activity was 252 times that of the wild-type enzyme activity. This indicates that modifying and mutating the wild-type glutamate dehydrogenase shown in SEQ ID NO.1 can significantly improve its catalytic activity for 2-carbonyl-4-(hydroxymethylphosphono)butyric acid, effectively solving the problems of inactivity or low activity exhibited by the wild-type glutamate dehydrogenase in the biocatalytic conversion of 2-carbonyl-4-(hydroxymethylphosphono)butyric acid as a substrate.

[0139] Example 2

[0140] (1) Cell disruption of wet cells: Take 40g of wild-type L-glutamate dehydrogenase wet cells and 10g of formate dehydrogenase wet cells, add 300g of water to dilute and stir, and then use a homogenizer to disrupt the cells of the mixed cell solution at an operating pressure of 950 bar.

[0141] (2) Removal of impurities from homogenized bacterial solution: The aqueous solution of ruptured bacterial cells obtained in step (1) is centrifuged at 4000 rpm for 15 min to remove bacterial residue. Then, 1000 g of water is added to the supernatant obtained by centrifugation, and the solution is filtered through a 5000D organic membrane. The retentate is the concentrated solution containing biological enzymes. Take 250 g of water and 0.3 g of NAD. + Add 100g of ammonium formate to the concentrated biological enzyme solution to obtain a homogeneous aqueous solution containing biological enzyme, coenzyme, and substrate for coenzyme regeneration. Finally, add water to adjust the total mass to 500g to obtain a biocatalyst solution.

[0142] (3) Conditions for continuous neutralization and continuous bioconversion of PPO: Take 500g of 100% pure PPO solid powder, prepare a 35wt% PPO aqueous solution in advance, use 26wt% ammonia water as the neutralization alkali raw material, and simultaneously pass the PPO aqueous solution and the 26wt% ammonia water solution into the micro mixer. The feed flow rate of the PPO aqueous solution is 4.2g / min, the feed flow rate of the ammonia water is 0.83g / min, the neutralization temperature in the micro mixer is controlled at 40℃, the molar ratio of NH3 to PPO is 1.5:1, and then the biocatalyst solution obtained in step (2) is simultaneously passed into the microchannel reactor at a rate of 1.47g / min along with the substrate solution from the micro mixer. The residence time in the microchannel reactor is 10min, and the reaction temperature of the microchannel reactor is controlled at 35℃.

[0143] (4) Collect the conversion liquid from the outlet of the microchannel reactor in step (3) and analyze the amount of L-glufosinate produced by HPLC.

[0144] Following the above process steps, the yield of PPO converted to phosphonium sulfate was 0.38%, and the space-time yield of phosphonium sulfate was 0.00567 kg. 精草铵膦 / (L·H)

[0145] Example 3

[0146] (1) Cell disruption of wet cells: Take 40g of L-glutamate dehydrogenase mutant wet cells and 10g of formic acid dehydrogenase wet cells, add 300g of water to dilute and stir, and then use a homogenizer to disrupt the cells of the mixed cell solution at an operating pressure of 950 bar.

[0147] (2) Removal of impurities from homogenized bacterial solution: The aqueous solution of ruptured bacterial cells obtained in step (1) is centrifuged at 4000 rpm for 15 min to remove bacterial residue. Then, 1000 g of water is added to the supernatant obtained by centrifugation, and the solution is filtered through a 5000D organic membrane. The retentate is the concentrated solution containing biological enzymes. Take 250 g of water and 0.3 g of NAD. + Add 100g of ammonium formate to the concentrated biological enzyme solution to obtain a homogeneous aqueous solution containing biological enzyme, coenzyme, and substrate for coenzyme regeneration. Finally, add water to adjust the total mass to 500g to obtain a biocatalyst solution.

