Process for the preparation of l-glufosinate or salts thereof

Description

1 / 26 PROCESS FOR PREPARING L-GLUFOSINATE OR SALTS THEREOF FIELD OF THE INVENTION:

[0001] The present invention relates to a green and cost-effective process for the preparation of L-glufosinate, esters or salts thereof. More particularly, the present invention relates to a biocatalytic process for the preparation of L-glufosinate, esters or salts thereof. BACKGROUND OF THE INVENTION

[0002] Glufosinate is a non-selective herbicide belonging to the organophosphate herbicide group; and it has been widely used throughout the world. It is generally used in the form of an ammonium salt for total vegetation control and to control 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, i.e., (S)-2-amino-4-(hydroxy(methyl)phosphoryl)butanoic acid, is much more potent than D-glufosinate. The L-isomer of glufosinate is a structural analog of glutamate and, therefore, is a competitive inhibitor of the glutamine synthetase (GS) enzyme in bacteria and plants. The L-enantiomer of glufosinate acts by inhibiting glutamine synthetase, thus causing the accumulation of toxic levels of ammonium ions and indirectly interrupting photosynthesis.

[0003] Primarily, there are three known methods in the state of the art for the preparation of optically pure L-glufosinate, namely, by asymmetric chemical synthesis, by chiral separation and by biocatalytic method.

[0004] The asymmetric chemical synthesis method is based on the synthesis of optically pure L-glufosinate, which is more common in laboratory research. One such process is provided in the Journal of Organic Chemistry, 1991, 56: 1783 to 1788; however, such processes involve many steps and provide low yields, and the asymmetric synthetic reagents used are Petition 870250087438, dated 09 / 26 / 2025, page 13 / 118 2 / 26 mainly expensive, resulting in high production costs, which is not feasible for large-scale production of L-glufosinate.

[0005] In the chiral separation method, the resolution of racemic glufosinate or its salts is performed using a chiral resolving reagent. Patent publication WO1995023805 provides a process in which racemic glufosinate or its salt is separated to obtain L-glufosinate using chiral bases such as quinine, cinchonine, cinchonidine, or brucine. The main disadvantage of this process is the use of an expensive chiral resolving reagent, which affects the overall cost of the process, thus hindering its use on an industrial scale.

[0006] Finally, the biocatalytic synthesis method is considered the perfect green technique. This method has many advantages, such as moderate reaction conditions, low toxicity, high stereoselectivity, and the production of ecological waste; and it is suitable for the industrial-scale production of optically pure L-glufosinate. The biocatalytic synthesis method involves the use of isolated enzymes or whole cells (such as bacteria, fungi, microalgae, and plants) as catalysts in organic reactions. Most publications on the biocatalytic synthesis method for the production of L-glufosinate or its salts focus on the use of isolated enzymes obtained by overexpression of enzymes in genetically modified microorganisms. However, gene identification, genetic modification of microorganisms to provide the desired enzymes, and isolation of such enzymes make this technique impractical and expensive.Furthermore, such enzyme isolation requires special procedures and resources.

[0007] Therefore, the inventors of the present invention have developed a biocatalytic process to obtain L-glufosinate, esters or salts thereof, using a biocatalyst, thus obtaining all the advantages of biocatalytic synthesis and additionally avoiding the aforementioned impediments of such processes. Petition 870250087438, dated 09 / 26 / 2025, page 14 / 118 3 / 26 OBJECTIVES OF THE INVENTION:

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

[0009] Another objective of the present invention is to provide a biocatalytic method for the preparation of L-glufosinate, esters or salts thereof, with high yield and purity.

[0010] Another objective of the present invention is to provide a simple, low-cost and efficient biocatalytic process for obtaining L-glufosinate, esters or salts thereof.

[0011] Yet another objective of the present invention is to provide an environmentally friendly process for obtaining L-glufosinate, esters or salts thereof. SUMMARY OF THE INVENTION:

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

[0013] According to one aspect of the present invention, a biocatalytic process is provided for the preparation of L-glufosinate, esters or salts thereof, wherein the process comprises converting 2-oxo-4(hydroxymethylphosphinyl)butyric acid into L-glufosinate, esters or salts thereof, using a biocatalyst derived from a microorganism of Amycolatopsis sp.

[0014] According to one aspect of the present invention, a biocatalytic process is provided for the preparation of L-glufosinate, esters or salts thereof, wherein the process comprises converting 2-oxo-4(hydroxymethylphosphinyl)butyric acid into L-glufosinate, esters or salts thereof, using a biocatalyst derived from a microorganism of Amycolatopsis sp. deposited as ATCC 39116.

[0015] According to another aspect of the present invention, a biocatalytic process is provided for the preparation of L-glufosinate, esters or salts thereof, the process comprising the conversion of 2-oxo-4 acid Petition 870250087438, dated 09 / 26 / 2025, p. 15 / 118 4 / 26 (hydroxymethylphosphinyl)butyric acid in L-glufosinate, esters or salts thereof, using a biocatalyst derived from the microorganism Amycolatopsis sp., in the presence of an amine donor.

[0016] According to another aspect of the present invention, a biocatalytic process is provided for the preparation of L-glufosinate, esters or salts thereof, wherein the process comprises converting 2-oxo-4(hydroxymethylphosphinyl)butyric acid into L-glufosinate, esters or salts thereof, using a biocatalyst derived from a microorganism of Amycolatopsis sp., in the presence of an amine donor and, optionally, a cofactor.

