WO2023232225A1 - Enzymatic method for the diastereoselective production of l-glufosinate p-esters - Google Patents

Enzymatic method for the diastereoselective production of l-glufosinate p-esters Download PDF

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WO2023232225A1
WO2023232225A1 PCT/EP2022/064676 EP2022064676W WO2023232225A1 WO 2023232225 A1 WO2023232225 A1 WO 2023232225A1 EP 2022064676 W EP2022064676 W EP 2022064676W WO 2023232225 A1 WO2023232225 A1 WO 2023232225A1
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seq
variants
carbamoylase
activity
range
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PCT/EP2022/064676
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French (fr)
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Ludger Lautenschütz
Steffen Osswald
Markus PÖTTER
Jakob Müller
Carsten ZIEM
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Evonik Operations Gmbh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
    • C07F9/6503—Five-membered rings
    • C07F9/6506—Five-membered rings having the nitrogen atoms in positions 1 and 3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/30—Phosphinic acids R2P(=O)(OH); Thiophosphinic acids, i.e. R2P(=X)(XH) (X = S, Se)
    • C07F9/32—Esters thereof
    • C07F9/3205—Esters thereof the acid moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/3211—Esters of acyclic saturated acids which can have further substituents on alkyl
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/86—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01087—N-Carbamoyl-L-amino-acid hydrolase (3.5.1.87)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/02—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amides (3.5.2)
    • C12Y305/02002—Dihydropyrimidinase (3.5.2.2), i.e. hydantoinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y501/00—Racemaces and epimerases (5.1)
    • C12Y501/99—Racemaces and epimerases (5.1) acting on other compounds (5.1.99)
    • C12Y501/99005—Hydantoin racemase (5.1.99.5)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers

Definitions

  • the present invention relates to a method for the production of an L-glufosinate P-ester from the corresponding L-glufosinate P-ester carbamoylate, which is enzymatically catalyzed by a carbamoylase.
  • the L-glufosinate P-ester and the L-glufosinate P-ester carbamoylate have a chiral a-carbon atom with a L-configuration and a chiral phosphorous atom with a D- or L-configuration.
  • both the L-glufosinate P-ester and the L-glufosinate P-ester carbamoylate each form two diastereoisomers.
  • the method allows for selective reaction of one diastereoisomer of the carbamoylate to the respective L-glufosinate P-ester.
  • One of the L-glufosinate P-ester diastereomer products is then at least partially separated from the other one and/or the L-glufosinate P-ester carbamoylate starting materials.
  • the L-glufosinate P-ester carbamoylate may be obtained by reaction from the corresponding hydantoin compound through catalysis by a hydantoinase.
  • the L-glufosinate hydantoin P-ester may be obtained from the corresponding D-glufosinate hydantoin P-ester, which reaction is preferably catalyzed by a hydantoin racemase.
  • Organic phosphorous compounds i.e. chemical agents comprising a carbon-phosphor bond
  • herbicides i.e. chemical agents comprising a carbon-phosphor bond
  • Agents such as the herbicides glyphosate (Roundup®, Touchdown®) and glufosinate (Basta®, Liberty®) as well as the growth regulator glyphosine (Polaris®) are used for this purpose (as described for example by G. Horlein, Rev. Environ. Contam. Toxicol. 1994, 138, 73 - 145).
  • esters of P-methyl phosphinic acid have a key role as synthetic building blocks in the synthesis of the non-selective herbicide glufosinate. These esters are accessible via two fundamental synthetic pathways (summarized in Figures 3 a and 3 b, page 130, of the article of K. Haack, Chem. Republicer Zeit 2003, 37, 128 - 138): a.
  • esters of P-methyl phosphinic acid add to carbon-carbon double bonds regioselectively. This property is used in the synthesis of glufosinate for the formation of the second phosphor-carbon bond.
  • Acrylic acid ester is a cheaper alternative starting material. It can react with the ester of P-methyl phosphinic acid to 3-[alkoxy(methyl)phosphinyl]propionic acid alkyl ester. Claisen reaction of this diester with diethyl oxalate, hydrolysis and decarboxylation provide the corresponding a-keto acid, which can be reductively aminated to give glufosinate.
  • L-glufosinate As there is no herbicidal activity of the D-enantiomer, L-glufosinate [hereinafter “LGA”; CAS-Nr. 35597-44-5; other names “(S)-glufosinate”, “(-)-glufosinate”] is the enantiomer of economical interest.
  • CN 111662325 A discloses a synthetic pathway in which L-homoserine is reacted to the respective hydantoin, followed by addition of methane phosphor dichloride, which results in a methane phosphane which is disubstituted with L-homoserine hydantoin. After a final Arbuzov reaction and hydrolysis, LGA is obtained.
  • ee a high enantiomeric excess
  • Drauz “A direct route from hydantoins to D-amino acids employing a resting cell biocatalyst with D-hydantoinase and D-carbamoylase acitivity” on page 164 and 167 in “Microbial Reagents in Organic Synthesis” Series C: Mathematical and Physical Sciences - Vol. 381 , S. Servi (Ed.), 1992, Springer Science+Business Media, B.V., Dordrecht.
  • WO 2020/145513 A1 and WO 2020/145514 A1 describe a chemical route to LGA.
  • an L-homoserine derivative such as O-acetyl L-homoserine or O-succinyl L-homoserine is used as starting material and L-glufosinate is obtained by a sequence of reactions including lactonization and halogenation.
  • WO 2020/145627 A1 describes a similar route, wherein during halogenation, a bromo derivative is obtained.
  • CN 108516991 A describes another synthetic pathway to LGA, starting from the azeotropic dehydration of L-homoserine to give L-3,6-bis(2-haloethyl)-2,5-diketopiperazine, followed by introduction of a methylphosphonate diester group, and hydrolysis.
  • WO 2017/151573 A1 discloses a two-step enzymatic synthesis of LGA from D-glufosinate.
  • D-glufosinate is oxidatively deaminated to give 2-oxo-4-[hydroxy(methyl)phosphinoyl]- butyric acid (“PPO”), followed by the specific amination of PPO to LGA as the second step.
  • PPO 2-oxo-4-[hydroxy(methyl)phosphinoyl]- butyric acid
  • the first step is carried out by catalysis of a D-amino acid oxidase
  • the second step is catalyzed by a transaminase.
  • WO 2020/051188 A1 discloses a similar method of converting racemic glufosinate to the L-enantiomer. In addition, it discloses a step in which the a-ketoacid or ketone byproduct formed during amination of PPO with an amine donor is converted by ketoglutarate decarboxylase to further shift the equilibrium to LGA.
  • WO 2019/018406 A1 discloses a method of purifying LGA from a mixture comprising LGA and glutamate. Glutamate is converted to pyroglutamate enzymatically by glutaminyl-peptidyl cyclotransferase, and LGA is then purified from the resulting mixture with an ion-exchange resin.
  • WO 2013/072486 A1 disclose hydantoinase mutants which have a greater activity towards D-amino acids.
  • WO 00/58449 A1 disclose hydantoinase mutants which have a greater activity towards L-amino acids.
  • a first object of the present invention is to provide a further enzymatic process for producing glufosinate or glufosinate P-esters.
  • This process should provide products with a high excess of the L-enantiomer over the D-enantiomer. Moreover, this process should allow to use new substrates which heretofore were not used in the enzymatic synthesis of glufosinate or glufosinate esters.
  • enzymes are used for racemic resolutions, e.g. as described by Schwarzwieser et al., Angewandte Chemie 2014, 126, 3805-3809 or in case of selective acylations using lipases as described by A. Ghanem & H.Y. Aboul-Enein, Tetrahedron: Asymmetry 2004, 15, 3331-3351 .
  • a further object of the present invention was hence the provision of such a method for synthesis of LL-(I) and DL-(I), wherein one of the diastereoisomers is preferably formed over the other.
  • L-glufosinate P-esters can be produced by an enzymatically catalyzed reaction from the corresponding carbamoylates of formula L-(ll), wherein R is an alkyl group or an aryl group:
  • LL-(II) LL-(I) and DL-(II) DL-(I) is kinetically favoured over the other, allowing for stereoselective synthesis of one of the two diastereoisomers LL-(I) and DL-(I) from a mixture comprising LL-(II) and DL-(II).
  • the present invention hence solves the problems mentioned above by providing a method for the production of a glufosinate P-ester according to formula LL-(I) and/or, preferably and, DL-(I), comprising steps (y), (6), and (e).
  • step (y) a mixture Mu comprising carbamoyl compounds according to formulae LL-(II) and DL- (II) is provided, wherein LL-(II) and DL-(II) have the formulae shown above.
  • step (6) in mixture Mu, a carbamoyl compound according to formula LL-(II) is reacted to give an amino acid compound according to formula LL-(I) and/or a carbamoyl compound according to formula DL-(II) is reacted to give an amino acid compound according to formula DL-(I), wherein LL-(I) and DL-(I) have the formulae shown above, wherein the reaction according step (6) is catalyzed by a carbamoylase Ei.
