Enzymatic process for preparation of l-glufosinate P-esters

Abstract

The present invention relates, in a first aspect, to an enzymatic process for the preparation of L-glufosinate P-esters. The method is characterized by a step (c) in which the L-glufosinate-ammonium P-ester carbamoylate is reacted to obtain the corresponding L-glufosinate-ammonium P-ester. The step (c) is catalyzed by a carbamoylase enzyme, preferably by an L-enantioselective carbamoylase enzyme. The L-glufosinate-ammonium P-ester carbamylate used in the step (c) can be obtained by a reaction of the corresponding L-glufosinate-ammonium hydantoin P-ester. The optional prior step (b) is catalyzed by a hydantoin enzyme, preferably an L-enantioselective hydantoin enzyme. Even more preferably, the L-glufosinate-ammonium hydantoin P-ester used in step (b) can be obtained by reacting the corresponding D-glufosinate-ammonium hydantoin P-ester. The optional prior step (a) is preferably catalyzed by a hydantoin racemase. In a second aspect, the invention relates to an enzymatic process for the enantioselective preparation of L-glufosinate P-ester from a mixture MIIIA of L-and D-glufosinate P-ester hydantoin. In the method of a second aspect of the invention, the mixture MIIIA is provided in step (i-A) and used in steps (ii) and (iii). Steps (ii) and (iii) correspond to the aforementioned steps (b) and (c), respectively, in which an L-carbamoylase is used in step (c). In an optional preferred step (i-B) of the method of a second aspect of the invention, corresponding to the preferred step (a) described above, at least a portion of the D-glufosinate P-ester hydantoin in the mixture MIIIA is converted to its corresponding L-isomer, preferably using a hydantoin racemase, resulting in a mixture MIIIB, which is subsequently used in steps (ii) and (iii). The L-glufosinate P-ester obtained in the process according to the first or second aspect of the present invention can be saponified to obtain L-glufosinate.

Description

[0001] The present invention relates in a first aspect to an enzymatic process for preparing L-glufosinate P-ester. The process is characterized by a step (c) in which a L-glufosinate P-ester carbamoylate is reacted to obtain the corresponding L-glufosinate P-ester. The step (c) is catalyzed by a carbamoylase, preferably a carbamoylase selective for the L-enantiomer.

[0002] The L-phosphine ammonium hydantoin P-ester carbamoylate used in step (c) can be obtained by reacting from the corresponding L-phosphine ammonium hydantoin P-ester. The optional previous step (b) is catalyzed by hydantoinase, preferably by the hydantoinase catalyzed by L-enantiomeric selectivity. Even more preferably, the L-phosphine ammonium hydantoin P-ester used in step (b) can be obtained by reacting from the corresponding D-phosphine ammonium hydantoin P-ester. The optional previous step (a) is preferably catalyzed by hydantoin racemase.

[0003] In a second aspect, the present invention relates to a mixture of L- and D-phosphinothricin P-ester hydantoins M IIIA An enzymatic method for preparing L-phosphinothricin ammonium P-ester with enantiomer selectivity. In the method of the second aspect of the invention, the mixture M IIIA Provided in step (iA) and used in steps (ii) and (iii). Steps (ii) and (iii) correspond to the above steps (b) and (c), respectively, wherein in step (c), L-carbamoylase is used.

[0004] In the optional preferred step (iB) of the method of the second aspect of the present invention, corresponding to the preferred step (a) above, the mixture M is preferably treated with a hydantoin racemase. IIIA At least a portion of the D-phosphinothricin P-ester hydantoin in the mixture is converted into its corresponding L-isomer to obtain a mixture M IIIB , which is subsequently used in steps (ii) and (iii).

[0005] The L-glufosinate P-ester obtained in the method according to the first or second aspect of the present invention may be saponified to obtain L-glufosinate. 1. Background Technology

[0006] Organophosphorus compounds, i.e. chemical agents containing carbon-phosphorus bonds, are widely used as herbicides in the field of plant protection. Herbicides such as glyphosate and glufosinate and the growth regulator glyphosate For this purpose (e.g. G. Rev. Environ. Contam. Toxicol. 1994, 138, 73-145).

[0007] Esters of P-methylphosphinic acid (e.g., butyl P-methylphosphinic acid ester; "MPBE"; CAS-No: 6172-80-1) play a key role as synthetic building blocks in the synthesis of the non-selective herbicide glufosinate. These esters can be obtained by two basic synthetic routes (summarized in Figures 3a and 3b on page 130 of the article K.Haack, Chem.Unserer Zeit 2003, 37, 128-138):

[0008] a. Diethyl chlorophosphite [ClP(OC2H5)2] is reacted with CH3MgCl to give methyldiethoxyphosphine [H3CP(OC2H5)2; "DEMP"; CAS-No.15715-41-0], which is partially hydrolyzed to give the corresponding methyl phosphinate (MPEE; CAS-Nr:16391-07-4).

[0009] b. Alternatively, methane can react with phosphorus trichloride at 500°C to form methyl dichlorophosphine H3CPCl2. The latter can undergo solvolysis in alcohol to give the corresponding methyl phosphinate.

[0010] The ester of P-methylphosphinic acid selectively adds carbon-carbon double bonds. This property is used in the synthesis of glufosinate-ammonium to form a second phosphorus-carbon bond. For example, H3CPH(O)OR (R=alkyl) reacts with 1-cyanoallyl acetate in an addition reaction to provide an intermediate. The acetate group is then exchanged with ammonia, and the ester group of the cyano and phosphinic acid moieties is hydrolyzed to obtain glufosinate-ammonium.

[0011] Acrylate is a cheaper alternative. It can react with esters of p-methylphosphinic acid to produce 3-[alkoxy(methyl)phosphino] alkyl propionate. The diester reacts with diethyl oxalate in a Claisen reaction, hydrolyzes, and decarboxylates to produce the corresponding α-keto acid, which can be reductively aminated to produce glufosinate.

[0012] These and other routes to the synthesis of glufosinate are also described in the art, for example in WO 1999 / 009039 A1, EP 0 508 296 A1.

[0013] A common disadvantage of all synthetic routes of glufosinate is that the glufosinate obtained is a racemic mixture. However, since the D-enantiomer has no herbicidal activity, L-glufosinate [hereinafter referred to as "LGA"; CAS-Nr.35597-44-5; other names "(S)-glufosinate", "(-)-glufosinate"] is the enantiomer with economic value.

[0014]

[0015] For example, CN 111662325 A discloses a synthetic route in which L-homoserine is reacted with the corresponding hydantoin, followed by the addition of methylphosphonium dichloride to produce methylphosphane disubstituted with L-homoserine hydantoin. After the final Arbuzov reaction and hydrolysis, LGA is obtained. Although the publication describes a high enantiomeric excess (= "ee"), this ee could not be reproduced by the inventors of the present invention. One reason may be that in this synthetic route, strongly acidic (HCl is used for the saponification of the hydantoin phosphorus bond) and alkaline conditions (NaOH, 100° C. is used for ring opening) are applied. This situation often leads to racemization, as described in M. Bovarnick & H. T. Clarke, Journal of the American Chemical Society 1938, 60, 2426-2430, RA Lazarus, J. Org. Chem. 1990, 55, 4755-4757, and AS Bommarius, M. Kottenhahn, H. Klenk, K. Drauz: "A direct route from hydantoins to D-amino acids employing a resting cell biocatalyst with D-hydantoinase and D-carbamoylase activity", in "Microbial Reagents in Organic Synthesis" Series C: Mathematical and Physical Sciences-Vol. 381, S. Servi (Ed.), 1992, Springer Science+Business Media, BV, Dordrecht, pages 164 and 167.

[0016] For the enantioselective synthesis of LGA, chemical and enzymatic routes are described in the art.

[0017] WO 2020 / 145513 A1 and WO 2020 / 145514 A1 describe a chemical route to LGA. In this route, L-homoserine derivatives such as O-acetyl L-homoserine or O-succinyl L-homoserine are used as starting materials, and L-phosphinothricin is obtained through a series of reactions including lactonization and halogenation.

[0018] WO 2020 / 145627 A1 describes a similar route, in which bromine derivatives are obtained during the halogenation.

[0019] CN 106083922 A discloses a pathway similar to these synthetic pathways, but starting from L-methionine.

[0020] CN 108516991 A describes another route to the synthesis of LGA starting from the azeotropic dehydration of L-homoserine to give L-3,6-di(2-haloethyl)-2,5-diketopiperazine, followed by the introduction of a methylphosphonic acid diester group and hydrolysis.

[0021] WO 2017 / 151573 A1 discloses a two-step enzymatic synthesis of LGA from D-phosphinothricin. In the first step, D-phosphinothricin is oxidatively deaminated to obtain 2-oxo-4-[hydroxy(methyl)phosphono]-butyric acid ("PPO"), and then as a second step, PPO is specifically aminated to LGA. The first step is catalyzed by D-amino acid oxidase, and the second step is catalyzed by transaminase.

[0022] WO 2020 / 051188 A1 discloses a similar method for converting racemic glufosinate to the L-enantiomer. In addition, it discloses a step in which the α-keto acid or ketone byproduct formed during the amination of PPO with an amine donor is converted by ketoglutarate decarboxylase, thereby further shifting the equilibrium toward LGA.

[0023] WO 2019 / 018406 A1 discloses a method for purifying LGA from a mixture comprising LGA and glutamate. Glutamic acid is enzymatically converted to pyroglutamic acid by glutamyl-peptidyl cyclotransferase, and then LGA is purified from the resulting mixture using an ion exchange resin.

[0024] WO 2013 / 072486 A1 discloses hydantoinase mutants having higher activity towards D-amino acids.

[0025] WO 00 / 58449 A1 discloses hydantoinase mutants having a higher activity towards L-amino acids.

[0026] The object of the present invention is to provide other enzymatic methods for preparing glufosinate or glufosinate P-ester. The method will provide a product of L-enantiomer with high excess relative to the D-enantiomer. In addition, the method will allow the use of new substrates that have not been used for the enzymatic synthesis of glufosinate or glufosinate ester so far. In addition, the art needs a kind of enantiomeric selectivity method for preparing L-glufosinate or L-glufosinate ester from the starting material (such as racemic mixture) comprising D- and L-enantiomers with a minimum number of synthesis steps. 2. Summary of the invention

[0027] That is, it was surprisingly found that the desired L-phosphinothricin P-ester can be prepared from the corresponding carbamoyl compound of formula L-(II) by an enzyme-catalyzed reaction:

[0028]

[0029] Where R 2L is a group selected from the following formulae (VIII), (IX), (X), preferably a group selected from the following formulae (IX), (X), more preferably R 2L Has the following formula (IX):

[0030]

[0031] When R 2L When the group is a group of formula (VIII), the group R 2L It can be selected from the formula L-(VIII) and D-(VIII), and preferably has the formula of L-(VIII):

[0032]

[0033] When R 2L When the group is a group of formula (IX), the group R 2L It can be selected from the formula L-(IX) and D-(IX), and preferably has the formula of L-(IX):

[0034]

[0035] When R 2L When the group is a group of formula (X), the group R 2L It can be selected from the formula L-(X) and D-(X), and preferably has the formula of L-(X):

[0036]

[0037] Previously, such carbamylated compounds have never been used for the enzymatic preparation of L-phosphinothricin or its P ester. This finding is even more surprising because it proves that the presence of a group selected from the following formulae (VIII), (IX), (X) is essential because R 2L =H of the compound of formula L-(II) has no corresponding reaction.

[0038] α.1) Therefore, the present invention solves the above-mentioned problem by providing a method for preparing glufosinate P-ester of formula L-(I) in the first aspect, the method comprising step (c), in which the compound of formula L-(II) is reacted to obtain a compound of formula L-(I):

[0039]

[0040] Where R 1L is a group selected from the above formula (VIII), (IX), (X), preferably a group selected from the above formula (IX), (X), more preferably R 1L Having formula (X).

[0041] When R 1L When the group is a group of formula (VIII), the group R 1L It can be selected from the above formulae L-(VIII) and D-(VIII), preferably L-(VIII).

[0042] When R 1L When the group is a group of formula (IX), the group R 1L It can be selected from the above formulae L-(IX) and D-(IX), preferably L-(IX).

[0043] When R 1L When the group is a group of formula (X), the group R 1L It can be selected from the above formulae L-(X) and D-(X), preferably L-(X).

[0044] The reaction of step (c) is catalyzed by a carbamoylase E1, preferably by an L-enantiomerically selective carbamoylase E1 ("L-enantiomerically selective carbamoylase"="L-carbamoylase").

[0045] The L-carbamoylase El is preferably classified in class EC 3.5.1.87.

[0046] The polypeptide sequence of L-carbamoylase E1 is preferably selected from SEQ ID NO: 1 and variants thereof, SEQ ID NO: 2 and variants thereof, SEQ ID NO: 3 and variants thereof, SEQ ID NO: 4 and variants thereof, SEQ ID NO: 5 and variants thereof, SEQ ID NO: 6 and variants thereof, SEQ ID NO: 7 and variants thereof, SEQ ID NO: 8 and variants thereof, SEQ ID NO: 9 and variants thereof.

[0047] α.2) In a preferred embodiment of the method of the first aspect of the present invention, the compound of formula L-(II) used in step (c) is obtained by step (b), in which the compound of formula L-(III) is reacted to obtain the compound of formula L-(II):

[0048]

[0049] The reaction of step (b) is catalyzed by a hydantoinase E2, preferably by an L-enantiomerically selective hydantoinase E2 ("L-enantiomerically selective hydantoinase"="L-hydantoinase").

[0050] R 3L is a group selected from the above formula (VIII), (IX), (X), preferably a group selected from the above formula (VIII), (IX), more preferably R 3L It has the formula (VIII).

[0051] When R 3L When the group is a group of formula (VIII), the group R 3L It can be selected from the above formulae L-(VIII) and D-(VIII), preferably L-(VIII).

[0052] When R 3L When the group is a group of formula (IX), the group R 3L It can be selected from the above formulae L-(IX) and D-(IX), preferably L-(IX).

[0053] When R 3L When the group is a group of formula (X), the group R 3L It can be selected from the above formulae L-(X) and D-(X), preferably L-(X).

[0054] Hydantoinase E2, in particular L-hydantoinase E2, is preferably classified in class EC 3.5.2.2.

[0055] The polypeptide sequence of hydantoinase E2, in particular L-hydantoinase E2, is preferably selected from SEQ ID NO: 10 and variants thereof, SEQ ID NO: 11 and variants thereof, SEQ ID NO: 12 and variants thereof, SEQ ID NO: 13 and variants thereof, SEQ ID NO: 14 and variants thereof, SEQ ID NO: 15 and variants thereof, SEQ ID NO: 16 and variants thereof.

[0056] α.3) In a more preferred embodiment of the method of the first aspect of the present invention, the compound of formula L-(III) used in step (b) is obtained by step (a), in which the compound of formula D-(III) is reacted to obtain the compound of formula L-(III):

[0057]

[0058] The reaction of step (a) is preferably catalyzed by hydantoin racemase E3.

[0059] R 3D is a group selected from the above formula (VIII), (IX), (X), preferably a group selected from the above formula (VIII), (IX), more preferably R 3D It has the formula (VIII).

[0060] When R3D When the group is a group of formula (VIII), the group R 3D It can be selected from the above formulae L-(VIII) and D-(VIII), preferably L-(VIII).

[0061] When R 3D When the group is a group of formula (IX), the group R 3D It can be selected from the above formulae L-(IX) and D-(IX), preferably L-(IX).

[0062] When R 3D When the group is a group of formula (X), the group R 3D It can be selected from the above formulae L-(X) and D-(X), preferably L-(X).

[0063] The hydantoin racemase E3 is preferably classified into class EC 5.1.99.5.

[0064] The polypeptide sequence of hydantoin racemase E3 in step (a) is preferably selected from SEQ ID NO: 17 and variants thereof, SEQ ID NO: 18 and variants thereof, SEQ ID NO: 19 and variants thereof, SEQ ID NO: 20 and variants thereof, SEQ ID NO: 21 and variants thereof, SEQ ID NO: 22 and variants thereof, SEQ ID NO: 23 and variants thereof, SEQ ID NO: 24 and variants thereof, SEQ ID NO: 25 and variants thereof, SEQ ID NO: 26 and variants thereof.

[0065] It has also surprisingly been found that the above findings can be advantageously used in a process for the selective preparation of L-phosphinothricin P-ester, in particular from a mixture of stereoisomers, preferably from a mixture of enantiomers, such as a racemic mixture of enantiomers.

[0066] β.1) Therefore, the present invention relates in a second aspect to a process for preparing L-phosphinothricin P-ester of formula L-(I):

[0067]

[0068] Where R 1L is a group selected from the above formula (VIII), (IX), (X), preferably a group selected from the above formula (IX), (X), more preferably R 1L Having formula (X).

[0069] When R 1L When the group is a group of formula (VIII), the group R 1L It can be selected from the above formulae L-(VIII) and D-(VIII), preferably L-(VIII).

[0070] When R1L When the group is a group of formula (IX), the group R 1L It can be selected from the above formulae L-(IX) and D-(IX), preferably L-(IX).

[0071] When R 1L When the group is a group of formula (X), the group R 1L It can be selected from the above formulae L-(X) and D-(X), preferably L-(X).

[0072] In this method, a mixture M comprising at least one compound L-(III) and at least one compound D-(III) is provided. IIIA [Step (i)-A]:

[0073]

[0074] R 3L is a group selected from the above formula (VIII), (IX), (X), preferably a group selected from the above formula (VIII), (IX), more preferably R 3L It has the formula (VIII).

[0075] When R 3L When the group is a group of formula (VIII), the group R 3L It can be selected from the above formulae L-(VIII) and D-(VIII), preferably L-(VIII).

[0076] When R 3L When the group is a group of formula (IX), the group R 3L It can be selected from the above formulae L-(IX) and D-(IX), preferably L-(IX).

[0077] When R 3L When the group is a group of formula (X), the group R 3L It can be selected from the above formulae L-(X) and D-(X), preferably L-(X).

[0078] R 3D is a group selected from the above formula (VIII), (IX), (X), preferably a group selected from the above formula (VIII), (IX), more preferably R 3D It has the formula (VIII).

[0079] When R 3D When the group is a group of formula (VIII), the group R 3D It can be selected from the above formulae L-(VIII) and D-(VIII), preferably L-(VIII).

[0080] When R 3D When the group is a group of formula (IX), the group R 3DIt can be selected from the above formulae L-(IX) and D-(IX), preferably L-(IX).

[0081] When R 3D When the group is a group of formula (X), the group R 3D It can be selected from the above formulae L-(X) and D-(X), preferably L-(X).

[0082] β.2) In step (ii), the mixture M IIIA Reaction with a hydantoinase E2, which is more preferably as defined in item α.2), to obtain a composition M comprising at least one compound of formula L-(II) and optionally at least one compound of formula D-(II) II , wherein L-(II) and D-(II) have the following formula:

[0083]

[0084] R 2L is a group selected from the above formula (VIII), (IX), (X), preferably a group selected from the above formula (IX), (X), more preferably R 2L It has the formula (IX).

[0085] R 2D is a group selected from the above formula (VIII), (IX), (X), preferably a group selected from the above formula (IX), (X), more preferably R 2D It has the formula (IX).

[0086] When R 2L When the group is a group of formula (VIII), the group R 2L It can be selected from the above formulae L-(VIII) and D-(VIII), preferably L-(VIII).

[0087] When R 2D When the group is a group of formula (VIII), the group R 2D It can be selected from the above formulae L-(VIII) and D-(VIII), preferably L-(VIII).

[0088] When R 2L When the group is a group of formula (IX), the group R 2L It can be selected from the above formulae L-(IX) and D-(IX), preferably L-(IX).

[0089] When R 2D When the group is a group of formula (IX), the group R 2D It can be selected from the above formulae L-(IX) and D-(IX), preferably L-(IX).

[0090] When R 2L When the group is a group of formula (X), the group R2L It can be selected from the above formulae L-(X) and D-(X), preferably L-(X).

[0091] When R 2D When the group is a group of formula (X), the group R 2D It can be selected from the above formulae L-(X) and D-(X), preferably L-(X).

[0092] β.3) Then, in a further step (iii), M II A reaction is carried out, which is catalyzed by an L-carbamoylase E1, preferably as defined in item α.1), to obtain a mixture comprising at least one compound of formula L-(I) and optionally at least one compound of formula D-(I) I , wherein D-(I) has the following formula:

[0093]

[0094] Where R 1D is a group selected from the above formula (VIII), (IX), (X), preferably a group selected from the above formula (IX), (X), more preferably R 1D Having formula (X).

[0095] When R 1D When the group is a group of formula (VIII), the group R 1D It can be selected from the above formulae L-(VIII) and D-(VIII), preferably L-(VIII).

[0096] When R 1D When the group is a group of formula (IX), the group R 1D It can be selected from the above formulae L-(IX) and D-(IX), preferably L-(IX).

[0097] When R 1D When the group is a group of formula (X), the group R 1D It can be selected from the above formulae L-(X) and D-(X), preferably L-(X).

[0098] The process of the second aspect of the invention is enantioselective because M I Contains only L-(I), or in M I In the case where both stereoisomers L-(I) and D-(I) are included, M I The molar ratio of L-(I) to D-(I) is greater than M IIIA The molar ratio of L-(III) to D-(III).

[0099] β.4) In addition, surprisingly, it was found that the enantioselective preparation method according to the second aspect of the present invention can be further improved. That is, in the optional step (iB) performed after step (iA) [which can be performed before or simultaneously with steps (ii) and (iii), preferably simultaneously with steps (ii) and (iii)], the mixture M IIIA At least a portion of the compound D-(III) contained is reacted, preferably catalyzed by a hydantoin racemase E3 enzyme, to produce an isomer L-(III) of D-(III), thereby obtaining a composition M comprising L-(III) and optionally an isomer D-(III) thereof. IIIB .

[0100] If the reaction of step (iB) is enzymatically catalyzed, it is preferably catalyzed by hydantoin racemase E3.

[0101] The hydantoin racemase E3 is preferably as defined in item α.3). 3. Description of the drawings

[0102] Figure 1 The pOM21c plasmid map is shown.

[0103] Figure 2 The pOM22c plasmid map is shown. 4. Detailed description of the invention

[0105] 4.1 Definitions

[0106] Any enzyme used according to any aspect of the present invention may be an isolated enzyme. In particular, the enzyme used according to any aspect of the present invention may be used in an active state and in the presence of all cofactors, substrates, auxiliary and / or activating factors necessary for the polypeptide or its activity. These factors may be metal ions, such as Mn 2+ or Co 2+ .

[0107] Specifically, the enzyme according to the present application may be carbamoylase E1, hydantoinase E2 or hydantoin racemase E3.

[0108] "Enzyme catalyzed" means that the corresponding reaction is catalyzed by an enzyme, which may be carbamoylase E1, hydantoinase E2 or hydantoin racemase E3.

[0109] The enzyme used according to any aspect of the present invention may be recombinant. The term "recombinant" as used herein refers to a molecule or a molecule encoded by such a molecule, particularly a polypeptide or nucleic acid, which itself is not naturally occurring but is the result of genetic engineering, or to a cell containing the recombinant molecule. For example, if a nucleic acid molecule comprises a promoter functionally associated with a sequence encoding a catalytically active polypeptide, and the promoter has been engineered so that the catalytically active polypeptide is overexpressed relative to the polypeptide level in a corresponding wild-type cell containing the original unaltered nucleic acid molecule, then the nucleic acid molecule is recombinant.

[0110] A "polypeptide" (one or more peptides) is a chain of chemical building blocks called amino acids, linked together by chemical bonds called peptide bonds. A protein or polypeptide, including an enzyme, may be "native" or "wild-type," meaning that it occurs in nature or has the amino acid sequence of a naturally occurring protein, respectively. These terms are sometimes used interchangeably. A polypeptide may or may not be glycosylated.

[0111] As used herein, the term "overexpression" refers to an expression level or activity of a corresponding polypeptide that is encoded or expressed that is higher than the level or activity normally found in a cell under the same conditions, in the absence of genetic modification to increase expression, such as in a corresponding wild-type cell.

[0112] 4.2 Methods for obtaining enzymes

[0113] The enzymes that can be used in the methods of the present invention can be synthesized by methods known to the skilled person.

[0114] A preferred method according to the invention is to express the enzyme in a microorganism such as Escherichia coli (= "E. coli"), Saccharomyces cerevisiae, Pichia pastoris, etc., and to add the whole cell as a whole cell biocatalyst to the reaction. Another method is to express the enzyme, lyse the microorganism, and add the cell lysate. Another method is to purify or partially purify the enzyme from the lysate and to add the pure or partially purified enzyme to the reaction. If a reaction requires multiple enzymes, these enzymes can be expressed in one or several microorganisms, including expressing all enzymes in a single microorganism.