[0017] Yet another aspect provides L-glufosinate, esters or salts thereof obtainable by a process comprising the conversion of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid into L-glufosinate, esters or salts thereof, using a biocatalyst derived from a microorganism of Amycolatopsis sp.

[0018] In another aspect, an agrochemical composition is provided comprising L-glufosinate, esters or salts thereof prepared by the biocatalytic process of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS:

[0019] Figure 1 illustrates the high-performance liquid chromatography (HPLC) profile of reagents and products on day 14 according to Example 4 of the present invention.

[0020] Figure 2 illustrates the chiral HPLC analysis of the product. DETAILED DESCRIPTION OF THE INVENTION:

[0021] Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It should be understood that the invention described herein includes all such variations and modifications. The invention also includes all steps, features, compositions and methods mentioned or indicated in this descriptive report, individually or collectively, and any and all combinations of any two or more of said steps or features. Petition 870250087438, dated 09 / 26 / 2025, page 16 / 118 5 / 26

[0022] For convenience, before providing a further description of the present invention, certain terms used in the descriptive report and examples will be described herein. These definitions should be read in light of the remainder of the disclosure and understood from the perspective of a person skilled in the art. Except where otherwise defined, all technical and scientific terms used in the present invention have the meaning commonly understood by a person skilled in the art. The terms used throughout this descriptive report are defined as follows, unless otherwise limited in specific occurrences. The terms used in the present invention are defined as follows.

[0023] As used in the descriptive report and claims, the singular forms a, an, and the include plural references unless the context clearly indicates otherwise. This disclosure should not be limited in scope by the specific modalities described herein, which are intended for illustrative purposes only.

[0024] The term ambient temperature, unless otherwise indicated, means a temperature essentially in the range of about 20 °C to 35 °C.

[0025] The term purity means purity as determined by HPLC.

[0026] The term "approximately" should be interpreted as meaning approximately or reasonably close to and any statistically insignificant variations thereof. "Approximately" or "approximately," as used herein, includes the stated value and means within an acceptable range of deviation for the specific value, as determined by one skilled in the art considering the measurement in question and the error associated with measuring the specific quantity (i.e., the limitations of the measuring system). For example, "approximately" may mean within one or more standard deviations or within ±10% or ±5% of the stated value. The use of any and all examples, or illustrative language (e.g., such as) is merely for the purpose of better illustrating the invention and does not represent a limitation on the scope of the invention unless otherwise claimed. No language in Petition 870250087438, dated 09 / 26 / 2025, page 17 / 118 6 / 26 descriptive report should be interpreted as indicating any unclaimed element essential to the practice of the invention, as used herein.

[0027] As used herein, the terms comprising, including, having, containing, involving and the like should be understood as open-ended, that is, with a meaning that includes, but is not limited to.

[0028] The terms preferred and preferred refer to embodiments of the invention that may provide certain benefits under certain circumstances. In an embodiment, the aspects and embodiments described herein should also be interpreted as replacing the clause comprising "because it consists of," or "because it essentially consists of," or "because it substantially consists of."

[0029] Except where defined otherwise, all technical and scientific terms used in the present invention have the same meaning as is commonly understood by those skilled in the art to which this invention pertains. The terminology used in the description of the present invention is for the purpose of describing only specific embodiments and is not intended to limit the invention.

[0030] The term glufosinate refers to an isomeric mixture of Lglufosinate and D-glufosinate.

[0031] As used herein, the term L-glufosinate includes the L-isomer of glufosinate or a salt and derivatives thereof. The term may also refer to L-glufosinate, wherein the L-glufosinate content is 70% or more, preferably 80% or more, and more preferably 90% or more. Typically, the ratio of L-glufosinate:D-glufosinate may be in the range of about 90:10 to about 100:0.

[0032] L-glufosinate is also known as L-phosphinothricin or (S)-2-amino-4-(hydroxy(methyl)phosphoryl)butanoic acid. The term can refer generically to any form of L-glufosinate, such as solvates, hydrates, anhydrous form, polymorphic forms, pseudopolymorphic forms, amorphous form or mixtures thereof, and derivatives, such as esters; and salts. The term L-glufosinate should Petition 870250087438, dated 09 / 26 / 2025, page 18 / 118 7 / 26 should be interpreted as meaning L-glufosinate or salts thereof. The definition includes salts of L-glufosinate, such as 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) salt. Agronomically acceptable salts include L-glufosinate-ammonium, L-glufosinate-sodium, and L-glufosinate-potassium.

[0033] The term % enantiomeric excess or % ee means the enantiomeric purity of a sample, that is, the percentage of one enantiomer that exceeds the other enantiomer in the sample. For example, the enantiomeric excess of L-glufosinate is the percentage of L-glufosinate that exceeds D-glufosinate in glufosinate.

[0034] It should be noted that, as used herein, a derivative or analogue of a molecule refers to a derived portion of, or a modified version of, the molecule.

[0035] As used herein, biocatalyst refers to a whole cell, particularly a microorganism, comprising a natural catalyst or one or more enzymes to perform chemical transformations on organic compounds; or isolated enzymes, partially purified enzymes, cell-free extracts or crude cell extract in liquid / powder / immobilized or fixed form, permeabilized cells, whole cells, whole fermentation broths, lyophilized cells, of such microorganisms.

