Preparation process of L-glufosinate or its salts

Abstract

The present invention relates to an environmentally friendly and cost-effective biocatalytic process for preparing L-glufosinate, its esters, or salts. The biocatalytic process comprises converting 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, its 5-ester, or salts under the action of a biocatalyst.

Description

Technical Field

[0001] The present invention relates to an environmentally friendly and cost-effective process for preparing L-glufosinate, its esters or salts. More specifically, the present invention relates to a biocatalytic process for preparing L-glufosinate, its esters or salts.

Background Art

[0002] Glufosinate is a non-selective herbicide belonging to the group of organophosphate herbicides and is widely used worldwide. Glufosinate is generally used in the form of ammonium salts for general weed control to suppress the growth of weeds and grasses. Glufosinate is used as a racemic mixture of L-glufosinate and D-glufosinate. However, it is well known that L-glufosinate, namely (S)-2-amino-4-(hydroxy(methyl)phosphoryl)butanoic acid), is much more potent than D-glufosinate. The L-isomer of glufosinate is a structural analogue of glutamate and is thus a competitive inhibitor of the bacterial and plant enzyme glutamine synthetase (GS). The L-enantiomer of glufosinate acts by inhibiting glutamine synthetase, thereby causing the accumulation of toxic levels of ammonium ions and indirectly stopping photosynthesis.

[0003] In the prior art for preparing optically pure L-glufosinate, mainly three methods are known, namely, asymmetric chemical synthesis, chiral separation, and biocatalysis.

[0004] Asymmetric chemical synthesis is based on the synthesis of optically pure L-glufosinate, which is more common in laboratory studies. One such process is provided in the Journal of Organic Chemistry, 1991, 56:J.1783-1788, but such processes involve many steps, have low yields, and the asymmetric synthesis reagents used are mostly expensive, resulting in high production costs and making them unsuitable for large-scale production of L-glufosinate.

[0005] In chiral resolution methods, the resolution of racemic glufosinate or its salts is performed using chiral resolution reagents. International Publication No. 1995023805 provides a process for obtaining L-glufosinate by resolving racemic glufosinate or its salts using chiral bases such as quinine, cinconine, cinconidine, or brucine. The main drawback of this process is the use of expensive chiral resolution reagents, which affects the overall cost of the process and makes it difficult to use on an industrial scale.

[0006] Finally, biocatalytic synthesis is considered a completely "environmentally friendly" technology. This method has many advantages, including mild reaction conditions, low toxicity, high stereoselectivity, and the generation of environmentally friendly waste, making it suitable for the industrial-scale production of optically pure L-glufosinate. Biocatalytic synthesis involves the use of isolated enzymes or whole cells (such as bacteria, fungi, microalgae, and plants) as catalysts in organic reactions. Most publications on biocatalytic synthesis for producing L-glufosinate or its salts focus on the use of isolated enzymes obtained by overexpression of the enzyme in genetically modified microorganisms. However, the identification of the gene, the genetic modification of the microorganism to provide the desired enzyme, and the isolation of such an enzyme make this technology cumbersome and expensive. Furthermore, such isolation of enzymes requires special procedures and resources.

[0007] Therefore, the inventors of the present invention have developed a biocatalytic process for obtaining L-glufosinate, its esters, or salts using a biocatalyst, thereby obtaining all the advantages of biocatalytic synthesis while still avoiding the aforementioned obstacles to such a process. [Overview of the Initiative]

[0008] The main objective of the present invention is to provide a green process for obtaining L-glufosinate, its esters, or salts.

[0009] Another object of the present invention is to provide a biocatalytic method for preparing L-glufosinate, its esters, or salts having high yield and high purity.

[0010] Another object of the present invention is to provide a simple, inexpensive, and efficient biocatalytic process for obtaining L-glufosinate, its esters, or salts.

[0011] Another object of the present invention is to provide an environmentally friendly process for obtaining L-glufosinate, its esters, or salts. [Means for solving the problem]

[0012] According to one aspect of the present invention, a biocatalytic process for preparing L-glufosinate, its esters, or salts is provided.

[0013] According to one aspect of the present invention, a biocatalytic process for preparing L-glufosinate, its esters, or salts is provided, the process comprising converting 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, its esters, or salts using a biocatalyst derived from a microorganism of the species Amicolatopsis.

[0014] According to one aspect of the present invention, a biocatalytic process for preparing L-glufosinate, its esters, or salts is provided, the process comprising converting 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, its esters, or salts using a biocatalyst derived from a microorganism of the species Amicolatopsis deposited as ATCC39116.

[0015] According to another aspect of the present invention, a biocatalytic process for preparing L-glufosinate, its esters, or salts is provided, the process comprising converting 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, its esters, or salts using a biocatalyst derived from a microorganism of the species Amicolatopsis in the presence of an amine donor.