  • a mixture Mi is obtained comprising at least one amino acid compound of DL-(I), LL-(I), wherein, if mixture Mi comprises both amino acid compounds DL-(I) and LL-(I), the molar ratio of DL-(I) to LL-(I) in Mi is different from the molar ratio of DL-(II) to LL-(II) in Mu, and wherein mixture Mi comprises at least one of the carbamoyl compounds DL-(II), LL-(II).
  • one of the amino acid compounds selected from DL-(I), LL-(I) comprised by mixture Mi is at least partially separated from at least one of the carbamoyl compounds DL-(II), LL-(II) and/or, more preferably and, from the other amino acid compound selected from DL-(I), LL-(I), wherein preferably the separation of at least one of the amino acid compounds selected from DL-(I), LL-(I) comprised by mixture Mi is performed by crystallizing the respective amino acid compound.
  • the L-carbamoylase Ei is preferably categorized in the EC class 3.5.1.87.
  • polypeptide sequence of the L-carbamoylase Ei 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.
  • the mixture Mu provided in step (y) is obtained by a step (p).
  • a step (p) in a mixture Mm comprising compounds according to LL-(III) and DL-(III), at least a part of the compounds according to formula LL-(III) are reacted to give compounds according to LL-(II), and at least a part of the compounds according to formula DL-(III) are reacted to give compounds according to DL-(II), wherein LL-(III) and DL-(III) have the following formulae: wherein the reaction according to step (p) is catalyzed by a hydantoinase E 2 , and wherein R has the same meaning as described for LL-(I) and DL-(I).
  • the hydantoinase E 2 in particular the L-hydantoinase E 2 , is preferably categorized in the EC class 3.5.2.2.
  • the polypeptide sequence of the hydantoinase E 2 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.
  • the compound according to formula L-(lll) and/or the compound according to formula DL-(III) is obtained by a step (a) in which a compound according to formula LD-(III) is reacted to give a compound according to formula LL-(III) and/or a compound according to formula DD-(III) is reacted to give a compound according to formula DL-(III), wherein LD-(III) and DD-(III) have the following formulae:
  • the reaction according to step (a) is catalyzed by a hydantoin racemase E 3 , and the hydantoin racemase E 3 is more preferably categorized in the EC class 5.1 .99.5.
  • the polypeptide sequence of the hydantoin racemase E 3 in step (a) is even more 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.
  • Figure 1 shows the pOM21 c plasmid map.
  • Figure 2 shows the pOM22c plasmid map.
  • Figure 3 shows the pOM17c plasmid map.
  • Figure 4 shows the pOM17c ⁇ Prha ⁇ [amaB_Gst] plasmid map.
  • Figure 5 shows the pOM17c ⁇ Prha ⁇ [atc_Ps] plasmid map.
  • Figure 6 shows the pOM17c ⁇ Prha ⁇ [hyuC_Pau] plasmid map.
  • Figure 7 shows the pOM17c ⁇ Prha ⁇ [hyuC_Asp (co_Ec)] plasmid map.
  • the x-axis gives the reaction time in minutes.
  • the y-axis gives the ratio ni/nu for each diastereomer LL-(I) and DL-(I) in percent.
  • nu is the total amount of substance (in mole) of each diastereomers LL-(II) and DL-(II) initially employed.
  • ni is the total amount of substance (in mole) of each one of the two diastereomers LL-(I) and DL-(I) obtained.
  • the broken graph gives the ratio ni/nu for one diastereomer, whereas the continuous graph gives the ratio ni/nu for the other diastereomer, wherein m is the molar amount of one of the two diastereoisomers LL-(I) or DL-(I).
  • the values of the x- and the y-axis are as described for Figure 8.
  • the values of the x- and the y-axis are as described for Figure 8.
  • Plad name: pOM17c ⁇ Prha ⁇ [hyuc_Pau] The values of the x- and the y-axis are as described for Figure 8. 4.
  • any of the enzymes used according to any aspect of the present invention may be an isolated enzyme.
  • the enzymes 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 polypeptides or factors essential for its activity.
  • factors may be metal ions such as Mn 2+ or Co 2+ .
  • an enzyme according to the present application may be a carbamoylase Ei, a hydantoinase E 2 , or a hydantoin racemase E 3 .
  • Enzymatically catalyzed means that the respective reaction is catalyzed by an enzyme, which may be a carbamoylase Ei, a hydantoinase E 2 , or a hydantoin racemase E 3 .
  • 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 is encoded by such a molecule, particularly a polypeptide or nucleic acid that, as such, does not occur naturally but is the result of genetic engineering or refers to a cell that comprises a recombinant molecule.
  • a nucleic acid molecule is recombinant if it comprises a promoter functionally linked to a sequence encoding a catalytically active polypeptide and the promoter has been engineered such that the catalytically active polypeptide is overexpressed relative to the level of the polypeptide in the corresponding wild type cell that comprises the original unaltered nucleic acid molecule.
  • a “polypeptide” (one or more peptides) is a chain of chemical building blocks called amino acids that are 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 native protein, respectively. These terms are sometimes used interchangeably.
  • a polypeptide may or may not be glycosylated.
  • overexpressed means that the respective polypeptide encoded or expressed is expressed at a level higher or at higher activity than would normally be found in the cell under identical conditions in the absence of genetic modifications carried out to increase the expression, for example in the respective wild type cell.
  • the enzymes that can be used in the method according to the present invention can be synthesized by methods that are known to the skilled person.
  • Another approach is to express the enzyme(s), lyse the microorganisms, and add the cell lysate.
  • Yet another approach is to purify, or partially purify, the enzyme(s) from a lysate and add pure or partially pure enzyme(s) to the reaction. If multiple enzymes are required for a reaction, the enzymes can be expressed in one or several microorganisms, including expressing all enzymes within a single microorganism.
  • the skilled person can obtain the enzymes according to the invention by expression, in particular, overexpression, [hereinafter, “expression, in particular overexpression” is abbreviated as (over)expression”, and “express, in particular overexpress” is abbreviated as (over)express”] of these enzymes in a cell and subsequent isolation thereof, e.g. as described in DE 100 31 999 A1.
  • Episomal plasmids are employed for increasing the expression of the respective genes.
  • the nucleic acid molecule to be (over)expressed or encoding the polypeptide or enzyme to be (over)expressed may be placed under the control of a strong inducible promoter such as the lac promoter, located upstream of the gene.
  • a promoter is a DNA sequence consisting of about 40 to 50 base pairs which constitutes the binding site for an RNA polymerase holoenzyme and the transcriptional start point (M. Patek, J. Holatko, T. Busche, J. Kalinowski, J. Nesvera, Microbial Biotechnology 2013, 6, 103-117), whereby the strength of expression of the controlled polynucleotide or gene can be influenced.
  • a “functional linkage” is obtained by the sequential arrangement of a promoter with a gene, which leads to a transcription of the gene.
  • Suitable strong promoters or methods of producing such promoters for increasing expression are known from the literature (e.g. S. Lisser & H. Margalit, Nucleic Acid Research 1993, 21, 1507-1516; M. Patek and J. Nesvera in H. Yukawa and M Inui (eds.), Corynebacterium glutamicum, Microbiology Monographs 23, Springer Verlag Berlin Heidelberg 2013, 51-88; B. J. Eikmanns, E. Kleinertz, W. Liebl, H. Sahm, Gene 1991 , 102, 93-98).
  • native promoters may be optimized by altering the promoter sequence in the direction of known consensus sequences with respect to increasing the expression of the genes functionally linked to these promoters (M.
  • Patek B.J. Eikmanns, J. Patek, H. Sahm, Microbiology 1996, 142, 1297-1309; M. Patek, J. Holatko, T. Busche, J. Kalinowski, J. Nesvera, Microbial Biotechnology 2013, 6, 103-117).
  • Constitutive promoters are also suitable for the (over)expression, in which the gene encoding the enzyme activity is expressed continuously under the control of the promoter such as, for example, the glucose dependent dec promoter.
  • Chemically induced promoters are also suitable, such as tac, lac, rha or trp.
  • tac, lac, rha or trp The most widespread system for the induction of promoters is the lac operon of E. coli. In this case, either lactose or isopropyl B-D-thiogalactopyranoside (IPTG) is used as inducer.
  • IPTG isopropyl B-D-thiogalactopyranoside
  • systems using arabinose e.g. the pBAD system
  • rhamnose e.g. E. coli KRX
  • a system for physical induction is, for example, the temperature-induced cold shock promoter system based on the E. coli cspA promoter from Takara or Lambda PL and also osmotically inducible promoters, for example, osmB (e.g. WO 95/25785 A1).
  • Suitable plasmids or vectors are in principle all embodiments available for this purpose to the person skilled in the art.
  • the state of the art describes standard plasmids that may be used for this purpose, for example the pET system of vectors exemplified by pET-3a or pET-26b(+) (commercially available from Novagen).
  • Further plasmids and vectors can be taken, for example, pOM21 described in WO 2004/111227 A2, pOM22 described in WO 00/058449 A1 or pOM18 described in WO 2013/072486 A1 or from the brochures of the companies Novagen, Promega, New England Biolabs, Clontech or Gibco BRL.