[0036] As used herein, Amycolatopsis sp. ATCC 39116 refers to the organism Amycolatopsis sp. deposited in the American Type Culture Collection (ATCC) patent application in the USA under collection number 39116.

[0037] As used herein, transaminase / amino acid transferase refers to an enzyme, a catalytically active portion, a derivative or analogue thereof, that catalyzes the interconversion of amino acids and oxoacids by transfer of amino groups. Petition 870250087438, dated 09 / 26 / 2025, p. 19 / 118 8 / 26

[0038] As used herein, amino acid dehydrogenase refers to an enzyme belonging to the group of oxidoreductases that catalyzes the oxidative deamination of primary amines by reducing an electron acceptor, usually NAD / NADP, to form aldehyde and ammonia.

[0039] As used herein, aldo-keto reductases refers to a superfamily of NAD(P)H-dependent oxidoreductases that reduce aldehydes and ketones to their respective primary and secondary alcohols.

[0040] The term pre-incubation, as used herein, means incubating (microorganisms / whole-cell catalyst) for 1 to 20 hours prior to a biocatalytic process according to the present invention.

[0041] The term 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is also called PPO or substrate, and both terms are used interchangeably throughout the description.

[0042] The term % assimilation, as used herein, means the percentage of substrate consumed by the biocatalyst relative to the total substrate added at the start of the reaction.

[0043] The term % conversion, as used herein, means the percentage of total product formed by the biocatalyst relative to the total substrate added at the start of the reaction.

[0044] According to one aspect of the present invention, a biocatalytic process is provided for obtaining L-glufosinate, esters or salts thereof.

[0045] According to one aspect of the present invention, a process is provided for the preparation of L-glufosinate, esters or salts thereof, and the process comprises:

[0046] convert 2-oxo-4-(hydroxymethylphosphinyl)butyric acid into L-glufosinate, esters or salts thereof using a biocatalyst derived from a microorganism of Amycolatopsis sp.

[0047] In another embodiment, the microorganism Amycolatopsis sp. is deposited as ATCC 39116. Petition 870250087438, dated 09 / 26 / 2025, p. 20 / 118 9 / 26

[0048] In one embodiment, the substrate 2-oxo-4(hydroxymethylphosphinyl)butyric acid is prepared by conventionally known processes, such as an enzymatic process or a chemical process.

[0049] In one embodiment, the process is carried out at a substrate concentration of about 20 g / l to about 400 g / l. In another embodiment, the process is carried out at substrate concentrations of about 80 g / l to about 400 g / l. In yet another embodiment, the process is carried out at substrate concentrations of about 100 g / l to about 300 g / l.

[0050] In one embodiment, the total amount of substrate is added at the beginning of the reaction. In another embodiment, the substrate is measured in batches during the reaction.

[0051] In one embodiment, the microorganism Amycolatopsis sp. can produce multiple enzymes, preferably one or more enzymes. According to one embodiment, Amycolatopsis sp. produces at least one enzyme capable of catalyzing the conversion of a substrate into L-glufosinate, esters, or salts thereof.

[0052] According to one embodiment, the biocatalyst comprises one or more enzymes produced by the microorganism Amycolatopsis sp. selected from among aminotransferase and / or amino acid dehydrogenase and / or aldoketo reductases.

[0053] 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-keto reductase, or combinations thereof. Aminotransferase enzymes belong to the class of transferases, which catalyze the transfer of an amino group from an amino donor to a prochiral acceptor ketone to obtain a chiral amine and a corresponding ketone or α-keto acid. The microorganism Amycolatopsis sp. additionally produces other transferases besides transaminases. The amino acid dehydrogenase enzyme refers to an enzyme belonging to the group of oxidoreductases that catalyzes the oxidative deamination of primary amines. Petition 870250087438, dated 09 / 26 / 2025, page 21 / 118 10 / 26 reducing an electron acceptor, usually NAD / NADP, to form aldehyde and ammonia. The microorganism Amycolatopsis sp. additionally produces dehydrogenases beyond amino acid dehydrogenases. Aldo-keto reductase enzymes refer to a superfamily of NAD(P)H-dependent oxidoreductases that reduce carbonyl substrates.

[0054] According to one embodiment, the biocatalyst is selected from, but not limited to, isolated enzymes; partially purified enzymes; cell-free extract or crude cell extract in liquid, powder or immobilized / fixed form; permeabilized cells, whole cells, whole fermentation broths, lyophilized cells or combinations thereof.

[0055] In a preferred embodiment, the biocatalyst comprises whole cells, whole fermentation broths, permeabilized cells, or lyophilized cells. In a more preferred embodiment, the biocatalyst comprises whole cells or whole fermentation broths.

[0056] In one embodiment, Amycolatopsis sp. is enriched and cultivated using culture media, and then the cells are centrifuged, collected, and used as a whole-cell catalyst for the biocatalytic process as provided in the present invention. The microorganism culture process and culture media selection are performed using conventional processes known to one skilled in the art.

[0057] In one embodiment, the amount of Amycolatopsis sp. in terms of wet cell weight is in a range of about 50 g / l to about 600 g / l.