[0016] According to another aspect of the present invention, a biocatalytic process for preparing L-glufosinate, its esters, or salts is provided, the process comprising converting 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, its esters, or salts using a biocatalyst derived from a microorganism of the species Amicolatopsis, in the presence of an amine donor and optionally a cofactor.

[0017] Another embodiment provides L-glufosinate, its ester, or salt, which can be obtained by a process comprising using a biocatalyst derived from a microorganism of the species Amicolatopsis to convert 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, its ester, or salt.

[0018] In another embodiment, a pesticide composition is provided comprising L-glufosinate, its ester, or a salt prepared by the biocatalytic process of the present invention. [Brief explanation of the drawing]

[0019] [Figure 1]The high-performance liquid chromatography (HPLC) profile of the reactants and products on the 14th day according to Example 4 of the present invention is shown. [Figure 2] The chiral HPLC analysis of the product is shown.

Mode for Carrying Out the Invention

[0020] Those skilled in the art will recognize that the invention described herein is subject to changes and modifications other than those specifically described. It should be understood that the invention described herein includes all such changes and modifications. The present invention also includes all the steps, features, compositions, and methods mentioned or shown in this specification, either individually or collectively, as well as any combination of any two or more of such steps or features.

[0021] For convenience, before providing a further description of the present invention, specific terms used in this specification and the examples are explained herein. These definitions should be read in light of the remainder of the disclosure and should be understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art. The terms used throughout this specification are defined as follows, unless specifically limited in a particular case. The terms used in this specification are defined as follows.

[0022] As used in this specification and the claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. The present disclosure is not limited by the specific embodiments described herein, and the specific embodiments are intended for illustrative purposes only.

[0023] Unless otherwise specified, the term "room temperature" essentially means a temperature in the range of about 20 to 35°C.

[0024] The term "purity" means the purity determined by HPLC.

[0025] The term "about" shall be interpreted to mean "approximately" or "reasonably close" and any statistically insignificant variation therefrom. As used herein, "about" or "approximately" includes the recited value and is within the allowable deviation range of a particular value determined by one of ordinary skill in the art considering the measured value in question and the error associated with the measurement of a particular quantity (i.e., the limits of the measurement system). For example, "about" can mean within the range of one or more standard deviations, or within the range of ±10% or ±5% of the recited value. The use of any examples or illustrative language (e.g., "such as") is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed elements essential to the practice of the invention as used herein.

[0026] As used herein, the terms "comprising", "including", "having", "containing", "involving" and the like are to be understood to mean without limitation, i.e., including but not limited to.

[0027] The terms "preferred" and "preferably" refer to embodiments of the invention that can provide certain benefits under certain circumstances. In one embodiment, the aspects and embodiments described herein are also to be interpreted as replacing the phrase "comprising" with any of "consisting of", "consisting essentially of", or "consisting substantially of".

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which the present invention pertains. Terms used herein in describing the present invention are for the purpose of illustrating only specific embodiments and are not intended to limit the invention.

[0029] The term "glufosinate" refers to a mixture of isomers of L-glufosinate and D-glufosinate.

[0030] As used herein, the term "L-glufosinate" includes the L-isomer of glufosinate, its salts, and derivatives. The term may also refer to L-glufosinate, with an L-glufosinate content of 70% or more, preferably 80% or more, and more preferably 90% or more. Typically, the L-glufosinate:D-glufosinate ratio may be in the range of about 90:10 to about 100:0.

[0031] "L-glufosinate" is also known as L-phosphinotricin or (S)-2-amino-4-(hydroxy(methyl)phosphoryl)butanoic acid. This term can generally refer to L-glufosinate in any form, such as solvates, hydrates, anhydrous forms, polymorphs, pseudopolymorphs, amorphous forms, or mixtures thereof, as well as derivatives such as esters and salts. The term "L-glufosinate" shall be interpreted as meaning L-glufosinate or its salts. Monosodium salt, disodium salt, monopotassium salt, dipotassium salt, calcium salt, ammonium salt, -NH3(CH3) + Salt, -NH2(CH3)2 + Salt, -NH(CH3)3 + Salt, -NH(CH3)2(C2H4OH) + Salt, and -NH2(CH3)(C2H4OH) + The definition includes salts of L-glufosinate, such as salts. Agriculturally acceptable salts include L-glufosinate-ammonium, L-glufosinate-sodium, and L-glufosinate-potassium.

[0032] The term "enantiomeric excess" or "%ee (enantiomeric excess)" refers to the enantiomeric purity of a sample, that is, the proportion of one enantiomer in the sample that exceeds the proportion of the other enantiomer. For example, the enantiomeric excess of L-glufosinate is the proportion of L-glufosinate in glufosinate that exceeds D-glufosinate.