[0148] (3) Conditions for continuous neutralization and continuous bioconversion of PPO: Take 500g of 100% pure PPO solid powder, prepare a 35wt% PPO aqueous solution in advance, use 26wt% ammonia water as the neutralization alkali raw material, and simultaneously pass the PPO aqueous solution and the 26wt% ammonia water solution into the micro mixer. The feed flow rate of the PPO aqueous solution is 4.2g / min, the feed flow rate of the ammonia water is 0.83g / min, the neutralization temperature in the micro mixer is controlled at 40℃, the molar ratio of NH3 to PPO is 1.5:1, and then the biocatalyst solution obtained in step (2) is simultaneously passed into the microchannel reactor at a rate of 1.47g / min along with the substrate solution from the micro mixer. The residence time in the microchannel reactor is 10min, and the reaction temperature of the microchannel reactor is controlled at 35℃.

[0149] (4) The conversion liquid at the outlet of the microchannel reactor in step (3) is collected and filtered with a 3000D organic membrane. The permeate is then filtered with a 150D organic membrane. The retentate is an aqueous solution containing glufosinate-ammonium salt. After dehydration at 80°C and 5KPa absolute pressure, 1000g of methanol is added. The crystallization temperature is controlled at 35°C. After 10h, the solution is filtered and dried to obtain glufosinate-ammonium salt.

[0150] Following the above process steps, the yield of PPO converted to phosphonium sulfate was 99.5%, and the space-time yield of phosphonium sulfate was 1.484 kg.精草铵膦 / (L·H), the crystal purity of glufosinate-ammonium salt is 99.2%, and the crystallization yield is approximately 98.0%.

[0151] Example 4

[0152] Steps (1)-(2) are the same as steps (1)-(2) in Example 3;

[0153] Step (3) Conditions for continuous neutralization and continuous bioconversion of PPO: Take 500g of 100% pure PPO solid powder, prepare a 35wt% PPO aqueous solution in advance, use 26wt% ammonia water as the neutralizing alkali raw material, and simultaneously pass the PPO aqueous solution and the 26wt% ammonia water solution into the micro mixer. The feed flow rate of the PPO aqueous solution is 4.39g / min, the feed flow rate of the ammonia water is 0.58g / min, the neutralization temperature in the micro mixer is controlled at 40℃, the molar ratio of NH3 to PPO is 1:1, and then the biocatalyst solution obtained in step (2) is simultaneously passed into the microchannel reactor at a rate of 1.53g / min along with the substrate solution from the micro mixer. The residence time in the microchannel reactor is 10min, and the reaction temperature of the microchannel reactor is controlled at 35℃.

[0154] Step (4) is the same as step (4) in Example 3.

[0155] Following the above process steps, the yield of PPO converted to phosphonium sulfate was 90.1%, and the space-time yield of phosphonium sulfate was 1.344 kg. 精草铵膦 The crystallization purity of glufosinate-ammonium salt was 92.8% (L·H), and the crystallization yield was approximately 87.0%.

[0156] Example 5

[0157] Steps (1)-(2) are the same as steps (1)-(2) in Example 3;

[0158] Step (3) Conditions for continuous neutralization and continuous bioconversion of PPO: Take 500g of 100% pure PPO solid powder, prepare a 35wt% PPO aqueous solution in advance, use 26wt% ammonia water as the neutralization alkali raw material, and simultaneously pass the PPO aqueous solution and the 26wt% ammonia aqueous solution into the micro mixer. The feed flow rate of the PPO aqueous solution is 4.03g / min, the feed flow rate of the ammonia water is 1.06g / min, the neutralization temperature in the micro mixer is controlled at 40℃, the molar ratio of NH3 to PPO is 2:1, and then the biocatalyst solution obtained in step (2) is simultaneously passed into the microchannel reactor at a rate of 1.41g / min along with the substrate solution from the micro mixer. The residence time in the microchannel reactor is 10min, and the reaction temperature of the microchannel reactor is controlled at 35℃.

[0159] Step (4) is the same as step (4) in Example 3.

[0160] Following the above process steps, the yield of keto acid to phosphonium sulfate was 94.0%, and the space-time yield of phosphonium sulfate was 1.402 kg. 精草铵膦 / (L·H), the crystal purity of glufosinate-ammonium salt is 98.5%, and the crystallization yield is approximately 90.8%.