[0058] In one embodiment, the amount of Amycolatopsis sp. in terms of wet cell weight is about 100 g / l to about 200 g / l. In one embodiment, the amount of Amycolatopsis sp. in terms of wet cell weight is about 200 g / l to about 300 g / l. In one embodiment, the amount of Amycolatopsis sp. in terms of wet cell weight is about 300 g / l to about 400 g / l. In one embodiment, the amount of Amycolatopsis sp. in terms of wet cell weight is about 400 g / l to about Petition 870250087438, dated 09 / 26 / 2025, p. 22 / 118 11 / 26 of 500 g / l. In one embodiment, the amount of Amycolatopsis sp. in terms of wet cell weight is about 500 g / l to about 600 g / l.

[0059] In one embodiment, the process of converting 2-oxo-4(hydroxymethylphosphinyl)butyric acid (PPO) into L-glufosinate, esters or salts thereof involves specific amination of PPO into L-glufosinate using an amine group from one or more amine donors.

[0060] In one embodiment, 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is converted to L-glufosinate, esters or salts thereof using a biocatalyst derived from the microorganism Amycolatopsis sp. and in the presence of an amine donor. The process of the present invention is carried out in the presence of an amine donor.

[0061] According to another aspect of the present invention, a process is provided for preparing L-glufosinate, esters or salts thereof, wherein 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is converted into L-glufosinate, esters or salts thereof using a biocatalyst derived from a microorganism of Amycolatopsis sp., in the presence of an amine donor.

[0062] In one embodiment, the amine donor is selected from, but not limited to, L-aspartate or racemic aspartate or salts thereof, L-glutamate or racemic glutamate or salts thereof, L-alanine or racemic alanine or salts thereof, L-phenylalanine or racemic phenylalanine or salts thereof, L-glycine or racemic glycine or salts thereof, L-lysine or racemic lysine or salts thereof, L-valine or racemic valine or salts thereof, L-serine or racemic serine or salts thereof, L-glutamine or racemic glutamine or salts thereof; inorganic ammonia source, ammonia; organic amine, such as isopropylamine, sec-butylamine, ethanolamine, 2-aminobutyric acid or salts thereof and diaminopropionic acid or salts thereof.

[0063] In a preferred embodiment, the amine donor is selected from L-glutamate or racemic glutamate or salts thereof. In another preferred embodiment, the amine donor is monosodium L-glutamate. Petition 870250087438, dated 09 / 26 / 2025, page 23 / 118 12 / 26

[0064] In another embodiment, the process is carried out in the presence of an amine donor present at a concentration of about 100 g / l to about 800 g / l in the reaction solution. In a preferred embodiment, the process is carried out in the presence of an amine donor present at a concentration of about 200 g / l to about 600 g / l in the reaction solution. In a preferred embodiment, the process is carried out in the presence of an amine donor present at a concentration of about 200 g / l in the reaction solution. In another preferred embodiment, the process is carried out in the presence of an amine donor present at a concentration of about 500 g / l in the reaction solution.

[0065] In one embodiment, Amycolatopsis sp. is pre-incubated before contact with the substrate. In another embodiment, Amycolatopsis sp. is pre-incubated for about 1 hour to about 20 hours with the amine donor.

[0066] In one embodiment, the process for the preparation of L-glufosinate, esters or salts thereof is optionally carried out in the presence of a cofactor. In one embodiment, the process for the preparation of L-glufosinate, esters or salts thereof is carried out in the presence of a cofactor. The cofactor is pyridoxal 5'-phosphate.

[0067] In one embodiment, the cofactor is added externally or produced by Amycolatopsis sp. In a preferred embodiment, the cofactor is added externally. In another preferred embodiment, the cofactor is produced by Amycolatopsis sp.

[0068] In another embodiment, the process of the present invention is carried out without the external addition of a cofactor. This means that the organisms themselves contain, or are capable of generating, a cofactor suitable for converting the substrate into L-glufosinate, esters or salts thereof.

[0069] In one embodiment, the step of converting 2-oxo-4-(hydroxymethylphosphinyl)butyric acid into L-glufosinate, esters or salts thereof comprises treating 2-oxo-4-(hydroxymethylphosphinyl)butyric acid with an alkaline solution. Petition 870250087438, dated 09 / 26 / 2025, p. 24 / 118 13 / 26

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

[0071] In one embodiment, 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is first treated with an alkaline solution followed by the addition of all reagents.

[0072] In one embodiment, 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is first treated with an alkaline solution followed by the addition of a biocatalyst, amine donor and, optionally, a cofactor.

[0073] In another embodiment, 2-oxo-4(hydroxymethylphosphinyl)butyric acid and the amine donor are first treated with an alkaline solution followed by the addition of a biocatalyst and, optionally, a cofactor.

[0074] In one embodiment, the treatment of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid with an alkaline solution comprises the neutralization of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid.

[0075] In another embodiment, all reagents together with the substrate are added to the reaction medium followed by treatment with an alkaline solution.

[0076] In another embodiment, 2-oxo-4(hydroxymethylphosphinyl)butyric acid, biocatalyst, amine donor and, optionally, cofactor are added to the reaction medium and then the reaction medium is treated with an alkaline solution. The reaction medium comprises water, preferably DM water.