[0033] As used herein, “derivative” or “analog” of a molecule should be noted as referring to a portion of the molecule or a modified version of the molecule.

[0034] As used herein, “biocatalyst” refers to whole cells, particularly microorganisms, containing a natural catalyst or one or more enzymes for chemical transformation of organic compounds, or isolated enzymes, partially purified enzymes, cell-free extracts or crude cell extracts / powder / immobilized or fixed forms, permeabilized cells, whole cells, whole fermentation broth, or freeze-dried cells.

[0035] As used herein, “Amicolatopsis species ATCC39116” refers to the microorganism of the species Amicolatopsis, which has collection number 39116 and is deposited with the American Type Culture Collection (ATCC) in the United States.

[0036] As used herein, "transaminase / amino acid transferase" refers to an enzyme that catalyzes the interconversion of amino acids and oxoacids by the transfer of an amino group, its catalytically active moiety, derivatives, or analogues.

[0037] As used herein, “amino acid dehydrogenase” refers to an enzyme belonging to the group of oxidoreductases that catalyze the oxidative deamination of primary amines by reducing an electron acceptor, usually NAD / NADP, to form aldehydes and ammonia.

[0038] As used herein, “aldo-ketereductase” refers to the superfamily of NAD(P)H-dependent oxidoreductases that reduce aldehydes and ketones to primary and secondary alcohols, respectively.

[0039] As used herein, the term “pre-incubation” means incubating (microorganisms / whole cell catalysts) for 1 to 20 hours prior to the biocatalytic process according to the present invention.

[0040] The term "2-oxo-4-(hydroxymethylphosphinyl)butyrate" is also referred to as "PPO" or "substrate," and both terms are used interchangeably throughout this explanation.

[0041] As used herein, the term "% assimilation rate" refers to the proportion of the substrate consumed by the biocatalyst relative to the total substrate added at the start of the reaction.

[0042] As used herein, the term "% conversion rate" refers to the ratio of the total product formed by the biocatalyst to the total substrate added at the start of the reaction.

[0043] According to one aspect of the present invention, a biocatalytic process for obtaining L-glufosinate, its ester, or salt is provided.

[0044] According to one aspect of the present invention, a process is provided for preparing L-glufosinate, its esters, or salts, the process being: This method involves using a biocatalyst derived from a microorganism of the Amicolatopsis species to convert 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, its ester, or salt.

[0045] In another embodiment, the microorganism of the Amicolatopsis species is deposited as ATCC39116.

[0046] In one embodiment, the substrate 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is prepared by a conventionally known process, such as an enzymatic or chemical process.

[0047] In one embodiment, the process is carried out with a substrate concentration of approximately 20 g / L to approximately 400 g / L. In another embodiment, the process is carried out with a substrate concentration of approximately 80 g / L to approximately 400 g / L. In yet another embodiment, the process is carried out with a substrate concentration of approximately 100 g / L to approximately 300 g / L.

[0048] In one embodiment, the total amount of substrate is added at the start of the reaction. In another embodiment, the substrate is supplied in batches by metering during the reaction.

[0049] In one embodiment, a microorganism of the Amycolatopsis species may produce multiple enzymes, preferably one or more. According to one embodiment, the Amycolatopsis species produces at least one enzyme capable of catalyzing the conversion of a substrate to L-glufosinate, its ester, or salt.

[0050] According to one embodiment, the biocatalyst comprises one or more enzymes produced by a microorganism of the Amycolatopsis species selected from aminotransferase and / or amino acid dehydrogenase and / or aldo-ketoreductase.

[0051] According to one embodiment, the biocatalyst comprises at least one aminotransferase and / or at least one amino acid dehydrogenase and / or at least one aldo-ketereductase, or a combination thereof. Aminotransferase enzymes belong to the class of transferases that catalyze the transfer of an amino group from an amino donor to a prochiral acceptor ketone to obtain chiral amines and the corresponding ketones or α-keto acids. Microorganisms of the Amicolatopsis species further produce additional transferases other than transaminases. Amino acid dehydrogenase enzymes refer to enzymes belonging to the group of oxidoreductases that catalyze the oxidative deamination of primary amines by reducing an electron acceptor, usually NAD / NADP, to form aldehydes and ammonia. Microorganisms of the Amicolatopsis species further produce additional dehydrogenases other than amino acid dehydrogenases. Aldo-ketereductase enzymes refer to the superfamily of NAD(P)H-dependent oxidoreductases that reduce carbonyl substrates.

[0052] According to one embodiment, the biocatalyst is selected from, but is not limited to, isolated enzymes; partially purified enzymes; cell-free extracts or crude cell extracts in liquid, powder, or immobilized / fixed form; permeabilized cells; whole cells; whole fermentation broth; freeze-dried cells; or a combination thereof.