  • plasmids and vectors can be found in: Glover, D.M. (1985) DNA cloning: a practical approach, Vol. I-III, IRL Press Ltd., Oxford; Rodriguez, R.L. and Denhardt, D. T (eds) (1988) Vectors: a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham; Goeddel, D. V. (1990) Systems for heterologous gene expression, Methods Enzymol. 185, 3-7; Sambrook, J.; Fritsch, E. F. and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York. Of these plasmids, pOM21 and pOM22 are preferred.
  • the plasmid vector which contains the gene to be amplified, is then converted to the desired strain, e.g. by conjugation or transformation.
  • the method of conjugation is described, for example, by A. Schafer, J. Kalinowski, A. Piihler, Applied and Environmental Microbiology 1994, 60, 756 - 759.
  • Methods for transformation are described, for example, in G. Thierbach, A. Schwarzer, A. Piihler, Applied Microbiology and Biotechnology 1988, 29, 356 - 362, L.K. Dunican & E. Shivnan, Bio/Technology 1989, 7, 1067-1070 and A. Tauch, O. Kirchner, L. Wehmeier, J. Kalinowski, A. Piihler, FEMS Microbiology Letters 1994, 123, 343-347. After homologous recombination by means of a “cross-over” event, the resulting strain contains at least two copies of the gene concerned.
  • the desired enzyme can be isolated by disrupting cells which contain the desired activity in a manner known to the person skilled in the art, for example with the aid of a ball mill, a French press or of an ultrasonic disintegrator and subsequently separating off cells, cell debris and disruption aids, such as, for example, glass beads, by centrifugation for 10 minutes at 13,000 rpm and 4 °C. Using the resulting cell-free crude extract, enzyme assays with subsequent LC-ESI-MS detection of the products can then be carried out.
  • the enzyme can be enriched in the manner known to the person skilled in the art by chromatographic methods (such as nickel-nitrilotriacetic acid affinity chromatography, streptavidin affinity chromatography, gel filtration chromatography or ion-exchange chromatography) or else purified to homogeneity. Quantification of the enzyme can be performed by methods known to the person skilled in the art, for example by determination of the concentration of the respective polypeptide of the enzyme (e.g. carbamoylase, hydantoinase and racemase) in the obtained solution by SDS page and analysis of the respective bands via the software GelQuant® (BiochemLabSolutions).
  • chromatographic methods such as nickel-nitrilotriacetic acid affinity chromatography, streptavidin affinity chromatography, gel filtration chromatography or ion-exchange chromatography
  • Quantification of the enzyme can be performed by methods known to the person skilled in the art, for example by determination of the concentration of the respective polypeptide of the enzyme (
  • nucleic acid or polypeptide may be determined by way of quantitative PCR reaction in the case of a nucleic acid molecule, SDS polyacrylamide electrophoreses, Western blotting or comparative activity assays in the case of a polypeptide. Genetic modifications may be directed to transcriptional, translational, and/or post-translational modifications that result in a change of enzyme activity and/or selectivity under selected and/or identified culture conditions.
  • the term “variant” with respect to polypeptide sequences refers to a polypeptide sequence with a degree of identity to the reference sequence (“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
  • Such variants may be prepared by introducing deletions, insertions, substitutions, or combinations thereof, in particular in amino acid sequences, as well as fusions comprising such macromolecules or variants thereof.
  • 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 that comprises the complete polypeptide sequence 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: 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, or at least the amino acids of the respective sequence 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 ,
  • the other amino acids may be deleted, substituted or replaced by insertions or essential amino acids are replaced in a conservative manner to the effect that the activity of the enzyme, in particular the 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,
  • BLASTP BLASTN
  • FASTA Altschul et al., Journal of Molecular Biology 1990, 215, 403 - 410.
  • the BLAST program can be obtained from the National Center for Biotechnology Information (NCBI) and from other sources (BLAST Handbook, S. Altschul et al., NCBI NLM NIH Bethesda ND 22894; S. Altschul et al., above).
  • the percentage identity between two polypeptide sequences can be determined by the algorithm developed by S. B. 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 either a BLOSUM62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1 , 2, 3, 4, 5 or 6.
  • the person skilled in the art will recognize that the use of different parameters will lead to slightly different results, but that the percentage identity between two polypeptide overall will not be significantly different.
  • the BLOSUM62 matrix is typically used applying the default settings (gap weight: 12, length weight: 1).
  • a sequence identity of 60% means 60% homology. The same applies to higher sequence identities.
  • the degree of identity between sequences is determined in the context of the present invention by the programme “Needle” using the substitution matrix BLOSUM62, the gap opening penalty of 10, and the gap extension penalty of 0.5.
  • the Needle program implements the global alignment algorithm described in S. B. Needleman & C. D. Wunsch, J. Mol. Biol. 1970, 48, 443-453.
  • the substitution matrix used according to the present invention is BLOSUM62, gap opening penalty is 10, and gap extension penalty is 0.5.
  • the preferred version used in the context of this invention is the one presented by F. Madeira, Y.M. Park, J. Lee, N. Buso, T. Gur, N. Madhusoodanan, P. Basutkar, A.R.N.
  • the percentage of identity of an amino acid sequence of a polypeptide with, or to, a reference polypeptide sequence is determined by i) aligning the two amino acid sequences using the Needle program, with the BLOSUM62 substitution matrix, a gap opening penalty of 10, and a gap extension penalty of 0.5; ii) counting the number of exact matches in the alignment; iii) dividing the number of exact matches by the length of the longest of the two amino acid sequences, and iv) converting the result of the division of iii) into percentage.
  • the present invention relates to a method for the production of an L-glufosinate P-ester according to formula LL-(I) and/or DL-(I):
  • the method according to the invention comprises steps (y), (5), and (e).
  • step (y) a mixture Mu comprising carbamoyl compounds according to formulae LL-(II) and DL-(II) is provided, wherein LL-(II) and DL-(II) have the following formulae:
  • the molar ratio of all carbamoyl compounds according to formula LL-(II) in mixture Mu provided in step (Y) to all carbamoyl compounds according to formula DL-(II) in mixture Mu provided in step (y) is not specifically limited.
  • the molar ratio of all carbamoyl compounds according to formula LL-(II) in mixture Mu provided in step (y) to all carbamoyl compounds according to formula DL-(II) provided in step (Y) in mixture Mu is in the range of 999 : 1 to 1 : 999, preferably 99 : 1 to 1 : 99, more preferably 9 : 1 to 1 : 9, more preferably 4 : 1 to 1 : 4, more preferably 7 : 3 to 3 : 7, more preferably 3 : 2 to 2 : 3, most preferably 1 : 1.
  • mixture Mu provided in step (y) further comprises the diastereoisomers LD-(II) and DD-(II), wherein LD-(II) and DD-(II) have the following formulae:
  • mixture Mu provided in step (y) further comprises the diastereoisomers LD-(II) and DD-(II)
  • the molar ratio of all carbamoyl compounds according to formula LD-(II) in mixture Mu provided in step (y) to all carbamoyl compounds according to formula DD-(II) in mixture Mu provided in step (y) is not specifically limited.
  • the molar ratio of all carbamoyl compounds according to formula LD-(II) in mixture Mu provided in step (y) to all carbamoyl compounds according to formula DD-(II) in mixture Mu provided in step (y) is in this case in the range of 999 : 1 to 1 : 999, preferably 99 : 1 to 1 : 99, more preferably 9 : 1 to 1 : 9, more preferably 4 : 1 to 1 : 4, more preferably 7 : 3 to 3 : 7, more preferably 3 : 2 to 2 : 3, most preferably 1 : 1.
  • mixture Mu provided in step (y) further comprises the diastereoisomers LD-(II) and DD-(II)
  • the molar ratio of all carbamoyl compounds LL-(II) and DL-(II) to all carbamoyl compounds LD-(II) and DD-(II) in mixture Mu provided in step (y) is not specifically limited.
  • the molar ratio of all carbamoyl compounds according to formulae LL-(II) and DL-(II) in mixture Mu provided in step (y) to all carbamoyl compounds according to formulae LD-(II) and DD-(II) in mixture Mu provided in step (y) is in this case in the range of 999 : 1 to 1 : 999, preferably 99 : 1 to 1 : 99, more preferably 9 : 1 to 1 : 9, more preferably 4 : 1 to 1 : 4, more preferably 7 : 3 to 3 : 7, more preferably 3 : 2 to 2 : 3, most preferably 1 : 1.
  • R is an alkyl group or an aryl group.
  • R is the same for LL-(II) and DL-(II), and, in those embodiments in which mixture Mu provided in step (y) comprises compounds according to formulae LL-(H), DL-(II) and at least one, preferably both of LD-(II) and DD-(II), is, in particular, the same in all the compounds according to these formulae in mixture Mu provided in step (y).
  • the method according to the invention comprises a step (5).