[0077] In one embodiment, the alkaline solution comprises, but is not limited to, sodium hydroxide, sodium carbonate, sodium bicarbonate, potassium hydroxide, potassium carbonate, potassium bicarbonate, ammonium hydroxide, ammonium carbonate, ammonium bicarbonate, or combinations thereof. Petition 870250087438, dated 09 / 26 / 2025, page 25 / 118 14 / 26

[0078] In one embodiment, the alkaline solution is an aqueous solution of an alkali. In a preferred embodiment, the alkaline solution is aqueous sodium hydroxide. In another preferred embodiment, the alkaline solution is aqueous sodium carbonate. In yet another preferred embodiment, the alkaline solution is aqueous ammonium carbonate.

[0079] In one embodiment, the process of converting 2-oxo-4(hydroxymethylphosphinyl)butyric acid into L-glufosinate, esters or salts thereof is carried out at a pH in the range of about 6 to about 9. In another embodiment, optionally, the pH is adjusted with a buffer solution. The buffer solution is selected from acetate buffer, phosphate buffer or TrisHCl buffer.

[0080] 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.

[0081] In one embodiment, a process is provided for the preparation of L-glufosinate, esters or salts thereof, the process comprising:

[0082] converting 2-oxo-4-(hydroxymethylphosphinyl)butyric acid into L-glufosinate, esters or salts thereof using a biocatalyst derived from a microorganism of Amycolatopsis sp., in the presence of monosodium L-glutamate and, optionally, pyridoxal 5'-phosphate.

[0083] In one embodiment, a process is provided for the preparation of L-glufosinate, esters or salts thereof, the process comprising:

[0084] converting 2-oxo-4-(hydroxymethylphosphinyl)butyric acid into L-glufosinate, esters or salts thereof using a biocatalyst derived from a microorganism of Amycolatopsis sp., in the presence of monosodium L-glutamate and pyridoxal 5'-phosphate.

[0085] In one embodiment, before coming into contact with the substrate, the Amycolatopsis sp. is pre-incubated for approximately 1 hour to approximately 20 hours with the amine donor and cofactor. In another embodiment, Amycolatopsis sp. Petition 870250087438, dated 09 / 26 / 2025, page 26 / 118 15 / 26 is not pre-incubated before coming into contact with the substrate; it is used directly.

[0086] In one embodiment, the process of the present invention optionally comprises the discontinuous addition of the substrate 2-oxo-4(hydroxymethylphosphinyl)butyric acid.

[0087] In another embodiment, Amycolatopsis sp. is capable of producing enzyme(s) necessary for the so-called multiple conversion and, therefore, can be reused or recycled. A single batch of Amycolatopsis sp. can be reused or recycled at least about 5 times for the biocatalytic process according to the present invention. In another embodiment, the biocatalyst is recycled at least about 5 times.

[0088] In another embodiment, a single batch of Amycolatopsis sp. can be reused or recycled approximately 5 to approximately 15 times for the biocatalytic process according to the present invention. In another embodiment, a single batch of Amycolatopsis sp. can be recycled approximately 5 to approximately 8 times for the biocatalytic process according to the present invention.

[0089] The inventors of the present invention have discovered that the microorganisms used remain catalytically competent for several days and can be reused or recycled several times to catalyze the biotransformation of the substrate into L-glufosinate, esters or salts thereof.

[0090] In one embodiment, the microorganisms used in the present process are easily separated from the reaction system by conventionally known processes such as centrifugation and filtration. The separation technique comprises centrifugation, filtration and the like.

[0091] Thus, 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.

[0092] In one embodiment, the general scheme of the biocatalytic process according to the present invention can be represented as in scheme (I): Petition 870250087438, dated 09 / 26 / 2025, p. 27 / 118 16 / 26 Scheme (I)

[0093] In one embodiment, the present invention provides a biocatalytic process for the preparation of L-glufosinate, esters or salts thereof, wherein the process comprises: To convert 2-oxo-4-(hydroxymethylphosphinyl)butyric acid into L-glufosinate, esters or salts thereof and a corresponding ketone or α-keto acid, using a biocatalyst derived from the microorganism Amycolatopsis sp. and in the presence of an amine donor.

[0094] In one embodiment, the present invention provides a biocatalytic process for the preparation of L-glufosinate, esters or salts thereof, wherein 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is converted into L-glufosinate, esters or salts thereof and a corresponding ketone or α-keto acid, using a biocatalyst derived from a microorganism of Amycolatopsis sp., in the presence of an amine donor and, optionally, a cofactor.

[0095] In one embodiment, the present invention provides a biocatalytic process for the preparation of L-glufosinate, esters or salts thereof, wherein 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is converted into L-glufosinate, esters or salts thereof and a corresponding ketone or α-keto acid, using a biocatalyst derived from a microorganism of Amycolatopsis sp., in the presence of an amine donor and a cofactor.

[0096] In one embodiment, the ketone or corresponding α-keto acids obtained in the biocatalytic process can be enzymatically converted into Petition 870250087438, dated 09 / 26 / 2025, p. 28 / 118 17 / 26 an amine donor by processes conventionally known and used in the biocatalytic process of the present invention.

[0097] In one embodiment, the biocatalytic process according to the present invention can be represented as in scheme (II): alpha-ketoglutaric acid Scheme (II)

[0098] In one embodiment, the amine donor is monosodium L-glutamate. In another embodiment, the corresponding keto compound formed is α-ketoglutaric acid.