[0115] For example, the technician can obtain the enzyme according to the invention by expressing, in particular overexpressing [hereinafter, "expression, in particular overexpression" is abbreviated as (over) expression, and "expression, in particular overexpression" is abbreviated as (over) expression] these enzymes in cells and then isolating them, for example as described in DE 100 31 999A1. For example, episomal plasmids are used to increase the expression of each gene. In such plasmids, the nucleic acid molecule to be (over) expressed or encoding the polypeptide or enzyme to be (over) expressed can be placed under the control of a strong inducible promoter such as the lac promoter upstream of the gene. The promoter is a DNA sequence consisting of about 40 to 50 base pairs, which constitutes the binding site and transcription start point of the RNA polymerase holoenzyme (M. Pátek, J. Holátko, T. Busche, J. Kalinowski, J. Microbial Biotechnology 2013, 6, 103-117), thereby affecting the expression intensity of the controlled polynucleotide or gene. "Functional connection" is obtained by the sequential arrangement of the promoter and the gene, which leads to the transcription of the gene.

[0116] Suitable strong promoters for increasing expression or methods for producing such promoters are known from the literature (e.g. S. Lisser & H. Margalit, Nucleic Acid Research 1993, 21, 1507-1516; M. Pátek and J. Nesvera in H. Yukawa and M Inui (eds.), Corynebacterium glutamicum, Microbiology Monographs 23, Springer Verlag Berlin Heidelberg 2013, 51-88; BJ Eikmanns, E. Kleinertz, W. Liebl, H. Sahm, Gene 1991, 102, 93-98). For example, natural promoters can be optimized by changing the promoter sequence in the direction of known consensus sequences that increase the expression of genes linked to the function of these promoters (M. Pátek, B. J. Eikmanns, J. Pátek, H. Sahm, Microbiology 1996, 142, 1297-1309; M. Pátek, J. Holátko, T. Busche, J. Kalinowski, J. Microbial Biotechnology 2013,6,103-117).

[0117] Constitutive promoters are also suitable for (over) expression, in which genes encoding enzyme activity are continuously expressed under the control of promoters such as glucose-dependent deo promoters. Chemically induced promoters are also suitable, such as tac, lac, rha or trp. The most extensive system for inducing promoters is the lac operon of E. coli. In this case, lactose or isopropyl β-D-thiogalactopyranoside (IPTG) is used as an inducer. In addition, systems using arabinose (e.g., pBAD system) or rhamnose (e.g., E. coli KRX) as inducers are common. Systems for physical induction are, for example, temperature-induced cold shock promoter systems based on E. coli cspA promoters from Takara or Lambda PL and osmotic inducible promoters such as osmB (e.g., WO 95 / 25785A1).

[0118] Suitable plasmids or vectors are in principle all embodiments that can be used for this purpose by those skilled in the art. The prior art describes standard plasmids that can be used for this purpose, such as the pET system (commercially available from Novagen) of the vectors illustrated by pET-3a or pET-26b (+). Further plasmids and vectors can be taken from, for example, pOM21 described in WO 2004 / 111227 A2, pOM22 described in WO 00 / 058449A1 or pOM18 described in WO 2013 / 072486 A1, or from the brochures of Novagen, Promega, New England Biolabs, Clontech or Gibco BRL. Further preferred plasmids and vectors can be found in the following literature: Glover, DM (1985) DNA cloning: a practical approach, Vol. I-III, IRL Press Ltd., Oxford; Rodriguez, RL and Denhardt, DT (eds) (1988) Vectors: a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham; Goeddel, DV (1990) Systems for heterologous gene expression, Methods Enzymol. 185, 3-7; Sambrook, J.; Fritsch, EF and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York. Among these plasmids, pOM21 and pOM22 are preferred.

[0119] The plasmid vector containing the gene to be amplified is then transformed into the desired strain, for example, by conjugation or transformation. Conjugation methods such as A. J. Kalinowski, A. Pühler, Applied and Environmental Microbiology 1994, 60, 756-759. Transformation methods are described, for example, in G. Thierbach, A. Schwarzer, A. Pühler, Applied Microbiology and Biotechnology 1988, 29, 356-362, LK Dunican & E. Shivnan, Bio / Technology 1989, 7, 1067-1070 and A. Tauch, O. Kirchner, L. Wehmeier, J. Kalinowski, A. Pühler, FEMS Microbiology Letters 1994, 123, 343-347. After homologous recombination by a "crossover" event, the resulting strain contains at least two copies of the relevant gene.

[0120] The desired enzyme can be isolated by crushing cells containing the desired activity in a manner known to those skilled in the art, for example by means of a ball mill, a French press or an ultrasonic pulverizer, followed by separation of cells, cell fragments and a crushing aid, such as glass beads, by centrifugation at 13,000 rpm and 4° C. for 10 minutes. The resulting cell-free crude extract can then be subjected to enzyme analysis and subsequent LC-ESI-MS detection of the product. Alternatively, the enzyme can be enriched in a manner known to those skilled in the art by chromatographic methods (e.g., nickel-nitrile triacetic acid affinity chromatography, streptavidin affinity chromatography, gel filtration chromatography or ion exchange chromatography), or purified to homogeneity. The quantification of the enzyme can be carried out by methods known to those skilled in the art, for example, by determining the concentration of the corresponding polypeptide of the enzyme (e.g., carbamoylase, hydantoinase and racemase) in the obtained solution by SDS PAGE, and by software The corresponding bands were analyzed by DNA sequencing (Biochem Lab Solutions).

[0121] In addition, whether nucleic acid or polypeptide is (over) expressed can be determined by quantitative PCR reaction (for nucleic acid molecules), SDS polyacrylamide electrophoresis, Western blotting or comparative activity assay (for polypeptides).Genetic modification can refer to transcription, translation and / or post-translational modification, which results in changes in enzyme activity and / or selectivity under selected and / or identified culture conditions.

[0122] 4.3 Variants

[0123] In the context of the present invention, the term "variant" with respect to a polypeptide sequence refers to a polypeptide having a degree of identity ("sequence identity") of at least 60%, preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.9% to the reference sequence. In further embodiments, the degree of identity is at least 98.0%, more preferably at least 98.2%, more preferably at least 98.4%, more preferably at least 98.6%, more preferably at least 98.8%, more preferably at least 99.0%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8% or at least more preferably at least 99.9%.

[0124] It goes without saying that a "variant" of a polypeptide sequence is not identical to that polypeptide sequence.

[0125] The variants can be prepared by introducing deletions, insertions, substitutions or a combination thereof, particularly in the amino acid sequence, as well as fusions comprising such macromolecules or variants thereof.

[0126] The person skilled in the art knows modifications of the amino acid residues of a given polypeptide sequence which do not lead to significant changes in the properties and functions of a given polypeptide. Thus, for example, many amino acids can often be interchanged with one another without problems; examples of such suitable amino acid substitutions are: Ala is replaced by Ser; Arg is replaced by Lys; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Pro; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Met or Val; Lys is replaced by Arg or Gln or Glu; Met is replaced by Leu or Ile; Phe is replaced by Met or Leu or Tyr; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp or Phe; Val is replaced by Ile or Leu. It is also known that modifications, particularly at the N- or C-terminus of a polypeptide, in the form of, for example, amino acid insertions or deletions, generally have no significant effect on the function of the polypeptide.

[0127] According to this, preferred variants according to the invention of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, respectively, have a polypeptide sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 complete polypeptide sequence,Or at least contain the amino acids of the corresponding sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 that are essential for the function, such as the catalytic activity of the protein or the folding or structure of the protein. Other amino acids may be deleted, substituted or replaced by insertion, or essential amino acids may be substituted in a conservative manner, the effect of which is to maintain the activity of the enzyme, in particular the activity of L-carbamoylase (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9), hydantoinase (SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16), hydantoin racemase (SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26).

[0128] 3.4 Sequence identity

[0129] Those skilled in the art will be aware that a variety of computer programs are available for calculating the similarity or identity between two nucleotide or polypeptide sequences.

[0130] Preferred methods for determining sequence identity initially produce maximum alignment between the sequences being compared. Computer programs for determining sequence identity include, but are not limited to, the GCG program package, which includes

[0131] -GAP [J. Deveroy et al., Nucleic Acid Research 1984, 12, page 387, Genetics Computer Group University of Wisconsin, Medicine (WI)], and

[0132] - BLASTP, BLASTN and FASTA (S. Altschul et al., Journal of Molecular Biology 1990, 215, 403-410). The BLAST program is available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Handbook, S. Altschul et al., NCBI NLM NIH Bethesda ND 22894; S. Altschul et al., see above).

[0133] For example, the percent identity between two polypeptide sequences can be determined by the algorithm developed by SB Needleman & C.D. Wunsch, J. Mol. Biol. 1970, 48, 443-453, which has been integrated into the GAP program in the GCG software package, using the BLOSUM62 matrix or the PAM250 matrix, with a gap weight of 16, 14, 12, 10, 8, 6 or 4, and a length weight of 1, 2, 3, 4, 5 or 6. Those skilled in the art will recognize that using different parameters will result in slightly different results, but the percent identity between the two polypeptides will not be significantly different overall. The BLOSUM62 matrix is ​​usually used with default settings (gap weight: 12, length weight: 1).

[0134] In the context of the present invention, a sequence identity of 60% according to the above algorithm means 60% homology. The same applies to higher sequence identities.

[0135] Most preferably, in the context of the present invention, the degree of identity between sequences is determined by the program "Needle" using the substitution matrix BLOSUM62, a gap opening penalty of 10 and a gap extension penalty of 0.5. The Needle program implements the overall alignment algorithm described in SB Needleman & C.D.Wunsch, J.Mol.Biol.1970,48,443-453. The substitution matrix used according to the present invention is BLOSUM62, a gap opening penalty of 10, and a gap extension penalty of 0.5. The preferred version used in the context of the present invention is the one proposed by F. Madeira, YM Park, J. Lee, N. Buso, T. Gur, N. Madhusoodanan, P. Basutkar, ARN Tivey, S Cotter, R D Finn, Nucleic Acids Research 2019, 47, W636–W641, Web Server issue (preferred version accessible online on March 4, 2022, at the following URL https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ).

[0136] In a specific embodiment, the percent identity of a polypeptide's amino acid sequence to a reference polypeptide sequence is determined by:

[0137] i) The two amino acid sequences were aligned using the Needle program, the BLOSUM62 substitution matrix was used, the gap opening penalty was 10, and the gap extension penalty was 0.5;

[0138] ii) counting the number of exact matches in the alignment;

[0139] iii) dividing the number of exact matches by the length of the longest of the two amino acid sequences, and

[0140] iv) Convert the result of division in iii) into a percentage.

[0141] 4.5 First aspect: Preparation method of glufosinate P-ester

[0142] 4.5.1 Step (c)

[0143] 4.5.1.1 Enzyme catalysis in step (c)

[0144] The present invention relates in a first aspect to a method for preparing L-phosphinothricin P-ester of formula L-(I):

[0145]

[0146] The method according to the first aspect of the present invention comprises step (c).

[0147] In step (c), a compound of formula L-(II) is reacted to obtain a compound of formula L-(I). Formula L-(II) has the following formula:

[0148]

[0149] The reaction of step (c) is enzymatically catalyzed, ie, it is catalyzed by carbamoylase E1.

[0150] R 1L is a group selected from the group consisting of formula (VIII), (IX), (X), preferably a group selected from the group consisting of formula (IX), (X), more preferably R 1L Having formula (X).

[0151] R 2L is a group selected from the group consisting of formula (VIII), (IX), (X), preferably a group selected from the group consisting of formula (IX), (X), more preferably R 2L Having formula (X).

[0152] Formulas (VIII), (IX), and (X) are as follows:

[0153]

[0154] When R 1L When the group is a group of formula (VIII), the group R 1L It can be selected from the formula L-(VIII) and D-(VIII), preferably L-(VIII).

[0155] When R 1L When the group is a group of formula (IX), the group R 1L It can be selected from formula L-(IX) and D-(IX), preferably L-(IX).

[0156] When R 1L When the group is a group of formula (X), the group R 1L It can be selected from the formula L-(X) and D-(X), preferably L-(X).

[0157] When R 2L When the group is a group of formula (VIII), the group R 2L It can be selected from the formula L-(VIII) and D-(VIII), preferably L-(VIII).

[0158] When R 2L When the group is a group of formula (IX), the group R 2L It can be selected from formula L-(IX) and D-(IX), preferably L-(IX).

[0159] When R 2LWhen the group is a group of formula (X), the group R 2L It can be selected from the formula L-(X) and D-(X), preferably L-(X).

[0160] Formulas L-(VIII), D-(VIII), L-(IX), D-(IX), L-(X) and D-(X) are as follows:

[0161]

[0162] 4.5.1.2 Enantioselective and enantiospecific catalysis in step (c)

[0163] Step (c) of the process according to the first aspect of the invention is preferably L-enantiomerically selective, even more preferably L-enantiomerically specific.

[0164] In such a preferred embodiment, in step (c) in particular the mixture M is used II L-(II), the mixture comprises at least one compound L-(II) and at least one compound D-(II), wherein D-(II) has the following formula:

[0165]

[0166] R 2D is a group selected from the group consisting of formula (VIII), (IX), (X), preferably a group selected from the group consisting of formula (IX), (X), more preferably R 2D Having formula (X).

[0167] When R 2D When the group is a group of formula (VIII), the group R 2D It can be selected from the formula L-(VIII) and D-(VIII), preferably L-(VIII).

[0168] When R 2D When the group is a group of formula (IX), the group R 2D It can be selected from formula L-(IX) and D-(IX), preferably L-(IX).

[0169] When R 2D When the group is a group of formula (X), the group R 2D It can be selected from the formula L-(X) and D-(X), preferably L-(X).

[0170] Mixture M II Preferably, it comprises at least one compound L-(II) and at least one compound D-(II), wherein R 2L =R 2D .

[0171] For such mixtures MII , it can be observed that at least one compound D-(II) undergoes a reaction according to step (c)*, i.e., a reaction in parallel with step (c). That is, in the reaction according to step (c)*, in the mixture M II At least one compound D-(II) is reacted to obtain a compound of formula D-(I):

[0172]

[0173] R 1D is a group selected from the above formula (VIII), (IX), (X), preferably a group selected from the above formula (IX), (X), more preferably R 1D Having formula (X).

[0174] When R 1D When the group is a group of formula (VIII), the group R 1D It can be selected from the above formulae L-(VIII) and D-(VIII), preferably L-(VIII).

[0175] When R 1D When the group is a group of formula (IX), the group R 1D It can be selected from the above formulae L-(IX) and D-(IX), preferably L-(IX).

[0176] When R 1D When the group is a group of formula (X), the group R 1D It can be selected from the above formulae L-(X) and D-(X), preferably L-(X).

[0177] In case step (c) is "L-enantiomerically selective", this means that in step (c) a mixture M is used. II In the case of

[0178] - In the mixture M II In the step (c)* reaction of any compound of formula D-(II) to produce a compound of formula D-(I) does not occur, or,

[0179] - In the case of the reaction of step (c)*, the mixture M II There is at least one pair of compounds D- in produce The reaction rate of step (c)* is produce The reaction rate of step (c) is low.

[0180] is a compound of formula D-(II), is a compound of formula L-(II), wherein The group R 2L and The group R 2D same.

[0181] is a compound of formula D-(I), is a compound of formula L-(I), wherein The group R 2L and The group R 2D same.

[0182] Preferably, The group R 2L and The group R 1L Preferably, The group R 2D and The group R 1D same.

[0183] If the reaction rate of step (c)* is substantially zero, that is, the mixture M II Step (c) is "L-enantiomer-specific" if the reaction of step (c)* to produce a compound of formula D-(I) from any compound of formula D-(II) does not occur.

[0184] In a preferred embodiment, the mixture M II All compounds and mixtures of formula L-(II) II The molar ratio of all compounds of formula D-(II) is substantially 1:1.

[0185] In other preferred embodiments, the mixture M II All compounds and mixtures of formula L-(II) II The molar ratio of all compounds of formula D-(II) is in the range of 3:2 to 1:99, more preferably in the range of 1.01:1 to 1:99, more preferably in the range of 1:1 to 1:99, more preferably in the range of 1:1.01 to 1:99, more preferably in the range of 1:1.01 to 1:9, more preferably in the range of 1:1.01 to 1:8, more preferably in the range of 1:1.01 to 1:3.

[0186] Alternatively, D-(II) in the mixture M II The content of L-(II) exceeds that of L-(II), which means that when at least one compound of formula L-(II) is present in the mixture M II When the mixture M II All compounds and mixtures of formula L-(II) IIThe molar ratio of all compounds of formula D-(II) is <1:1, preferably <0.9:1, more preferably <0.75:1, more preferably <0.5:1, more preferably <0.2:1, more preferably <0.1:1, more preferably <0.01:1.

[0187] If step (c) is catalyzed by L-carbamoylase El, step (c) is in particular L-enantiomerically selective, which can be determined by the skilled person as described in 4.5.4.3.

[0188] In case step (c) is L-enantiomer selective, the compound given is For produce The reaction of step (c) is preferably carried out at a ratio of produce The reaction rate of step (c)* is at least 2 times higher, preferably at least 10 times higher, more preferably at least 100 times higher, even more preferably at least 10 times higher 3 times, even more preferably at least 10 times higher 4 times, even more preferably at least 10 times higher 5 times the reaction rate.

[0189] For a given compound Yes, in order to quantify the factor by which the reaction rate in step (c) is compared to the reaction rate in step (c)*, the following test can be performed:

[0190] (1) An equimolar mixture [two compounds and The molar ratio of 1:1] was placed under the respective reaction conditions and the two products were monitored over time. and Development of (e.g., by LC-MS as described under 5.4).

[0191] (2) When initially used n LI10 = 10 mol% reaction product When the measurement is The reaction formed The amount [relative to the initial use mol%](=n DI10 ).

[0192] (3)n LI10 / n DI10 =10 / n DI10 The ratio of gives the given compound A factor that affects the rate of reaction of step (c) relative to the rate of reaction of step (c)*.

[0193] →If n LI10 / n DI10 >1, then the given compound The reaction rate of step (c) of is greater than the reaction rate of step (c)*.

[0194] →If n LI10 / n DI10 <1, then the given compound The reaction rate of step (c) of is less than the reaction rate of step (c)*.

[0195] →If n LI10 / n DI10 =1, then the given compound The reaction rates of steps (c) and (c)* are the same.

[0196] 4.5.2 Carbamoylase

[0197] The reaction of step (c) of the method according to the first aspect of the present invention is catalyzed by carbamoylase E1.

[0198] That is, surprisingly, it was found that carbamoylase accepts compounds of formula L-(II) as substrates and converts them into product L-(I), which can therefore be used to catalyze the reaction of step (c). This discovery has high scientific and economic value because it opens up new synthetic routes based on new raw materials for the preparation of L-phosphinothricin P-esters and L-phosphinothricin. Even more surprisingly, it was found that L-phosphinothricin carbamoylates, i.e. compounds of L-(II) where R=H, do not undergo the reaction of carbamoylase to produce L-phosphinothricin.

[0199] In practice, carbamoylases usually catalyze the following reaction <1> , where R X It may be an organic group, such as the side chain of one of the naturally occurring amino acids.

[0200] <1> :

[0201] It has now surprisingly been found that carbamoylase also accepts of substrate, where R = R 2L .

[0202] Surprisingly, they do not accept And a substrate wherein R=H.

[0203] In the context of the present invention, "carbamoylase E1" is an enzyme that catalyzes the conversion of carbamoyl substrate S L The corresponding amino acid product P LThe following reaction <1A> carbamoylase, wherein Where R = R 2L .

[0204] <1A>:

[0205] In particular, carbamoylase E1 is an "L-carbamoylase", i.e., it has a greater catalytic activity for reaction <1A> than for reaction <1B>, wherein the substrate S in reaction <1B> D R X and the substrate S in reaction <1A> L R X are the same, and Where R = R 2L .

[0206] <1B>:

[0207] Since the L-carbamoylase has a higher catalytic activity for reaction <1A> than for reaction <1B>, it is "L-enantiomer selective". An L-carbamoylase that has no catalytic activity for reaction <1B> and therefore has catalytic activity only for reaction <1A> is "L-enantiomer specific".

[0208] A "D-carbamoylase" is defined as a carbamoylase that is "D-enantiomerically selective", i.e., it has a higher catalytic activity for reaction <1B> than for reaction <1A>. A D-carbamoylase that does not catalyze reaction <1A> and therefore has catalytic activity only for reaction <1B> is "D-enantiomerically specific".

[0209] A carbamoylase having the same catalytic activity for reaction <1B> and reaction <1A> is referred to as a "diastereoselective carbamoylase".

[0210] To determine whether a carbamoylase in the context of the present invention can be indicated as "L-carbamoylase", "D-carbamoylase" or "non-enantiomerically selective carbamoylase", the method according to Test B (item 4.5.4) can preferably be used.

[0211] The carbamoylase E1, in particular the L-carbamoylase E1, which can be used in step (c) of the method according to the first aspect of the present invention can be derived from Achromobacter sp., in particular Achromobacter xylosoxidans; Agrobacterium sp., in particular Agrobacterium tumefaciens; Arthrobacter sp., in particular Arthrobacter crystallopoietes, Arthrobacter aurescens, Arthrobacter sp. BT801; Bacillus sp., in particular Bacillus fordii; Blastobacter sp.; Bradyrhizobium sp., in particular Bradyrhizobium tumefaciens; japonicum; Brevibacillus sp., in particular Brevibacillus reuszeri; Comamonas sp.; Ensifer sp., in particular Ensifer adhaerens; Flavobacterium sp., Geobacillus sp., in particular Geobacillus kaustophilus, Geobacillus stearothermophilus; Microbacterium sp., in particular Microbacterium liquefaciens strain AJ3912; Paenarthrobacter sp., in particular Paenarthrobacter aurescens; Pasteurella sp. sp.); Pseudomonas sp.; Ralstonia sp., in particular Ralstonia pickettii; Sinorhizobium sp., in particular Sinorhizobium meliloti.

[0212] The L-carbamoylase E1 suitable for the method of the present invention may be the enzyme HyuC derived from Arthrobacter. Other enzymes are AmaB, AtcC, Inc, SinmeB_2280.

[0213] WO 01 / 23582 A1 discloses examples of the enzyme having carbamoylase activity of the present invention.

[0214] The carbamoylase E1 that can be used in step (c) of the method of the present invention may be an L-carbamoylase classified in EC classification EC 3.5.1.87.

[0215] L-carbamoylases are described, for example, in J. Ogawa, H. Miyake, S. Shimizu, Appl. Microbiol. Biotechnol. 1995 43, 1039-1043 and WO 01 / 23582 A1.

[0216] The L-carbamoylase E1 preferably used in step (c) of the first aspect of the present invention can be derived from the genus Arthrobacter, in particular Arthrobacter crystallinum, Arthrobacter golden yellow, Arthrobacter species BT801, Arthrobacter golden yellow DSM 3747; Bacillus, in particular Bacillus fordy; Geobacillus, in particular Geobacillus stearothermophilus, Geobacillus thermophilus; Microbacterium, in particular Microbacterium liquefaciens strain AJ3912; Arthrobacter genus, in particular Arthrobacter golden yellow; Pseudomonas, in particular Pseudomonas species QR-101; Sinorhizobium, in particular Sinorhizobium alfalfa. Even more preferably, the L-carbamoylase E1 preferably used in step (c) of the first aspect of the present invention can be derived from the genus Arthrobacter, in particular Arthrobacter crystallinum, Arthrobacter golden yellow, Arthrobacter species BT801, Arthrobacter golden yellow DSM 3747, most preferably from Arthrobacter golden yellow DSM 3747.

[0217] The individual sequences can be obtained from databases such as Br aunschweig En zyme Da tabase (BRENDA, Germany, its website is www.brenda-enzymes.org / index.php), the N ational C enter for B iotechnological I nformation (NCBI, its website is https: / / www.ncbi.nlm.nih.gov / ) or K yoto Encyclopedia of G enes and G enomes (KEGG, Japan, its website is www.https: / / www.genome.jp / kegg / ).

[0218] Table 1 below gives preferred examples of polypeptide sequences of L-carbamoylase E1, which can be preferably used in step (c) of the method of the first aspect of the present invention. The genes encoding the corresponding L-carbamoylase E1 and the corresponding accession numbers are known.

[0219] Table 1

[0220]

[0221] In a preferred embodiment of the method of the first aspect of the invention, the reaction of step (c) is catalyzed by L-carbamoylase E1, wherein the polypeptide sequence of E1 is selected from SEQ ID NO: 1 and variants thereof, SEQ ID NO: 2 and variants thereof, SEQ ID NO: 3 and variants thereof, SEQ ID NO: 4 and variants thereof, SEQ ID NO: 5 and variants thereof, SEQ ID NO: 6 and variants thereof, SEQ ID NO: 7 and variants thereof, SEQ ID NO: 8 and variants thereof, SEQ ID NO: 9 and variants thereof, preferably SEQ ID NO: 1 and variants thereof.