[0161] Example 6

[0162] Steps (1)-(2) are the same as steps (1)-(2) in Example 3;

[0163] Step (3) Conditions for continuous neutralization and continuous bioconversion of PPO: Take 500g of 100% pure PPO solid powder, prepare a 35wt% PPO aqueous solution in advance, use 26wt% ammonia water as the neutralization alkali raw material, and simultaneously pass the PPO aqueous solution and the 26wt% ammonia water solution into the micro mixer. The feed flow rate of the PPO aqueous solution is 4.2g / min, the feed flow rate of the ammonia water is 0.83g / min, the neutralization temperature in the micro mixer is controlled at 45℃, the molar ratio of NH3 to PPO is 1.5:1, and then the biocatalyst solution obtained in step (2) is simultaneously passed into the microchannel reactor at a rate of 1.47g / min along with the substrate solution from the micro mixer. The residence time in the microchannel reactor is 10min, and the reaction temperature of the microchannel reactor is controlled at 35℃.

[0164] Step (4) is the same as step (4) in Example 3.

[0165] Following the above process steps, the yield of PPO converted to phosphonium sulfate was 93.0%, and the space-time yield of phosphonium sulfate was 1.387 kg. 精草铵膦 The crystallization purity of glufosinate-ammonium salt was 97.8% (L·H), and the crystallization yield was approximately 90.2%.

[0166] Example 7

[0167] Steps (1)-(2) are the same as steps (1)-(2) in Example 3.

[0168] Step (3) Conditions for continuous neutralization and continuous bioconversion of PPO: Take 500g of 100% pure PPO solid powder, prepare a 35wt% PPO aqueous solution in advance, use 26wt% ammonia water as the neutralization alkali raw material, and simultaneously pass the PPO aqueous solution and the 26wt% ammonia water solution into the micro mixer. The feed flow rate of the PPO aqueous solution is 4.2g / min, the feed flow rate of the ammonia water is 0.83g / min, the neutralization temperature in the micro mixer is controlled at 35℃, the molar ratio of NH3 to PPO is 1.5:1, and then the biocatalyst solution obtained in step (2) is simultaneously passed into the microchannel reactor at a rate of 1.47g / min along with the substrate solution from the micro mixer. The residence time in the microchannel reactor is 10min, and the reaction temperature of the microchannel reactor is controlled at 35℃.

[0169] Step (4) is the same as step (4) in Example 3.

[0170] Following the above process steps, the yield of PPO converted to phosphonium sulfate was 93.5%, and the space-time yield of phosphonium sulfate was 1.395 kg. 精草铵膦 / (L·H), the crystal purity of glufosinate-ammonium salt is 98.2%, and the crystallization yield is approximately 90.5%.

[0171] Example 8

[0172] Steps (1)-(2) are the same as steps (1)-(2) in Example 3.

[0173] Step (3) Conditions for continuous neutralization and continuous bioconversion of PPO: Take 500g of 100% pure PPO solid powder, prepare a 35wt% PPO aqueous solution in advance, use 26wt% ammonia water as the neutralizing alkali raw material, and simultaneously pass the PPO aqueous solution and the 26wt% ammonia water solution into the micro mixer. The feed flow rate of the PPO aqueous solution is 4.2g / min, the feed flow rate of the ammonia water is 0.83g / min, the neutralization temperature in the micro mixer is controlled at 40℃, the molar ratio of NH3 to PPO is 1.5:1, and pass the biocatalyst solution obtained in step (2) into the microchannel reactor at 1.47g / min along with the substrate solution from the micro mixer. The residence time in the microchannel reactor is 10min, and the reaction temperature of the microchannel reactor is controlled at 30℃.

[0174] Step (4) is the same as step (4) in Example 3;

[0175] Following the above process steps, the yield of PPO converted to phosphonium sulfate was 94.1%, and the space-time yield of phosphonium sulfate was 1.403 kg. 精草铵膦The crystallization purity of glufosinate-ammonium salt was 96.9% (L·H), and the crystallization yield was approximately 91.2%.

[0176] Example 9

[0177] Steps (1)-(2) are the same as steps (1)-(2) in Example 3.