[0099] In one embodiment, the enantiomeric excess of L-glufosinate, esters or salts thereof obtained that have the L-shape 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, % 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.

[0100] In one embodiment, the assimilation percentage of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is at least 30%. In one embodiment, the assimilation percentage of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is at least 40%. In one embodiment, the assimilation percentage of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is at least 50%. Petition 870250087438, dated 09 / 26 / 2025, p. 29 / 118 18 / 26

[0101] In one embodiment, the % conversion of 2-oxo-4(hydroxymethylphosphinyl)butyric acid to L-glufosinate, esters or salts thereof is at least 1%.

[0102] In one embodiment, the process of the present invention is capable of converting 30% to 85% of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid into L-glufosinate. In one embodiment, the process of the present invention is capable of converting more than 30% of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid into L-glufosinate. In one embodiment, the process of the present invention is capable of converting more than 40% of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid into L-glufosinate. In one embodiment, the process of the present invention is capable of converting more than 50% of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid into L-glufosinate. In one embodiment, the process of the present invention is capable of converting more than 60% of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid into L-glufosinate. In another embodiment, the process of the present invention is capable of converting more than 70% of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid into L-glufosinate.

[0103] In one embodiment, the present invention provides L-glufosinate, esters or salts thereof, wherein L-glufosinate, esters or salts thereof are produced by a process that uses a biocatalyst derived from a microorganism of Amycolatopsis sp.

[0104] In one embodiment, the present invention provides an agrochemical composition comprising L-glufosinate, esters or salts thereof, wherein L-glufosinate, esters or salts thereof are produced by a process using a biocatalyst derived from a microorganism of Amycolatopsis sp.

[0105] In another embodiment, the agrochemical composition comprises L-glufosinate, esters or salts thereof produced by a biocatalytic process; wherein the process comprises converting 2-oxo-4-(hydroxymethylphosphinyl)butyric acid into L-glufosinate, esters or salts thereof, using a biocatalyst derived from a microorganism of Amycolatopsis sp. Petition 870250087438, dated 09 / 26 / 2025, p. 30 / 118 19 / 26 in the presence of an amine donor and, optionally, a cofactor and, optionally, an agrochemically acceptable excipient.

[0106] In one embodiment, the present invention provides the use of L-glufosinate, esters or salts thereof; or an agrochemical composition comprising L-glufosinate, esters or salts thereof, prepared in accordance with the present process, as described herein, to control unwanted plants or weeds. Advantages of the present invention: 1. The present invention provides a green technique for obtaining L-glufosinate, esters or salts thereof. 2. The present invention provides a simple and efficient method for obtaining L-glufosinate, esters or salts thereof. 3. The biocatalytic process of the present invention efficiently converts 2-oxo-4-(hydroxymethylphosphinyl)butyric acid into L-glufosinate, esters or salts thereof, at a high substrate concentration. 4. The present invention provides L-glufosinate, esters or salts thereof in high yields and enantiomeric excess (% ee). 5. The present invention provides a method for obtaining L-glufosinate, esters or salts thereof, using a biocatalyst derived from a microorganism of Amycolatopsis sp., capable of producing enzyme(s) and cofactor. 6. The biocatalyst used in the present invention can be reused or recycled several times without affecting the efficiency of the process. Examples:

[0107] The present invention is explained more specifically by the examples below. However, it should be understood that the scope of the present invention is by no means limited to the examples. It will be recognized by those skilled in the art that the present invention includes the examples below and, additionally, may be modified and altered within the technical scope of the present invention. Petition 870250087438, dated 09 / 26 / 2025, p. 31 / 118 20 / 26 Analytical method details:

[0108] For the reagent and product detection profile, samples were analyzed on a high-performance liquid chromatograph with a UV detector using a C-18 column (inert seal, ODS - 35 μm, 4.6 x 250 mm).

[0109] For qualitative analysis of the L isomer and the D isomer of glufosinate, samples were analyzed on a high-performance liquid chromatograph with UV detector using a Chirex 3126 (D) - penicillamine LC column (150 x 4.6 mm). Example 1: Cultivation of a strain for the biocatalytic process

[0110] In the present study, for biocatalyst growth, a culture of Amycolatopsis sp. (deposited as ATCC 39116) from glycerol stock was inoculated into a modified tryptone soy broth (5 ml) and incubated for 32 hours at approximately 37 °C. The culture was then transferred to a 100 ml modified tryptone soy broth for 24 hours at approximately 37 °C. The culture was inoculated at 2% v / v to scale up to the desired cell count for biotransformation.

[0111] The cultured cells were centrifuged at 4000 rpm for 5 minutes and washed once with 0.1 M Tris HCl, pH 8, to remove excess medium to obtain a biocatalyst with 85% moisture content. This biocatalyst, derived from a microorganism of Amycolatopsis sp., was used for the preparation of L-glufosinate, esters or salts thereof, as represented in the examples below. Example 2: Process for the preparation of L-glufosinate