[0222] 4.5.3 Test A for determining carbamoylase activity L and A D

[0223] The skilled person is aware of carbamoylases, in particular L-carbamoylases, which can be used in step (c) of the process of the first aspect of the invention.

[0224] Specifically, Test A described below L Can be used to determine the given enzyme E X The invention can be advantageously used to determine the carbamoylase and L-carbamoylase activities in variants of SEQ ID NO: 1, variants of SEQ ID NO: 2, variants of SEQ ID NO: 3, variants of SEQ ID NO: 4, variants of SEQ ID NO: 5, variants of SEQ ID NO: 6, variants of SEQ ID NO: 7, variants of SEQ ID NO: 8, and variants of SEQ ID NO: 9.

[0225] For comparison reasons, Experiment A D Can be used to determine the E XD-carbamoylase activity.

[0226] To test A L and Test A D For the purpose of testing, the enzyme E X The molar mass is calculated as E X The molar mass of the polypeptide sequence.

[0227] In addition, the substrate of formula L-(II), wherein R 2L = n-butyl, used in test A L Therefore, there is only one chiral carbon atom in the substrate. The compound of formula L-(II), wherein R 2L = n-butyl, expressed as

[0228] The product of formula L-(I), wherein R 1L = n-butyl, in test A L Detected in.

[0229] A compound of formula L-(I), wherein R 1L = n-butyl, expressed as

[0230] In addition, the substrate of formula D-(II), wherein R 2D = n-butyl, used in test A D , so there is only one chiral carbon atom in the substrate. The compound of formula D-(II), wherein R 2D = n-butyl, expressed as

[0231] The product of formula D-(I), wherein R 1D = n-butyl, in test A D Detected in.

[0232] A compound of formula D-(I), wherein R 1D = n-butyl, expressed as

[0233] 4.5.3.1 Test A L :

[0234] Add 0.9 ml of 50 mM To the reaction aqueous solution of the compound (phosphate buffer, pH 7.2, 10 mM MnCl2) was added 400 nmol of E in 0.1 ml of phosphate buffer (pH 7.2, 10 mM MnCl2). X The resulting solution was incubated at 25°C and the pH was maintained at pH 7.2 by adding 0.5 M NaOH. After 300 min, the reaction was terminated by adding 2 M HCl to obtain a pH of 2.5 and the formula was determined. The molar amount of the corresponding compound. It can be detected by the LC-MS method described in the Examples section (item 5.4) for detecting LGA.

[0235] 4.5.3.2 Test A D :

[0236] Add 0.9 ml of 50 mM To the reaction aqueous solution of the compound (phosphate buffer, pH 7.2, 10 mM MnCl2) was added 400 nmol of E in 0.1 ml of phosphate buffer (pH 7.2, 10 mM MnCl2). X The resulting solution was incubated at 25°C and the pH was maintained at pH 7.2 by adding 0.5 M NaOH. After 300 min, the reaction was terminated by adding 2 M HCl to obtain a pH of 2.5 and the formula was determined. The molar amount of the corresponding compound. It can be detected by the LC-MS method described in the Examples section (item 5.4) for detecting LGA.

[0237] 4.5.4 Test B for the identification of carbamoylase, L-carbamoylase, D-carbamoylase, L- and D-enantiomer specificity

[0238] The carbamoylase E1 according to the present invention is preferably an L-carbamoylase, more preferably L-enantiomer-specific.

[0239] A given enzyme E x Whether a enzyme can be regarded as a carbamoylase E1, in particular an L-carbamoylase, can be determined in the context of the present invention by the following test B.

[0240] 4.5.4.1 Test B:

[0241] B-1. First, perform test A described in 4.5.3.1. L , and according to test A L The obtained formula The molar amount of the compound.

[0242] B-2. Next, perform test A as described in 4.5.3.2. D , and according to test A D The obtained formula The molar amount of the compound.

[0243] B-3. ​​Then, step B-1 was repeated, except that 400 nmol E in 0.1 ml of phosphate buffer solution (pH 7.2, 10 mM nCl2) was not added. x , but add x 0.1 ml of phosphate buffered saline (pH 7.2, 10 mM MnCl2).

[0244] B-4. Then, step B-2 was repeated, except that 400 nmol E in 0.1 ml of phosphate buffer solution (pH 7.2, 10 mM nCl2) was not added. x , but add x 0.1 ml of phosphate buffered saline (pH 7.2, 10 mM MnCl2).

[0245] 4.5.4.2 Carbamoylase activity

[0246] If the formula determined in step B-1 is The molar amount of the compound is greater than the formula The molar amount of the compound, then E x It is considered to have carbamoylase activity and can therefore be considered to be carbamoylase E1 in the context of the present invention.

[0247] 4.5.4.3 L-Carbamoylase Activity

[0248] 4.5.4.3.1 L-Carbamoylase

[0249] (i) If the formula determined in step B-1 is The molar amount of the compound is greater than the formula The molar amount of the compound, and

[0250] (ii) If the formula determined in step B-2 is The molar amount of the compound is greater than or equal to the formula The molar amount of the compound, and

[0251] (iii) In addition, if the formula determined in step B-1 is The molar amount of the compound is greater than the formula The molar amount of the compound,

[0252] → Then E x is considered to have L-carbamoylase activity and can therefore be considered to be an L-carbamoylase in the context of the present invention. x In the context of the present invention this is considered to be "L-enantiomerically selective".

[0253] For the sake of clarity, it is indicated that the formula determined in step B-2 The molar amount of the compound is the same as that of the formula determined in step B-4. When the molar amounts of the compounds are the same, condition (iii) is automatically satisfied.

[0254] (i) If the formula determined in step B-1 is The molar amount of the compound is greater than the formula The molar amount of the compound, and

[0255] (ii) If the formula determined in step B-2 is The molar amount of the compound is the same as that of the formula determined in step B-4. The molar amounts of the compounds are the same.

[0256] → Then E x is considered to have L-carbamoylase activity and can therefore be considered to be an L-carbamoylase in the context of the present invention. x Not only "L-enantiomerically selective", but also "L-enantiomerically specific" in the context of the present invention.

[0257] For L-carbamoylase E which is not L-enantiomer specific x , which can then be obtained by converting the formula determined in step B-1 The molar amount of the compound is divided by the formula determined in step B-2 The molar amount of the compound is then multiplied by 100 to obtain E in %. x The enantiomeric selectivity of the L-enantiomer was measured to quantify the L-enantiomer selectivity.

[0258] 4.5.4.3.2 D-Carbamoylase

[0259] (i) If the formula determined in step B-1 is The molar amount of the compound is greater than or equal to the formula The molar amount of the compound, and

[0260] (ii) If the formula determined in step B-2 is The molar amount of the compound is greater than the formula The molar amount of the compound, and

[0261] (iii) In addition, if the formula determined in step B-2 is The molar amount of the compound is greater than the formula The molar amount of the compound,

[0262] → Then E x is considered to have D-carbamoylase activity and can therefore be considered a D-carbamoylase. In this case, E x In the context of the present invention it is "D-enantiomerically selective".

[0263] For the sake of clarity, it is indicated that the formula determined in step B-1 The molar amount of the compound is the same as that of the formula determined in step B-3. When the molar amounts of the compounds are the same, condition (iii) is automatically satisfied.

[0264] (i) If the formula determined in step B-1 is The molar amount of the compound is the same as that of the formula determined in step B-3. The molar amounts of the compounds are the same, and

[0265] (ii) If the formula determined in step B-2 is The molar amount of the compound is greater than the formula The molar amount of the compound,

[0266] → Then E x is considered to have D-carbamoylase activity and can therefore be considered to be a D-carbamoylase. In this case, in the context of the present invention, E x Not only "D-enantiomer selective", but also "D-enantiomer specific".

[0267] For D-carbamoylase E which is not D-enantiomer specific x , the formula determined in step B-2 can be The molar amount of the compound is divided by the formula determined in step B-1 The molar amount of the compound is then multiplied by 100 to obtain E in %. x The D-enantiomer selectivity of the reaction was measured to quantify the D-enantiomer selectivity.

[0268] 4.5.4.3.3 Non-enantiomerically selective carbamoylases

[0269] (i) If the formula determined in step B-1 is The molar amount of the compound is greater than the formula The molar amount of the compound, and

[0270] (ii) If the formula determined in step B-2 is The molar amount of the compound is greater than the formula The molar amount of the compound, and

[0271] (iii) In addition, if the formula determined in step B-2 is The molar amount of the compound is the same as that of the formula determined in step B-1. The molar amounts of the compounds are the same.

[0272] → Then E x In the context of the present invention it is considered to be a non-enantiomerically selective carbamoylase. x is "diastereoselective".

[0273] 4.5.5 Test C for Identifying Preferred Carbamoylase Variants of SEQ ID NOs: 1-9

[0274] 4.5.5.1 L- and D-enantiomerically selective carbamoylase variants

[0275] The enzyme whose polypeptide sequence is selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 has carbamoylase and L-carbamoylase activities.

[0276] In a preferred embodiment of the method of the first aspect of the present invention, the polypeptide sequence of L-carbamoylase E1 is selected from SEQ ID NO: 1 and variants thereof, SEQ ID NO: 2 and variants thereof, SEQ ID NO: 3 and variants thereof, SEQ ID NO: 4 and variants thereof, SEQ ID NO: 5 and variants thereof, SEQ ID NO: 6 and variants thereof, SEQ ID NO: 7 and variants thereof, SEQ ID NO: 8 and variants thereof, SEQ ID NO: 9 and variants thereof, more preferably SEQ ID NO: 1 and variants thereof.

[0277] The term "variant" is defined in Section 4.3.

[0278] In the context of the present invention, the polypeptide sequence of enzyme E1 is a variant of one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, which has carbamoylase activity, preferably L-carbamoylase activity, more preferably L-enantiomer specific.

[0279] For a given enzyme E X, whose polypeptide sequence is a variant of one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, whether it has carbamoylase activity, L-carbamoylase activity and / or L-enantiomer specificity can be determined according to the methods described in 4.5.4.2 and 4.5.4.3.1, respectively.

[0280] Given L-carbamoylase E 1V The polypeptide sequence is a variant of one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, and the carbamoylase activity thereof is relative to that of L-carbamoylase E 1S (E 1S The carbamoylase activity of a polypeptide having a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9) can be quantified in the context of the present invention by the following test C:

[0281] 4.5.5.2 Test C

[0282] C-1. Carry out test A as described in 4.5.3.1. L , where E 1S is the enzyme to be tested. L The determined formula The molar amount of the compound.

[0283] C-2. Repeat step C-1, except use E 1V Replace E 1S as the enzyme to be tested.

[0284] C-3. Then, the formula determined in step C-2 The molar amount of the compound is divided by the formula determined in step C-1 The molar amount of the compound was calculated and the obtained ratio was multiplied by 100 to obtain the activity of L-carbamoylase E expressed in %. 1S L-Carbamoylase E 1V of carbamoylase activity.

[0285] 4.5.6 Preferred carbamoylase variants of SEQ ID NOs: 1-9

[0286] In the context of the present invention, L-carbamoylase E1, whose polypeptide sequence is selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, is generally represented by “E 1S ”.

[0287] L-carbamoylase E1, whose polypeptide sequence is selected from a variant of a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, generally represented by “E 1V ”.

[0288] In a preferred embodiment of the method of the first aspect of the present invention, the reaction in step (c) is catalyzed by L-carbamoylase E1, and the polypeptide sequence of L-carbamoylase E1 is selected from SEQ ID NO: 1 and variants thereof, SEQ ID NO: 2 and variants thereof, SEQ ID NO: 3 and variants thereof, SEQ ID NO: 4 and variants thereof, SEQ ID NO: 5 and variants thereof, SEQ ID NO: 6 and variants thereof, SEQ ID NO: 7 and variants thereof, SEQ ID NO: 8 and variants thereof, SEQ ID NO: 9 and variants thereof. More preferably, the reaction in step (c) is catalyzed by L-carbamoylase E1, and the polypeptide sequence of L-carbamoylase E1 is selected from SEQ ID NO: 1 and variants thereof.

[0289] 4.5.6.1 Preferred variants of SEQ ID NO: 1

[0290] According to the present invention, the polypeptide sequence of L-carbamoylase E1 may also be a variant of SEQ ID NO:1.

[0291] L-carbamoylase E1, whose polypeptide sequence is SEQ ID NO: 1, is represented by “E 101S ”.

[0292] L-carbamoylase E1, whose polypeptide sequence is selected from a variant of SEQ ID NO: 1, is generally represented by "E 101V ”.

[0293] A variant of the polypeptide sequence of SEQ ID NO: 1 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO: 1.

[0294] A variant of the polypeptide sequence SEQ ID NO:1 has a polypeptide sequence that is not identical to SEQ ID NO:1.

[0295] According to the present invention, L-carbamoylase E 101V It has carbamoylase activity and L-carbamoylase activity as determined as described in 4.5.4.2 and 4.5.4.3.1.

[0296] According to the present invention, L-carbamoylase E 101V Preferably, the L-carbamoylase E has at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100%. 101S The carbamoylase activity of 101S The carbamoylase activity of E 101V The carbamoylase activity of the enzyme is determined by Test C as described under 4.5.5.2.

[0297] According to the present invention, even more preferably, L-carbamoylase E 101V The carbamoylase activity of L-carbamoylase E 101S The activity of L-carbamoylase E is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of L-carbamoylase E 101S The carbamoylase activity of 101S The carbamoylase activity of E 101V The carbamoylase activity of the protein was determined by assay C as described in item 4.5.5.2.

[0298] 4.5.6.2 Preferred variants of SEQ ID NO: 2

[0299] According to the present invention, the polypeptide sequence of L-carbamoylase E1 may also be a variant of SEQ ID NO:2.

[0300] L-carbamoylase E1, whose polypeptide sequence is SEQ ID NO: 2, is represented by "E 102S ”.

[0301] L-carbamoylase E1, whose polypeptide sequence is selected from a variant of SEQ ID NO: 2, is generally represented by "E 102V ”.

[0302] A variant of the polypeptide sequence of SEQ ID NO: 2 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO: 2.

[0303] A variant of the polypeptide sequence SEQ ID NO:2 has a polypeptide sequence that is not identical to SEQ ID NO:2.

[0304] According to the present invention, L-carbamoylase E 102V It has carbamoylase activity and L-carbamoylase activity as determined as described in 4.5.4.2 and 4.5.4.3.1.

[0305] According to the present invention, L-carbamoylase E 102V Preferably, the L-carbamoylase E has at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100%. 102S The carbamoylase activity of 102S The carbamoylase activity of E 102V The carbamoylase activity of the protein was determined by assay C as described in item 4.5.5.2.

[0306] According to the present invention, even more preferably, L-carbamoylase E 102V The carbamoylase activity is in L-carbamoylase E 102SThe activity of L-carbamoylase E is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of L-carbamoylase E 102S The carbamoylase activity of 102S The carbamoylase activity of E 102V The carbamoylase activity of the protein was determined by assay C as described in item 4.5.5.2.

[0307] 4.5.6.3 Preferred variants of SEQ ID NO: 3

[0308] According to the present invention, the polypeptide sequence of L-carbamoylase E1 may also be a variant of SEQ ID NO:3.

[0309] L-carbamoylase E1, whose polypeptide sequence is SEQ ID NO: 3, is represented by "E 103S ”.

[0310] L-carbamoylase E1, whose polypeptide sequence is selected from a variant of SEQ ID NO: 3, is generally represented by "E 103V ”.

[0311] A variant of the polypeptide sequence of SEQ ID NO:3 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO:3.

[0312] A variant of the polypeptide sequence SEQ ID NO:3 has a polypeptide sequence that is not identical to SEQ ID NO:3.

[0313] According to the present invention, L-carbamoylase E 103V It has carbamoylase activity and L-carbamoylase activity as determined as described in 4.5.4.2 and 4.5.4.3.1.

[0314] According to the present invention, L-carbamoylase E 103VPreferably, the L-carbamoylase E has at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100%. 103S The carbamoylase activity of 103S The carbamoylase activity of E 103V The carbamoylase activity of the protein was determined by assay C as described in item 4.5.5.2.

[0315] According to the present invention, even more preferably, L-carbamoylase E 103V The carbamoylase activity is in L-carbamoylase E 103S The activity of L-carbamoylase E is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of L-carbamoylase E 103S The carbamoylase activity of 103S The carbamoylase activity of E 103V The carbamoylase activity of the protein was determined by assay C as described in item 4.5.5.2.

[0316] 4.5.6.4 Preferred variants of SEQ ID NO: 4

[0317] According to the present invention, the polypeptide sequence of L-carbamoylase E1 may also be a variant of SEQ ID NO:4.

[0318] L-carbamoylase E1, whose polypeptide sequence is SEQ ID NO: 4, is represented by “E 104S ”.

[0319] L-carbamoylase E1, whose polypeptide sequence is selected from a variant of SEQ ID NO: 4, is generally represented by "E 104V ”.

[0320] A variant of the polypeptide sequence of SEQ ID NO:4 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO:4.

[0321] A variant of the polypeptide sequence SEQ ID NO:4 has a polypeptide sequence that is not identical to SEQ ID NO:4.

[0322] According to the present invention, L-carbamoylase E 104V It has carbamoylase activity and L-carbamoylase activity as determined as described in 4.5.4.2 and 4.5.4.3.1.

[0323] According to the present invention, L-carbamoylase E 104V Preferably, the L-carbamoylase E has at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100%. 104S The carbamoylase activity of 104S The carbamoylase activity of E 104V The carbamoylase activity of the protein was determined by assay C as described in item 4.5.5.2.

[0324] According to the present invention, even more preferably, L-carbamoylase E 104V The carbamoylase activity is in L-carbamoylase E 104S The activity of L-carbamoylase E is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of L-carbamoylase E 104S The carbamoylase activity of 104S The carbamoylase activity of E 104V The carbamoylase activity of the protein was determined by assay C as described in item 4.5.5.2.

[0325] 4.5.6.5 Preferred variants of SEQ ID NO: 5

[0326] According to the present invention, the polypeptide sequence of L-carbamoylase E1 may also be a variant of SEQ ID NO:5.

[0327] L-carbamoylase E1, whose polypeptide sequence is SEQ ID NO: 5, is represented by “E 105S ”.

[0328] L-carbamoylase E1, whose polypeptide sequence is selected from a variant of SEQ ID NO: 5, is generally represented by "E 105V ”.

[0329] A variant of the polypeptide sequence of SEQ ID NO:5 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO:5.

[0330] A variant of the polypeptide sequence SEQ ID NO:5 has a polypeptide sequence that is not identical to SEQ ID NO:5.

[0331] According to the present invention, L-carbamoylase E 105V It has carbamoylase activity and L-carbamoylase activity as determined as described in 4.5.4.2 and 4.5.4.3.1.

[0332] According to the present invention, L-carbamoylase E 105V Preferably, the L-carbamoylase E has at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100%. 105S The carbamoylase activity of 105S The carbamoylase activity of E 105V The carbamoylase activity of the protein was determined by assay C as described in item 4.5.5.2.

[0333] According to the present invention, even more preferably, L-carbamoylase E 105V The carbamoylase activity is in L-carbamoylase E 105SThe activity of L-carbamoylase E is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of L-carbamoylase E 105S The carbamoylase activity of 105S The carbamoylase activity of E 105V The carbamoylase activity of the protein was determined by assay C as described in item 4.5.5.2.

[0334] 4.5.6.6 Preferred variants of SEQ ID NO: 6

[0335] According to the present invention, the polypeptide sequence of L-carbamoylase E1 may also be a variant of SEQ ID NO:6.

[0336] L-carbamoylase E1, whose polypeptide sequence is SEQ ID NO: 6, is represented by “E 106S ”.

[0337] L-carbamoylase E1, whose polypeptide sequence is selected from a variant of SEQ ID NO: 6, is generally represented by "E 106V ”.

[0338] A variant of the polypeptide sequence of SEQ ID NO:6 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO:6.

[0339] A variant of the polypeptide sequence SEQ ID NO:6 has a polypeptide sequence that is not identical to SEQ ID NO:6.

[0340] According to the present invention, L-carbamoylase E 106V It has carbamoylase activity and L-carbamoylase activity as determined as described in 4.5.4.2 and 4.5.4.3.1.

[0341] According to the present invention, L-carbamoylase E 106VPreferably, the L-carbamoylase E has at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100%. 106S The carbamoylase activity of 106S The carbamoylase activity of E 106V The carbamoylase activity of the protein was determined by assay C as described in item 4.5.5.2.

[0342] According to the present invention, even more preferably, L-carbamoylase E 106V The carbamoylase activity is in L-carbamoylase E 106S The activity of L-carbamoylase E is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of L-carbamoylase E 106S The carbamoylase activity of 106S The carbamoylase activity of E 106V The carbamoylase activity of the protein was determined by assay C as described in item 4.5.5.2.

[0343] 4.5.6.7 Preferred variants of SEQ ID NO: 7

[0344] According to the present invention, the polypeptide sequence of L-carbamoylase E1 may also be a variant of SEQ ID NO:7.

[0345] L-carbamoylase E1, whose polypeptide sequence is SEQ ID NO: 7, is represented by “E 107S ”.

[0346] L-carbamoylase E1, whose polypeptide sequence is selected from a variant of SEQ ID NO: 7, is generally represented by "E 107V ”.

[0347] A variant of the polypeptide sequence of SEQ ID NO:7 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO:7.

[0348] A variant of the polypeptide sequence SEQ ID NO:7 has a polypeptide sequence that is not identical to SEQ ID NO:7.

[0349] According to the present invention, L-carbamoylase E 107V It has carbamoylase activity and L-carbamoylase activity as determined as described in 4.5.4.2 and 4.5.4.3.1.

[0350] According to the present invention, L-carbamoylase E 107V Preferably, the L-carbamoylase E has at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100%. 107S The carbamoylase activity of 107S The carbamoylase activity of E 107V The carbamoylase activity of the protein was determined by assay C as described in item 4.5.5.2.

[0351] According to the present invention, even more preferably, L-carbamoylase E 107V The carbamoylase activity is in L-carbamoylase E 107S The activity of L-carbamoylase E is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of L-carbamoylase E 107S The carbamoylase activity of 107S The carbamoylase activity of E 107V The carbamoylase activity of the protein was determined by assay C as described in item 4.5.5.2.

[0352] 4.5.6.8 Preferred variants of SEQ ID NO: 8

[0353] According to the present invention, the polypeptide sequence of L-carbamoylase E1 may also be a variant of SEQ ID NO:8.

[0354] L-carbamoylase E1, whose polypeptide sequence is SEQ ID NO: 8, is represented by “E 108S ”.

[0355] L-carbamoylase E1, whose polypeptide sequence is selected from a variant of SEQ ID NO: 8, is generally represented by "E 108V ”.

[0356] A variant of the polypeptide sequence of SEQ ID NO:8 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO:8.

[0357] A variant of the polypeptide sequence SEQ ID NO:8 has a polypeptide sequence that is not identical to SEQ ID NO:8.

[0358] According to the present invention, L-carbamoylase E 108V It has carbamoylase activity and L-carbamoylase activity as determined as described in 4.5.4.2 and 4.5.4.3.1.

[0359] According to the present invention, L-carbamoylase E 108V Preferably, the L-carbamoylase E has at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100%. 108S The carbamoylase activity of 108S The carbamoylase activity of E 108V The carbamoylase activity of the protein was determined by assay C as described in item 4.5.5.2.

[0360] According to the present invention, even more preferably, L-carbamoylase E 108V The carbamoylase activity is in L-carbamoylase E 108SThe activity of L-carbamoylase E is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of L-carbamoylase E 108S The carbamoylase activity of 108S The carbamoylase activity of E 108V The carbamoylase activity of the protein was determined by assay C as described in item 4.5.5.2.

[0361] 4.5.6.9 Preferred variants of SEQ ID NO: 9

[0362] According to the present invention, the polypeptide sequence of L-carbamoylase E1 may also be a variant of SEQ ID NO:9.

[0363] L-carbamoylase E1, whose polypeptide sequence is SEQ ID NO: 9, is represented by “E 109S ”.

[0364] L-carbamoylase E1, whose polypeptide sequence is selected from a variant of SEQ ID NO: 9, is generally represented by "E 109V ”.

[0365] A variant of the polypeptide sequence of SEQ ID NO:9 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO:9.

[0366] A variant of the polypeptide sequence SEQ ID NO:9 has a polypeptide sequence that is not identical to SEQ ID NO:9.