[0178] Step (3) Conditions for continuous neutralization and continuous bioconversion of PPO: Take 500g of 100% pure PPO solid powder, prepare a 35wt% PPO aqueous solution in advance, use 26wt% ammonia water as the neutralization alkali raw material, and simultaneously pass the PPO aqueous solution and the 26wt% ammonia aqueous solution into the micro mixer. The feed flow rate of the PPO aqueous solution is 4.2g / min, the feed flow rate of the ammonia water is 0.83g / min, the neutralization temperature in the micro mixer is controlled at 40℃, the molar ratio of NH3 to PPO is 1.5:1, and the biocatalyst solution obtained in step (2) is simultaneously passed into the microchannel reactor at a rate of 1.47g / min along with the substrate solution from the micro mixer. The residence time in the microchannel reactor is 10min, and the reaction temperature of the microchannel reactor is controlled at 25℃.

[0179] Step (4) is the same as step (4) in Example 3;

[0180] Following the above process steps, the yield of PPO converted to phosphonium sulfate was 91.7%, and the space-time yield of phosphonium sulfate was 1.368 kg. 精草铵膦 The crystallization purity of glufosinate-ammonium salt was 94.3% (L·H), and the crystallization yield was approximately 87.5%.

[0181] Example 10

[0182] Steps (1)-(2) are the same as steps (1)-(2) in Example 3.

[0183] Step (3) Conditions for continuous neutralization and continuous bioconversion of PPO: Take 500g of 100% pure PPO solid powder, prepare a 35wt% PPO aqueous solution in advance, use 26wt% ammonia water as the neutralization alkali raw material, and simultaneously pass the PPO aqueous solution and the 26wt% ammonia water solution into the micro mixer. The feed flow rate of the PPO aqueous solution is 4.2g / min, the feed flow rate of the ammonia water is 0.83g / min, the neutralization temperature in the micro mixer is controlled at 40℃, the molar ratio of NH3 to PPO is 1.5:1, and pass the biocatalyst solution obtained in step (2) into the microchannel reactor at 1.47g / min along with the substrate solution from the micro mixer. The residence time in the microchannel reactor is 10min, and the reaction temperature of the microchannel reactor is controlled at 40℃.

[0184] Step (4) is the same as step (4) in Example 3;

[0185] Following the above process steps, the yield of PPO converted to phosphonium sulfate was 92.0%, and the space-time yield of phosphonium sulfate was 1.373 kg. 精草铵膦 The crystallization purity of glufosinate-ammonium salt was 94.5% (L·H), and the crystallization yield was approximately 89.5%.

[0186] Example 11

[0187] Steps (1)-(2) are the same as steps (1)-(2) in Example 3.

[0188] Step (3) Conditions for continuous neutralization and continuous bioconversion of PPO: Take 500g of 100% pure PPO solid powder, prepare a 35% PPO aqueous solution in advance, use 26% ammonia water as the neutralization alkali raw material, and simultaneously pass the PPO aqueous solution and the 26% ammonia water solution into the micro mixer. The feed flow rate of the PPO aqueous solution is 13.99g / min, the feed flow rate of the ammonia water is 2.78g / min, the neutralization temperature in the micro mixer is controlled at 40℃, the molar ratio of NH3 to PPO is 1.5:1, and then the biocatalyst solution obtained in step (2) is simultaneously passed into the microchannel reactor at a flow rate of 4.90g / min along with the substrate solution from the micro mixer. The residence time in the microchannel reactor is 6min, and the reaction temperature of the microchannel reactor is controlled at 35℃.

[0189] Step (4) is the same as step (4) in Example 3;

[0190] Following the above process steps, the yield of PPO converted to phosphonium sulfate was 93.1%, and the space-time yield of phosphonium sulfate was 2.314 kg. 精草铵膦 The crystallization purity of glufosinate-ammonium salt was 93.5% (L·H), and the crystallization yield was approximately 91.1%.

[0191] Example 12

[0192] Steps (1)-(2) are the same as steps (1)-(2) in Example 3.

[0193] Step (3) Conditions for continuous neutralization and continuous bioconversion of PPO: Take 500g of 100% pure PPO solid powder, prepare a 35% PPO aqueous solution in advance, use 26% ammonia water as the neutralization alkali raw material, and simultaneously pass the PPO aqueous solution and the 26% ammonia water solution into the micro mixer. The feed flow rate of the PPO aqueous solution is 1.40g / min, the feed flow rate of the ammonia water is 0.28g / min, the neutralization temperature in the micro mixer is controlled at 40℃, the molar ratio of NH3 to PPO is 1.5:1, and then the biocatalyst solution obtained in step (2) is simultaneously passed into the microchannel reactor at a rate of 0.49g / min along with the substrate solution from the micro mixer. The residence time in the microchannel reactor is 20min, and the reaction temperature of the microchannel reactor is controlled at 35℃.