  • step (5) in mixture Mu, a carbamoyl compound according to formula LL-(II) is reacted to give an amino acid compound according to formula LL-(I) and/or a carbamoyl compound according to formula DL-(II) is reacted to give an amino acid compound according to formula DL-(I):
  • step (6) in mixture Mu, a carbamoyl compound according to formula LL-(II) is reacted to give an amino acid compound according to formula LL-(I) and a carbamoyl compound according to formula DL-(II) is reacted to give an amino acid compound according to formula DL-(I).
  • the reaction according step (6) is enzymatically catalyzed, namely it is catalyzed by a carbamoylase Ei.
  • Step (6) of the method according to the invention is preferably L-enantioselective, even more preferably L-enantiospecific.
  • a mixture Mu is in particular employed in step (6), wherein the mixture Mu comprises, besides compounds according to formulae LL-(II) and DL-(II), the respective enantiomers according to formulae LD-(II) and DD-(II).
  • Such mixtures comprising all four diastereomers LL-(II), DL-(II), LD-(II), and DD-(II) are referred to as “mixture Mu* hereinafter.
  • a parallel reaction according to step (6)* may be observed. Namely, in the reaction according to step (6)*, LD-(II), i.e. the enantiomer of DL-(II), in mixture Mu* is reacted to give a compound according to formula LD-(I), and/or, preferably and, DD-(II), i.e. the enantiomer of LL-(II), in mixture Mu* is reacted to give a compound according to formula DD-(I), wherein
  • step (6) is “L-enantioselective”, this means that in case a mixture Mu* is employed in step (5), then there is either no reaction according to step (5)* or, in case there is a reaction according to step (5)*, then the rate of reaction according to step (5)* is lesser than the rate of the reaction according to step (5).
  • Step (5) is “L-enantiospecific”, if the rate of reaction according to step (5)* is essentially zero, i.e. there is no reaction according to step (5)*.
  • Step (6) is in particular L-enantioselective, if it is catalyzed by an L-carbamoylase Ei, which may be determined by the skilled person as set forth under 4.5.4.3.
  • step (6) is L-enantioselective
  • the reaction according to step (6) proceeds preferably at a reaction rate that is at least 2 times greater, preferably at least 10 times greater, more preferably at least 100 times greater, even more preferably at least 10 3 times greater, even more preferably at least 10 4 times greater, even more preferably at least 10 5 times greater than the reaction rate at which step (6)* proceeds.
  • n L no I n D no > 1 the reaction rate of step (5) is greater than the reaction rate of step (5)*. If n L no I n D no ⁇ 1 , the reaction rate of step (5) is lesser than the reaction rate of step (5)*.
  • step (5) of the method according to the invention is catalyzed by a carbamoylase Ei.
  • carbamoylases accept compounds of formulae LL-(II) and DL-(II) as substrates and convert them to the respective product LL-(I) and DL-(I), and hence can be used to catalyze the reaction according to step (6).
  • This finding is of high scientific and economic value, as it opens new synthetic routes based on new starting materials for the production of L-glufosinate P-esters and L-gluofsinate.
  • carbamoylases generally catalyze the following reaction ⁇ 1>, wherein R x may be an organic residue, e.g. a side chain of one of the naturally occurring amino acids.
  • the carbamoylase Ei is a “L-carbamolyase”, i.e. it has a greater catalytic activity for reaction ⁇ 1A> than for reaction ⁇ 1 B>, wherein the substrate S D in the reaction ⁇ 1 B> is the enantiomer of the substrate S L in the reaction ⁇ 1A>:
  • L-carbamoylase As an L-carbamoylase has a higher catalytic activity for reaction ⁇ 1A> than for reaction ⁇ 1 B>, it is “L-enantioselective”. An L-carbamoylase that has no catalytic activity for reaction ⁇ 1 B> and thus only has catalytic activity for reaction ⁇ 1A> is “L-enantiospecific”.
  • a “D-carbamoylase” is defined as a carbamoylase which is “D-enantioselective”, 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 thus only has catalytic activity for reaction ⁇ 1B> is “D-enantiospecific”.
  • a carbamoylase may be denoted as “L-carbamoylase”, “D-carbamoylase” or “non-enantioselective carbamoylase” in the context of the present invention.
  • the procedure according to Assay B (item 4.5.4) may preferably be used.
  • the carbamoylase Ei in particular the L-carbamoylase Ei, that may be used in step (6) of the method according to invention may originate 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 japonicum
  • Brevibacillus sp. in particular Brevibacillus reuszeri
  • Comamonas sp. Ensifer sp., in particular Ensifer adhaerens
  • Geobacillus sp. in particular Geobacillus kaustophilus
  • Geobacillus stearothermophilus Microbacterium sp. in particular Microbacterium liquefaciens strain AJ3912; Paenarthrobacter sp., in particular Paenarthrobacter aurescens; Pasteurella sp.; Pseudomonas sp.; Ralstonia sp., in particular Ralstonia pickettii; Sinorhizobium sp., in particular Sinorhizobium meliloti.
  • An L-carbamoylase Ei suitable for the method according to the present invention may be the enzyme HyuC, which originates from Arthrobacter.
  • Other enzymes are AmaB, AtcC, Inc, SinmeB_2280.
  • WO 01/23582 A1 discloses an example of an enzyme having carbamoylase activity according to the invention.
  • the carbamoylase Ei that may be used in step (6) of the method according to the present invention may be an L-carbamoylase categorized in the EC class EC 3.5.1 .87.
  • L-carbamoylase enzymes are for example described by J. Ogawa, H. Miyake, S. Shimizu, Appl. Microbiol. Biotechnol. 1995 43, 1039 - 1043 and in WO 01/23582 A1 .
  • a L-carbamoylase Ei that may preferably be used in step (6) according to the first aspect of the invention may originate from Arthrobacter sp., in particular Arthrobacter crystallopoietes, Arthrobacter aurescens, Arthrobacter sp. BT801 , Arthrobacter aurescens DSM 3747; Bacillus sp.
  • Microbacterium sp. in particular Microbacterium liquefaciens strain AJ3912; Paenarthrobacter sp., in particular Paenarthrobacter aurescens Pseudomonas sp. , in particular Pseudomonas sp. QR-101 Sinorhizobium sp., in particular Sinorhizobium meliloti.
  • the L-carbamoylase Ei that may preferably be used in step (6) according to the first aspect of the invention may originate from Arthrobacter sp., in particular Arthrobacter crystallopoietes, Arthrobacter aurescens, Arthrobacter sp. BT801 , Arthrobacter aurescens DSM 3747, most preferably from Arthrobacter aurescens DSM 3747.
  • the respective sequences can be derived from databases such as the Braunschweig Enzyme Database (BRENDA, Germany, available underwww.brenda-enzymes.org/index.php), the National Center for Biotechnological Information (NCBI, available under https://www.ncbi.nlm.nih.gov/) or the Kyoto Encyclopedia of Genes and Genomes (KEGG, Japan, available under www. https://www.genome.jp/kegg/).
  • BRENDA Braunschweig Enzyme Database
  • NCBI National Center for Biotechnological Information
  • KEGG Kyoto Encyclopedia of Genes and Genomes
  • the following table 1 gives preferred examples for polypeptide sequences of L-carbamoylases Ei that may be preferably used in step (6) of the method according to the invention.
  • the genes encoding the respective L-carbamoylase Ei and the respective accession code are indicated as far as known.
  • the reaction according to step (5) is catalyzed by an L-carbamoylases Ei, wherein the polypeptide sequence of Ei is selected from the group consisting of 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.
  • carbamoylases in particular L-carbamoylases, that may be used in step (6) of the method according to the invention.
  • Assay A L may be used to determine carbamolyase and L-carbamoylase activity of a given enzyme Ex and may advantageously be used according to the invention to determine carbamoylase and L-carbamoylase activity 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, variants of SEQ ID NO: 9.
  • Assay A D may be used to determine D-carbamoylase activity of a given enzyme E x .
  • the molar mass of the enzyme Ex to be tested is calculated as the molar mass of the polypeptide sequence of E x .
  • R n-butyl
  • LD-(II) and DD-(II) are equimolar, are added 400 nmol of Ex in 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh).
  • the resulting solution is incubated at 25 °C, and the pH is held at pH 7.2 by addition of 0.5 M NaOH.
  • the carbamoylase Ei according to the invention is preferably an L-carbamoylase, more preferably L-enantiospecific.
  • Whether a given enzyme E x may be considered a carbamoylase Ei, in particular an L-carbamoylase, may be determined in the context of the present invention by the following Assay B.
  • step B-1 is repeated, except that instead of the addition of 400 nmol E x in 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh), 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh) without E x is added.
  • step B-2 is repeated, except that instead of the addition of 400 nmol E x in 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh), 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh) without E x is added.
  • E x is deemed to have L-carbamoylase activity, and hence may be considered an L-carbamoylase in the context of the invention. In this case, E x is deemed to be “L-enantioselective” in the context of the invention.
  • E x is deemed to have L-carbamoylase activity, and hence may be considered to be an L- carbamoylase in the context of the invention.
  • E x is not only “L-enantioselective”, but also “L-enantiospecific” in the context of the invention.
  • E x is deemed to have D-carbamoylase activity, and hence may be considered a D-carbamoylase.
  • E x is “D-enantioselective” in the context of the invention.