[0367] According to the present invention, L-carbamoylase E 109V It has carbamoylase activity and L-carbamoylase activity as determined as described in 4.5.4.2 and 4.5.4.3.1.

[0368] According to the present invention, L-carbamoylase E 109VPreferably, the L-carbamoylase E has at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100%. 109S The carbamoylase activity of 109S The carbamoylase activity of E 109V The carbamoylase activity of the protein was determined by assay C as described in item 4.5.5.2.

[0369] According to the present invention, even more preferably, L-carbamoylase E 109V The carbamoylase activity is in L-carbamoylase E 109S The activity of L-carbamoylase E is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of L-carbamoylase E 109S The carbamoylase activity of 109S The carbamoylase activity of E 109V The carbamoylase activity of the protein was determined by assay C as described in item 4.5.5.2.

[0370] 4.5.7 Preferred process conditions in step (c)

[0371] The reaction in step (c) of the process of the first aspect of the present invention can be carried out under conditions known to the skilled person.

[0372] The reaction medium is preferably aqueous, more preferably an aqueous buffer.

[0373] Exemplary buffers commonly used in biotransformation reactions and advantageously used herein include Tris, phosphate or any zwitterionic buffer, such as 2-(N-morpholino)ethanesulfonic acid ("MES"), N-(2-acetamido)iminodiacetic acid ("ADA"), piperazine-N,N'-bis(2-ethanesulfonic acid) ("PIPES"), N-(2-acetamido)-2-aminoethanesulfonic acid ("ACES"), p-hydroxy-4-morpholinopropanesulfonic acid ("MOPSO"), cholestyramine chloride, 3-(N-morpholino)propanesulfonic acid ("MOPS"), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid ("BES"), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid ("TES"), 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid ("HEPES"), 3-(bis(2-hydroxyethyl)amino)-2-hydroxypropane-1-sulfonic acid ("DIPSO"), acetylaminoglycine, 3-(N-tris(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid ("TAPSO"), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) ("POPSO"), 4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid) ("HEPPSO"), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid ("HEPPS"), tris(hydroxymethyl)methylglycine, glycinamide, N,N-bis(2-hydroxyethyl)glycine, or 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonic acid ("TAPS").

[0374] In some embodiments, ammonium can act as a buffer.One or more organic solvents can also be added to the reaction.

[0375] The buffer preferably comprises a metal salt, more preferably a metal salt such as a metal halide, preferably a monovalent or divalent or trivalent metal halide, preferably a monovalent or divalent metal chloride, preferably CoCl2 or MnCl2, preferably CoCl2.

[0376] The concentration of these metal salts in the reaction medium is preferably from 1 μM to 1 M, more preferably from 1 mM to 100 mM, even more preferably from 1 to 10 mM.

[0377] Preferably, step (c) of the method of the first aspect of the invention is carried out in phosphate buffer.

[0378] The pH of the reaction medium in step (c) of the process of the first aspect of the invention is preferably in the range of 2 to 10, more preferably in the range of 5 to 8, more preferably in the range of 7.2 to 7.5, most preferably 7.5.

[0379] Preferably, step (c) of the method of the first aspect of the invention is carried out at a temperature in the range of 20°C to 70°C, more preferably in the range of 30°C to 55°C, most preferably 50°C.

[0380] Preferably, the total concentration of all carbamoylases E1 in the reaction solution in step (c) is in the range of 1 μM to 10 mM, preferably 10 μM to 1 mM, more preferably 0.1 mM to 0.5 mM, most preferably 0.4 mM.

[0381] In an alternative preferred embodiment, the total concentration of all carbamoylases E1 in the reaction solution in step (c) is in the range of 1 μg / l to 10 g / l, preferably 0.1 mg / l to 5 g / l, more preferably 1 mg / l to 1 g / l, more preferably 5 mg / l to 500 mg / l.

[0382] Preferably, the initial concentration of all compounds of formula L-(II) in the reaction medium of step (c) is in the range of 1 μM to 1 M, preferably in the range of 10 μM to 0.5 M, more preferably in the range of 0.1 mM to 0.1 M, more preferably in the range of 1 mM to 10 mM, and most preferably 1.25 mM.

[0383] If a compound of formula D-(II) is present in the reaction medium of step (c), the initial concentration of all compounds of formula D-(II) in the reaction medium is preferably 1% to 100% of the concentration of all compounds of formula L-(II), more preferably 10% to 100% of the concentration of all compounds of formula L-(II), even more preferably 50 to 100%, even more preferably 100% of the concentration of all compounds of formula L-(II).

[0384] "The initial concentration of all compounds of formula L-(II) / D-(II)" refers to the respective concentrations of the corresponding compounds L-(II) or D-(II) in the reaction medium when the corresponding compounds are used in step (c).

[0385] 4.5.8 Step (b)

[0386] 4.5.8.1 Enzyme catalysis in step (b)

[0387] In a preferred embodiment of the method of the first aspect of the present invention, the compound of formula L-(II) is obtained by step (b), wherein the compound of formula L-(III) is reacted to obtain the compound of formula L-(II):

[0388]

[0389] Step (b) provides the starting material for step (c).

[0390] R3L is a group selected from the above formula (VIII), (IX), (X), preferably a group selected from the above formula (VIII), (IX), more preferably R 3L It has the formula (VIII).

[0391] When R 3L When the group is a group of formula (VIII), the group R 3L It can be selected from the above formulae L-(VIII) and D-(VIII), preferably L-(VIII).

[0392] When R 3L When the group is a group of formula (IX), the group R 3L It can be selected from the above formulae L-(IX) and D-(IX), preferably L-(IX).

[0393] When R 3L When the group is a group of formula (X), the group R 3L It can be selected from the above formulae L-(X) and D-(X), preferably L-(X).

[0394] The reaction of step (b) is enzymatically catalyzed, ie, catalyzed by hydantoinase E2.

[0395] 4.5.8.2 Enantioselective and enantiospecific catalysis of step (b)

[0396] Step (b) of the process of the first aspect of the invention is preferably L-enantiomerically selective, even more preferably L-enantiomerically specific.

[0397] In such a preferred embodiment, in step (b) in particular the mixture M is used III L-(III), the mixture comprises at least one compound L-(III) and at least one compound D-(III), wherein D-(III) has the following formula:

[0398]

[0399] R 3D is a group selected from the above formula (VIII), (IX), (X), preferably a group selected from the above formula (VIII), (IX), more preferably R 3D It has the formula (VIII).

[0400] When R 3D When the group is a group of formula (VIII), the group R 3D It can be selected from the above formulae L-(VIII) and D-(VIII), preferably L-(VIII).

[0401] When R 3DWhen the group is a group of formula (IX), the group R 3D It can be selected from the above formulae L-(IX) and D-(IX), preferably L-(IX).

[0402] When R 3D When the group is a group of formula (X), the group R 3D It can be selected from the above formulae L-(X) and D-(X), preferably L-(X).

[0403] Mixture M III Preferably, it comprises at least one compound L-(III) and at least one compound D-(III), wherein R 3L =R 3D .

[0404] For this mixture M III , it can be observed that at least one compound D-(II) undergoes a reaction of step (b)*, ie in parallel with step (b).

[0405] That is, in the reaction of step (b)*, the mixture M III At least one compound D-(III) is reacted to obtain a compound of formula D-(II), wherein the compound of formula D-(II) is as explained above (especially under item 4.5.1.2).

[0406] In case step (b) is "L-enantiomerically selective", this means that in step (b) a mixture M is used. III In the case of

[0407] - In the mixture M III In the step (b)*, the reaction of any compound of formula D-(III) to produce a compound of formula D-(II) does not occur, or,

[0408] - In the case of the reaction of step (b)*, the mixture M III There is at least one pair of compounds in produce The reaction rate of step (b)* is produce The reaction rate of step (b) is small.

[0409] is a compound of formula D-(III), is a compound of formula L-(III), wherein The group R 3L and The group R 3D same.

[0410] is a compound of formula D-(II), is a compound of formula L-(II), wherein The group R 2L and The group R 2D same.

[0411] Preferably, The group R 2L and The group R 3L Preferably, The group R 2D and The group R 3D same.

[0412] If the reaction rate of step (b)* is substantially zero, that is, the mixture M III If the reaction of step (b)* without any compound of formula D-(III) produces a compound of formula D-(II), then step (b) is "L-enantiomer-specific".

[0413] In a preferred embodiment, the mixture M III All compounds and mixtures of formula L-(III) III The molar ratio of all compounds of formula D-(III) is substantially 1:1.

[0414] In other preferred embodiments, the mixture M III All compounds and mixtures of formula L-(III) III The molar ratio of all compounds of formula D-(III) is in the range of 3:2 to 1:99, more preferably in the range of 1.01:1 to 1:99, more preferably in the range of 1:1 to 1:99, more preferably in the range of 1:1.01 to 1:99, more preferably in the range of 1:1.01 to 1:9, more preferably in the range of 1:1.01 to 1:9, more preferably in the range of 1:1.01 to 1:8, more preferably in the range of 1:1.01 to 1:3.

[0415] Alternatively, D-(III) in the mixture M III The content of L-(III) exceeds that of L-(III), which means that when at least one compound of formula L-(III) is present in the mixture M III When the mixture M III All compounds and mixtures of formula L-(III) IIIThe molar ratio of all compounds of formula D-(III) is <1:1, preferably <0.9:1, more preferably <0.75:1, more preferably <0.5:1, more preferably <0.2:1, more preferably <0.1:1, more preferably <0.01:1.

[0416] If step (b) is preferably catalyzed by L-hydantoinase E2, step (b) is in particular L-enantiomerically selective, which can be determined by the skilled person as described in 4.5.10.3.

[0417] In case step (b) is L-enantiomer selective, the compound For produce The reaction of step (b) is preferably carried out at a ratio of produce The reaction rate of step (b)* is at least 2 times higher, preferably at least 10 times higher, more preferably at least 100 times higher, even more preferably at least 10 times higher 3 times, even more preferably at least 10 times higher 4 times, even more preferably at least 10 times higher 5 times the reaction rate.

[0418] For a given compound Yes, to quantify the factor of the reaction rate of step (b) relative to the reaction rate of step (b)*, the following test can be performed:

[0419] (1) An equimolar mixture [two compounds and The molar ratio of 1:1] was placed under the respective reaction conditions and the two products were monitored over time. and Development (e.g., by LC-MS as described in Item 5.4).

[0420] (2) When n LII10 = 10 mol% of the initial use The reaction product When the measurement is The reaction formed The molar amount [relative to the initial use mol%](=n DII10 ).

[0421] (3)n LII10 / n DII10 =10 / n DII10 The ratio of gives the given compound The factor by which the reaction rate of step (b) is compared to the reaction rate of step (b)*.

[0422] →If n LII10 / n DII10 >1, then the given compound The reaction rate of step (b)* of is greater than the reaction rate of step (b)*.

[0423] →If n LII10 / n DII10 <1, then the given compound The reaction rate of step (b) of is less than the reaction rate of step (b)*.

[0424] →If n LII10 / n DII10 =1, then the given compound The reaction rates of steps (b) and (b)* are the same.

[0425] The reaction of step (b) of a preferred embodiment of the first aspect of the invention is catalysed by a hydantoinase ("dihydropyrimidinase") E2.

[0426] That is, surprisingly, it was found that hydantoinase accepts compounds of formula L-(III) as substrates and converts them into products of formula L-(II), thereby catalyzing the reaction of step (b). This discovery has high scientific and economic value because it further broadens the scope of novel raw material-based synthetic routes for preparing L-phosphinothricin P-esters and L-phosphinothricin. Even more surprisingly, it is suggested that L-phosphinothricin hydantoin, i.e. compounds of formula L-(III) where R=H, will not undergo the reaction of hydantoinase to produce the corresponding LGA carbamoylates.

[0427] In practice, hydantoinases ("dihydropyrimidinases") typically catalyze the reaction of 5,6-dihydrouracil to produce ureidopropionic acid (see reaction <2A> below):

[0428] <2A>:

[0429] They also catalyze the ring opening of monosubstituted hydantoins to give the corresponding carbamoylamino acids according to the following reaction <2B>, where R* can be an organic group, such as the side chain of one of the naturally occurring amino acids.

[0430] <2B>:

[0431] Thesis "Untersuchungen zur und An overview of hydantoinases is given in Chapter 1.3 (pages 7 to 11) of "Microbial Hydantoinases / Investigations of substrate specificity and enantioselectivity of microbial hydantoinases" by T. Waniek, University of Stuttgart, 2000 (URL: https: / / elib.uni-stuttgart.de / bitstream / 11682 / 1511 / 1 / Diss.pdf).

[0432] It has now surprisingly been found that hydantoinase also accepts And where R = R 3L substrate.

[0433] Surprisingly, they do not accept And a substrate wherein R=H.

[0434] In the context of the present invention, "hydantoinase E2" is an enzyme that catalyzes the conversion of carbamoyl substrates S' L To the corresponding amino acid product P' L The following reaction <2C> hydantoinase, where Where R = R 3L :

[0435] <2C>:

[0436] In particular, hydantoinase E2 is an "L-hydantoinase", ie, it has a greater catalytic activity for reaction <2C> than for reaction <2D>, wherein the substrate S' in reaction <2D> D R* in reaction <2C> and substrate S' L The R* in is the same, and Where R = R 3L .

[0437] <2D>:

[0438] Since the L-hydantoinase has a higher catalytic activity for the <2C> reaction than for the <2D> reaction, it is "L-enantiomer selective". An L-hydantoinase that has no catalytic activity for the <2D> reaction and therefore is only catalytically active for the <2C> reaction is "L-enantiomer specific".

[0439] A "D-hydantoinase" is defined as a hydantoinase that is "D-enantiomerically selective", i.e. it has a higher catalytic activity towards reaction <2D> than towards reaction <2C>. A D-carbamoylase that does not catalyze reaction <2C> and therefore has catalytic activity only towards reaction <2D> is "D-enantiomerically specific".

[0440] A hydantoinase having the same catalytic activity for reaction <2C> and reaction <2D> is referred to as a "non-enantiomerically selective hydantoinase".

[0441] In order to determine whether a hydantoinase in the context of the present invention can be indicated as "L-hydantoinase", "D-hydantoinase" or "non-enantiomerically selective hydantoinase", the method according to Test E (item 4.5.10) can preferably be used.

[0442] The hydantoinase E2, especially L-hydantoinase E2, that can be used in step (b) of the preferred embodiment of the method of the first aspect of the present invention can be derived from Arthrobacter, especially Arthrobacter crystallomorpha, Arthrobacter golden yellow, Arthrobacter species BT801; Alcaligenes sp.; especially Alcaligenes faecalis subsp. faecalis; Bacillus, especially Bacillus fordy; Microbacterium, especially Microbacterium liquefaciens strain AJ3912; Pseudomonas, especially Pseudomonas fluorescens, Pseudomonas aeruginosa.

[0443] A hydantoinase E2, in particular an L-hydantoinase E2 suitable for use in the method of the present invention may be the enzyme HyuH derived from Arthrobacter. Another enzyme may be Dht.

[0444] Even more preferably, the hydantoinase E2, in particular L-hydantoinase E2, which may be preferably used in step (b) of the first aspect of the present invention, may be derived from Arthrobacter, in particular Arthrobacter crystallomorpha, Arthrobacter aureus, Arthrobacter species BT801, Arthrobacter aureus DSM 9771, most preferably from Arthrobacter aureus DSM 9771.

[0445] Hydantoinases suitable for use in the process of the invention are described, for example, in WO 01 / 23582 A1 and in JM Clemente-Jiménez, S. Martínez-Rodríguez, F. Rodríguez-Vico, FJL Heras-Vázquez, Recent Pat. Biotechnology 2008, 2, 35-46; G. Latacz, E. Pekala, K. Kiec-Kononowicz, Biotechnologia 2006, 2, 189-205.

[0446] Other suitable hydantoinases are described by K. Yokozeki, H. Yoshiteru, K. Kubota, Agric. Biol. Chem. 1987, 51, 737-746.

[0447] The hydantoinase E2 that can be used in preferred step (b) of the method of the present invention may be a hydantoinase classified in EC classification EC 3.5.2.2.

[0448] Table 2 below gives preferred examples of hydantoinase E2 polypeptide sequences, which can be preferably used in step (b) of the preferred embodiment of the method of the first aspect of the present invention. The genes encoding the corresponding hydantoinase E2 and the corresponding accession numbers are known.

[0449] Table 2

[0450]

[0451] In a preferred embodiment of the method of the first aspect of the invention, the reaction of step (b) is catalyzed by hydantoinase E2, wherein the polypeptide sequence of E2 is selected from SEQ ID NO: 10 and variants thereof, SEQ ID NO: 11 and variants thereof, SEQ ID NO: 12 and variants thereof, SEQ ID NO: 13 and variants thereof, SEQ ID NO: 14 and variants thereof, SEQ ID NO: 15 and variants thereof, SEQ ID NO: 16 and variants thereof, preferably SEQ ID NO: 10 and variants thereof.

[0452] 4.5.9 Test D for determination of hydantoinase activity L and D D

[0453] The skilled person is aware of hydantoinases, in particular L-hydantoinases, which may be used in step (b) of the preferred method of the first aspect of the invention.

[0454] In particular, Test D described below L Can be used to determine the given enzyme EY The invention can be advantageously used to determine the hydantoinase and L-hydantoinase activities in variants of SEQ ID NO: 10, variants of SEQ ID NO: 11, variants of SEQ ID NO: 12, variants of SEQ ID NO: 13, variants of SEQ ID NO: 14, variants of SEQ ID NO: 15, and variants of SEQ ID NO: 16.

[0455] For comparison reasons, Experiment D D Can be used to determine the E Y D-hydantoinase activity.

[0456] To test D L and Test D D For the purpose of testing, the enzyme E Y The molar mass is calculated as E Y The molar mass of the polypeptide sequence.

[0457] In addition, the substrate of formula L-(III), wherein R 3L = n-butyl, used in test D L Therefore, there is only one chiral carbon atom in the substrate. The compound of formula L-(III), wherein R 3L = n-butyl, expressed as

[0458] The product of formula L-(II), wherein R 2L = n-butyl, in test A L Detected in.

[0459] A compound of formula L-(II), wherein R 2L = n-butyl, expressed as

[0460] In addition, the substrate of formula D-(III), wherein R 3D = n-butyl, used in test A D , so there is only one chiral carbon atom in the substrate. The compound of formula D-(III), wherein R 3D = n-butyl, expressed as

[0461] The product of formula D-(II), wherein R 2D = n-butyl, in test A D Detected in.

[0462] A compound of formula D-(II), wherein R 2D = n-butyl, expressed as

[0463] 4.5.9.1 Test D L :

[0464] To the mixture containing 50 mM To 0.9 ml of a reaction aqueous solution (phosphate buffer, pH 7.2, 10 mM MnCl2) of the compound, 400 nmol of E in 0.1 ml of a phosphate buffer aqueous solution (pH 7.2, 10 mM MnCl2) was added. Y The resulting solution was incubated at 25°C and the pH was maintained at pH 7.2 by adding 1 M NaOH. After 300 min, the reaction was terminated by adding 2 M HCl to obtain a pH of 2.5 and the formula was determined. The molar amount of the compound. Detection can be carried out by LC-MS methods, as described in the Examples section (item 5.4) for the detection of LGA.

[0465] 4.5.9.2 Test D D :

[0466] To the mixture containing 50 mM To 0.9 ml of a reaction aqueous solution (phosphate buffer, pH 7.2, 10 mM MnCl2) of the compound, 400 nmol of E in 0.1 ml of a phosphate buffer aqueous solution (pH 7.2, 10 mM MnCl2) was added. Y The resulting solution was incubated at 25°C and the pH was maintained at 7.2 by adding 1 M NaOH. After 300 min, the reaction was terminated by adding 2 M HCl to obtain a pH of 2.5 and the formula was determined. The molar amount of the compound. Detection can be carried out by LC-MS methods, as described in the Examples section (item 5.4) for the detection of LGA.

[0467] 4.5.10 Test E for the identification of the specificity of the L- and D-enantiomers of hydantoinase, L-hydantoinase, D-hydantoinase

[0468] The hydantoinase E2 of the present invention is preferably an L-hydantoinase, more preferably L-enantiomer-specific.

[0469] A given enzyme E Y Whether or not a hydantoinase E2, in particular an L-hydantoinase, can be considered, can be determined in the context of the present invention by the following test E:

[0470] 4.5.10.1 Test E:

[0471] E-1. First, perform test D described in 4.5.9.1. L , and according to test D LThe obtained formula The molar amount of the compound.

[0472] E-2. Next, perform test D as described in 4.5.9.2 D , and according to test D D The obtained formula The molar amount of the compound.

[0473] E-3. Then, step E-1 was repeated, except that 400 nmol E in 0.1 ml of phosphate buffer solution (pH 7.2, 10 mM NCl2) was not added. Y , but add Y 0.1 ml of phosphate buffered saline (pH 7.2, 10 mM MnCl2).

[0474] E-4. Then, step E-2 was repeated, except that 400 nmol E in 0.1 ml of phosphate buffer solution (pH 7.2, 10 mM nCl2) was not added. Y , but add Y 0.1 ml of phosphate buffered saline (pH 7.2, 10 mM MnCl2).

[0475] 4.5.10.2 Hydantoinase activity:

[0476] If the formula determined in step E-1 is The molar amount of the compound of formula The molar amount of the compound, then E Y It is considered to have hydantoinase activity and can therefore be considered to be a hydantoinase E2 in the context of the present invention.

[0477] 4.5.10.3 L-Hydantoinase Activity

[0478] 4.5.10.3.1 L-Hydantoinase

[0479] (i) If the formula determined in step E-1 is The molar amount of the compound of formula The molar amount of the compound, and

[0480] (ii) If the formula determined in step E-2 is The molar amount of the compound of formula The molar amount of the compound, and

[0481] (iii) In addition, if the formula determined in step E-1 is The molar amount of the compound of formula The molar amount of the compound,

[0482] → Then E Y is considered to have L-hydantoinase activity and can therefore be considered to be an L-hydantoinase in the context of the present invention. In this case, in the context of the present invention, E Y It is considered to be "L-enantiomerically selective".

[0483] For the sake of clarity, it is indicated that the formula determined in step E-2 The molar amount of the compound of formula When the molar amounts of the compounds are the same, condition (iii) is automatically satisfied.

[0484] (i) If the formula determined in step E-1 is The molar amount of the compound of formula The molar amount of the compound, and

[0485] (ii) If the formula determined in step E-2 is The molar amount of the compound of formula The molar amounts of the compounds are the same,

[0486] → Then E Y is considered to have L-hydantoinase activity and can therefore be considered to be an L-hydantoinase in the context of the present invention. In this case, in the context of the present invention, E Y It is considered not only "L-enantiomerically selective" but also "L-enantiomerically specific".

[0487] For L-hydantoinase E which is not L-enantiomer specific Y , the L-enantiomeric selectivity can then be quantified by the following method: The molar amount of the compound is divided by the formula determined in E-2 The molar amount of the compound is then multiplied by 100 to obtain E in %. Y L-enantiomer selectivity.

[0488] 4.5.10.3.2 D-Carbamoylase

[0489] (i) If the formula determined in step E-1 is The molar amount of the compound of formula The molar amount of the compound, and

[0490] (ii) If the formula determined in step E-2 is The molar amount of the compound of formula The molar amount of the compound, and

[0491] (iii) In addition, if the formula determined in step E-2 is The molar amount of the compound of formula The molar amount of the compound,

[0492] → Then E Y is considered to have D-hydantoinase activity and can therefore be considered a D-hydantoinase. In this case, E Y In the context of the present invention it is "D-enantiomerically selective".

[0493] For the sake of clarity, it is indicated that the formula determined in step E-1 The molar amount of the compound of formula When the molar amounts of the compounds are the same, condition (iii) is automatically satisfied.

[0494] (i) If the formula determined in step E-1 is The molar amount of the compound of formula The molar amounts of the compounds are the same, and

[0495] (ii) If the formula determined in step E-2 is The molar amount of the compound of formula The molar amount of the compound,

[0496] → Then E Y is considered to have D-hydantoinase activity and can therefore be considered to be a D-hydantoinase in the context of the present invention. In this case, in the context of the present invention, E Y It is considered not only "D-enantiomer selective" but also "D-enantiomer specific".

[0497] For D-hydantoinase E which is not D-enantiomer specific Y , the D-enantiomeric selectivity can be quantified by the following method: The molar amount of the compound of formula The molar amount of the compound is then multiplied by 100 to obtain E in %. Y The D-enantiomer selectivity.