[0194] Step (4) is the same as step (4) in Example 3;

[0195] Following the above process steps, the yield of PPO converted to phosphonium sulfate was 99.7%, and the space-time yield of phosphonium sulfate was 0.742 kg. 精草铵膦 / (L·H), the crystal purity of glufosinate-ammonium salt is 99.6%, and the crystallization yield is approximately 98.6%.

[0196] Comparative Example 1

[0197] Bioconversion in a batch reactor using homogenized bacterial broth as a catalyst.

[0198] (1) Cell disruption of wet cells: Take 112g of L-glutamate dehydrogenase mutant wet cells and 28g of formate dehydrogenase wet cells, add 900g of water to dilute and stir, and then use a homogenizer to disrupt the cells of the mixed cell solution at an operating pressure of 950 bar.

[0199] (2) Removal of impurities from homogenized bacterial solution: The aqueous solution of ruptured bacterial cells obtained in step (1) was centrifuged at 4000 rpm for 15 min to remove bacterial residue. Then, 3000 g of water was added to the supernatant obtained after centrifugation, and the solution was filtered through a 5000D organic membrane. 600 g of water and 0.6 g of NAD were added to the retentate. + Add 250g of ammonium formate to obtain a homogeneous aqueous solution containing biological enzymes, coenzymes, and substrates for coenzyme regeneration. Add water to adjust the total mass to 1000g to obtain a biocatalyst solution, which is then poured into a 5L bioconversion tank.

[0200] (3) PPO discontinuous neutralization reaction: Take 500g of 100% pure PPO solid powder, prepare a 35wt% PPO aqueous solution in advance, use 26wt% ammonia water as the neutralization alkali raw material, add the PPO aqueous solution dropwise to the neutralization tank containing 26wt% ammonia water under stirring conditions for mixing and neutralization reaction, control the neutralization reaction temperature at 40℃, and the molar ratio of NH3 to PPO is 1.5:1 to obtain the substrate solution.

[0201] (4) PPO discontinuous biotransformation reaction: The substrate solution obtained in step (3) was added dropwise to a 5L biotransformation tank for 10 hours. The reaction temperature in the tank was controlled at 35°C. The substrate concentration was detected by liquid chromatography (HPLC). The total time for substrate transformation was 12 hours.

[0202] (5) After the conversion liquid in the conversion tank in step (4) is treated with a 3000D organic membrane, the permeate is treated with a 150D organic membrane, and the retentate is used to obtain an aqueous solution containing glufosinate. After dehydration under absolute pressure of 80°C and 5KPa, 1000g of methanol is added, the crystallization temperature is controlled at 35°C, and after 10h, the solution is filtered and dried to obtain glufosinate ammonium salt.

[0203] Following the above process steps, the yield of PPO converted to phosphonium sulfate was 88.2%, and the space-time yield of phosphonium sulfate was 0.008085 kg. 精草铵膦 / (L·H), the crystal purity of glufosinate-ammonium salt is 90.8%, and the crystallization yield is approximately 85.0%.

[0204] Comparative Example 2

[0205] Bioconversion in a batch reactor using microbial cells as catalysts.

[0206] (1) Preparation of biocatalyst solution containing wet bacterial cells: Take 112g of L-glutamate dehydrogenase mutant wet bacterial cells and 28g of formate dehydrogenase wet bacterial cells, add 600g of water to dilute and stir, and then add 0.6g of NAD to it. + Add 250g of ammonium formate and add water to adjust the total mass to 1000g, thus obtaining a biocatalyst solution containing bacterial cells, coenzymes, and substrates for coenzyme regeneration. Then pour it into a 5L bioconversion tank.