  • E x is deemed to have D-carbamoylase activity, and hence may be considered to be a D-carbamoylase.
  • E x is not only “D-enantioselective”, but also “D-enantiospecific” in the context of the invention.
  • E x is deemed to be a non-enantioselective carbamoylase in the context of the invention. In this case, E x is “non-enantioselective”.
  • An enzyme the polpypetide sequence of which is 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 has carbamoylase, and L-carbamoylase activity.
  • the polypeptide sequence of the L-carbamoylase Ei is selected from the group consisting of 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.
  • the polypeptide sequence of the L-carbamoylase Ei is selected from the group consisting of SEQ ID NO: 1 and variants thereof, SEQ ID NO: 2 and variants thereof, SEQ ID NO: 3 and variants thereof, SEQ ID NO: 5 and variants thereof, SEQ ID NO: 8 and variants thereof, more preferably, the polypeptide sequence of the L-carbamoylase Ei is selected from the group consisting of SEQ ID NO: 1 and variants thereof, SEQ ID NO: 2 and variants thereof, SEQ ID NO: 3 and variants thereof, SEQ ID NO: 5 and variants thereof, more preferably the polypeptide sequence of the L-carbamoylase Ei is selected from SEQ ID NO: 1 and variants thereof.
  • an enzyme Ei the polypeptide sequence of which 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 has carbamoylase activity, preferably L-carbamoylase activity, more preferably is L-enantiospecific.
  • the polypeptide sequence of which 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, has carbamoylase activity, L carbamoylase activity and/or is L-enantiospecific may be determined as set forth under items 4.5.4.2 and 4.5.4.3.1 , respectively.
  • the carbamoylase activity of a given L-carbamoylase Ei V may be quantified in the context of the present invention by the following Assay C:
  • Step C-2 Step C-1 is repeated, except that, instead of Eis, Eiv is used as the enzyme to be tested.
  • L-carbamoylases Ei the polypeptide sequence of which is 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, are generally denoted as tis ⁇
  • L-carbamoylases Ei the polypeptide sequence of which is selected from variants of 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, are generally denoted as “Eiv”.
  • the reaction in step (6) is catalyzed by an L-carbamoylase Ei, and the polypeptide sequence of the L-carbamoylase Ei is selected from the group consisting of 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,
  • reaction in step (5) is catalyzed by an L-carbamoylase Ei, and the polypeptide sequence of the L-carbamoylase Ei is selected from the group consisting of SEQ ID NO: 1 and variants of SEQ ID NO: 1.
  • polypeptide sequence of the L-carbamoylase Ei may also be a variant of SEQ ID NO: 1 .
  • L-carbamoylase Ei the polypeptide sequence of which is SEQ ID NO: 1
  • Eiois the polypeptide sequence of which is selected from variants of SEQ ID NO: 1
  • Eioiv the polypeptide sequence of which is selected from variants of SEQ ID NO: 1
  • a variant of the polypeptide sequence of SEQ ID NO: 1 is a polypeptide with sequence identity of at least 60 %, preferably > 65 %, more preferably > 70 %, more preferably > 75 %, more preferably
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 1 is not identical to SEQ ID NO: 1.
  • an L-carbamoylase Eioiv has carbamoylase activity and L-carbamoylase activity, determined as described under items 4.5.4.2 and 4.5.4.3.1 .
  • an L-carbamoylase Eioiv preferably has carbamoylase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least
  • an L-carbamoylase Eioiv has carbamoylase activity 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 % the carbamoylase activity of the
  • L-carbamoylase Eiois wherein the carbamoylase activity of Eioiv, relative to the carbamoylase activity of Eiois, is determined by Assay C described under item 4.5.5.2.
  • polypeptide sequence of the L-carbamoylase Ei may also be a variant of SEQ ID NO: 2.
  • L-carbamoylase Ei the polypeptide sequence of which is SEQ ID NO: 2
  • EI 0 2S The L-carbamoylases Ei, the polypeptide sequence of which is selected from variants of SEQ ID NO: 2, are generally denoted as “EI 0 2V”.
  • a variant of the polypeptide sequence of SEQ ID NO: 2 is a polypeptide with sequence identity of at least 60 %, preferably > 65 %, more preferably > 70 %, more preferably > 75 %, more preferably
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 2 is not identical to SEQ ID NO: 2.
  • an L-carbamoylase EI 0 2V has carbamoylase activity and L-carbamoylase activity, determined as described under items 4.5.4.2 and 4.5.4.3.1 .
  • an L-carbamoylase EI 0 2V preferably has carbamoylase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least
  • an L-carbamoylase EI 0 2V has carbamoylase activity 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 % the carbamoylase activity of the L-carbamoylase EI 0 2S, wherein the carbamoylase activity of EI 0 2V, relative to the carbamoylase activity of EI 0 2S, is determined by Assay C described under item 4.5.5.2.
  • polypeptide sequence of the L-carbamoylase Ei may also be a variant of SEQ ID NO: 3.
  • L-carbamoylase Ei the polypeptide sequence of which is SEQ ID NO: 3, is denoted as “Eioss”.
  • L-carbamoylases Ei the polypeptide sequence of which is selected from variants of SEQ ID NO: 3, are generally denoted as “Eiosv”.
  • a variant of the polypeptide sequence of SEQ ID NO: 3 is a polypeptide with sequence identity of at least 60 %, preferably > 65 %, more preferably > 70 %, more preferably > 75 %, more preferably
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 3 is not identical to SEQ ID NO: 3.
  • an L-carbamoylase Ewsv has carbamoylase activity and L-carbamoylase activity, determined as described under items 4.5.4.2 and 4.5.4.3.1 .
  • an L-carbamoylase Ewsv preferably has carbamoylase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least
  • an L-carbamoylase Ewsv has carbamoylase activity 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 % the carbamoylase activity of the L-carbamoylase Ewss, wherein the carbamoylase activity of Ewsv, relative to the carbamoylase activity of Ewss, is determined by Assay C described under item 4.5.5.2.
  • polypeptide sequence of the L-carbamoylase Ei may also be a variant of SEQ ID NO: 4.
  • L-carbamoylase Ei the polypeptide sequence of which is SEQ ID NO: 4, is denoted as “Ems”.
  • L-carbamoylases Ei the polypeptide sequence of which is selected from variants of SEQ ID NO: 4, are generally denoted as “Emv”.
  • a variant of the polypeptide sequence of SEQ ID NO: 4 is a polypeptide with sequence identity of at least 60 %, preferably > 65 %, more preferably > 70 %, more preferably > 75 %, more preferably
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 4 is not identical to SEQ ID NO: 4.
  • an L-carbamoylase Emv has carbamoylase activity and L-carbamoylase activity, determined as described under items 4.5.4.2 and 4.5.4.3.1 .
  • an L-carbamoylase Emv preferably has carbamoylase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least
  • an L-carbamoylase Emv has carbamoylase activity 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 % the carbamoylase activity of the L-carbamoylase Ems, wherein the carbamoylase activity of E , relative to the carbamoylase activity of Ems, is determined by Assay C described under item 4.5.5.2.
  • polypeptide sequence of the L-carbamoylase Ei may also be a variant of SEQ ID NO: 5.
  • L-carbamoylase Ei the polypeptide sequence of which is SEQ ID NO: 5 is denoted as “Ems”.
  • L-carbamoylases Ei the polypeptide sequence of which is selected from variants of SEQ ID NO: 5, are generally denoted as “Emv”.
  • a variant of the polypeptide sequence of SEQ ID NO: 5 is a polypeptide with sequence identity of at least 60 %, preferably > 65 %, more preferably > 70 %, more preferably > 75 %, more preferably
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 5 is not identical to SEQ ID NO: 5.
  • an L-carbamoylase E v has carbamoylase activity and L-carbamoylase activity, determined as described under items 4.5.4.2 and 4.5.4.3.1 .
  • an L-carbamoylase E v preferably has carbamoylase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least
  • an L-carbamoylase E v has carbamoylase activity 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 % the carbamoylase activity of the
  • polypeptide sequence of the L-carbamoylase Ei may also be a variant of SEQ ID NO: 6.
  • L-carbamoylase Ei the polypeptide sequence of which is SEQ ID NO: 6, is denoted as “Ewes”.
  • L-carbamoylases Ei the polypeptide sequence of which is selected from variants of SEQ ID NO: 6, are generally denoted as “Eioev”.
  • a variant of the polypeptide sequence of SEQ ID NO: 6 is a polypeptide with sequence identity of at least 60 %, preferably > 65 %, more preferably > 70 %, more preferably > 75 %, more preferably
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 6 is not identical to SEQ ID NO: 6.
  • an L-carbamoylase Eioev has carbamoylase activity and L-carbamoylase activity, determined as described under items 4.5.4.2 and 4.5.4.3.1 .
  • an L-carbamoylase Eioev preferably has carbamoylase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least
  • an L-carbamoylase Eioev has carbamoylase activity 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 % the carbamoylase activity of the
  • L-carbamoylase Ewes wherein the carbamoylase activity of Eioev, relative to the carbamoylase activity of Ewes, is determined by Assay C described under item 4.5.5.2. 4.5.6.7 Preferred variants of SEQ ID NO: 7
  • polypeptide sequence of the L-carbamoylase Ei may also be a variant of SEQ ID NO: 7.