[0498] 4.5.10.3.3 Non-enantiomerically selective carbamoylases

[0499] (i) If the formula determined in step E-1 is The molar amount of the compound of formula The molar amount of the compound, and

[0500] (ii) If the formula determined in step E-2 is The molar amount of the compound of formula The molar amount of the compound, and

[0501] (iii) In addition, if the formula determined in step E-2 is The molar amount of the compound of formula The molar amounts of the compounds are the same,

[0502] → Then E Y is considered to be a non-enantiomerically selective hydantoinase in the context of the present invention. Y is "diastereoselective".

[0503] 4.5.11 Test F for identifying preferred hydantoinase variants of SEQ ID NOs: 10-16

[0504] 4.5.11.1 L- and D-enantiomerically selective hydantoinase variants

[0505] The enzyme whose polypeptide sequence is selected from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 has hydantoinase activity, in particular L-hydantoinase activity.

[0506] In a preferred embodiment of the method of the first aspect of the present invention, the polypeptide sequence of hydantoinase, in particular L-hydantoinase E2, is selected from SEQ ID NO: 10 and variants thereof, SEQ ID NO: 11 and variants thereof, SEQ ID NO: 12 and variants thereof, SEQ ID NO: 13 and variants thereof, SEQ ID NO: 14 and variants thereof, SEQ ID NO: 15 and variants thereof, SEQ ID NO: 16 and variants thereof, more preferably SEQ ID NO: 10 and variants thereof.

[0507] The term "variant" is defined in Section 4.3.

[0508] In the context of the present invention, an enzyme whose polypeptide sequence is a variant of one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 has hydantoinase activity, preferably L-hydantoinase activity, more preferably is L-enantiomer specific.

[0509] For a given enzyme E Y , whose polypeptide sequence is a variant of one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, whether it has hydantoinase activity, L-hydantoinase activity and whether it is L-enantiomer specific can be determined according to the methods described in 4.5.10.2 and 4.5.10.3.1, respectively.

[0510] Hydantoinase E 2S Hydantoinase activity (E 2S The polypeptide sequence is selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16) of a given hydantoinase E 2V The hydantoinase activity of a polypeptide of the type A described herein (whose polypeptide sequence is a variant of one of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16) can be determined in the context of the present invention by the following test F:

[0511] 4.5.11.2 Test F

[0512] F-1. Carry out test D as described in 4.5.9.1. L , where E 2S is the enzyme to be tested. L The determined formula The molar amount of the compound.

[0513] F-2. Repeat step F-1, except use E 2V Replace E 2S As the enzyme to be tested.

[0514] F-3. Then, the formula determined in step F-2 The molar amount of the compound of formula The molar amount of the compound was calculated and the obtained ratio was multiplied by 100 to obtain the activity of hydantoinase E expressed in %. 2S Hydantoinase E 2V of hydantoinase activity.

[0515] 4.5.12 Preferred hydantoinase variants of SEQ ID NOS: 10-16

[0516] In the context of the present invention, hydantoinase E2, whose polypeptide sequence is selected from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, is generally denoted as "E 2S ”.

[0517] Hydantoinase E2, whose polypeptide sequence is selected from the sequence variants of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, generally represented by "E 2V ”.

[0518] In a preferred embodiment of the method of the first aspect of the invention, the reaction in step (b) is catalyzed by hydantoinase E2, preferably L-hydantoinase E2, and the polypeptide sequence of hydantoinase E2, preferably L-hydantoinase E2, is selected from SEQ ID NO: 10 and variants thereof, SEQ ID NO: 11 and variants thereof, SEQ ID NO: 12 and variants thereof, SEQ ID NO: 13 and variants thereof, SEQ ID NO: 14 and variants thereof, SEQ ID NO: 15 and variants thereof, SEQ ID NO: 16 and variants thereof.

[0519] More preferably, the reaction in step (b) is catalyzed by hydantoinase E2, preferably L-hydantoinase E2, and the polypeptide sequence of hydantoinase E2, preferably L-hydantoinase E2 is selected from SEQ ID NO: 10 and variants of SEQ ID NO: 10.

[0520] 4.5.12.1 Preferred variants of SEQ ID NO: 10

[0521] According to the present invention, the polypeptide sequence of hydantoinase E2, preferably the polypeptide sequence of L-hydantoinase E2, may also be a variant of SEQ ID NO:10.

[0522] Hydantoinase E2, whose polypeptide sequence is SEQ ID NO: 10, is represented by "E 210S ”.

[0523] Hydantoinase E2, whose polypeptide sequence is selected from a variant of SEQ ID NO: 10, is generally represented by "E 210V ”.

[0524] A variant of the polypeptide sequence of SEQ ID NO: 10 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO: 10.

[0525] A variant of the polypeptide sequence SEQ ID NO:10 has a polypeptide sequence that is not identical to SEQ ID NO:10.

[0526] According to the present invention, hydantoinase E 210V It has hydantoinase activity, preferably L-hydantoinase activity, as determined as described in Items 4.5.10.2 and 4.5.10.3.1.

[0527] According to the present invention, hydantoinase E 210V Preferably, it has hydantoinase E 210S at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100% of the hydantoinase activity of E 210S The hydantoinase activity of E 210V The hydantoinase activity of the strain was determined by test F as described in item 4.5.11.2.

[0528] According to the present invention, even more preferably, hydantoinase E 210V The hydantoinase activity is 210SThe hydantoinase activity of the present invention is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of the hydantoinase E 210S The hydantoinase activity relative to hydantoinase E 210S The hydantoinase activity of E 210V The hydantoinase activity of the strain was determined by test F as described in item 4.5.11.2.

[0529] 4.5.12.2 Preferred variants of SEQ ID NO: 11

[0530] According to the present invention, the polypeptide sequence of hydantoinase E2, preferably the polypeptide sequence of L-hydantoinase E2, may also be a variant of SEQ ID NO:11.

[0531] Hydantoinase E2, whose polypeptide sequence is SEQ ID NO: 11, is represented by "E 211S ”.

[0532] Hydantoinase E2, whose polypeptide sequence is selected from a variant of SEQ ID NO: 11, is generally represented by "E 211V ”.

[0533] A variant of the polypeptide sequence of SEQ ID NO: 11 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO: 11.

[0534] A variant of the polypeptide sequence SEQ ID NO:11 has a polypeptide sequence that is not identical to SEQ ID NO:11.

[0535] According to the present invention, hydantoinase E 211V It has hydantoinase activity, preferably L-hydantoinase activity, as determined as described in Items 4.5.10.2 and 4.5.10.3.1.

[0536] According to the present invention, hydantoinase E 211V Preferably, it has hydantoinase E 211Sat least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100% of the hydantoinase activity of E 211S The hydantoinase activity of E 211V The hydantoinase activity of the enzyme is determined by test F as described in item 4.5.11.2.

[0537] According to the present invention, even more preferably, hydantoinase E 211V The hydantoinase activity is 211S The hydantoinase activity of the present invention is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of the hydantoinase E 211S The hydantoinase activity relative to hydantoinase E 211S The hydantoinase activity of E 211V The hydantoinase activity of is determined by test F as described in item 4.5.11.2.

[0538] 4.5.12.3 Preferred variants of SEQ ID NO: 12

[0539] According to the present invention, the polypeptide sequence of hydantoinase E2, preferably the polypeptide sequence of L-hydantoinase E2, may also be a variant of SEQ ID NO:12.

[0540] Hydantoinase E2, whose polypeptide sequence is SEQ ID NO: 12, is represented by "E 212S ”.

[0541] Hydantoinase E2, whose polypeptide sequence is selected from a variant of SEQ ID NO: 12, is generally represented by "E 212V ”.

[0542] A variant of the polypeptide sequence of SEQ ID NO: 12 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO: 12.

[0543] A variant of the polypeptide sequence SEQ ID NO:12 has a polypeptide sequence that is not identical to SEQ ID NO:12.

[0544] According to the present invention, hydantoinase E 212V It has hydantoinase activity, preferably L-hydantoinase activity, as determined as described in Items 4.5.10.2 and 4.5.10.3.1.

[0545] According to the present invention, hydantoinase E 212V Preferably, hydantoinase E 212S at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100% of the hydantoinase activity of E 212S The hydantoinase activity of E 212V The hydantoinase activity of the enzyme is determined by test F as described in item 4.5.11.2.

[0546] According to the present invention, even more preferably, hydantoinase E 212V The hydantoinase activity is 212S The hydantoinase activity of the present invention is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of the hydantoinase E 212S The hydantoinase activity relative to hydantoinase E 212S The hydantoinase activity of E 212V The hydantoinase activity of is determined by test F as described in item 4.5.11.2.

[0547] 4.5.12.4 Preferred variants of SEQ ID NO: 13

[0548] According to the present invention, the polypeptide sequence of hydantoinase E2, preferably the polypeptide sequence of L-hydantoinase E2, may also be a variant of SEQ ID NO:13.

[0549] Hydantoinase E2, whose polypeptide sequence is SEQ ID NO: 13, is represented by "E 213S ”.

[0550] Hydantoinase E2, whose polypeptide sequence is selected from a variant of SEQ ID NO: 13, is generally represented by "E 213V ”.

[0551] A variant of the polypeptide sequence of SEQ ID NO: 13 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO: 13.

[0552] A variant of the polypeptide sequence SEQ ID NO:13 has a polypeptide sequence that is not identical to SEQ ID NO:13.

[0553] According to the present invention, hydantoinase E 213V It has hydantoinase activity, preferably L-hydantoinase activity, as determined as described in Items 4.5.10.2 and 4.5.10.3.1.

[0554] According to the present invention, hydantoinase E 213V Preferably, it has hydantoinase E 213S at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100% of the hydantoinase activity of E 213S The hydantoinase activity of E 213V The hydantoinase activity of the strain was determined by test F as described in item 4.5.11.2.

[0555] According to the present invention, even more preferably, hydantoinase E 213V The hydantoinase activity is 213SThe hydantoinase activity of the present invention is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of the hydantoinase E 213S The hydantoinase activity relative to hydantoinase E 213S The hydantoinase activity of E 213V The hydantoinase activity of is determined by test F as described in item 4.5.11.2.

[0556] 4.5.12.5 Preferred variants of SEQ ID NO: 14

[0557] According to the present invention, the polypeptide sequence of hydantoinase E2, preferably the polypeptide sequence of L-hydantoinase E2, may also be a variant of SEQ ID NO:14.

[0558] Hydantoinase E2, whose polypeptide sequence is SEQ ID NO: 14, is represented by "E 214S ”.

[0559] Hydantoinase E2, whose polypeptide sequence is selected from a variant of SEQ ID NO: 14, is generally represented by "E 214V ”.

[0560] A variant of the polypeptide sequence of SEQ ID NO: 14 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO: 14.

[0561] A variant of the polypeptide sequence SEQ ID NO:14 has a polypeptide sequence that is not identical to SEQ ID NO:14.

[0562] According to the present invention, hydantoinase E 214V It has hydantoinase activity, preferably L-hydantoinase activity, as determined as described in Items 4.5.10.2 and 4.5.10.3.1.

[0563] According to the present invention, hydantoinase E 214V Preferably, it has hydantoinase E 214Sat least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100% of the hydantoinase activity of E 214S The hydantoinase activity of E 214V The hydantoinase activity of the enzyme is determined by test F as described in item 4.5.11.2.

[0564] According to the present invention, even more preferably, hydantoinase E 214V The hydantoinase activity is 214S The hydantoinase activity of the present invention is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of the hydantoinase E 214S The hydantoinase activity relative to hydantoinase E 214S The hydantoinase activity of E 214V The hydantoinase activity of is determined by test F as described in item 4.5.11.2.

[0565] 4.5.12.6 Preferred variants of SEQ ID NO: 15

[0566] According to the present invention, the polypeptide sequence of hydantoinase E2, preferably the polypeptide sequence of L-hydantoinase E2, may also be a variant of SEQ ID NO:15.

[0567] Hydantoinase E2, whose polypeptide sequence is SEQ ID NO: 15, is represented by "E 215S ”.

[0568] Hydantoinase E2, whose polypeptide sequence is selected from a variant of SEQ ID NO: 15, is generally represented by "E 215V ”.

[0569] A variant of the polypeptide sequence of SEQ ID NO: 15 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO: 15.

[0570] A variant of the polypeptide sequence SEQ ID NO:15 has a polypeptide sequence that is not identical to SEQ ID NO:15.

[0571] According to the present invention, hydantoinase E 215V It has hydantoinase activity, preferably L-hydantoinase activity, as determined as described in Items 4.5.10.2 and 4.5.10.3.1.

[0572] According to the present invention, hydantoinase E 215V Preferably, hydantoinase E 215S at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100% of the hydantoinase activity of E 215S The hydantoinase activity of E 215V The hydantoinase activity of the enzyme is determined by test F as described in item 4.5.11.2.

[0573] According to the present invention, even more preferably, hydantoinase E 215V The hydantoinase activity is 215S The hydantoinase activity of the present invention is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of the hydantoinase E 215S The hydantoinase activity relative to hydantoinase E 215S The hydantoinase activity of E 215V The hydantoinase activity of is determined by test F as described in item 4.5.11.2.

[0574] 4.5.12.7 Preferred variants of SEQ ID NO: 16

[0575] According to the present invention, the polypeptide sequence of hydantoinase E2, preferably the polypeptide sequence of L-hydantoinase E2, may also be a variant of SEQ ID NO:16.

[0576] Hydantoinase E2, whose polypeptide sequence is SEQ ID NO: 16, is represented by "E 216S ”.

[0577] Hydantoinase E2, whose polypeptide sequence is selected from a variant of SEQ ID NO: 16, is generally represented by "E 216V ”.

[0578] A variant of the polypeptide sequence of SEQ ID NO: 16 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO: 16.

[0579] A variant of the polypeptide sequence SEQ ID NO:16 has a polypeptide sequence that is not identical to SEQ ID NO:16.

[0580] According to the present invention, hydantoinase E 216V It has hydantoinase activity, preferably L-hydantoinase activity, as determined as described in Items 4.5.10.2 and 4.5.10.3.1.

[0581] According to the present invention, hydantoinase E 216V Preferably, it has hydantoinase E 216S at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100% of the hydantoinase activity of E 216S The hydantoinase activity of E 216V The hydantoinase activity of the strain was determined by test F as described in item 4.5.11.2.

[0582] According to the present invention, even more preferably, hydantoinase E 216V The hydantoinase activity is 216SThe hydantoinase activity of the present invention is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of the hydantoinase E 216S The hydantoinase activity relative to hydantoinase E 216S The hydantoinase activity of E 216V The hydantoinase activity of is determined by test F as described in item 4.5.11.2.

[0583] 4.5.13 Preferred process conditions in step (b)

[0584] The reaction in step (b) of the process of the first aspect of the present invention can be carried out under conditions known to the skilled person.

[0585] The reaction medium is preferably aqueous, more preferably an aqueous buffer.

[0586] Exemplary buffers commonly used in biotransformation reactions and advantageously used herein include Tris, phosphate or any zwitterionic buffer, such as 2-(N-morpholino)ethanesulfonic acid ("MES"), N-(2-acetamido)iminodiacetic acid ("ADA"), piperazine-N,N'-bis(2-ethanesulfonic acid) ("PIPES"), N-(2-acetamido)-2-aminoethanesulfonic acid ("ACES"), p-hydroxy-4-morpholinopropanesulfonic acid ("MOPSO"), cholestyramine chloride, 3-(N-morpholino)propanesulfonic acid ("MOPS"), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid ("BES"), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid ("TES"), 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid ("HEPES"), 3-(bis(2-hydroxyethyl)amino)-2-hydroxypropane-1-sulfonic acid ("DIPSO"), acetylaminoglycine, 3-(N-tris(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid ("TAPSO"), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) ("POPSO"), 4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid) ("HEPPSO"), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid ("HEPPS"), tris(hydroxymethyl)methylglycine, glycinamide, N,N-bis(2-hydroxyethyl)glycine, or 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonic acid ("TAPS").

[0587] In some embodiments, ammonium can act as a buffer.One or more organic solvents can also be added to the reaction.

[0588] The buffer preferably comprises a metal salt, more preferably a metal salt such as a metal halide, preferably a monovalent or divalent or trivalent metal halide, preferably a monovalent or divalent metal chloride, preferably CoCl2 or MnCl2, preferably CoCl2.

[0589] The concentration of these metal salts in the reaction medium is preferably from 1 μM to 1 M, more preferably from 1 mM to 100 mM, even more preferably from 1 to 10 mM.

[0590] Preferably, step (b) of the method of the first aspect of the invention is carried out in phosphate buffer.

[0591] The pH of the reaction medium in step (b) of the process of the first aspect of the invention is preferably in the range of 2 to 10, more preferably in the range of 5 to 8, more preferably in the range of 7.2 to 7.5, most preferably 7.5.

[0592] Preferably, step (b) of the method of the first aspect of the invention is carried out at a temperature in the range of 20°C to 70°C, more preferably in the range of 30°C to 55°C, most preferably 50°C.

[0593] Preferably, the total concentration of all hydantoinases E2 in the reaction solution in step (b) is in the range of 1 μM to 10 mM, preferably 10 μM to 1 mM, more preferably 0.1 mM to 0.5 mM, most preferably 0.4 mM.

[0594] In an alternative preferred embodiment, the total concentration of all hydantoinase E2 in the reaction solution in step (b) is in the range of 1 μg / l to 10 g / l, preferably 0.1 mg / l to 5 g / l, more preferably 1 mg / l to 1 g / l, more preferably 5 mg / l to 500 mg / l.

[0595] Preferably, the initial concentration of all compounds of formula L-(III) in the reaction medium of step (b) is in the range of 1 μM to 1 M, preferably in the range of 10 μM to 0.5 M, more preferably in the range of 0.1 mM to 0.1 M, more preferably in the range of 1 mM to 10 mM, and most preferably 1.25 mM.

[0596] If a compound of formula D-(III) is present in the reaction medium of step (b), the initial concentration of all compounds of formula D-(III) in the reaction medium is preferably 1% to 100% of the concentration of all compounds of formula L-(III), more preferably 10% to 100% of the concentration of all compounds of formula L-(III), even more preferably 50 to 100%, even more preferably 100% of the concentration of all compounds of formula L-(III).

[0597] Preferably, step (b) is carried out in the same reaction medium as step (c).

[0598] In this case, preferably, the initial concentration of all compounds of formula L-(III) in the reaction medium in step (b) is in the range of 1 μM to 1 M, preferably in the range of 10 μM to 0.5 M, more preferably in the range of 0.1 mM to 0.1 M, more preferably in the range of 1 mM to 10 mM, and most preferably 1.25 mM.

[0599] "The initial concentration of all compounds of formula L-(III) / D-(III)" refers to the respective concentration of the corresponding compound L-(III) or D-(III) in the reaction medium when the corresponding compound is used in step (b).

[0600] 4.5.14 Step (a)

[0601] In a preferred embodiment of the method of the first aspect of the present invention, the compound of formula L-(III) is obtained by step (a), wherein a compound of formula D-(III) is reacted to obtain a compound of formula L-(III):

[0602]

[0603] Step (a) provides the starting material for step (b).

[0604] R 3D is a group selected from the above formula (VIII), (IX), (X), preferably a group selected from the above formula (VIII), (IX), more preferably R 3D It has the formula (VIII).

[0605] When R 3D When the group is a group of formula (VIII), the group R 3D It can be selected from the above formulae L-(VIII) and D-(VIII), preferably L-(VIII).

[0606] When R 3D When the group is a group of formula (IX), the group R 3D It can be selected from the above formulae L-(IX) and D-(IX), preferably L-(IX).

[0607] When R 3D When the group is a group of formula (X), the group R 3D It can be selected from the above formulae L-(X) and D-(X), preferably L-(X).

[0608] The reaction of step (a) can be performed enzymatically or non-enzymatically, preferably enzymatically. More preferably, the reaction of step (a) is catalyzed by hydantoin racemase E3.

[0609] In a preferred step (a), the compound of formula D-(III) is prepared in step (a) as a mixture of at least one compound D-(III) and at least one compound L-(III). III Use, where R 3L =R 3D .

[0610] In a preferred embodiment, the mixture M III All compounds and mixtures of formula L-(III) III The molar ratio of all compounds of formula D-(III) is substantially 1:1.

[0611] In other preferred embodiments, the mixture M II All compounds and mixtures of formula L-(III) III The molar ratio of all compounds of formula D-(III) is in the range of 3:2 to 1:99, more preferably in the range of 1.01:1 to 1:99, more preferably in the range of 1:1 to 1:99, more preferably in the range of 1:1.01 to 1:99, more preferably in the range of 1:1.01 to 1:9, more preferably in the range of 1:1.01 to 1:9, more preferably in the range of 1:1.01 to 1:8, more preferably in the range of 1:1.01 to 1:3.

[0612] Alternatively, D-(III) in the mixture M III The content of L-(III) in the mixture M III At least one compound of formula L-(III) is greater than the mixture M III All compounds of formula D-(III) are <1:1, preferably <0.9:1, more preferably <0.75:1, more preferably <0.5:1, more preferably <0.2:1, more preferably <0.1:1, more preferably <0.01:1.

[0613] 4.5.14.1 Step (a) without enzyme catalysis

[0614] Step (a) may be performed in a non-enzymatic manner, ie without the use of enzymes. The reaction of the compound of step D-(III) to form the compound according to L-(III) is carried out in an alkaline solution, as known to the skilled person, as described by Slomka et al., M. Bovarnick & H. T. Clarke, Journal of the American Chemical Society 1938, 60, 2426-2430, RA Lazarus, J. Org. Chem. 1990, 55, 4755-4757, and ASBommarius, M. Kottenhahn, H. Klenk, K. Drauz: "A direct route from hydantoins to D-amino acids employing aresting cell biocatalyst with D-hydantoinase and D-carbamoylase activity", which is described in "Microbial Reagents in Organic Synthesis" Series C: Mathematical and Physical Sciences-Vol. 381, S. Servi (Ed.), 1992, Springer Science + Business Media, BV, Dordrecht, pp. 164 and 167.

[0615] Therefore, if step (a) is carried out non-enzymatically, the conditions preferably applied to the reaction medium for carrying out the non-enzymatic step (a) are preferably the conditions described for the preferred conditions of step (b) (item 4.5.13), except that the pH is ≥ 8, preferably the pH is in the range of 8 to 12, more preferably in the range of 8 to 11, more preferably in the range of 8 to 10, even more preferably in the range of 8 to 9. Since in these embodiments, the preferred conditions regarding the pH range in steps (a) and (b) are different, it is preferred that the reaction media in steps (a) and (b) are different.

[0616] 4.5.14.2 Enzymatic conversion of D-(III) to L-(III)

[0617] Step (a) is preferably carried out enzymatically, ie the reaction of step (a) is preferably catalyzed by hydantoin racemase E3.

[0618] That is, it was surprisingly found that hydantoin racemase accepts compounds of formula D-(III) as substrates and converts them into products of formula L-(III), thereby catalyzing the reaction of step (a), but has no catalytic effect on the corresponding compounds of formula D-(III) wherein R=H.

[0619] This discovery has high scientific and economic value because it further expands the range of new raw materials for preparing L-phosphinothricin alkyl esters and L-phosphinothricin by new synthetic routes. In addition, this discovery also opens up new possibilities for enantioselective preparation of LGA from a racemic mixture of L-(III) and D-(III), as embodied in the method of the second aspect of the invention.

[0620] In fact, hydantoin racemase catalyzes the conversion of two hydantoin enantiomers H L and H R One of the two is converted into another (see the following reaction <3> ):

[0621] <3> :

[0622] It has now surprisingly been found that hydantoin racemase also accepts of substrate, where R = R 3D .

[0623] Surprisingly, they do not accept And a substrate wherein R=H.

[0624] Suitable hydantoin racemases are described, for example, in WO 01 / 23582 A1 and in U. Engel, J. Rudat, C. Syldatk, in "The hydantoinase process: recent developments for the production of non-canonical amino acids" in "Industrial biocatalysis" by P. Grunwald (ed.), Pan Stanford Series on Biocatalysis, 2015, pp. 817-862, and in FJ Las Heras-Vazquez, JM Clemente-Jimenez, S. Martinez-Rodriguez, F. Rodriguez-Vico, in "Modern Biocatalysis: Stereoselective and environmentally friendly reactions" by W. Fessner, T. Anthonsen (ed.), Weinheim: WILEY-VCH Verlag GmbH & Co, 2009, Chapter 12, pp. 173-193. enzyme for the production of optically pure α-amino acids”.