[0053] In a preferred embodiment, the biocatalyst comprises whole cells, whole fermentation broth, permeabilized cells, or freeze-dried cells. In a more preferred embodiment, the biocatalyst comprises whole cells or whole fermentation broth.

[0054] In one embodiment, Amycolatopsis species are concentrated and cultured in a culture medium, and the cells are then centrifuged, collected, and used as whole cell catalysts for the biocatalytic process provided in the present invention. The process for culturing the microorganisms and the selection of the culture medium are carried out using conventional processes known to those skilled in the art.

[0055] In one embodiment, the amount of Amicolatopsis species is in the range of approximately 50 g / L to approximately 600 g / L in terms of wet cell weight units.

[0056] In one embodiment, the amount of Amicolatopsis species is approximately 100 g / L to approximately 200 g / L in terms of wet cell weight units. In one embodiment, the amount of Amicolatopsis species is approximately 200 g / L to approximately 300 g / L in terms of wet cell weight units. In one embodiment, the amount of Amicolatopsis species is approximately 300 g / L to approximately 400 g / L in terms of wet cell weight units. In one embodiment, the amount of Amicolatopsis species is approximately 400 g / L to approximately 500 g / L in terms of wet cell weight units. In one embodiment, the amount of Amicolatopsis species is approximately 500 g / L to approximately 600 g / L in terms of wet cell weight units.

[0057] In one embodiment, the process of converting 2-oxo-4-(hydroxymethylphosphinyl)butyric acid (PPO) to L-glufosinate, its ester, or salt involves specific amination from PPO to L-glufosinate using amine groups from one or more amine donors.

[0058] In one embodiment, 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is converted to L-glufosinate, its ester, or salt in the presence of an amine donor using a biocatalyst derived from a microorganism of the species Amicolatopsis. The process of the present invention is carried out in the presence of an amine donor.

[0059] According to another aspect of the present invention, a process is provided for preparing L-glufosinate, its esters, or salts, wherein 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is converted to L-glufosinate, its esters, or salts using a biocatalyst derived from a microorganism of the species Amicolatopsis in the presence of an amine donor.

[0060] In one embodiment, the amine donor is selected from, but is not limited to, L-aspartic acid (L-aspatratate) or racemic aspartic acid or its salts, L-glutamic acid (L-glutamate) or racemic glutamic acid or its salts, L-alanine or racemic alanine or its salts, L-phenylalanine or racemic phenylalanine or its salts, L-glycine or racemic glycine or its salts, L-lysine or racemic lysine or its salts, L-valine or racemic valine or its salts, L-serine or racemic serine or its salts, L-glutamine or racemic glutamine or its salts; inorganic ammonia source, ammonia; organic amines such as isopropylamine, sec-butylamine, ethanolamine, 2-aminobutyric acid or its salts, and diaminopropionic acid or its salts.

[0061] In a preferred embodiment, the amine donor is selected from L-glutamic acid, racemic glutamic acid, or a salt thereof. In another preferred embodiment, the amine donor is monosodium L-glutamic acid.

[0062] In another embodiment, the process is carried out in the presence of an amine donor present in the reaction solution at a concentration of about 100 g / L to about 800 g / L. In a preferred embodiment, the process is carried out in the presence of an amine donor present in the reaction solution at a concentration of about 200 g / L to about 600 g / L. In a preferred embodiment, the process is carried out in the presence of an amine donor present in the reaction solution at a concentration of about 200 g / L. In another preferred embodiment, the process is carried out in the presence of an amine donor present in the reaction solution at a concentration of about 500 g / L.

[0063] In one embodiment, the Amicolatopsis species is pre-incubated before contact with the substrate. In another embodiment, the Amicolatopsis species is pre-incubated with an amine donor for about 1 to 20 hours.

[0064] In one embodiment, the process for preparing L-glufosinate, its ester, or salt is optionally carried out in the presence of a cofactor. In one embodiment, the process for preparing L-glufosinate, its ester, or salt is carried out in the presence of a cofactor. The cofactor is pyridoxal 5'-phosphate.

[0065] In one embodiment, the cofactor is added externally or produced by the Amicolatopsis species. In a preferred embodiment, the cofactor is added externally. In another preferred embodiment, the cofactor is produced by the Amicolatopsis species.

[0066] In another embodiment, the process of the present invention is carried out without the external addition of cofactors. This means that the organism itself may contain or produce cofactors suitable for converting the substrate to L-glufosinate, its ester, or salt.

[0067] In one embodiment, the step of converting 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, its ester, or salt includes treating 2-oxo-4-(hydroxymethylphosphinyl)butyric acid with an alkaline solution.

[0068] In one embodiment, 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is treated with an alkaline solution before the addition of a biocatalyst, an amine donor, and optionally a cofactor.

[0069] In one embodiment, 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is first treated with an alkaline solution, followed by the addition of all reagents.