  • L-carbamoylase Ei the polypeptide sequence of which is SEQ ID NO: 7, is denoted as “Eiozs”.
  • L-carbamoylases Ei the polypeptide sequence of which is selected from variants of SEQ ID NO: 7, are generally denoted as “Eiozv”.
  • a variant of the polypeptide sequence of SEQ ID NO: 7 is a polypeptide with sequence identity of at least 60 %, preferably > 65 %, more preferably > 70 %, more preferably > 75 %, more preferably
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 7 is not identical to SEQ ID NO: 7.
  • an L-carbamoylase EI 0 7V has carbamoylase activity and L-carbamoylase activity, determined as described under items 4.5.4.2 and 4.5.4.3.1 .
  • an L-carbamoylase EI 0 7V preferably has carbamoylase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least
  • an L-carbamoylase EI 0 7V has carbamoylase activity 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 % the carbamoylase activity of the
  • polypeptide sequence of the L-carbamoylase Ei may also be a variant of SEQ ID NO: 8.
  • L-carbamoylase Ei the polypeptide sequence of which is SEQ ID NO: 8 is denoted as “Eioss”.
  • L-carbamoylases Ei the polypeptide sequence of which is selected from variants of SEQ ID NO: 8, are generally denoted as “Eiosv”.
  • a variant of the polypeptide sequence of SEQ ID NO: 8 is a polypeptide with sequence identity of at least 60 %, preferably > 65 %, more preferably > 70 %, more preferably > 75 %, more preferably
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 8 is not identical to SEQ ID NO: 8.
  • an L-carbamoylase Eiosv has carbamoylase activity and L-carbamoylase activity, determined as described under items 4.5.4.2 and 4.5.4.3.1 .
  • an L-carbamoylase Eiosv preferably has carbamoylase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least
  • an L-carbamoylase Eiosv has carbamoylase activity 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 % the carbamoylase activity of the
  • L-carbamoylase Eioss wherein the carbamoylase activity of Ews, relative to the carbamoylase activity of Eioss, is determined by Assay C described under item 4.5.5.2. 4.5.G.9 Preferred variants of SEQ ID NO: 9
  • polypeptide sequence of the L-carbamoylase Ei may also be a variant of SEQ ID NO: 9.
  • L-carbamoylase Ei the polypeptide sequence of which is SEQ ID NO: 9, is denoted as “Eioss”.
  • L-carbamoylases Ei the polypeptide sequence of which is selected from variants of SEQ ID NO: 9, are generally denoted as “Eiosv”.
  • a variant of the polypeptide sequence of SEQ ID NO: 9 is a polypeptide with sequence identity of at least 60 %, preferably > 65 %, more preferably > 70 %, more preferably > 75 %, more preferably
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 9 is not identical to SEQ ID NO: 9.
  • an L-carbamoylase Ei O gv has carbamoylase activity and L-carbamoylase activity, determined as described under items 4.5.4.2 and 4.5.4.3.1 .
  • an L-carbamoylase Ei O gv preferably has carbamoylase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least
  • an L-carbamoylase Ei O gv has carbamoylase activity 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 % the carbamoylase activity of the
  • step (6) of the method according to the present invention may be carried out under conditions known to the skilled person.
  • the reaction medium is preferably aqueous, more preferably an aqueous buffer.
  • Exemplary buffers commonly used in biotransformation reactions and advantageously used herein include Tris, phosphate, or any of Good's buffers, such as 2-(/V-morpholino)ethanesulfonic acid (“MES”), /V-(2-acetamido)iminodiacetic acid (“ADA”), piperazine-/V,/ ⁇ /'-bis(2-ethanesulfonic acid) (“PIPES”), /V-(2-acetamido)-2- aminoethanesulfonic acid (“ACES”), P-hydroxy-4- morpholinepropanesulfonic acid (“MOPSO”), cholamine chloride, 3-(/V-morpholino)propanesulfonic acid (“MOPS”), /V,/V-Bis(2-hydroxyethyl)- 2-aminoethanesulfonic acid (“BES”), 2-[[1 ,3-dihydroxy-MES”), /V-(2-acetamido)iminodiacetic acid
  • HEPES 1-piperazineethanesulfonic acid
  • DIPSO 3-(Bis(2-hydroxyethyl)amino)-2-hydroxypropane- 1 -sulfonic acid
  • acetamidoglycine
  • TAPSO 3-(/V-Tris(hydroxymethyl)methylamino(-2-hydroxypropane)sulfonic acid
  • POPSO piperazine- /V,/ ⁇ /'-bis(2-hydroxypropanesulfonic acid)
  • POPSO 4-(2- Hydroxyethyl)piperazine- 1-(2-hydroxypropanesulfonic acid)
  • HEPPSO 3-[4-(2-Hydroxyethyl)-1-piperazinyl]propanesulfonic acid
  • HPPS 3-[4-(2-Hydroxyethyl)-1-piperazinyl]propanesulfonic acid
  • TAPS 3-[[1 ,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1 -sulfonic acid
  • ammonium can act as a buffer.
  • One or more organic solvents can also be added to the reaction.
  • the buffer preferably contains metal salts, more preferably metal salts such as halogenides of metals, preferably halogenides of monovalent or bivalent or trivalent metals, preferably chlorides of monovalent or bivalent metals, preferably C0CI2 or MnCh, preferably C0CI2.
  • metal salts more preferably metal salts such as halogenides of metals, preferably halogenides of monovalent or bivalent or trivalent metals, preferably chlorides of monovalent or bivalent metals, preferably C0CI2 or MnCh, preferably C0CI2.
  • the concentration of these metal salts in the reaction medium is preferably in the range from 1 pM to 1 M, more preferably 1 mM to 100 mM, even more preferably 1 to 10 mM.
  • step (6) of the method according to the invention is carried out in a phosphate buffer.
  • the pH of the reaction medium in step (6) of the method according to the invention is preferably in the range of from 2 to 10, more preferably in the range of from 5 to 8, more preferably 7.2 to 7.5, most preferably 7.5.
  • step (6) of the method according to the invention is carried out at a temperature in the range of from 20 °C to 70 °C, more preferably in the range of from 30 °C to 55 °C, most preferably 50 °C.
  • the total concentration of all carbamoylases Ei in the reaction solution in step (6) is in the range of from 1 pM to 10 mM, preferably 10 pM to 1 mM, more preferably 0.1 mM to 0.5 mM, most preferably 0.4 mM.
  • the total concentration of all carbamoylases Ei in the reaction solution in step (6) is in the range of from 1 pg/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.
  • the initial concentration of all the compounds according to formula L-(ll) in the reaction medium in step (6) is in the range of from 1 pM to 1 M, preferably of from 10 pM to 0.5 M, more preferably of from 0.1 mM to 0.1 M, more preferably of from 1 mM to 10 mM, most preferably 1.25 mM.
  • the initial concentration of all the compounds according to formula D-(ll) in the reaction medium is preferably from 1 % to 100 % the concentration of all the compounds according to formula L-(ll), more preferably 10 % to 100 % the concentration, even more preferably 50 to 100 %, even more preferably 100 % the concentration of all the compounds according to formula L-(ll).
  • “Initial concentration of all the compounds according to formula L-(ll)/ D-(ll)” refers to the concentration of the respective compound L-(ll) or D-(ll) respectively, in the reaction medium when the respective compounds are employed in step (6).
  • the mixture Mu provided in step (y) is obtained by a step (p) wherein in a mixture Mm comprising compounds according to LL- (III) and DL-(III), at least a part of the compounds according to formula LL-(III) are reacted to give compounds according to LL-(II), and at least a part of the compounds according to formula DL-(III) are reacted to give compounds according to DL-(II), wherein LL-(III) and DL-(III) have the following formulae:
  • step (p) The reaction according to step (p) is catalyzed by a hydantoinase E 2 .
  • R in LL-(IH) and DL-(III) has the same meaning as described for LL-(I) and DL-(I).
  • Step (p) gives the starting material for step (6)
  • the reaction according step (p) is enzymatically catalyzed, namely it is catalyzed by a hydantoinase E 2 .
  • Step (p) of the method according to the present invention is preferably L-enantioselective, even more preferably L-enantiospecific.
  • Mixture Mm comprises, besides LL-(III) and DL-(III), the respective enantiomer of LL-(III) and DL-(III), which is DD-(III) and LD-(III):
  • mixtures comprising all four diastereomers LL-(III), DL-(III), LD-(III), and DD-(III) are referred to as “mixture Mm* hereinafter.
  • step (p)* a parallel reaction according to step (p)* may be observed. Namely, in the reaction according to step (p)*, LD-(III), i.e. the enantiomer of DL-(III), in mixture M * is reacted to give a compound according to formula LD-(II), and/or, preferably and, DD-(III), i.e. the enantiomer of LL-(III), in mixture Mm* is reacted to give a compound according to formula DD-(II).