[0625] The hydantoin racemase E3 that can be used in the optional step (a) of the method of the first aspect of the present invention can be derived from Agrobacterium species, in particular Agrobacterium strain IP_I-671; Arthrobacter, in particular Arthrobacter aureus, more particularly Arthrobacter aureus DSM 3745 or Arthrobacter species BT801; Flavobacterium sp., in particular Flavobacterium species AJ 11199; Microbacterium, in particular Microbacterium liquefaciens, preferably Microbacterium liquefaciens AJ 3912; Pasteurella, in particular Pasteurella species AJ11221; Pseudomonas, in particular Pseudomonas species NS671; Pyrococcus sp., in particular Pyrococcus horikoshii OT3; Rhodococcus sp. sp.), in particular Rhodococcus sp. R04; Sinorhizobium spp., in particular Sinorhizobium meliloti, more particularly Sinorhizobium meliloti CECT 4114, most preferably from Arthrobacter aureus DSM 3745.

[0626] A hydantoin racemase E3 suitable for use in the method of the invention may be the enzyme HyuR derived from Arthrobacter aureus DSM 3745. Another enzyme may be selected from HyuE, Hyu2, HRase, HyuA, PH1054.

[0627] The hydantoin racemase E3 which may be preferably used in the preferred step (a) of the method of the first aspect of the present invention may be classified in the class EC 5.1.99.5.

[0628] Table 3 below gives preferred examples of polypeptide sequences of hydantoin racemase E3, which can be preferably used in step (a) of the method of the first aspect of the present invention. The genes encoding the corresponding hydantoin racemase E3 and the corresponding accession numbers are known.

[0629] Table 3

[0630]

[0631]

[0632] In a preferred embodiment of the preferred method according to the second aspect of the present invention, the reaction of step (a) is catalyzed by a hydantoin racemase E3, wherein the polypeptide sequence of E3 is selected from the group consisting of SEQ ID NO: 17 and variants thereof, SEQ ID NO: 18 and variants thereof, SEQ ID NO: 19 and variants thereof, SEQ ID NO: 20 and variants thereof, SEQ ID NO: 21 and variants thereof, SEQ ID NO: 22 and variants thereof, SEQ ID NO: 23 and variants thereof, SEQ ID NO: 24 and variants thereof, SEQ ID NO: 25 and variants thereof and SEQ ID NO: 26 and variants thereof.

[0633] 4.5.14.3 Test G for determination of hydantoin racemase activity

[0634] The skilled person is aware of hydantoin racemases which may be used in the preferred step (a) of the method of the first aspect of the invention.

[0635] In particular, the assay G described below can be used to determine the activity of a given enzyme E Z and can be advantageously used according to the present invention to determine the carbamoylase and L-carbamoylase activities in variants of SEQ ID NO: 17, variants of SEQ ID NO: 18, variants of SEQ ID NO: 19, variants of SEQ ID NO: 20, variants of SEQ ID NO: 21, variants of SEQ ID NO: 22, variants of SEQ ID NO: 23, variants of SEQ ID NO: 24, variants of SEQ ID NO: 25, and variants of SEQ ID NO: 26.

[0636] For the purpose of test G, the enzyme E to be tested Z The molar mass is calculated as E Z The molar mass of the polypeptide sequence.

[0637] In addition, the substrate of formula D-(III), wherein R 3D = n-butyl, used in test G, so there is only one chiral carbon atom in the substrate. Compounds of formula D-(III) wherein R 3D = n-butyl, expressed as

[0638] The product of formula L-(III), wherein R 3L =n-Butyl, detected in Test G.

[0639] A compound of formula L-(III), wherein R 3L = n-butyl, expressed as

[0640] Test G:

[0641] Add 0.9 ml of 50 mM To an aqueous reaction solution (phosphate buffer, pH 7.2, 10 mM Mg2Cl) of the pure D-enantiomer of the n-butyl ester of phosphinothricin ammonium was added 400 nmol of E in 0.1 ml of aqueous phosphate buffer (pH 7.2, 10 mM Mg2Cl). Z .

[0642] The resulting solution was incubated at 25°C and the pH was maintained at pH 7.2 by adding 0.5 M. After 300 min, the reaction was terminated by adding 2 M HCl to bring the pH to 2.5. The formula was measured at least every 3 min. The molar amount of the L-enantiomer of the compound (determined by LC-MS, for example by the LC-MS method for detecting LGA described in the Examples section, item 5.4).

[0643] 4.5.14.4 Test for identification of hydantoin racemase

[0644] A given enzyme E Z Whether it can be considered as a hydantoin racemase E3 can be determined in the context of the present invention by the following test H:

[0645] H-1 First, perform Test G described under 4.5.14.3 and determine the obtained Molar amount.

[0646] H-2 Then, step H-1 was repeated, except that instead of adding 400 nmol E in 0.1 ml of phosphate buffer solution (pH 7.2, 10 mM NCl2), Z , but add Z 0.1 ml of phosphate buffered saline (pH 7.2, 10 mM MnCl2).

[0647] 4.5.14.5 Hydantoin racemase activity

[0648] If the formula determined in step H-1 is The molar amount of the compound of formula The molar amount of the compound, then E Z It is believed to have hydantoin racemase activity and can therefore be considered as a hydantoin racemase E3 in the context of the present invention.

[0649] 4.5.14.6 Assay for Identifying Preferred Hydantoin Racemase Variants of SEQ ID NOs: 17-26

[0650] The enzyme whose polypeptide sequence is selected from SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 has hydantoin racemase activity.

[0651] In a preferred embodiment of the method of the first aspect of the invention, the polypeptide sequence of hydantoin racemase E3 is selected from SEQ ID NO: 17 and variants thereof, SEQ ID NO: 18 and variants thereof, SEQ ID NO: 19 and variants thereof, SEQ ID NO: 20 and variants thereof, SEQ ID NO: 21 and variants thereof, SEQ ID NO: 22 and variants thereof, SEQ ID NO: 23 and variants thereof, SEQ ID NO: 24 and variants thereof, SEQ ID NO: 25 and variants thereof, SEQ ID NO: 26 and variants thereof.

[0652] The term "variant" is defined in Section 4.3.

[0653] In the context of the present invention, an enzyme whose polypeptide sequence is a variant of one of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 has hydantoin racemase activity.

[0654] For a given enzyme E Z , whose polypeptide sequence is a variant of one of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, and whether it has hydantoin racemase activity can be determined according to the methods described in Items 4.5.14.4 and 4.5.14.5.

[0655] Relative to hydantoin racemase E 3S Hydantoin racemase activity (E 3S The polypeptide sequence is selected from SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26), the given hydantoin racemase E 3V The hydantoin racemase activity of a polypeptide whose polypeptide sequence is a variant of one of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 can be quantified in the context of the present invention by the following assay J.

[0656] Test J:

[0657] J-1. First, perform test G as described in 4.5.14.3, where E 3S is the enzyme to be tested. The molar amount of the compound.

[0658] J-2. Repeat step J-1, except use E 3VReplace E 3S As the enzyme to be tested.

[0659] J-3. Then, the formula determined in step J-2 is The molar amount of the compound of formula L-(III) (wherein R = n-butyl) determined in step J-1 is divided by the molar amount of the compound of formula L-(III) (wherein R = n-butyl) determined in step J-1, and the obtained ratio is multiplied by 100 to obtain the relative hydantoin racemase E expressed in %. 3S Hydantoin racemase E 3V of hydantoin racemase activity.

[0660] 4.5.14.7 Preferred hydantoin racemase variants SEQ ID NOs: 17-26

[0661] In the context of the present invention, the hydantoin racemase E3, whose polypeptide sequence is selected from SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, is generally denoted as “E 3S ”.

[0662] A hydantoin racemase E3, whose polypeptide sequence is selected from the sequence of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, a variant of SEQ ID NO: 26, generally represented by “E 3V ”.

[0663] In a preferred embodiment of the method of the first aspect of the present invention, the reaction in step (a) is catalyzed by a hydantoin racemase E3, and the polypeptide sequence of the hydantoin racemase E3 is selected from the group consisting of SEQ ID NO: 17 and variants thereof, SEQ ID NO: 18 and variants thereof, SEQ ID NO: 19 and variants thereof, SEQ ID NO: 20 and variants thereof, SEQ ID NO: 21 and variants thereof, SEQ ID NO: 22 and variants thereof, SEQ ID NO: 23 and variants thereof, SEQ ID NO: 24 and variants thereof, SEQ ID NO: 25 and variants thereof, SEQ ID NO: 26 and variants thereof. More preferably, the reaction in step (a) is catalyzed by a hydantoin racemase E3, and the polypeptide sequence of the hydantoin racemase E3 is selected from the group consisting of SEQ ID NO: 1 and variants thereof.

[0664] 4.5.14.7.1 Preferred variants of SEQ ID NO: 17

[0665] According to the present invention, the polypeptide sequence of hydantoin racemase E3 may also be a variant of SEQ ID NO:17.

[0666] Hydantoin racemase E3, whose polypeptide sequence is SEQ ID NO: 17, is represented by “E 317S The hydantoin racemase E3, whose polypeptide sequence is selected from a variant of SEQ ID NO: 17, is generally represented by “E 317V ”.

[0667] A variant of the polypeptide sequence of SEQ ID NO: 17 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO: 17.

[0668] A variant of the polypeptide sequence SEQ ID NO:17 has a polypeptide sequence that is not identical to SEQ ID NO:17.

[0669] According to the present invention, hydantoin racemase E 317V Having hydantoin racemase activity as determined as described in Items 4.5.14.4 and 4.5.14.5.

[0670] According to the present invention, hydantoin racemase E 317V Preferably, the hydantoin racemase E 317S at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100% of the hydantoin racemase activity of E 317S Hydantoin racemase activity of E 317V The hydantoin racemase activity of is determined by Assay J as described in Item 4.5.14.6.

[0671] According to the present invention, more preferably, hydantoin racemase E 317V The hydantoin racemase activity is in the hydantoin racemase E 317SThe hydantoin racemase activity of the present invention is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of the hydantoin racemase activity of the present invention. 317S The hydantoin racemase activity was 317S Hydantoin racemase activity of E 317V The hydantoin racemase activity of is determined by Assay J as described in Item 4.5.14.6.

[0672] 4.5.14.7.2 Preferred variants of SEQ ID NO: 18

[0673] According to the present invention, the polypeptide sequence of hydantoin racemase E3 may also be a variant of SEQ ID NO:18.

[0674] Hydantoin racemase E3, whose polypeptide sequence is SEQ ID NO: 18, is represented by “E 318S The hydantoin racemase E3, whose polypeptide sequence is selected from a variant of SEQ ID NO: 18, is generally represented by “E 318V ”.

[0675] A variant of the polypeptide sequence of SEQ ID NO: 18 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO: 18.

[0676] A variant of the polypeptide sequence SEQ ID NO:18 has a polypeptide sequence that is not identical to SEQ ID NO:18.

[0677] According to the present invention, hydantoin racemase E 318V Having hydantoin racemase activity as determined as described in Items 4.5.14.4 and 4.5.14.5.

[0678] According to the present invention, hydantoin racemase E 318V Preferably, the hydantoin racemase E 318Sat least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100% of the hydantoin racemase activity of E 318S Hydantoin racemase activity of E 318V The hydantoin racemase activity of is determined by Assay J as described in Item 4.5.14.6.

[0679] According to the present invention, even more preferably, the hydantoin racemase E 318V The hydantoin racemase activity is in the hydantoin racemase E 318S The hydantoin racemase activity of the present invention is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of the hydantoin racemase activity of the present invention. 318S The hydantoin racemase activity was 318S Hydantoin racemase activity of E 318V The hydantoin racemase activity of is determined by Assay J as described in Item 4.5.14.6.

[0680] 4.5.14.7.3 Preferred variants of SEQ ID NO: 19

[0681] According to the present invention, the polypeptide sequence of hydantoin racemase E3 may also be a variant of SEQ ID NO:19.

[0682] Hydantoin racemase E3, whose polypeptide sequence is SEQ ID NO: 19, represented by “E 319S Hydantoin racemase E3, whose polypeptide sequence is selected from a variant of SEQ ID NO: 19, is generally represented by “E 319V ”.

[0683] A variant of the polypeptide sequence of SEQ ID NO: 19 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO: 19.

[0684] A variant of the polypeptide sequence SEQ ID NO:19 has a polypeptide sequence that is not identical to SEQ ID NO:19.

[0685] According to the present invention, hydantoin racemase E 319V Having hydantoin racemase activity as determined as described in Items 4.5.14.4 and 4.5.14.5.

[0686] According to the present invention, hydantoin racemase E 319V Preferably, the hydantoin racemase E 319S The hydantoin racemase activity of E is at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100%, wherein E 319V The hydantoin racemase activity was relative to that of E 319S The hydantoin racemase activity of is determined by Assay J as described in Item 4.5.14.6.

[0687] According to the present invention, even more preferably, the hydantoin racemase E 319V The hydantoin racemase activity is in the hydantoin racemase E 319S The hydantoin racemase activity of the present invention is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of the hydantoin racemase activity of the present invention. 319S The hydantoin racemase activity was 319S Hydantoin racemase activity of E 319V The hydantoin racemase activity of is determined by Assay J as described in Item 4.5.14.6.

[0688] 4.5.14.7.4 Preferred variants of SEQ ID NO: 20

[0689] According to the present invention, the polypeptide sequence of hydantoin racemase E3 may also be a variant of SEQ ID NO:20.

[0690] Hydantoin racemase E3, whose polypeptide sequence is SEQ ID NO: 20, is represented by “E 320S Hydantoin racemase E3, whose polypeptide sequence is selected from a variant of SEQ ID NO: 20, is generally represented by “E 320V ”.

[0691] A variant of the polypeptide sequence of SEQ ID NO:20 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO:20.

[0692] A variant of the polypeptide sequence SEQ ID NO:20 has a polypeptide sequence that is not identical to SEQ ID NO:20.

[0693] According to the present invention, hydantoin racemase E 320V Having hydantoin racemase activity as determined as described in Items 4.5.14.4 and 4.5.14.5.

[0694] According to the present invention, hydantoin racemase E 320V Preferably, the hydantoin racemase E 320S at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100% of the hydantoin racemase activity of E 320S Hydantoin racemase activity of E 320V The hydantoin racemase activity of is determined by Assay J as described in Item 4.5.14.6.

[0695] According to the present invention, even more preferably, the hydantoin racemase E 320V The hydantoin racemase activity is in the hydantoin racemase E 320SThe hydantoin racemase activity of the present invention is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of the hydantoin racemase activity of the present invention. 320S The hydantoin racemase activity was 320S Hydantoin racemase activity of E 320V The hydantoin racemase activity of is determined by Assay J as described in Item 4.5.14.6.

[0696] 4.5.14.7.5 Preferred variants of SEQ ID NO: 21

[0697] According to the present invention, the polypeptide sequence of hydantoin racemase E3 may also be a variant of SEQ ID NO:21.

[0698] Hydantoin racemase E3, whose polypeptide sequence is SEQ ID NO: 21, represented by “E 321S The hydantoin racemase E3, whose polypeptide sequence is selected from a variant of SEQ ID NO: 21, is generally represented by “E 321V ”.

[0699] A variant of the polypeptide sequence of SEQ ID NO:21 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO:21.

[0700] A variant of the polypeptide sequence SEQ ID NO:21 has a polypeptide sequence that is not identical to SEQ ID NO:21.

[0701] According to the present invention, hydantoin racemase E 321V Having hydantoin racemase activity as determined as described in Items 4.5.14.4 and 4.5.14.5.

[0702] According to the present invention, hydantoin racemase E 321V Preferably, the hydantoin racemase E 321Sat least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100% of the hydantoin racemase activity of E 321S Hydantoin racemase activity of E 321V The hydantoin racemase activity of is determined by Assay J as described in Item 4.5.14.6.

[0703] According to the present invention, even more preferably, the hydantoin racemase E 321V The hydantoin racemase activity is in the hydantoin racemase E 321S The hydantoin racemase activity of the present invention is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of the hydantoin racemase E 321S The hydantoin racemase activity was 321S Hydantoin racemase activity of E 321V The hydantoin racemase activity of is determined by Assay J as described in Item 4.5.14.6.

[0704] 4.5.14.7.6 Preferred variants of SEQ ID NO: 22

[0705] According to the present invention, the polypeptide sequence of hydantoin racemase E3 may also be a variant of SEQ ID NO:22.

[0706] Hydantoin racemase E3, whose polypeptide sequence is SEQ ID NO: 22, is represented by “E 322S Hydantoin racemase E3, whose polypeptide sequence is selected from a variant of SEQ ID NO: 22, is generally represented by “E 322V ”.

[0707] A variant of the polypeptide sequence of SEQ ID NO:22 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO:22.

[0708] A variant of the polypeptide sequence SEQ ID NO:22 has a polypeptide sequence that is not identical to SEQ ID NO:22.

[0709] According to the present invention, hydantoin racemase E 322V Having hydantoin racemase activity as determined as described in Items 4.5.14.4 and 4.5.14.5.

[0710] According to the present invention, hydantoin racemase E 322V Preferably, the hydantoin racemase E 322S at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100% of the hydantoin racemase activity of E 322S Hydantoin racemase activity of E 322V The hydantoin racemase activity of is determined by Assay J as described in Item 4.5.14.6.

[0711] According to the present invention, even more preferably, the hydantoin racemase E 322V The hydantoin racemase activity is in the hydantoin racemase E 322S The hydantoin racemase activity of the present invention is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of the hydantoin racemase activity of the present invention. 322S The hydantoin racemase activity was 322S Hydantoin racemase activity of E 322V The hydantoin racemase activity of is determined by Assay J as described in Item 4.5.14.6.

[0712] 4.5.14.7.7 Preferred variants of SEQ ID NO: 23

[0713] According to the present invention, the polypeptide sequence of hydantoin racemase E3 may also be a variant of SEQ ID NO:23.

[0714] Hydantoin racemase E3, whose polypeptide sequence is SEQ ID NO: 23, is represented by “E 323S The hydantoin racemase E3, whose polypeptide sequence is selected from a variant of SEQ ID NO: 23, is generally represented by “E 323V ”.

[0715] A variant of the polypeptide sequence of SEQ ID NO:23 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO:23.

[0716] A variant of the polypeptide sequence SEQ ID NO:23 has a polypeptide sequence that is not identical to SEQ ID NO:23.

[0717] According to the present invention, hydantoin racemase E 323V Having hydantoin racemase activity as determined as described in Items 4.5.14.4 and 4.5.14.5.

[0718] According to the present invention, hydantoin racemase E 323V Preferably, the hydantoin racemase E 323S at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100% of the hydantoin racemase activity of E 323S Hydantoin racemase activity of E 323V The hydantoin racemase activity of is determined by Assay J as described in Item 4.5.14.6.

[0719] According to the present invention, even more preferably, the hydantoin racemase E 323V The hydantoin racemase activity is in the hydantoin racemase E 323SThe hydantoin racemase activity of the present invention is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of the hydantoin racemase E 323S The hydantoin racemase activity was 323S Hydantoin racemase activity of E 323V The hydantoin racemase activity of is determined by Assay J as described in Item 4.5.14.6.

[0720] 4.5.14.7.8 Preferred variants of SEQ ID NO: 24

[0721] According to the present invention, the polypeptide sequence of hydantoin racemase E3 may also be a variant of SEQ ID NO:24.

[0722] Hydantoin racemase E3, whose polypeptide sequence is SEQ ID NO: 24, is represented by “E 324S The hydantoin racemase E3, whose polypeptide sequence is selected from a variant of SEQ ID NO: 24, is generally represented by “E 324V ”.

[0723] A variant of the polypeptide sequence of SEQ ID NO:24 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO:24.

[0724] A variant of the polypeptide sequence SEQ ID NO:24 has a polypeptide sequence that is not identical to SEQ ID NO:24.

[0725] According to the present invention, hydantoin racemase E 324V Having hydantoin racemase activity as determined as described in Items 4.5.14.4 and 4.5.14.5.

[0726] According to the present invention, hydantoin racemase E 324V Preferably, the hydantoin racemase E 324Sat least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100% of the hydantoin racemase activity of E 324S Hydantoin racemase activity of E 324V The hydantoin racemase activity of is determined by Assay J as described in Item 4.5.14.6.

[0727] According to the present invention, even more preferably, the hydantoin racemase E 324V The hydantoin racemase activity is in the hydantoin racemase E 324S The hydantoin racemase activity of the present invention is in the range of 1 to 1000%, preferably in the range of 5 to 500%, more preferably in the range of 10 to 400%, more preferably in the range of 40 to 200%, more preferably in the range of 50 to 150%, more preferably in the range of 60 to 140%, more preferably in the range of 70 to 130%, more preferably in the range of 80 to 120%, more preferably in the range of 90 to 110%, more preferably 100% of the hydantoin racemase activity of the present invention. 324S The hydantoin racemase activity was 324S Hydantoin racemase activity of E 324V The hydantoin racemase activity of is determined by Assay J as described in Item 4.5.14.6.

[0728] 4.5.14.7.9 Preferred variants of SEQ ID NO: 25

[0729] According to the present invention, the polypeptide sequence of hydantoin racemase E3 may also be a variant of SEQ ID NO:25.

[0730] Hydantoin racemase E3, whose polypeptide sequence is SEQ ID NO: 25, is represented by “E 325S Hydantoin racemase E3, whose polypeptide sequence is selected from a variant of SEQ ID NO: 25, is generally represented by “E 325V ”.

[0731] A variant of the polypeptide sequence of SEQ ID NO:25 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO:25.

[0732] A variant of the polypeptide sequence SEQ ID NO:25 has a polypeptide sequence that is not identical to SEQ ID NO:25.

[0733] According to the present invention, hydantoin racemase E 325V Having hydantoin racemase activity as determined as described in Items 4.5.14.4 and 4.5.14.5.

[0734] According to the present invention, hydantoin racemase E 325V Preferably, the hydantoin racemase E 325S at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100% of the hydantoin racemase activity of E 325S Hydantoin racemase activity of E 325V The hydantoin racemase activity of is determined by Assay J as described in Item 4.5.14.6.

[0735] According to the present invention, even more preferably, the hydantoin racemase E 325V The hydantoin racemase activity is in the hydantoin racemase E 325S The hydantoin racemase activity of the present invention is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of the hydantoin racemase activity of the present invention. 325S The hydantoin racemase activity was 325S Hydantoin racemase activity of E 325V The hydantoin racemase activity of is determined by Assay J as described in Item 4.5.14.6.

[0736] 4.5.14.7.10 Preferred variants of SEQ ID NO: 26

[0737] According to the present invention, the polypeptide sequence of hydantoin racemase E3 may also be a variant of SEQ ID NO:26.

[0738] Hydantoin racemase E3, whose polypeptide sequence is SEQ ID NO: 26, is represented by “E 326S The hydantoin racemase E3, whose polypeptide sequence is selected from a variant of SEQ ID NO: 26, is generally represented by “E 326V ”.

[0739] A variant of the polypeptide sequence of SEQ ID NO:26 is a polypeptide having at least 60%, preferably ≥65%, more preferably ≥70%, more preferably ≥75%, more preferably ≥80%, more preferably ≥85%, more preferably ≥90%, more preferably ≥91%, more preferably ≥92%, more preferably ≥93%, more preferably ≥94%, more preferably ≥95%, more preferably ≥96%, more preferably ≥97%, more preferably ≥98%, more preferably ≥99%, more preferably ≥99.9% sequence identity to the polypeptide sequence of SEQ ID NO:26.

[0740] A variant of the polypeptide sequence SEQ ID NO:26 has a polypeptide sequence that is not identical to SEQ ID NO:26.

[0741] According to the present invention, hydantoin racemase E 326V Having hydantoin racemase activity as determined as described in Items 4.5.14.4 and 4.5.14.5.

[0742] According to the present invention, hydantoin racemase E 326V Preferably, the hydantoin racemase E 326S at least 1%, preferably at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, more preferably at least 100% of the hydantoin racemase activity of E 326S Hydantoin racemase activity of E 326V The hydantoin racemase activity of is determined by Assay J as described in Item 4.5.14.6.

[0743] According to the present invention, even more preferably, the hydantoin racemase E 326V Hydantoin racemase activity in hydantoin racemase E 326SThe hydantoin racemase activity of the present invention is in the range of 1-1000%, preferably in the range of 5-500%, more preferably in the range of 10-400%, more preferably in the range of 40-200%, more preferably in the range of 50-150%, more preferably in the range of 60-140%, more preferably in the range of 70-130%, more preferably in the range of 80-120%, more preferably in the range of 90-110%, more preferably 100% of the hydantoin racemase activity of the present invention. 326S The hydantoin racemase activity was 326S Hydantoin racemase activity of E 326V The hydantoin racemase activity of is determined by Assay J as described in Item 4.5.14.6.

[0744] 4.5.15 Preferred process conditions in step (a)

[0745] In the preferred embodiment, wherein step (a) is catalyzed by hydantoin racemase E3, the reaction in step (a) can be carried out under conditions known to the skilled person.

[0746] The reaction medium is preferably aqueous, more preferably an aqueous buffer.