[0070] In one embodiment, 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is first treated with an alkaline solution, followed by the addition of a biocatalyst, an amine donor, and optionally a cofactor.

[0071] In another embodiment, 2-oxo-4-(hydroxymethylphosphinyl)butyric acid and an amine donor are first treated with an alkaline solution, followed by the addition of a biocatalyst and optionally a cofactor.

[0072] In one embodiment, treatment of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid with an alkaline solution includes neutralization of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid.

[0073] In another embodiment, all reagents are added to the reaction medium along with the substrate, and then treated with an alkaline solution.

[0074] In another embodiment, 2-oxo-4-(hydroxymethylphosphinyl)butyric acid, a biocatalyst, an amine donor, and optionally a cofactor are added to the reaction medium, and the reaction medium is then treated with an alkaline solution. The reaction medium comprises water, preferably DM water.

[0075] In one embodiment, the alkaline solution includes, but is not limited to, sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate, potassium bicarbonate, ammonium hydroxide, ammonium carbonate, ammonium bicarbonate, or a combination thereof.

[0076] In one embodiment, the alkaline solution is an aqueous solution of an alkali. In a preferred embodiment, the alkaline solution is an aqueous solution of sodium hydroxide. In another preferred embodiment, the alkaline solution is an aqueous solution of sodium carbonate. In yet another preferred embodiment, the alkaline solution is an aqueous solution of ammonium carbonate.

[0077] In one embodiment, the process of converting 2-oxo-4-(hydroxymethylphosphinyl)butyrate to L-glufosinate, its ester, or salt is carried out at a pH in the range of about 6 to about 9. In one embodiment, optionally, the pH is adjusted with a buffer. The buffer is selected from acetate buffer, phosphate buffer, or Tris-HCl buffer.

[0078] In another embodiment, the biocatalytic process according to the present invention is carried out at a temperature in the range of about 20°C to about 60°C.

[0079] In one embodiment, a process is provided for preparing L-glufosinate, its ester, or salt, and the process is as follows: The process involves using a biocatalyst derived from a microorganism of the Amicolatopsis species to convert 2-oxo-4-(hydroxymethylphosphinyl)butyrate to L-glufosinate, its ester, or salt in the presence of monosodium L-glutamate and optionally pyridoxal 5'-phosphate.

[0080] In one embodiment, a process is provided for preparing L-glufosinate, its ester, or salt, and the process is as follows: This method involves using a biocatalyst derived from a microorganism of the Amicolatopsis species to convert 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, its ester, or salt in the presence of monosodium L-glutamate and pyridoxal 5'-phosphate.

[0081] In one embodiment, the Amicolatopsis species is pre-incubated with an amine donor and cofactors for approximately 1 to 20 hours before contact with the substrate. In another embodiment, the Amicolatopsis species is not pre-incubated before contact with the substrate and is used directly.

[0082] In one embodiment, the process of the present invention optionally includes batch addition of the substrate 2-oxo-4-(hydroxymethylphosphinyl)butyrate.

[0083] In another embodiment, the Amicolatopsis species can produce the enzymes necessary for the conversion multiple times and can therefore be reused or recycled. A single batch of Amicolatopsis species can be reused or recycled at least about five times for the biocatalytic process according to the present invention. In another embodiment, the biocatalyst is recycled at least about five times.

[0084] In another embodiment, a single batch of Amycolatopsis species can be reused or recycled about 5 to about 15 times for the biocatalytic process according to the present invention. In another embodiment, a single batch of Amycolatopsis species can be recycled about 5 to about 8 times for the biocatalytic process according to the present invention.

[0085] The inventors of this invention have found that the microorganisms used can maintain their catalytic activity for several days and can be reused or recycled multiple times to catalyze the biotransformation of a substrate to L-glufosinate, its ester, or salt.

[0086] In one embodiment, the microorganisms used in this process are easily separated from the reaction system by conventionally known processes such as centrifugation and filtration. Separation techniques include centrifugation and filtration.

[0087] Therefore, in one embodiment, the biocatalyst used in the process described in the present invention can be recovered and / or recycled by conventional processes such as centrifugation and / or filtration.

[0088] In one embodiment, a general scheme of the biocatalytic process according to the present invention can be represented as scheme (I).

[0089] [ka]

[0090] In one embodiment, the present invention provides a biocatalytic process for preparing L-glufosinate, its esters, or salts, the process being: The process involves using a biocatalyst derived from a microorganism of the Amicolatopsis species to convert 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, its ester or salt, and the corresponding ketone or α-keto acid in the presence of an amine donor.

[0091] In one embodiment, the present invention provides a biocatalytic process for preparing L-glufosinate, its esters or salts, wherein 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is converted to L-glufosinate, its esters or salts, and the corresponding ketones or α-keto acids using a biocatalyst derived from a microorganism of the Amicolatopsis species, in the presence of an amine donor and optionally a cofactor.