  • LD-(III) i.e. the enantiomer of DL-(III)
  • mixture M * is reacted to give a compound according to formula LD-(II)
  • DD-(III) i.e. the enantiomer of LL-(III
  • step (p) is “L-enantioselective”, this means that in case a mixture M * is employed in step (p), then there is either no reaction according to step (p)* or, in case there is a reaction according to step (p)*, then the rate of reaction according to step (p)* is lesser than the rate of the reaction according to step (p).
  • Step (p) is “L-enantiospecific”, if the rate of reaction according to step (p)* is essentially zero, i.e. there is no reaction according to step (p)*.
  • the mixture Mm* is a racemic mixture of enantiomer L-(lll) and enantiomer D-(lll), meaning that the molar ratio of all compounds according to L-(lll) to compounds according to D-(lll) is essentially 1 : 1 , wherein:
  • the molar ratio of all compounds L-(lll) to all compounds D-(lll) in mixture Mm* is in the range of from 99 : 1 to 1 : 99, more preferably in the range of from 1.01 : 1 to 1 : 99, more preferably in the range of from 1 : 1 to 1 : 99, more preferably in the range of from 1 : 1.01 to 1 : 99, more preferably in the range of from 1 : 1.01 to 1 : 9, more preferably in the range of from 1 : 1.01 to 1 : 8, more preferably in the range of from 1 : 1.01 to 1 : 3.
  • Step (p) is in particular L-enantioselective, if it is preferably catalyzed by an L-hydantoinase E 2 , which may be determined by the skilled person as set forth under 4.5.10.3.
  • step (p) is L-enantioselective
  • the reaction according to step (p) proceeds preferably at a reaction rate that is at least 2 times greater, preferably at least 10 times greater, more preferably at least 100 times greater, even more preferably at least 10 3 times greater, even more preferably at least 10 4 times greater, even more preferably at least 10 5 times greater than the reaction rate at which step (p)* proceeds.
  • step (p) is greater than the reaction rate of step (p)*.
  • step (p) is lesser than the reaction rate of step (p)*.
  • step (p) of the preferred embodiment of the invention is catalyzed by a hydantoinase (“dihydropyrimidinase”) E 2 .
  • hydantoinases accept compounds of formula L-(lll) as substrates and convert them to products according to formulae L-(ll), and hence catalyze the reaction according to step (p).
  • This finding is of high scientific and economic value, as it further broadens the scope of synthetic routes based on new starting materials for the production of L-glufosinate P-esters.
  • hydantoinases (“dihydropyrimidinases”) generally catalyze the reaction of 5,6-dihydrouracil to produce ureidopropionate (see the following reaction ⁇ 2A>):
  • R* may be an organic residue, e.g. a side chain of one of the naturally occurring amino acids.
  • the hydantoinase E 2 is a “L-hydantoinase”, i.e. it has a greater catalytic activity for reaction ⁇ 20 than for reaction ⁇ 20, wherein the substrate S’ D in the reaction ⁇ 20 is the enantiomer of the substrate S L in the reaction ⁇ 20:
  • L-hydantoinase As an L-hydantoinase has a higher catalytic activity for reaction ⁇ 20 than for reaction ⁇ 2D>, it is “L-enantioselective”. An L-hydantoinase that has no catalytic activity for reaction ⁇ 20 and thus only has catalytic activity for reaction ⁇ 20 is “L-enantiospecific”.
  • a “D-hydantoinase” is defined as a hydantoinase which is “D-enantioselective”, i.e. it has a higher catalytic activity for reaction ⁇ 20 than for reaction ⁇ 20.
  • a D-carbamoylase that does not catalyze reaction ⁇ 20 and thus only has catalytic activity for reaction ⁇ 20 is “D-enantiospecific”.
  • a hydantoinase which has the same catalytic activity for reaction ⁇ 20 as for reaction ⁇ 2D>, is referred to as “non enantioselective hydantoinase”.
  • hydantoinase For determination whether a hydantoinase may be denoted as “L-hydantoinase”, “D-hydantoinase” or “non-enantioselective hydantoinase” in the context of the present invention, the procedure according to Assay E (item 4.5.10) may preferably be used.
  • the hydantoinase E 2 in particular the L-hydantoinase E 2 , that may be used in step (p) of the preferred embodiment of the method according to the invention may originate from Arthrobacter sp., in particular Arthrobacter crystallopoietes, Arthrobacter aurescens, Arthrobacter sp. BT801 ; Alcaligenes sp., in particular Alcaligenes faecalis subsp.
  • Bacillus sp. in particular Bacillus fordii
  • Microbacterium sp. in particular Microbacterium liquefaciens strain AJ3912
  • Pseudomonas sp. in particular Pseudomonas fluorescens, Pseudomonas aeruginosa.
  • a hydantoinase E 2 in particular an L-hydantoinase E 2 suitable for the method according to the present invention may be the enzyme HyuH, which originates from Arthrobacter. Another enzyme may be Dht.
  • the hydantoinase E 2 in particular the L-hydantoinase E 2 , that may preferably be used in step (p) according to the first aspect of the invention may originate from Arthrobacter sp., in particular Arthrobacter crystallopoietes, Arthrobacter aurescens, Arthrobacter sp. BT801 , Arthrobacter aurescens DSM 9771 , most preferably from Arthrobacter aurescens DSM 9771.
  • a hydantoinase suitable for the method according to the present invention is described e.g. in WO 01/23582 A1 and by J.M. Clemente-Jimenez, S. Martinez-Rodriguez, F. Rod rig uez- Vico, F.J.L. Heras-Vazquez, Recent Pat. Biotechnology 2008, 2, 35 - 46; G. Latacz, E. Pekala, K. Kiec- Kononowicz, Biotechnologia 2006, 2, 189 - 205.
  • the hydantoinase E 2 that may be used in preferred step (p) of the method according to the present invention may be a hydantoinase categorized in the EC class EC 3.5.2.2.
  • the reaction according to step (p) is catalyzed by an hydantoinase E 2 , wherein the polypeptide sequence of E 2 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, preferably SEQ ID NO: 10 and variants thereof.
  • hydantoinases in particular L-hydantoinases, that may be used in step (p) of the preferred method according to the invention.
  • Assay D L may be used to determine hydantoinase and L-hydantoinase activity of a given enzyme E Y and may advantageously be used according to the invention to determine hydantoinase and L-hydantoinase activity 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, variants of SEQ ID NO: 16.
  • Assay D D may be used to determine D-hydantoinase activity of a given enzyme E Y .
  • the molar mass of the enzyme E Y to be tested is calculated as the molar mass of the polypeptide sequence of E Y . 4.5.9.1 Assay DL:
  • the hydantoinase E 2 according to the invention is preferably an L-hydantoinase, more preferably L-enantiospecific.
  • a given enzyme E Y may be considered a hydantoinase E 2 , in particular an L-hydantoinase, may be determined in the context of the present invention by the following Assay E:
  • step E-1 is repeated, except that instead of the addition of 400 nmol E Y in 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh), 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh) without E Y is added.
  • step E-2 is repeated, except that instead of the addition of 400 nmol E Y in 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh), 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh) without E Y is added.
  • E Y is deemed to have hydantoinase activity, and hence may be considered a hydantoinase E 2 in the context of the invention.
  • E Y is deemed to have L-hydantoinase activity, and hence may be considered an L-hydantoinase in the context of the invention. In this case, E Y is deemed to be “L-enantioselective” in the context of the invention.
  • E Y is deemed to have L-hydantoinase activity, and hence may be considered a L-hydantoinase in the context of the invention.
  • E Y is deemed to be not only “L-enantioselective”, but also “L-enantiospecific” in the context of the invention.
  • E Y is deemed to have D-hydantoinase activity, and hence may be considered a D-hydantoinase.
  • E Y is “D-enantioselective” in the context of the invention.
  • E Y is deemed to have D-hydantoinase activity, and hence may be considered a D-hydantoinase in the context of the invention.
  • E Y is deemed to be not only “D-enantioselective”, but also “D-enantiospecific” in the context of the invention.
  • R n-butyl, that is determined in step E-1 and then multiplying the obtained value by 100, giving the D-enantioselectivity of E Y in %.
  • E Y is deemed to be a non-enantioselective hydantoinase in the context of the invention. In this case, E Y is “non-enantioselective”.
  • An enzyme the polpypetide sequence of which 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 has hydantoinase, in particular L-hydantoinase activity.
  • the polypeptide sequence of the hydantoinase in particular the L-hydantoinase E 2 is selected from the group consisting 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.
  • an enzyme in the context of the invention, has hydantoinase activity, preferably L-hydantoinase activity, more preferably is L-enantiospecific.
  • a given enzyme E Y has hydantoinase activity, L-hydantoinase activity and is L-enantiospecific may be determined as set forth under items 4.5.10.2 and 4.5.10.3.1 , respectively.
  • the hydantoinase activity of a given hydantoinase E 2V may be determined in the context of the present invention by the following Assay F:
  • F-1 Assay D L as set forth under item 4.5.9.1 is conducted, wherein E 2S is the enzyme to be tested.