[0747] Exemplary buffers commonly used in biotransformation reactions and advantageously used herein include Tris, phosphate or any zwitterionic buffer, such as 2-(N-morpholino)ethanesulfonic acid ("MES"), N-(2-acetamido)iminodiacetic acid ("ADA"), piperazine-N,N'-bis(2-ethanesulfonic acid) ("PIPES"), N-(2-acetamido)-2-aminoethanesulfonic acid ("ACES"), p-hydroxy-4-morpholinopropanesulfonic acid ("MOPSO"), cholestyramine chloride, 3-(N-morpholino)propanesulfonic acid ("MOPS"), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid ("BES"), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid ("TES"), 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid ("HEPES"), 3-(bis(2-hydroxyethyl)amino)-2-hydroxypropane-1-sulfonic acid ("DIPSO"), acetylaminoglycine, 3-(N-tris(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid ("TAPSO"), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) ("POPSO"), 4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid) ("HEPPSO"), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid ("HEPPS"), tris(hydroxymethyl)methylglycine, glycinamide, N,N-bis(2-hydroxyethyl)glycine, or 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonic acid ("TAPS").

[0748] In some embodiments, ammonium can act as a buffer.One or more organic solvents can also be added to the reaction.

[0749] The buffer preferably comprises a metal salt, more preferably a metal salt such as a halide of a monovalent or divalent metal (eg, CoCl2, MnCl2).

[0750] The concentration of these metal salts in the reaction medium is preferably from 1 μM to 1 M, more preferably from 1 mM to 100 mM, even more preferably from 1 to 10 mM.

[0751] Preferably, step (a) of the method of the first aspect of the invention is carried out in phosphate buffer.

[0752] The pH of the reaction medium in step (a) of the process of the first aspect of the invention is preferably in the range of 2 to 10, more preferably in the range of 5 to 8, more preferably in the range of 7.2 to 7.5, most preferably 7.5.

[0753] Preferably, step (a) of the method of the first aspect of the invention is carried out at a temperature in the range of 20°C to 70°C, more preferably in the range of 30°C to 55°C, most preferably 50°C.

[0754] In this preferred embodiment, wherein step (a) is catalyzed by hydantoin racemase E3, the preferred reaction conditions in step (a) are the same as those described in steps (b) and (c), with reference to items 4.5.7 and 4.5.13, respectively. Even more preferably, step (a) is performed simultaneously with steps (b) and (c).

[0755] Preferably, the total concentration of all hydantoin racemase E3 in the reaction solution in step (a) is in the range of 1 μM-10 mM, preferably 10 μM-1 mM, more preferably 0.1 mM to 0.5 mM, most preferably 0.4 mM.

[0756] In an alternative preferred embodiment, the total concentration of all hydantoin racemase E3 in the reaction solution in step (a) is in the range of 1 μg / l to 10 g / l, preferably 0.1 mg / l to 5 g / l, more preferably 1 mg / l to 1 g / l, more preferably 5 mg / l to 500 mg / l.

[0757] Preferably, step (a) is carried out in the same reaction medium as steps (c) and (b). The advantage is that this allows a one-pot synthesis of all steps (a), (b) and (c).

[0758] Preferably, the initial concentration of all compounds of formula D-(III) in the reaction medium of step (a) is in the range of 1 μM to 1 M, preferably 10 μM to 0.5 M, more preferably 0.1 mM to 0.1 M, more preferably 1 mM to 10 mM, most preferably 1.25 mM.

[0759] If a compound of formula L-(III) is present in the reaction medium of step (a), the initial concentration of all compounds of formula L-(III) in the reaction medium is preferably 1 to 100 times the concentration of all compounds of formula D-(III), more preferably 1 to 10 times the concentration of all compounds of formula D-(III), even more preferably 1 to 2 times the concentration of all compounds of formula D-(III), even more preferably the same as the concentration of all compounds of formula D-(III).

[0760] "The initial concentration of all compounds of formula L-(III) / D-(III)" refers to the respective concentration of the corresponding compound L-(III) or D-(III) in the reaction medium when the corresponding compound is used in step (a).

[0761] 4.6 Second Aspect: Method for Preparing L-phosphinothricin P-Ester

[0762] In a second aspect, the invention relates to a mixture M comprising at least one compound L-(III) and at least one compound D-(III). IIIA A further method for preparing L-phosphinothricin P-ester of formula L-(I):

[0763]

[0764]

[0765] Where R 1L is a group selected from the above formula (VIII), (IX), (X), preferably a group selected from the above formula (IX), (X), more preferably R 1L Having formula (X).

[0766] When R 1L When the group is a group of formula (VIII), the group R 1L It can be selected from the above formulae L-(VIII) and D-(VIII), preferably L-(VIII).

[0767] When R 1L When the group is a group of formula (IX), the group R 1L It can be selected from the above formulae L-(IX) and D-(IX), preferably L-(IX).

[0768] When R 1L When the group is a group of formula (X), the group R 1L It can be selected from the above formulae L-(X) and D-(X), preferably L-(X).

[0769] R 3L is a group selected from the above formula (VIII), (IX), (X), preferably a group selected from the above formula (VIII), (IX), more preferably R 3L It has the formula (VIII).

[0770] When R 3L When the group is a group of formula (VIII), the group R 3L It can be selected from the above formulae L-(VIII) and D-(VIII), preferably L-(VIII).

[0771] When R 3L When the group is a group of formula (IX), the group R 3L It can be selected from the above formulae L-(IX) and D-(IX), preferably L-(IX).

[0772] When R 3L When the group is a group of formula (X), the group R 3L It can be selected from the above formulae L-(X) and D-(X), preferably L-(X).

[0773] R 3D is a group selected from the above formula (VIII), (IX), (X), preferably a group selected from the above formula (VIII), (IX), more preferably R 3D It has the formula (VIII).

[0774] When R 3D When the group is a group of formula (VIII), the group R 3D It can be selected from the above formulae L-(VIII) and D-(VIII), preferably L-(VIII).

[0775] When R 3D When the group is a group of formula (IX), the group R 3D It can be selected from the above formulae L-(IX) and D-(IX), preferably L-(IX).

[0776] When R 3D When the group is a group of formula (X), the group R 3D It can be selected from the above formulae L-(X) and D-(X), preferably L-(X).

[0777] In the method of the second aspect of the invention, a mixture M comprising at least one compound L-(III) and at least one compound D-(III) is provided. IIIA , where R is preferred 3L =R 3D and subjecting it to at least two enzymatic reactions (ii) and (iii) to obtain a composition M comprising at least one compound L-(I) and optionally D-(I) I Therefore, it is preferred that the reaction according to the second aspect of the invention is enzymatic.

[0778] According to a second aspect of the present invention, the method for preparing L-phosphinothricin P-ester of formula L-(I) is L-enantiomer selective, in particular L-enantiomer specific, because M I contains a compound of formula L-(I), but does not contain a compound of formula D-(I) (in which case it is "L-enantiomer specific"), or when M I When the compound of formula L-(I) and D-(I) is included, M I All compounds of formula L-(I) and M I The molar ratio of all compounds of formula D-(I) is greater than M IIIA All compounds of formula L-(III) and M IIIA The molar ratio of all compounds of formula D-(III) (in this case, it is "L-enantiomerically selective, but not L-enantiomerically specific").

[0779] In a specific embodiment of the method of the second aspect of the invention, M I comprising at least one compound of formula L-(I) and at least one compound of formula D-(I), and M I All compounds of formula L-(I) and M I The molar ratio of all compounds of formula D-(I) is greater than M IIIA All compounds of formula L-(III) and M IIIA The molar ratio of all compounds of formula D-(III) (in this case, the process of the second aspect of the invention is "L-enantiomerically selective, but not L-enantiomerically specific").

[0780] In a preferred embodiment of the method of the second aspect of the present invention, in step (a) a mixture M is provided IIIA , which comprises at least one compound L-(III) and at least one compound D-(III), wherein R 3L =R 3D .

[0781] In the method of the second aspect of the present invention, the product composition M I The amount of the compound of formula L-(I) is enriched relative to the amount of the compound of formula D-(I), which means that M I All compounds of formula L-(I) and M I The molar ratio of all compounds of formula D-(I) is greater than the molar ratio of M provided in step (iA). IIIA All compounds of formula L-(III) and M IIIA The molar ratio of all compounds of formula D-(III).

[0782] In particular, M I The molar amount of the compound of formula L-(I) / the compound of formula D-(I) is greater than M IIIA The molar amount of the compound of formula L-(III) / the compound of formula D-(III), in particular x L times larger, where x L is at least 1.1, preferably at least 1.5, more preferably at least 2, more preferably at least 5, even more preferably at least 9, even more preferably at least 99. In another preferred embodiment, x L In the range of 1.1 to 99, preferably in the range of 1.5 to 9, even more preferably in the range of 2 to 5.

[0783] 4.6.1 Step (iA)

[0784] In the first step of the method of the second aspect of the present invention, a mixture M comprising at least one compound L-(III) and at least one compound D-(III) is provided. IIIA .

[0785] In a preferred embodiment, the mixture M IIIA All compounds and mixtures of formula L-(III) III The molar ratio of all compounds of formula D-(III) is substantially 1:1.

[0786] Alternatively, the compound of formula D-(III) is in the mixture M IIIA The content of L-(III) in the mixture M exceeds that of the compound of formula L-(III), which means that when L-(III) is present in the mixture M IIIA When the mixture M IIIA The molar ratio of all compounds of formula L-(III) to all compounds of formula D-(III) is <1:1, preferably <0.9:1, more preferably <0.75:1, more preferably <0.5:1, more preferably <0.2:1, more preferably <0.1:1, more preferably <0.01:1.

[0787] In other preferred embodiments, the mixture M IIIA All compounds and mixtures of formula L-(III) IIIA The molar ratio of all compounds of formula D-(III) is in the range of 3:2 to 1:99, more preferably in the range of 1.01:1 to 1:99, more preferably in the range of 1:1 to 1:99, more preferably in the range of 1:1.01 to 1:99, more preferably in the range of 1:1.01 to 1:9, more preferably in the range of 1:1.01 to 1:9, more preferably in the range of 1:1.01 to 1:8, more preferably in the range of 1:1.01 to 1:3.

[0788] Alternatively, D-(III) in the mixture M IIIA The content of L-(III) exceeds that of L-(III), which means that when at least one compound of formula L-(III) is present in the mixture M IIIA When the mixture M IIIA All compounds and mixtures of formula L-(III) IIIA The molar ratio of all compounds of formula D-(III) is <1:1, preferably <0.9:1, more preferably <0.75:1, more preferably <0.5:1, more preferably <0.2:1, more preferably <0.1:1, more preferably <0.01:1.

[0789] Mixtures of these compounds can be obtained by the skilled worker, for example, by organic syntheses as described in DE 31 42 036 A1 and by Slomka et al.

[0790] E.Ware,Chem.Rev.1950,46,403-470 and C. & J.C.Menendez, “Hydantoin and its derivates” in Kirk-Othmer Encyclopedia of Chemical Technology 2000 give a general review of hydantoin chemistry.

[0791] Preferably, in step (iA) a mixture M is provided in a reaction medium IIIA .

[0792] That is, the reaction medium in step (iA) is preferably aqueous, more preferably an aqueous buffer.

[0793] Exemplary buffers commonly used in biotransformation reactions and advantageously used in step (iA) include: Tris, phosphate or any zwitterionic buffer, such as 2-(N-morpholino)ethanesulfonic acid ("MES"), N-(2-acetamido)iminodiacetic acid ("ADA"), piperazine-N,N'-bis(2-ethanesulfonic acid) ("PIPES"), N-(2-acetamido)-2-aminoethanesulfonic acid ("ACES"), p-hydroxy-4-morpholinopropanesulfonic acid ("MOPSO"), cholestyramine chloride, 3-(N-morpholino)propanesulfonic acid ("MOPS"), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid ("BES"), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid ("TES"), 4-(2-hydroxyethyl)-2-aminoethanesulfonic acid ("MOPSO"), cholestyramine chloride, 3-(N-morpholino)propanesulfonic acid ("MOPS"), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid ("BES"), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid ("TES"), 4-(2-hydroxyethyl)-2-aminoethanesulfonic acid ("MOPSO"), -1-piperazineethanesulfonic acid ("HEPES"), 3-(bis(2-hydroxyethyl)amino)-2-hydroxypropane-1-sulfonic acid ("DIPSO"), acetylaminoglycine, 3-(N-tris(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid ("TAPSO"), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) ("POPSO"), 4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid) ("HEPPSO"), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid ("HEPPS"), tris(hydroxymethyl)methylglycine, glycinamide, N,N-bis(2-hydroxyethyl)glycine, or 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonic acid ("TAPS").

[0794] In some embodiments, ammonium can act as a buffer.One or more organic solvents can also be added to the reaction.

[0795] The buffer preferably comprises a metal salt, more preferably a metal salt such as a halide of a monovalent or divalent metal (eg, CoCl2, MnCl2).

[0796] The concentration of these metal salts in the reaction medium in step (iA) is preferably from 1 μM to 1 M, more preferably from 1 mM to 100 mM, even more preferably from 1 to 10 mM.

[0797] Preferably, step (iA) of the method of the second aspect of the invention is carried out in phosphate buffer.

[0798] The pH of the reaction medium in step (iA) of the process of the first aspect of the invention is preferably in the range of 2 to 10, more preferably in the range of 5 to 8, more preferably in the range of 7.2 to 7.5, most preferably 7.5.

[0799] More preferably, the concentration of all compounds of formula D-(III) in the reaction medium provided in step (iA) is in the range of 1 μM to 1 M, preferably in the range of 10 μM to 0.5 M, more preferably in the range of 0.1 mM to 0.1 M, more preferably in the range of 1 mM to 10 mM, and most preferably 1.25 mM.

[0800] Regarding the compounds of formula L-(III) present in the reaction medium preferably provided in step (iA), the concentration of all compounds of formula L-(III) in the reaction medium is preferably 1 to 100 times the concentration of all compounds of formula D-(III), more preferably 1 to 10 times the concentration of all compounds of formula D-(III), even more preferably 1 to 2 times the concentration of all compounds of formula D-(III), even more preferably the same as the concentration of all compounds of formula D-(III).

[0801] 4.6.2 Optional Step (iB)

[0802] In optional step (iB), the mixture M IIIA At least a portion of the compound D-(III) is reacted to form a compound of formula L-(III) to obtain a composition M comprising a compound of formula L-(III) and optionally a compound of formula D-(III) IIIB It goes without saying that the mixture M IIIB The molar ratio of all compounds of formula L-(III) to all compounds of formula D-(III) is greater than the molar ratio of all compounds of formula L-(III) to all compounds of formula D-(III). IIIA The molar ratio of all compounds of formula L-(III) to all compounds of formula D-(III).

[0803] In case step (iB) is performed, it is preferably carried out as described in step (a) of items 4.5.14 and 4.5.15.

[0804] In particular, the reaction of step (iB) is catalyzed by hydantoin racemase E3, as described for step (a).

[0805] Preferably, the hydantoin racemase E3 used in step (iB) is classified into EC 5.1.99.5.

[0806] In another embodiment, the polypeptide sequence of the hydantoin racemase E3 used in step (iB) is selected from SEQ ID NO: 17 and variants thereof, SEQ ID NO: 18 and variants thereof, SEQ ID NO: 19 and variants thereof, SEQ ID NO: 20 and variants thereof, SEQ ID NO: 21 and variants thereof, SEQ ID NO: 22 and variants thereof, SEQ ID NO: 23 and variants thereof, SEQ ID NO: 24 and variants thereof, SEQ ID NO: 25 and variants thereof, SEQ ID NO: 26 and variants thereof, most preferably selected from SEQ ID NO: 17 and variants thereof.

[0807] Preferably, the hydantoin racemase E3 used in step (iB) is selected from E 317S and E 317v (both described in 4.5.14.7.1), E 318S and E 318v (both described in 4.5.14.7.2), E 319S and E 319v (both described in 4.5.14.7.3), E 320S and E 320v (both described in 4.5.14.7.4), E 321S and E 321v (both described in 4.5.14.7.5), E 322S and E 322v (both described in 4.5.14.7.6), E 323S and E 323v (both described in 4.5.14.7.7), E 324S and E 324v (both described in 4.5.14.7.8), E 325S and E 325v (both described in 4.5.14.7.9), E 326S and E 326v (all described under 4.5.14.7.10), more preferably selected from E 317S and E 317v , even more preferably selected from E 317S .

[0808] The reaction conditions in step (iB) are preferably as described for step (a) under item 4.5.15.

[0809] That is, the reaction medium in step (iB) is preferably aqueous, more preferably an aqueous buffer.

[0810] Exemplary buffers commonly used in biotransformation reactions and advantageously used in step (iB) include: Tris, phosphate or any zwitterionic buffer, such as 2-(N-morpholino)ethanesulfonic acid ("MES"), N-(2-acetamido)iminodiacetic acid ("ADA"), piperazine-N,N'-bis(2-ethanesulfonic acid) ("PIPES"), N-(2-acetamido)-2-aminoethanesulfonic acid ("ACES"), p-hydroxy-4-morpholinopropanesulfonic acid ("MOPSO"), cholestyramine chloride, 3-(N-morpholino)propanesulfonic acid ("MOPS"), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid ("BES"), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid ("TES"), 4-(2-hydroxyethyl)-2-aminoethanesulfonic acid ("MOPSO"), cholestyramine chloride, 3-(N-morpholino)propanesulfonic acid ("MOPS"), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid ("BES"), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid ("TES"), 4-(2-hydroxyethyl)-2-aminoethanesulfonic acid ("MOPSO"), -1-piperazineethanesulfonic acid ("HEPES"), 3-(bis(2-hydroxyethyl)amino)-2-hydroxypropane-1-sulfonic acid ("DIPSO"), acetylaminoglycine, 3-(N-tris(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid ("TAPSO"), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) ("POPSO"), 4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid) ("HEPPSO"), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid ("HEPPS"), tris(hydroxymethyl)methylglycine, glycinamide, N,N-bis(2-hydroxyethyl)glycine, or 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonic acid ("TAPS").

[0811] In some embodiments, ammonium can act as a buffer.One or more organic solvents can also be added to the reaction.

[0812] The buffer preferably comprises a metal salt, more preferably a metal salt such as a halide of a monovalent or divalent metal (eg, CoCl2, MnCl2).

[0813] The concentration of these metal salts in the reaction medium in step (iB) is preferably from 1 μM to 1 M, more preferably from 1 mM to 100 mM, even more preferably from 1 to 10 mM.

[0814] Preferably, step (iB) of the method of the second aspect of the invention is carried out in phosphate buffer.

[0815] The pH of the reaction medium in step (iB) of the process of the first aspect of the invention is preferably in the range of 2 to 10, more preferably in the range of 5 to 8, more preferably in the range of 7.2 to 7.5, most preferably 7.5.

[0816] Preferably, step (iB) of the method of the first aspect of the invention is carried out at a temperature in the range of 20°C to 70°C, more preferably in the range of 30°C to 55°C, most preferably 50°C.

[0817] Preferably, the total concentration of all hydantoin racemase E3 in the reaction solution in step (iB) is in the range of 1 μM to 10 mM, preferably 10 μM to 1 mM, more preferably 0.1 mM to 0.5 mM, most preferably 0.4 mM.

[0818] In an alternative preferred embodiment, the total concentration of all hydantoin racemase E3 in the reaction solution in step (iB) is in the range of 1 μg / l to 10 g / l, preferably 0.1 mg / l to 5 g / l, more preferably 1 mg / l to 1 g / l, more preferably 5 mg / l to 500 mg / l.

[0819] Preferably, step (iB) is carried out in the same reaction medium as steps (ii) and (iii). This has the advantage that this allows a one-pot synthesis of all steps (iB), (ii) and (iii).

[0820] At the end of optional step (iB), a composition M is obtained comprising a compound of formula L-(III) and optionally a compound of formula D-(III) IIIB , preferably obtaining a composition M comprising a compound of formula L-(III) and a compound of formula D-(III) IIIB .

[0821] 4.6.3 Step (ii)

[0822] In step (ii) of the method according to the second aspect of the present invention, the mixture M IIIA Alternatively, when step (iB) is performed, the composition M IIIB Perform steps (b) as described in items 4.5.8 to 4.5.13 above.

[0823] In other words: in those embodiments in which step (iB) is not performed, M IIIA Go through steps (b) as described in items 4.5.8 to 4.5.13 above.

[0824] In the case where step (iB) is performed, M obtained after step (iB) IIIB Go through steps (b) as described in items 4.5.8 to 4.5.13 above.

[0825] The reaction of step (ii) is catalyzed by the hydantoinase E2 described in step (b), preferably by the L-hydantoinase E2 described in step (b).

[0826] More preferably, the hydantoinase E2, even more preferably the L-hydantoinase E2 used in step (ii) is classified in class EC 3.5.2.2.

[0827] In another preferred embodiment, the polypeptide sequence of hydantoinase E2, even more preferably the polypeptide sequence of L-hydantoinase E2 used in step (ii) is selected from SEQ ID NO: 10 and variants thereof, SEQ ID NO: 11 and variants thereof, SEQ ID NO: 12 and variants thereof, SEQ ID NO: 13 and variants thereof, SEQ ID NO: 14 and variants thereof, SEQ ID NO: 15 and variants thereof, SEQ ID NO: 16 and variants thereof, even more preferably selected from SEQ ID NO: 10 and variants thereof.

[0828] Preferably, the hydantoinase E2, even more preferably the L-hydantoinase E2 used in step (ii) is selected from the group consisting of E 210S and E 210v (both described in 4.5.12.1), E 211S and E 211v (both described in 4.5.12.2), E 212S and E 212v (both described in 4.5.12.3), E 213S and E 213v (both described in 4.5.12.4), E 214S and E 214v (both described in 4.5.12.5), E 215S and E 215v (both described in 4.5.12.6), E 216S and E 216v (all described under 4.5.12.7), more preferably selected from E 210S and E 210V , even more preferably selected from E 210S .

[0829] The reaction conditions in step (ii) are preferably as described in step (b) under item 4.5.13.

[0830] That is, the reaction medium in step (ii) is preferably aqueous, more preferably an aqueous buffer.

[0831] Exemplary buffers commonly used in biotransformation reactions and advantageously used in step (ii) include: Tris, phosphate or any zwitterionic buffer, such as 2-(N-morpholino)ethanesulfonic acid ("MES"), N-(2-acetamido)iminodiacetic acid ("ADA"), piperazine-N,N'-bis(2-ethanesulfonic acid) ("PIPES"), N-(2-acetamido)-2-aminoethanesulfonic acid ("ACES"), p-hydroxy-4-morpholinopropanesulfonic acid ("MOPSO"), cholestyramine chloride, 3-(N-morpholino)propanesulfonic acid ("MOPS"), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid ("BES"), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid ("TES"), 4-(2-hydroxyethyl)propanesulfonic acid ("TES"), )-1-piperazineethanesulfonic acid ("HEPES"), 3-(bis(2-hydroxyethyl)amino)-2-hydroxypropane-1-sulfonic acid ("DIPSO"), acetylaminoglycine, 3-(N-tris(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid ("TAPSO"), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) ("POPSO"), 4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid) ("HEPPSO"), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid ("HEPPS"), tris(hydroxymethyl)methylglycine, glycinamide, N,N-bis(2-hydroxyethyl)glycine, or 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonic acid ("TAPS").

[0832] In some embodiments, ammonium can act as a buffer.One or more organic solvents can also be added to the reaction.

[0833] The buffer preferably comprises a metal salt, more preferably a metal salt such as a halide of a monovalent or divalent metal (eg, CoCl2, MnCl2).

[0834] The concentration of these metal salts in the reaction medium is preferably from 1 μM to 1 M, more preferably from 1 mM to 100 mM, even more preferably from 1 to 10 mM.

[0835] Preferably, step (ii) of the method of the second aspect of the invention is carried out in phosphate buffer.

[0836] The pH of the reaction medium in step (ii) of the process of the first aspect of the invention is preferably in the range of 2 to 10, more preferably in the range of 5 to 8, more preferably in the range of 7.2 to 7.5, most preferably 7.5.

[0837] Preferably, step (ii) of the method of the first aspect of the invention is carried out at a temperature in the range of 20°C to 70°C, more preferably in the range of 30°C to 55°C, most preferably 50°C.

[0838] Preferably, the total concentration of all hydantoinases E2 in the reaction solution in step (ii) is in the range of 1 μM to 10 mM, preferably 10 μM to 1 mM, more preferably 0.1 mM to 0.5 mM, most preferably 0.4 mM.

[0839] In an alternative preferred embodiment, the total concentration of all hydantoinase E2 in the reaction solution in step (ii) is in the range of 1 μg / l to 10 g / l, preferably 0.1 mg / l to 5 g / l, more preferably 1 mg / l to 1 g / l, more preferably 5 mg / l to 500 mg / l.