[0092] In one embodiment, the present invention provides a biocatalytic process for preparing L-glufosinate, its esters or salts, wherein 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is converted to L-glufosinate, its esters or salts, and the corresponding ketones or α-keto acids using a biocatalyst derived from a microorganism of the species Amicolatopsis, in the presence of an amine donor and cofactors.

[0093] In one embodiment, the corresponding ketone or α-keto acid obtained by the biocatalytic process can be enzymatically converted to an amine donor by a conventionally known process and used in the biocatalytic process of the present invention.

[0094] In one embodiment, the biocatalytic process according to the present invention can be represented as shown in scheme (II).

[0095] [ka]

[0096] In one embodiment, the amine donor is monosodium L-glutamate. In one embodiment, the corresponding keto compound formed is α-ketoglutaric acid.

[0097] In one embodiment, the enantiomer excess of the obtained L-glufosinate, its ester, or salt having an L-form may be, for example, 10%ee or more, 20%ee or more, 30%ee or more, 40%ee or more, 50%ee or more, 60%ee or more, 70%ee or more, 80%ee or more, 90%ee or more, 91%ee or more, 92%ee or more, 93%ee or more, 94%ee or more, 95%ee or more, 96%ee or more, 97%ee or more, 98%ee or more, or 99%ee or more.

[0098] In one embodiment, the % assimilation rate of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is at least 30%. In one embodiment, the % assimilation rate of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is at least 40%. In one embodiment, the % assimilation rate of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is at least 50%.

[0099] In one embodiment, the percentage conversion rate from 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, its ester, or salt is at least 1%.

[0100] In one embodiment, the process of the present invention can convert 30% to 85% of 2-oxo-4-(hydroxymethylphosphinyl)butyrate to L-glufosinate. In one embodiment, the process of the present invention can convert more than 30% of 2-oxo-4-(hydroxymethylphosphinyl)butyrate to L-glufosinate. In one embodiment, the process of the present invention can convert more than 40% of 2-oxo-4-(hydroxymethylphosphinyl)butyrate to L-glufosinate. In one embodiment, the process of the present invention can convert more than 50% of 2-oxo-4-(hydroxymethylphosphinyl)butyrate to L-glufosinate. In one embodiment, the process of the present invention can convert more than 60% of 2-oxo-4-(hydroxymethylphosphinyl)butyrate to L-glufosinate. In one embodiment, the process of the present invention can convert more than 70% of 2-oxo-4-(hydroxymethylphosphinyl)butyrate to L-glufosinate.

[0101] In one embodiment, the present invention provides L-glufosinate, its ester, or salt, which is produced by a process using a biocatalyst derived from a microorganism of the species Amicolatopsis.

[0102] In one embodiment, the present invention provides a pesticide composition comprising L-glufosinate, its ester, or a salt thereof, wherein L-glufosinate, its ester, or salt is produced by a process using a biocatalyst derived from a microorganism of the species Amicolatopsis.

[0103] In another embodiment, the pesticide composition comprises L-glufosinate, its ester, or salt produced by a biocatalytic process, the process comprising converting 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, its ester, or salt using a biocatalyst derived from a microorganism of the species Amicolatopsis, in the presence of an amine donor and optionally a cofactor, and optionally an excipient that is pesticide-acceptable.

[0104] In one embodiment, the present invention provides the use of L-glufosinate, its ester or salt, or a pesticide composition comprising L-glufosinate, its ester or salt, prepared according to the process described herein, for controlling undesirable plants or weeds.

[0105] Advantages of the present invention: 1. The present invention provides an "environmentally friendly" technology for obtaining L-glufosinate, its esters, or salts. 2. The present invention provides a simple and efficient process for obtaining L-glufosinate, its esters, or salts. 3. The biocatalytic process of the present invention efficiently converts 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, its ester, or salt at high substrate concentrations. 4. The present invention provides L-glufosinate, its ester, or salt having high yield and enantiomer excess (%ee). 5. The present invention provides a method for obtaining L-glufosinate, its esters, or salts using a biocatalyst derived from a microorganism of the Amycolatopsis species that can produce enzymes and cofactors. 6. The biocatalyst used in the present invention can be reused or recycled multiple times without affecting the efficiency of the process. [Examples]

[0106] The present invention will be described more specifically by the following embodiments. However, it should be understood that the scope of the present invention is not limited in any way by the embodiments. Those skilled in the art will understand that the present invention includes the following embodiments and can be further modified and altered within the technical scope of the invention.

[0107] Details of the analysis method: To obtain detection profiles for reactants and products, samples were analyzed using a high-performance liquid chromatograph equipped with a UV detector, employing a C-18 column (inert seal, ODS-35 μm, 4.6 × 250 mm).