[0207] (2) PPO discontinuous neutralization reaction: Take 500g of 100% pure PPO solid powder, prepare a 35wt% PPO aqueous solution in advance, use 26wt% ammonia water as the neutralization alkali raw material, add the PPO aqueous solution dropwise to the neutralization tank containing 26wt% ammonia water under stirring conditions for mixing and neutralization reaction, control the neutralization temperature at 40℃, and the molar ratio of NH3 to PPO is 1.5:1 to obtain the substrate solution.

[0208] (3) PPO discontinuous biotransformation reaction: The substrate solution obtained in step (2) was added dropwise to a 5L biotransformation tank for 10 hours. The temperature in the transformation tank was controlled at 35°C. The substrate concentration was detected by HPLC. The total time for substrate transformation to terminate was 22 hours.

[0209] (4) After the conversion liquid in the conversion tank in step (4) is treated with a 3000D organic membrane, the permeate is treated with a 150D organic membrane, and the retentate is an aqueous solution containing glufosinate. After dehydration under absolute pressure of 80℃ and 5KPa, 1000g of methanol is added, the crystallization temperature is controlled at 35℃, and after 10h, the solution is filtered and dried to obtain glufosinate ammonium salt.

[0210] Following the above process steps, the yield of PPO converted to phosphonium sulfate was 85.1%, and the space-time yield of phosphonium sulfate was 0.00429 kg. 精草铵膦 The crystallization purity of glufosinate-ammonium salt was 90.3% (L·H), and the crystallization yield was approximately 82.6%.

[0211] A comparison of Examples 2 and 3 shows that:

[0212] Compared with wild-type glutamate dehydrogenase derived from halophilic archaea, using the L-glutamate dehydrogenase mutant as a catalyst significantly improved the yield of L-glufosinate-ammonia produced by microchannel continuous reactor biotransformation using 2-carbonyl-4-(hydroxymethylphosphono)butyric acid as a substrate. The space-time yield of glufosinate-ammonia was high, demonstrating that the glutamate dehydrogenase mutant provided by this invention has great industrial application value.

[0213] A comparison of Comparative Example 1, Comparative Example 2, and Example 3 shows that:

[0214] Comparative Example 1 was a batch reactor biotransformation using cell-wall-breaking homogenized liquid as a catalyst. 112g of L-glutamate dehydrogenase mutant wet cells, 28g of formate dehydrogenase wet cells, and 0.6g of NAD were added. + With 250g of ammonium formate, the biotransformation reaction was carried out for 12 hours, yielding L-glufosinate-ammonium salt with a yield of 88.2%. Comparative Example 2 was a batch reactor biotransformation using whole bacterial cells as a catalyst, with the following added: 112g of L-amino acid dehydrogenase mutant wet bacterial cells, 28g of formate dehydrogenase wet bacterial cells, and 0.6g of NAD+. + 250g of ammonium formate was added, and the biotransformation reaction was carried out for 22 hours, yielding L-glufosinate-ammonium salt with a yield of 85.1%. Example 3 is a microchannel continuous reactor biotransformation using cell cell disruption homogenate as a catalyst. 40g of L-glutamate dehydrogenase mutant wet cells, 10g of formate dehydrogenase wet cells, and 0.3g of NAD were added. +The product was reacted with 100g of ammonium formate and subjected to a biotransformation reaction for 10 minutes to obtain L-glufosinate-ammonium salt with a yield of 99.5%.

[0215] Comparative Example 2 uses a batch reactor for bioconversion with whole bacterial cells as the catalyst, requiring a reaction time of 22 hours. Comparative Example 1 uses a batch reactor for bioconversion with a homogenized bacterial cell solution as the catalyst, requiring a reaction time of 12 hours. Example 3 uses a microchannel continuous reactor for bioconversion with a homogenized bacterial cell solution as the catalyst, requiring a reaction time of 10 minutes. In batch reactor bioconversion, regardless of whether whole bacterial cells or a homogenized bacterial cell solution is used as the catalyst, the bioconversion time is 12 hours or more. The complete conversion time of PPO is relatively long. Because the PPO added dropwise to the bioconversion tank cannot be converted in time, PPO accumulates, leading to side reactions and the generation of more impurities in the system. Therefore, the yield of L-glufosinate is low. Compared with the batch reactor bioconversion process, this application uses a microchannel continuous bioconversion process, which significantly reduces the production of L-amino acid dehydrogenase wet cells, formic acid dehydrogenase wet cells, and coenzyme NAD. + The amount of ammonium formate substrate used for coenzyme regeneration was increased, the yield of L-glufosinate was improved, the reaction time was shortened, and the space-time yield of L-glufosinate ammonium salt was greatly improved.