[0112] 50 g (0.29 moles) of monosodium L-glutamate (at a concentration of A 500 g / L solution, 0.2 mM pyridoxal 5'-phosphate, and 20 g of the biocatalyst (at a concentration of 200 g / L) obtained in Example 1 were added to a reaction vessel. 25 g (0.138 mol) of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid (PPO) (at a concentration of 250 g / L) were added to the reaction mixture in a single batch. The pH of the reaction system was adjusted to approximately 8 using concentrated sodium hydroxide solution. The reaction system was then diluted to Petition 870250087438, dated 09 / 26 / 2025, page 32 / 118 21 / 26 100 ml of buffer solution (0.1 M Tris HCl) was added and further maintained at approximately pH 8 using buffer solution (0.1 M Tris HCl). The reaction was maintained at 30 °C with a rotation speed of 200 rpm for 1 hour. The conversion of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid (PPO) to L-glufosinate (LGF) was monitored using HPLC over a period of days, and the reaction progress was as shown in Table 1. Table 1 Number of days PPO (g / l) LGF (g / l) % assimilation % conversion 0 179.08 0.00 0.00 0.00 5 86.16 45.29 52.14 25.16 10 17.46 71.03 90.30 39.46 14 7.12 82.95 96.02 46.08

[0113] From the table above, it was concluded that the biocatalyst used in the present invention correctly converted 2-oxo-4(hydroxymethylphosphinyl)butyric acid at a concentration of 250 g / l into L-glufosinate (purity of 72%) with a total assimilation of 96.02% of 2-oxo-4(hydroxymethylphosphinyl)butyric acid and 46.08% conversion to L-glufosinate. The chiral ratio was approximately 99.60:0.4 (L:D). Example 3: Process for the preparation of L-glufosinate reusing the biocatalyst

[0114] The process according to Example 2 was performed to prepare ml of reaction system with a concentration of 25 g / l of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid, 0.2 mM of pyridoxal 5'-phosphate, and a concentration of 200 g / l of whole-cell biocatalyst obtained in Example 1. Cycle 1 was performed to convert 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate. After cycle 1, the biocatalyst was centrifuged, washed with water, and reused for cycle 2. The same process was used for recycling the biocatalyst. Petition 870250087438, dated 09 / 26 / 2025, p. 33 / 118 22 / 26 was repeated after each cycle until the process was complete. The biocatalyst was recycled for 6 cycles and the conversion of 2-oxo-4(hydroxymethylphosphinyl)butyric acid (PPO) to L-glufosinate (L-GF) was monitored using HPLC and the observations are listed in Table 2. Table 2 Cycle No. Time (hours) PPO (g / l) L-GF (g / l) % assimilation % conversion Cycle 1 0 23.36 0 0 0 48 10.97 4.90 53 20.97 96 2.09 6.98 91.05 29.88 120 1.13 9.20 95.46 R 48 6.25 7.36 72.5 32.33 96 1.36 10.02 94.01 44.08 120 0.37 11.25 98.51 49.49 Cycle 4 0 24.18 0 0 0 48 4.26 9.36 82.20 39.90 96 0.59 10.58 97.53 44.19 120 0.00 12.6 99.08 52.63 Petition 870250087438, dated 09 / 26 / 2025, p. 34 / 118 23 / 26 Cycle 5 0 23.59 0 0 0 48 5.79 6.48 75.45 27.47 96 3.06 8.31 87.03 35.22 120 1.92 10.94 91.86 46.37 Cycle 6 0 24.70 0 0 0 48 9.49 4.74 61.57 19.19 96 4.13 7.18 83.28 29.06 120 2.34 10.35 90.52 41.90

[0115] From the above observations, it can be concluded that the whole-cell catalyst used in the present invention can be used to prepare Lglufosinate, esters or salts thereof, consequently for at least 6 cycles with consistent conversion of about 40% to about 50%. Example 4: Process for preparing cofactor-free L-glufosinate

[0116] The process according to Example 2 was executed, without pyridoxal 5'-phosphate was used to prepare 10 ml of reaction system with a concentration of 25 g / l of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid and a concentration of 400 g / l of the biocatalyst obtained in Example 1. The biocatalyst was reused for 5 cycles and the conversion of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid (PPO) to L-glufosinate (L-GF) was monitored using HPLC and the observations are listed in Table 3. Table 3 Cycle No. Time (hours) PPO (g / l) L-GF (g / l) % assimilation % conversion Cycle 1 0 24.15 0.00 0.00 0.00 72 6.72 7.38 72.15 30.55 Petition 870250087438, dated 09 / 26 / 2025, p. 35 / 118 24 / 26 120 2.88 11.13 88.07 46.08 Cycle 2 0 23.64 0.00 0.00 0.00 72 4.72 9.14 80.08 38.66 120 1.91 11.93 91.94 50.46 Cycle 3 0 24.08 0.00 0.00 0.00 72 2.39 8.71 90.07 36.17 120 0.0 13.12 99.95 54.48 Cycle 4 0 23.96 0.00 0.00 0.00 72 5.24 7.62 78.13 31.8 120 1.39 11.71 94.19 48.77 Cycle 5 0 23.11 0.00 0.00 0.00 72 6.54 8.83 71.68 38.20 120 0.00 12.25 99.95 52.86 Example 5: Process for obtaining L-glufosinate by batch addition of the substrate.