[0108] For the qualitative analysis of L- and D-isomers of glufosinate, samples were analyzed using a high-performance liquid chromatograph equipped with a UV detector, employing a Chirex 3126(D)-penicillamine LC column (150 × 4.6 mm).

[0109] Example 1: Culture of bacterial strains for biocatalytic processes In this study, to grow the biocatalyst, cultures of Amicolatopsis species (deposited as ATCC39116) from glycerol stock were inoculated into modified tryptone soybean broth (5 mL) and incubated at approximately 37°C for 32 hours. The grown cultures were then transferred to 100 mL of modified tryptone soybean broth and incubated at approximately 37°C for 24 hours. The cultures were then inoculated at 2% v / v to scale up to the desired cell volume for biotransformation.

[0110] The proliferated cells were centrifuged at 4000 rpm for 5 minutes, washed once with 0.1 M Tris-HCl (pH 8) to remove excess culture medium, and a biocatalyst with an 85% water content was obtained. This biocatalyst, derived from the Amycolatopsis microorganism, was used to prepare L-glufosinate, its ester, or salt, as shown in the following examples.

[0111] Example 2: Process for the preparation of L-glufosinate 50 g (0.29 mol) of monosodium L-glutamate (concentration 500 g / L), 0.2 mM pyridoxal 5'-phosphate, and 20 g of the biocatalyst obtained in Example 1 (concentration 200 g / L) were added to the reaction vessel. 25 g (0.138 mol) of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid (PPO) (concentration 250 g / L) was added to the reaction mixture in a single lot. The pH of the reaction system was adjusted to approximately 8 using concentrated sodium hydroxide solution. The reaction system was then diluted to 100 mL volume with buffer (0.1 M Tris-HCl) and further maintained at approximately 8 pH using buffer (0.1 M Tris-HCl). The reaction was maintained at 30°C for 1 hour at a rotation speed of 200 rpm. The conversion of 2-oxo-4-(hydroxymethylphosphinyl)butyrate (PPO) to L-glufosinate (LGF) was monitored over several days using HPLC, and the progress of the reaction is shown in Table 1.

[0112] [Table 1]

[0113] From the table above, it was concluded that the biocatalyst used in the present invention successfully converted 2-oxo-4-(hydroxymethylphosphinyl)butyrate at a concentration of 250 g / L to L-glufosinate (purity 72%), with a total assimilation rate of 96.02% of 2-oxo-4-(hydroxymethylphosphinyl)butyrate and a conversion rate of 46.08% to L-glufosinate. The chiral ratio was approximately 99.60:0.4 (L:D).

[0114] Example 3: Process for preparing L-glufosinate by reusing a biocatalyst. The process was carried out according to Example 2 to prepare a 10 mL reaction system containing 25 g / L of 2-oxo-4-(hydroxymethylphosphinyl)butyrate, 0.2 mM pyridoxal 5'-phosphate, and 200 g / L of the whole-cell biocatalyst obtained in Example 1. Cycle 1 was performed to convert 2-oxo-4-(hydroxymethylphosphinyl)butyrate to L-glufosinate. After Cycle 1, the biocatalyst was centrifuged, washed with water, and reused in Cycle 2. The same process for biocatalyst recycling was repeated after each cycle until the process was complete. The biocatalyst was recycled for 6 cycles, and the conversion from 2-oxo-4-(hydroxymethylphosphinyl)butyrate (PPO) to L-glufosinate (L-GF) was monitored using HPLC. The observations are listed in Table 2.

[0115] [Table 2]

[0116] From the above observations, it can be concluded that the whole cell catalyst used in the present invention can be used to prepare L-glufosinate, its esters, or salts, resulting in a consistent conversion of approximately 40% to 50% over at least 6 cycles.

[0117] Example 4: Preparation process of L-glufosinate without cofactors The process was carried out according to Example 2 without using pyridoxal 5'-phosphate to prepare a 10 mL reaction system containing 25 g / L of 2-oxo-4-(hydroxymethylphosphinyl)butyrate and 400 g / L of the biocatalyst obtained in Example 1. The biocatalyst was reused for 5 cycles, and the conversion from 2-oxo-4-(hydroxymethylphosphinyl)butyrate (PPO) to L-glufosinate (L-GF) was monitored using HPLC. The observations are listed in Table 3.