[0840] Preferably, step (ii) is carried out in the same reaction medium as step (iii).

[0841] At the end of step (ii), a composition M is obtained comprising at least one compound of formula L-(II) and optionally at least one compound of formula D-(II) II , wherein L-(II) and D-(II) have the above formula.

[0842] 4.6.4 Step (iii)

[0843] In step (iii) of the method of the second aspect of the present invention, the M obtained in step (ii) is II Step (c) is performed as described in Items 4.5.1 to 4.5.7 above, wherein the reaction of step (iii) is catalyzed by L-carbamoylase E1.

[0844] This leads to the I The compound of formula L-(I) is enriched compared to the compound of formula D-(I), that is, compared to M IIIA The molar ratio of the compound of formula L-(I) to the compound of formula D-(I).

[0845] More preferably, the L-carbamoylase E1 used in step (iii) is classified into EC 3.5.1.87.

[0846] In another preferred embodiment, the polypeptide sequence of L-carbamoylase E1 used in step (iii) is selected from SEQ ID NO: 1 and variants thereof, SEQ ID NO: 2 and variants thereof, SEQ ID NO: 3 and variants thereof, SEQ ID NO: 4 and variants thereof, SEQ ID NO: 5 and variants thereof, SEQ ID NO: 6 and variants thereof, SEQ ID NO: 7 and variants thereof, SEQ ID NO: 8 and variants thereof, SEQ ID NO: 9 and variants thereof.

[0847] Preferably, the L-carbamoylase E1 used in step (iii) is selected from E 101S and E 101v (both described in 4.5.6.1), E 102S and E 102v (both described in 4.5.6.2), E 103S and E 103v (both described in 4.5.6.3), E 104S and E 104v (both described in 4.5.6.4), E 105S and E 105v (both described in 4.5.6.5), E 106S and E 106v (both described in 4.5.6.6), E 107S and E 107v (both described in 4.5.6.7), E 108S and E 108v (both described in 4.5.6.8), E 109S and E 109v (all described under 4.5.6.9), more preferably selected from E 101S and E 101v , even more preferably selected from E 101S .

[0848] The reaction conditions in step (iii) are preferably as described in step (c) under item 4.5.7.

[0849] That is, the reaction medium in step (iii) is preferably aqueous, more preferably an aqueous buffer.

[0850] Exemplary buffers commonly used in biotransformation reactions and advantageously used in step (iii) include: Tris, phosphate or any zwitterionic buffer, such as 2-(N-morpholino)ethanesulfonic acid ("MES"), N-(2-acetamido)iminodiacetic acid ("ADA"), piperazine-N,N'-bis(2-ethanesulfonic acid) ("PIPES"), N-(2-acetamido)-2-aminoethanesulfonic acid ("ACES"), p-hydroxy-4-morpholinopropanesulfonic acid ("MOPSO"), cholestyramine chloride, 3-(N-morpholino)propanesulfonic acid ("MOPS"), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid ("BES"), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid ("TES"), 4-(2-hydroxyethyl)propanesulfonic acid ("TES"), )-1-piperazineethanesulfonic acid ("HEPES"), 3-(bis(2-hydroxyethyl)amino)-2-hydroxypropane-1-sulfonic acid ("DIPSO"), acetylaminoglycine, 3-(N-tris(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid ("TAPSO"), piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) ("POPSO"), 4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid) ("HEPPSO"), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid ("HEPPS"), tris(hydroxymethyl)methylglycine, glycinamide, N,N-bis(2-hydroxyethyl)glycine, or 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonic acid ("TAPS").

[0851] In some embodiments, ammonium can act as a buffer.One or more organic solvents can also be added to the reaction.

[0852] The buffer preferably comprises a metal salt, more preferably a metal salt such as a halide of a monovalent or divalent metal (eg, CoCl2, MnCl2).

[0853] The concentration of these metal salts in the reaction medium is preferably from 1 μM to 1 M, more preferably from 1 mM to 100 mM, even more preferably from 1 to 10 mM.

[0854] Preferably, step (iii) of the method of the second aspect of the invention is carried out in phosphate buffer.

[0855] The pH of the reaction medium in step (iii) of the process of the first aspect of the invention is preferably in the range of 2 to 10, more preferably in the range of 5 to 8, more preferably in the range of 7.2 to 7.5, most preferably 7.5.

[0856] Preferably, step (iii) of the method of the first aspect of the invention is carried out at a temperature in the range of 20°C to 70°C, more preferably in the range of 30°C to 55°C, most preferably 50°C.

[0857] Preferably, the total concentration of all carbamoylases E1 in the reaction solution in step (iii) is in the range of 1 μM to 10 mM, preferably 10 μM to 1 mM, more preferably 0.1 mM to 0.5 mM, most preferably 0.4 mM.

[0858] In an alternative preferred embodiment, the total concentration of all carbamoylases E1 in the reaction solution in step (iii) is in the range of 1 μg / l to 10 g / l, preferably 0.1 mg / l to 5 g / l, more preferably 1 mg / l to 1 g / l, more preferably 5 mg / l to 500 mg / l.

[0859] At the end of step (i), a mixture M is obtained comprising a compound of formula L-(I) and optionally a compound of formula D-(I) I , preferably obtaining a mixture M comprising a compound of formula L-(I) and a compound of formula D-(I) I .

[0860] 4.7 Saponification

[0861] The L-phosphinothricin P-ester of formula L-(I) prepared according to the method of the first or second aspect can then be saponified to prepare LGA, for example in an acidic aqueous medium, preferably at pH <7, even more preferably between pH <6, more preferably at pH <3, even more preferably at pH <1. These saponification conditions are known to the skilled person and are described, for example, in HJ Zeiss, J. Org. Chem. 1991, 56, 1783-1788. 5. Examples

[0862] 5.1 Example 1 Identification of suitable enzymes and construction of plasmids

[0863] Genes from different sources encoding hydantoinase (dihydropyrimidinase, EC 3.5.2.2), L-carbamoylase (N-carbamoyl-L-aminoacid hydrolase, EC 3.5.1.87) and hydantoin racemase (EC 5.1.99.5) were tested for their ability to react with different hydantoin substrates according to formula L-(III) and D-(III) to form the respective enantioselective L-phosphinothricin derivatives according to formula L-(I).

[0864] 5.1.1 Tested enzymes

[0865] Table 4 summarizes the details of strains and genes for various enzymes used in the Examples.

[0866] Table 4

[0867]

[0868] 5.1.2 Enzyme cloning

[0869] The cloning of hydantoin racemase and the generation of plasmid pOM21c were performed as described in Example 1 of WO 2004 / 111227 A2 ( Figure 1 ). In particular, a polynucleotide comprising the corresponding gene (SEQ ID NO: 29) and additional sequences of NdeI and PstI restriction sites was used.

[0870] The cloning of hydantoinase and L-carbamoylase into the rhamnose expression vector pJOE4036 was performed in a plasmid derivative of the rhamnose expression vector pJOE4036.

[0871] The polynucleotides (SEQ ID NOs: 27, 28) containing the genes for each enzyme were synthesized by GeneArt (ThermoFisher Scientific (Waltham, USA)). The polynucleotides carried additional sequences for EcoR1 and HindIII restriction sites. These restriction sites were used to clone the two enzymes into pJOE4036 under the control of the rhamnose promoter, generating plasmid pOM22c ( Figure 2 ).

[0872] 5.2 Example 2: Preparation of strains positive for hydantoinase, L-carbamoylase and hydantoin racemase

[0873] Chemically competent E. coli ET5 cells (as described in WO 2004 / 042047 A1) were transformed with 10 ng of plasmid pOM22c produced according to Example 1 .

[0874] The resulting hydantoinase and carbamoylase positive strains were made competent and transformed with 10 ng of plasmid pOM21c.

[0875] E. coli ET5 strain transformed with pOM21c or pOM22c was cultured in LB medium containing ampicillin (100 μg / l), chloramphenicol (50 μg / l) and 2 g / l rhamnose at 30°C with shaking (250 U / min) for 18 hours.

[0876] The biomass was separated by centrifugation, resuspended in 50 mM phosphate buffer (pH 7.2), and used in the bioconversion tests in the following examples. The concentration of the biomass in the solution was 40-50 g / l. This solution was used as a catalyst ("Catalyst 1") hereinafter.

[0877] The concentrations of the corresponding polypeptide carbamoylase, hydantoinase and racemase in the resulting solution can be determined by SDSPAGE and analyzed by software The corresponding bands were analyzed by Biochem Lab Solutions.

[0878] 5.3 Example I1 of the present invention and comparative examples C1 to C4: Preparation of phosphine using the strains produced in Examples 1 and 2 P-alkyl esters of streptomycin

[0879] In the following examples, different hydantoin substrates were tested to determine whether the corresponding polypeptides enantiomerically selectively catalyze the reaction of the corresponding substrates to give the corresponding L-amino acids.

[0880] 5.3.1 Comparative Example C1

[0881] In Comparative Example C1, 1.25 mmol L-(IV) C1 and 1.25mmol D-(IV) C1 The racemic mixture M C1 As substrate, L-(IV) C1 and D-(IV) C1 is a hydantoin having the formula:

[0882]

[0883] The mixture M C1 Dissolve in a stirred reactor with 25 ml of water. Add 2.4 g of catalyst 1 and then 50 μl of CoCl2 solution. Set the suspension to pH 7.5 with 0.5 M NaOH. Make up the total volume to 50 ml with water so that each enantiomer L-(IV) C1 and D-(IV) C1 The concentration of 0.025 mol / l was maintained between pH 7.0 and 7.5 by HCl titration or NaOH titration. During the reaction, the temperature was maintained at 37° C. by a thermostat.

[0884] The reaction was terminated after 120 hours by adding 2N HCl until the pH reached 2.5. The biomass was separated by centrifugation or filtration.

[0885] The enzyme reaction was monitored by the ninhydrin test to determine the formation of amino acids. The ninhydrin test did not detect the formation of amino acids.

[0886] 5.3.2 Comparative Example C2

[0887] In Comparative Example C2, 1.25 mmol L-(V) C2 and 1.25mmol D-(V) C2 The racemic mixture MC2 As a substrate, L-(V) C2 and D-(V) C2 is a carbamoyl compound having the formula:

[0888]

[0889] The mixture M C2 Dissolve in a stirred reactor with 25 ml of water. Add 2.4 g of catalyst 1 and then 50 μl of CoCl2 solution. Set the suspension to pH 7.5 with 0.5 M NaOH. Make up the total volume to 50 ml with water so that each enantiomer L-(V) C2 and D-(V) C2 The concentration of was 0.025 mol / L. The pH was maintained between 7.0 and 7.5 by HCl titration or NaOH titration. During the reaction, the temperature was maintained at 37° C. by a thermostat.

[0890] The reaction was terminated after 120 hours by adding 2N HCl until the pH reached 2.5. The biomass was separated by centrifugation or filtration.

[0891] The enzyme reaction was monitored by the ninhydrin test to determine the formation of amino acids. The ninhydrin test did not detect the formation of amino acids.

[0892] 5.3.3 Example C3 (not according to the invention)

[0893] In Example C3, 1.25 mmol L-(VI) C3 and 1.25mmol D-(VI) C3 The racemic mixture M C3 As a substrate, L-(VI) C3 and D-(VI) C3 is a hydantoin having the formula:

[0894]

[0895] The mixture M I1 Dissolve in a stirred reactor with 25 ml of water. Add 2.4 g of catalyst 1 and then 50 μl of CoCl2 solution. Set the suspension to pH 7.5 with 0.5 M NaOH. Make up the total volume to 50 ml with water so that each enantiomer L-(VI) C3 and D-(VI) C3 The concentration of was 0.025 mol / L. The pH was maintained between 7.0 and 7.5 by HCl titration or NaOH titration. During the reaction, the temperature was maintained at 37° C. by a thermostat.

[0896] The reaction was terminated after 120 hours by adding 2N HCl until the pH reached 2.5. The biomass was separated by centrifugation or filtration.

[0897] The enzyme reaction was monitored by the ninhydrin test to determine the formation of amino acids. The formation of amino acids was monitored by the ninhydrin test.

[0898] In the last step, the reaction mixture was saponified at 100°C for 10 hours by adding 6M HCl to obtain L-phosphinothricin and D-phosphinothricin (if present). The final reaction mixture was analyzed by LC-MS using a CR-I column as described in Item 5.4 to determine the enantiomeric excess ("ee") of D- or L-phosphinothricin. An LGAee of 79% relative to the D-enantiomer was detected. The ee of the L-enantiomer ("ee L ”) is determined in the context of the present invention by the following formula, where m L and m D are the molar amounts of L- and D-glufosinate detected, respectively:

[0899]

[0900] 5.3.4 Example C4 (not according to the invention)

[0901] In Example C4, 1.25 mmol L-(VII) was used. C4 and 1.25mmol D-(VII) C4 The racemic mixture M C4 As a substrate, L-(VII) C4 and D-(VII) C4 Is a hydantoin of the formula:

[0902]

[0903] The mixture M C4 Dissolve in a stirred reactor with 25 ml of water. Add 2.4 g of catalyst 1 and then 50 μl of CoCl2 solution. Set the suspension to pH 7.5 with 0.5 M NaOH. Make up the total volume to 50 ml with water so that each enantiomer L-(VII) C4 and D-(VII) C4 The concentration of was 0.025 mol / L. The pH was maintained between 7.0 and 7.5 by HCl titration or NaOH titration. During the reaction, the temperature was maintained at 37° C. by a thermostat.

[0904] The reaction was terminated after 120 hours by adding 2N HCl until the pH reached 2.5. The biomass was separated by centrifugation or filtration.

[0905] The enzyme reaction was monitored by the ninhydrin test to determine the formation of amino acids. The formation of amino acids was monitored by the ninhydrin test.

[0906] In the last step, the reaction mixture was saponified at 100°C for 10 hours by adding 6M HCl to obtain L-phosphinothricin and D-phosphinothricin (if present). The final reaction mixture was analyzed by LC-MS using a CR-I column as described in Item 5.4 to determine the enantiomeric excess ("ee") of D- or L-phosphinothricin. 77% LGAee relative to the D-enantiomer was detected.

[0907] 5.3.5 Embodiment 11 of the present invention

[0908] In Example I1, 0.625 mmol of L-(VIII) was used. I1 、0.625mmol L-(VIII) I1’ 、0.625mmolD-(VIII) I1 and 0.625mmol D-(VIII) I1’ A mixture of M I1 As substrate, L(VIII) I1 、L(VIII) I1’ 、D(VIII) I1 and D(VIII) I1’ is a hydantoin having the formula:

[0909]

[0910] These compounds were obtained by the synthesis method described in CN 111662325 A from a mixture of L-homoserine / D-homoserine.

[0911] The mixture M I1 Dissolve in a stirred reactor with 25 ml of water. Add 2.4 g of catalyst 1 and then 50 μl of CoCl2 solution. Set the suspension to pH 7.5 with 0.5 M NaOH. Make up the total volume to 50 ml with water so that each isomer L-(VIII) I1 、L-(VIII) I1’ 、D-(VIII) I1 and D-(VIII) I1 The concentration of ' was 0.0125 mol / L. The pH was kept between 7.0 and 7.5 by HCl-titration or NaOH-titration. During the reaction, the temperature was kept at 37°C by a thermostat.

[0912] The reaction was terminated after 120 hours by adding 2N HCl until the pH reached 2.5. The biomass was separated by centrifugation or filtration.

[0913] The enzyme reaction is monitored by the ninhydrin test to determine the formation of amino acids. The formation of amino acids can be detected by the ninhydrin test.

[0914] In the final step, the reaction mixture was saponified at 100°C for 10 hours by adding 6M HCl to obtain L-phosphinothricin and D-phosphinothricin (if present). The final reaction mixture was analyzed by LC-MS using a CR-I column as described in Item 5.4 to determine the enantiomeric excess ("ee") of D- or L-phosphinothricin. LGAee relative to the D-enantiomer was detected (not quantified). The ee of the L-enantiomer ("ee L ”) is determined in the context of the present invention by the following formula, where m L and m D are the molar amounts of L- and D-glufosinate detected, respectively:

[0915]

[0916] 5.3.5 Results

[0917] The results of Examples C1, C2, C3, and C4 are summarized in Table 5.

[0918] Table 5

[0919] Example Substrate Ninhydrin test <![CDATA[ee L ]]> C1 <![CDATA[L-(IV) C1 ,D-(IV) C1 ]]> Negative - C2 <![CDATA[L-(V) C2 ,D-(V) C2 ]]> Negative - C3 <![CDATA[L-(VI) C3 ,D-(VI) C3 ]]> Positive 79％ C4 <![CDATA[L-(VII) C4 ,D-(VII) C4 ]]> Positive 77％

[0920] 5.3.6 Conclusion

[0921] The result of summarizing in Table 5 shows that some hydantoin esters, for example alkylated hydantoin esters, can be used as substrates (C3, C4) of enzymatic enantioselective synthesis of L-phosphinothricin ammonium. On the contrary, wherein the unprotected substrate of phosphonic acid group is not accepted by this catalytic system (C1, C2). In this respect, C2 shows that at least one reason is that L-carbamoylase does not accept the corresponding substrate that phosphonic acid functional group is not protected by ester group.

[0922] Similar results were obtained when using a mixture of compounds within the scope of the present invention, i.e. a mixture of compounds of L-(III) and D-(III), wherein R 3L and R 3D Preferably the same, such as in Example I1 of the present invention.

[0923] Furthermore, in contrast to the enantioselective preparation process according to the prior art (CN 111662325 A), the ee excess can be maintained throughout the process since the saponification is not carried out at the hydantoin stage but at the amino acid ester stage.

[0924] 5.4 Analytical methods

[0925] L-Glufosinate and D-Glufosinate were detected by LC-MS ("Liquid Chromatography-Mass Spectrometry") using a chiral column [Daicel CROWNPAKCR-I-(-)] as follows. For hydantoins, a Daicel Chiralpak IA-U column can also be used.

[0926] This assay can also be used to detect and quantify the following:

[0927] -Mode LGAP-(n-butyl) ester, according to Test A L (item 4.5.3.1);

[0928] -Mode D-phosphinothricin P-(n-butyl) ester, according to Test A D (item 4.5.3.2);

[0929] -Mode Carbamoyl LGA P-(n-butyl) ester, according to Test D L (item 4.5.9.1);

[0930] -Mode Carbamoyl D-phosphinothricin P-(n-butyl) ester, according to Test D D (item 4.5.9.2);

[0931] -Mode The L-enantiomer of the n-butyl ester of phosphinothricin hydantoin according to Test G (item 4.5.14.3).

[0932] -Mode The D-enantiomer of the n-butyl ester of phosphinothricin hydantoin according to Test G (Item 4.5.14.3).

[0933] 5.4.1 Collection method details

[0934] CR-I-(-),3.0mm IDx 150mm,5μm,Crownpak,Part.No.54784

[0935] MS QQQ Mass Spectrometer 6420,Agilent

[0936] ESI+

[0937] MS2 SIM,Glufosinat m / z 182.06Unit Positive

[0938] Centroid

[0939] 5.4.2 Source Parameters

[0940] Parameter values ​​(+); gas temperature (℃): 350; gas flow (l / min): 12; nebulizer (psi): 25; capillary (V): 3000; sampler module: G1329B; injection volume 1.00μL; 5℃

[0941] 5.4.3 Binary Pump Module

[0942] G1312B

[0943] Flow rate: 0.200 mL / min;

[0944] Channel A: H2O containing trifluoroacetic acid (TFA), pH 1.15; Channel B: ACN (acetonitrile);

[0945] Isocratic 80.0% A / 20.0% B; stop time 7.00 min Column Comp. Module: G1316A Temperature 5.0°C.

Claims

1. A method for preparing an L-phosphinothricin P-ester of formula L-(I), comprising step (c), wherein a compound of formula L-(II) is reacted to obtain a compound of formula L-(I): wherein the reaction according to step (c) is catalyzed by carbamoylase E1, And where R 1L With R 2L are groups independently selected from the following formulae (VIII), (IX), (X):

2. The method according to claim 1, wherein R 1L With R 2L are groups independently selected from the group consisting of formulae (IX) and (X).

3. The method according to claim 1 or 2, wherein the reaction according to step (c) is catalyzed by L-carbamoylase E1. The method according to claim 3 , wherein the L-carbamoylase E1 is classified into EC class 3.5.1.

87.

5. The method according to claim 3 or 4, wherein the polypeptide sequence of the L-carbamoylase E1 is selected from SEQ ID NO: 1 and variants thereof, SEQ ID NO: 2 and variants thereof, SEQ ID NO: 3 and variants thereof, SEQ ID NO: 4 and variants thereof, SEQ ID NO: 5 and variants thereof, SEQ ID NO: 6 and variants thereof, SEQ ID NO: 7 and variants thereof, SEQ ID NO: 8 and variants thereof, SEQ ID NO: 9 and variants thereof.

6. The method according to any one of claims 1 to 5, wherein the compound of formula L-(II) is obtained by step (b), wherein the compound of formula L-(III) is reacted to obtain the compound of formula L-(II): wherein said reaction according to step (b) is catalyzed by hydantoinase E2, And where R 3L It is a group selected from the group consisting of the formulae (VIII), (IX) and (X) described in claim 1.

7. The method according to claim 6, wherein R 3L is a group selected from the group consisting of the formula (VIII) described in claim 1.

8. The method according to claim 6 or 7, wherein the hydantoinase E2 is classified into EC class 3.5.2.

2.

9. The method according to one of claims 6 to 8, wherein the polypeptide sequence of the hydantoinase E2 is selected from the group consisting of SEQ ID NO: 10 and variants thereof, SEQ ID NO: 11 and variants thereof, SEQ ID NO: 12 and variants thereof, SEQ ID NO: 13 and variants thereof, SEQ ID NO: 14 and variants thereof, SEQ ID NO: 15 and variants thereof, SEQ ID NO: 16 and variants thereof.

10. The method according to one of claims 6 to 9, wherein the hydantoinase E2 is L-hydantoinase E2.

11. The process according to claim 6, wherein the compound of formula L-(III) is obtained by step (a), wherein a compound of formula D-(III) is reacted to give a compound of formula L-(III): And where R 3D It is a group selected from the group consisting of the formulae (VIII), (IX) and (X) described in claim 1.

12. The method according to claim 11, wherein R 3D is a group selected from the group consisting of the formula (VIII) described in claim 1.

13. The method according to claim 11 or 12, wherein the reaction according to step (a) is catalyzed by hydantoin racemase E3.

14. The method according to claim 13, wherein the hydantoin racemase E3 is classified into EC class 5.1.99.

5.

15. The method according to claim 13 or 14, wherein the polypeptide sequence of the hydantoin racemase E3 is selected from SEQ ID NO: 17 and variants thereof, SEQ ID NO: 18 and variants thereof, SEQ ID NO: 19 and variants thereof, SEQ ID NO: 20 and variants thereof, SEQ ID NO: 21 and variants thereof, SEQ ID NO: 22 and variants thereof, SEQ ID NO: 23 and variants thereof, SEQ ID NO: 24 and variants thereof, SEQ ID NO: 25 and variants thereof, SEQ ID NO: 26 and variants thereof.

16. A method for preparing L-phosphinothricin P-ester of formula L-(I): The following steps are involved: (iA) providing a mixture M comprising at least one compound L-(III) and at least one compound D-(III) IIIA , wherein L-(III) and D-(III) have the following formula: (iB) optionally by step (a) of one of claims 11 to 15, IIIA At least a portion of the compound of formula D-(III) is reacted to form a compound of formula L-(III) to obtain a composition M comprising a compound of formula L-(III) and optionally a compound of formula D-(III). IIIB ; (ii) making the mixture M IIIA or in the case where step (iB) is performed, the composition M IIIB After the reaction of step (b) according to one of claims 6 to 10, a composition M comprising at least one compound of formula L-(II) and optionally at least one compound of formula D-(II) is obtained. II , wherein L-(II) and D-(II) have the following formula: (iii) Make M II Carrying out the reaction of step (c) as claimed in claims 3 to 5 to obtain a composition M comprising at least one compound of formula L-(I) and optionally at least one compound of formula D-(I) I , Among them, in M I In the case of a compound of formula L-(I) and a compound of formula D-(I), M I All compounds of formula L-(I) and M I The molar ratio of all compounds of formula D-(I) is greater than M IIIA All compounds of formula L-(III) and M IIIA The molar ratio of all compounds of formula D-(III), wherein D-(I) has the following formula: Where R 1L , R 1D , R 2L , R 2D , R 3L and R 3D are groups independently selected from the group consisting of the formulae (VIII), (IX) and (X) described in claim 1.

17. The process according to claim 1, wherein the compound of formula L-(I) is saponified to give L-phosphinothricin.

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