[0117] 50 g (0.29 moles) of monosodium L-glutamate at a concentration of 500 g / L, 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 a volume of 65 mL using buffer solution (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) were added to the reaction mixture in 4 batches, each with a concentration of 62.5 g / L (the total PPO added is at a concentration of 250 g / L) over a 48-hour period at 12-hour intervals. Petition 870250087438, dated 09 / 26 / 2025, page 36 / 118 25 / 26 each. The conversion of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid (PPO) to L-glufosinate (L-GF) was monitored using HPLC over a period of days and the progress of the reaction was as shown below in Table 4. Table 4 Number of days PPO (g / l) L-GF (g / l) % assimilation % conversion 0 0.00 0.00 0.00 0.00 0.5 20.34 2.88 44.97 1.98 5 99.01 46.04 44.99 31.68 10 49.87 69.27 72.29 47.67 14 34.68 75.75 80.74 52.11

[0118] From the table above, it was concluded that when 2-oxo-4-(hydroxymethylphosphinyl)butyric acid was added in batches, 80.74% assimilation of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid was obtained and the conversion to L-glufosinate was 52.11%. Example 6: Process for the preparation of L-glufosinate using a neutralized substrate.

[0119] 7 g of PPO (75% purity) (0.28 M) were neutralized in a controlled manner using a 20% ammonium carbonate solution to pH 8 in an ice bath. 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 again adjusted to 8 using a 20% ammonium carbonate solution. The reaction was maintained at 40 °C with a rotation speed of 300 rpm for 90 hours. The conversion of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid (PPO) to L-glufosinate (L-GF) was monitored using HPLC over a period of days, and the reaction progress was as shown in Table 5. Petition 870250087438, dated 09 / 26 / 2025, page 37 / 118 26 / 26 Table 5 Time (hours) PPO (g / l) L-GF (g / l) % assimilation % conversion 0 51.00 0.00 0.00 0.00 48 15.70 28.90 69.22 56.67 72 7.20 36.50 85.88 71.57 96 4.50 39.50 91.18 77.45

[0120] From the table above, it was observed that the biocatalyst used in the present invention correctly converted 2-oxo-4(hydroxymethylphosphinyl)butyric acid (purity of 75%) at a concentration of 50 g / l into L-glufosinate with a total assimilation of 91.18% of 2-oxo-4(hydroxymethylphosphinyl)butyric acid and 77.45% conversion to L-glufosinate. The chiral ratio was approximately 99.68:0.32 (L:D). Petition 870250087438, dated 09 / 26 / 2025, p. 38 / 118

Claims

1 / 2 CLAIMS 1. Process for preparing L-glufosinate, esters or salts thereof, the process being characterized by comprising: converting 2-oxo-4-(hydroxymethylphosphinyl)butyric acid into L-glufosinate, esters or salts thereof using a biocatalyst derived from a microorganism of Amycolatopsis sp.

2. Process according to claim 1, characterized in that the microorganism Amycolatopsis sp. is deposited as ATCC 39116.

3. Process, according to claim 1, characterized in that the biocatalyst is selected from isolated enzymes; partially purified enzymes; cell-free extract or crude cell extract in liquid, powder or immobilized / fixed form; permeabilized cells, whole cells, whole fermentation broths, lyophilized cells or combinations thereof.

4. Process according to claim 3, characterized in that the biocatalyst comprises whole cells, whole fermentation broths, permeabilized cells or lyophilized cells.

5. Process according to claim 4, characterized in that the biocatalyst comprises one or more enzymes selected from aminotransferases, amino acid dehydrogenases and aldo-keto reductases.

6. Process according to claim 1, the process being characterized in that it is carried out in the presence of an amine donor.

7. Process, according to claim 6, characterized in that the amine donor is selected from L-aspartate or racemic aspartate or salts thereof, L-glutamate or racemic glutamate or salts thereof, L-alanine or racemic alanine or salts thereof, L-phenylalanine or racemic phenylalanine or salts thereof, L-glycine or racemic glycine or salts thereof, L-lysine or racemic lysine or salts thereof, L-valine or racemic valine or salts thereof, L-serine or racemic serine or salts thereof, L-glutamine or racemic glutamine or salts thereof; source of inorganic ammonia, ammonia; Petition 870250087438, dated 09 / 26 / 2025, p. 39 / 118 2 / 2 organic amine, such as isopropylamine, sec-butylamine, ethanolamine, 2-aminobutyric acid or salts thereof, diaminopropionic acid or salts thereof.

8. Process according to claim 1, the process being characterized in that it can optionally be performed in the presence of a cofactor.

9. Process according to claim 8, characterized in that the cofactor is added externally or produced by Amycolatopsis sp.

10. Process according to claim 9, characterized in that the cofactor is pyridoxal 5'-phosphate.

11. Process according to claim 1, the process being characterized in that it is carried out at a pH in the range of about 6 to about 9.

12. Process according to claim 1, characterized in that the biocatalyst is recycled at least about 5 times.

13. Process according to claim 1, the process being characterized by optionally comprising the batch addition of 2-oxo4-(hydroxymethylphosphinyl)butyric acid.

14. Process according to claim 1, characterized in that the assimilation percentage of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid is at least 30%.

15. Process according to claim 1, characterized in that the % conversion of 2-oxo-4-(hydroxymethylphosphinyl)butyric acid to L-glufosinate, esters or salts thereof is at least 1%.

16. L-glufosinate, esters or salts thereof, characterized for preparation by the process defined in claim 1.

17. Agrochemical composition characterized by comprising L-glufosinate, esters or salts thereof, wherein L-glufosinate, esters or salts thereof are prepared by the process as defined in claim 1. Petition 870250087438, dated 09 / 26 / 2025, p. 40 / 118

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