[0118] [Table 3]

[0119] Example 5: Process for obtaining L-glufosinate by batch addition of substrates 50 g (0.29 mol) of monosodium L-glutamate at a concentration of 500 g / L and 0.2 mM pyridoxal 5'-phosphate were added to the reaction flask, and the biocatalyst obtained in Example 1 at a concentration of 200 g / L was added to it. The pH of the reaction system was adjusted to approximately 8 using concentrated sodium hydroxide solution, and the system was diluted to 65 mL volume with buffer (0.1 M Tris-HCl). The reaction was maintained at 30°C with a rotation speed of 200 rpm, and 25 g (0.138 mol) of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid (PPO) was added to the reaction mixture in four batches, each with a concentration of 62.5 g / L (total PPO added to a concentration of 250 g / L), at 12-hour intervals over a period of 48 hours. The conversion of 2-oxo-4-(hydroxymethylphosphinyl)butyrate (PPO) to L-glufosinate (L-GF) was monitored over several days using HPLC, and the progress of the reaction is shown in Table 4 below.

[0120] [Table 4]

[0121] From the table above, it was concluded that when 2-oxo-4-(hydroxymethylphosphinyl)butyrate was added in batches, 80.74% of the 2-oxo-4-(hydroxymethylphosphinyl)butyrate was assimilated, and 52.11% was converted to L-glufosinate.

[0122] Example 6: Preparation process of L-glufosinate using neutralized substrate 7 g of (75% purity) PPO (0.28 M) was neutralized in an ice bath using a 20% ammonium carbonate solution in a controlled manner to pH 8. To this solution, 8 g of L-glutamic acid (0.54 M), 0.2 mM pyridoxal 5'-phosphate, and 20 g of biocatalyst were added. The pH of the reaction system was adjusted again to 8 using a 20% ammonium carbonate solution. The reaction was maintained at 40°C for 90 hours at a rotation speed of 300 rpm. The conversion of 2-oxo-4-(hydroxymethylphosphinyl)butyrate (PPO) to L-glufosinate (L-GF) was monitored over several days using HPLC, and the progress of the reaction is shown in Table 5.

[0123] [Table 5]

[0124] From the table above, it was observed that the biocatalyst used in the present invention successfully converted 2-oxo-4-(hydroxymethylphosphinyl)butyric acid (75% purity) at a concentration of 50 g / L to L-glufosinate. The total assimilation of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid was 91.18%, and the conversion to L-glufosinate was 77.45%. The chiral ratio was approximately 99.68:0.32 (L:D).

Claims

1. A process for preparing L-glufosinate, its esters, or salts, A process comprising using a biocatalyst derived from a microorganism of the species Amicolatopsis to convert 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, its ester, or salt.

2. The process according to claim 1, wherein the microorganism of the Amicolatopsis species is deposited as ATCC39116.

3. The process according to claim 1, wherein the biocatalyst is selected from isolated enzymes; partially purified enzymes; cell-free extracts or crude cell extracts in liquid, powder, or immobilized / fixed form; permeabilized cells; whole cells; whole fermentation broth; freeze-dried cells; or a combination thereof.

4. The process according to claim 3, wherein the biocatalyst comprises whole cells, whole fermentation broth, permeabilized cells, or freeze-dried cells.

5. The process according to claim 4, wherein the biocatalyst comprises one or more enzymes selected from aminotransferases, amino acid dehydrogenases, and aldo-ketoreductases.

6. The process according to claim 1, wherein the process is carried out in the presence of an amine donor.

7. The process according to claim 6, wherein the amine donor is selected from L-aspartic acid or racemic aspartic acid or a salt thereof, L-glutamic acid or racemic glutamic acid or a salt thereof, L-alanine or racemic alanine or a salt thereof, L-phenylalanine or racemic phenylalanine or a salt thereof, L-glycine or racemic glycine or a salt thereof, L-lysine or racemic lysine or a salt thereof, L-valine or racemic valine or a salt thereof, L-serine or racemic serine or a salt thereof, L-glutamine or racemic glutamine or a salt thereof; an inorganic ammonia source, ammonia; and organic amines such as isopropylamine, sec-butylamine, ethanolamine, 2-aminobutyric acid or a salt thereof, diaminopropionic acid or a salt thereof.

8. The process according to claim 1, wherein the process is optionally carried out in the presence of a cofactor.

9. The process according to claim 8, wherein the cofactor is added externally or produced by an Amicolatopsis species.

10. The process according to claim 9, wherein the cofactor is pyridoxal 5'-phosphate.

11. The process according to claim 1, wherein the process is carried out at a pH in the range of about 6 to about 9.

12. The process according to claim 1, wherein the biocatalyst is recycled at least about five times.

13. The process according to claim 1, wherein the process optionally comprises adding 2-oxo-4-(hydroxymethylphosphinyl)butyric acid in a batch manner.

14. The process according to claim 1, wherein the percentage assimilation rate of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is at least 30%.

15. The process according to claim 1, wherein the percentage conversion rate from 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, its ester, or salt is at least 1%.

16. L-glufosinate, its ester, or salt prepared by the process described in claim 1.

17. A pesticide composition comprising L-glufosinate, its ester, or a salt thereof, wherein the L-glufosinate, its ester, or a salt thereof is prepared by the process described in claim 1.

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