[0216] This invention achieves continuous neutralization and biotransformation of 2-oxo-4-(hydroxymethylphosphoryl)butyric acid, avoiding substrate accumulation and reducing side reactions. The continuous microchannel synthesis technology for L-glufosinate-ammonium salt used in this invention significantly reduces the levels of L-amino acid dehydrogenase, coenzyme regeneration enzyme, and coenzyme NAD. + The amount of substrate used for coenzyme regeneration was reduced, thus lowering production costs.

[0217] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing L-glufosinate-ammonium salt, characterized in that, Includes the following steps: (1) Mix L-amino acid dehydrogenase wet cells and coenzyme regeneration enzyme wet cells, and after cell disruption treatment, separate and remove solid bacterial residue to obtain an aqueous solution containing L-amino acid dehydrogenase and coenzyme regeneration enzyme. (2) Add coenzyme NAD to the aqueous solution containing L-amino acid dehydrogenase and coenzyme regeneration enzyme in step (1). + and for NAD + The raw materials from the NADH cycle are used to obtain a biocatalyst solution; (3) Neutralize the aqueous solution of 2-oxo-4-(hydroxymethylphosphoryl)butyric acid and the aqueous solution of ammonia in a microchannel mixer to obtain a neutralized reaction solution. Then, continuously pass the neutralized reaction solution and the biocatalyst solution obtained in step (2) into a microchannel reactor to carry out a bioconversion reaction to obtain a conversion solution containing L-glufosinate-ammonium salt. The L-amino acid dehydrogenase in step (1) is an L-glutamate dehydrogenase mutant with the amino acid sequence number SEQ ID NO.

2. The L-glutamate dehydrogenase mutant is derived from the wild-type L-glutamate dehydrogenase with the amino acid sequence number SEQ ID NO.

1.

2. The method for preparing L-glufosinate-ammonium salt according to claim 1, characterized in that, The cell disruption treatment in step (1) is homogenization at a pressure of 600-950 bar, followed by centrifugation at 4000-5000 rpm for 10-20 min to obtain an aqueous solution containing L-amino acid dehydrogenase and coenzyme regeneration enzyme. The aqueous solution is then concentrated through a membrane to obtain a concentrated aqueous solution of enzyme protein.

3. The method for preparing L-glufosinate-ammonium salt according to claim 1, characterized in that, The coenzyme regenerating enzyme in step (1) is formate dehydrogenase, and the amino acid sequence number of formate dehydrogenase is SEQ ID NO.

3.

4. The method for preparing L-glufosinate-ammonium salt according to claim 1, characterized in that, In the preparation of L-glufosinate from 500g of 2-oxo-4-(hydroxymethylphosphoryl)butyric acid via biotransformation, the amount of L-amino acid dehydrogenase wet cells added in step (1) is 30-40g, and the amount of coenzyme regeneration enzyme wet cells added is 8-10g. In step (2), the amount of coenzyme NAD... + The addition amount is 0.2-0.3g, used for NAD. + The feedstock for the NADH cycle is ammonium formate, and the amount added is 75-100g.

5. The method for preparing L-glufosinate-ammonium salt according to claim 1, characterized in that, In step (3), the concentration of the aqueous solution of 2-oxo-4-(hydroxymethylphosphoryl)butyric acid is 30-35wt%, the concentration of the aqueous solution of ammonia is 23-26wt%, and the molar ratio of ammonia to 2-oxo-4-(hydroxymethylphosphoryl)butyric acid is 1.5-2:

1.

6. The method for preparing L-glufosinate-ammonium salt according to claim 1, characterized in that, In step (3), the neutralization reaction is carried out in a microchannel mixer at a temperature of 35-45℃, and the biotransformation reaction is carried out in a microchannel reactor at a residence time of 6-20 min and a reaction temperature of 25-40℃.

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