  • the obtained molar amount of the compound according to formula L-(ll), wherein R n-butyl, is determined according to Assay D L .
  • Step F-1 is repeated, except that, instead of E 2S , E 2V is used as the enzyme to be tested.
  • hydantoinases E 2 the polypeptide sequence of which 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, are generally denoted as “E 2S ”.
  • Hydantoinases E 2 the polypeptide sequence of which is selected from variants of a sequence 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, are generally denoted as “E 2V ”.
  • the reaction in step (p) is catalyzed by a hydantoinase E 2 , preferably an L-hydantoinase E 2
  • the polypeptide sequence of the hydantoinase E 2 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.
  • the reaction in step (p) is catalyzed by a hydantoinase E 2 , preferably an L-hydantoinase E 2 , and the polypeptide sequence of the hydantoinase E 2 , preferably the L-hydantoinase E 2 , is selected from the group consisting of SEQ ID NO: 10 and variants of SEQ ID NO: 10.
  • the polypeptide sequence of the hydantoinase E 2 may also be a variant of SEQ ID NO: 10.
  • the hydantoinase E 2 is denoted as “E 2 IOS”.
  • the hydantoinase E 2 the polypeptide sequence of which is selected from variants of SEQ ID NO: 10, are generally denoted as “E 2 IOV”.
  • a variant of the polypeptide sequence of SEQ ID NO: 10 is a polypeptide with sequence identity of at least 60 %, preferably > 65 %, more preferably > 70 %, more preferably > 75 %, more preferably
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 10 is not identical to SEQ ID NO: 10.
  • a hydantoinase E 2 IOV has hydantoinase activity and preferably L-hydantoinase activity, determined as described under items 4.5.10.2 and 4.5.10.3.1.
  • a hydantoinase E 2 IOV preferably has hydantoinase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoinase activity of the hydantoinase E 2 IOS, wherein the hydantoinase activity of E 2 IOV, relative to the hydantoinase activity of E 2 IOS is determined by Assay F described under item 4.5.11 .2.
  • a hydantoinase E 2 IOV has hydantoinase activity 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 % the hydantoinase activity of the hydantoinase E 2 IOS, wherein the hydantoinase activity of E 2 IOV, relative to the hydantoinase activity E 2 IOS is determined by Assay F described under item 4.5.11 .2.
  • the polypeptide sequence of the hydantoinase E 2 may also be a variant of SEQ ID NO: 11 .
  • the hydantoinase E 2 is denoted as “E 2 n S ”.
  • the hydantoinase E 2 the polypeptide sequence of which is selected from variants of SEQ ID NO: 1 1 , are generally denoted as “E 2 n V ”.
  • a variant of the polypeptide sequence of SEQ ID NO: 11 is a polypeptide with sequence identity of at least 60 %, preferably > 65 %, more preferably > 70 %, more preferably > 75 %, more preferably
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 11 is not identical to SEQ ID NO: 11.
  • a hydantoinase E 2 n V has hydantoinase activity and preferably L-hydantoinase activity, determined as described under items 4.5.10.2 and 4.5.10.3.1.
  • a hydantoinase E 2 n V preferably has hydantoinase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoinase activity of the hydantoinase E 2 ns, wherein the hydantoinase activity of E 2 nv, relative to the hydantoinase activity of E 2 HS is determined by Assay F described under item 4.5.11 .2.
  • a hydantoinase E 2 n V has hydantoinase activity 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 % the hydantoinase activity of the hydantoinase E 2 n S , wherein the hydantoinase activity of E 2 n V , relative to the hydantoinase activity E 2 n S is determined by Assay F described under item 4.5.11 .2.
  • the polypeptide sequence of the hydantoinase E 2 may also be a variant of SEQ ID NO: 12.
  • the hydantoinase E 2 is denoted as “E 2 I 2S ”.
  • the hydantoinase E 2 is generally denoted as “E 2 I 2V ”.
  • a variant of the polypeptide sequence of SEQ ID NO: 12 is a polypeptide with sequence identity of at least 60 %, preferably > 65 %, more preferably > 70 %, more preferably > 75 %, more preferably
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 12 is not identical to SEQ ID NO: 12.
  • a hydantoinase E 2 I 2V has hydantoinase activity and preferably L-hydantoinase activity, determined as described under items 4.5.10.2 and 4.5.10.3.1.
  • a hydantoinase E 2 I 2V preferably has hydantoinase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoinase activity of the hydantoinase E212S, wherein the hydantoinase activity of E212V, relative to the hydantoinase activity of E212S is determined by Assay F described under item 4.5.11 .2.
  • a hydantoinase E212V has hydantoinase activity 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 % the hydantoinase activity of the hydantoinase E212S, wherein the hydantoinase activity of E212V, relative to the hydantoinase activity E212S is determined by Assay F described under item 4.5.11 .2.
  • the polypeptide sequence of the hydantoinase E 2 may also be a variant of SEQ ID NO: 13.
  • E 2 The hydantoinase E 2 , the polypeptide sequence of which is SEQ ID NO: 13, is denoted as “E 2 i3s”.
  • the hydantoinase E 2 the polypeptide sequence of which is selected from variants of SEQ ID NO: 13, are generally denoted as “E 2 i3v”.
  • a variant of the polypeptide sequence of SEQ ID NO: 13 is a polypeptide with sequence identity of at least 60 %, preferably > 65 %, more preferably > 70 %, more preferably > 75 %, more preferably
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 13 is not identical to SEQ ID NO: 13.
  • a hydantoinase E213V has hydantoinase activity and preferably L-hydantoinase activity, determined as described under items 4.5.10.2 and 4.5.10.3.1.
  • a hydantoinase E213V preferably has hydantoinase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoinase activity of the hydantoinase E213S, wherein the hydantoinase activity of E 2 isv, relative to the hydantoinase activity of E 2 i3s is determined by Assay F described under item 4.5.11 .2.
  • a hydantoinase E 2 I 3 V has hydantoinase activity 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 % the hydantoinase activity of the hydantoinase E 2 I 3 S, wherein the hydantoinase activity of E 2 I 3 V, relative to the hydantoinase activity E 2 I 3 S is determined by Assay F described under item 4.5.11 .2.
  • the polypeptide sequence of the hydantoinase E 2 may also be a variant of SEQ ID NO: 14.
  • the hydantoinase E 2 is denoted as “E 2 I 4S ”.
  • the hydantoinase E 2 is generally denoted as “E 2 I 4V ”.
  • a variant of the polypeptide sequence of SEQ ID NO: 14 is a polypeptide with sequence identity of at least 60 %, preferably > 65 %, more preferably > 70 %, more preferably > 75 %, more preferably
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 14 is not identical to SEQ ID NO: 14.
  • a hydantoinase E 2 I 4V has hydantoinase activity and preferably L-hydantoinase activity, determined as described under items 4.5.10.2 and 4.5.10.3.1.
  • a hydantoinase E 2 I 4V preferably has hydantoinase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoinase activity of the hydantoinase E 2 I 4S , wherein the hydantoinase activity of E 2 I 4V , relative to the hydantoinase activity of E 2 I 4S is determined by Assay F described under item 4.5.11 .2.
  • a hydantoinase E214V has hydantoinase activity 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 % the hydantoinase activity of the hydantoinase E 2 i4s, wherein the hydantoinase activity of E 2 I 4 V, relative to the hydantoinase activity E 2 I 4 S is determined by Assay F described under item 4.5.11 .2.
  • the polypeptide sequence of the hydantoinase E 2 may also be a variant of SEQ ID NO: 15.
  • the hydantoinase E 2 is denoted as “E 2 I 5 S”.
  • the hydantoinase E 2 is selected from variants of SEQ ID NO: 15, are generally denoted as “E 2 I 5 V”.
  • a variant of the polypeptide sequence of SEQ ID NO: 15 is a polypeptide with sequence identity of at least 60 %, preferably > 65 %, more preferably > 70 %, more preferably > 75 %, more preferably
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 15 is not identical to SEQ ID NO: 15.
  • a hydantoinase E 2 I 5 V has hydantoinase activity and preferably L-hydantoinase activity, determined as described under items 4.5.10.2 and 4.5.10.3.1.
  • a hydantoinase E 2 I 5 V preferably has hydantoinase activity of at least
  • a hydantoinase E 2 I 5 V has hydantoinase activity 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 % the hydantoinase activity of the hydantoinase E 2 I 5 S, wherein the hydantoinase activity of E 2 I 5 V, relative to the hydantoinase activity E 2 I 5 S is determined by Assay F described under item 4.5.11 .2.
  • the polypeptide sequence of the hydantoinase E 2 may also be a variant of SEQ ID NO: 16.
  • E 2 I 6 S The hydantoinase E 2 , the polypeptide sequence of which is SEQ ID NO: 16, is denoted as “E 2 I 6 S”.
  • the hydantoinase E 2 the polypeptide sequence of which is selected from variants of SEQ ID NO: 16, are generally denoted as “E 2 I 6 V”.
  • a variant of the polypeptide sequence of SEQ ID NO: 16 is a polypeptide with sequence identity of at least 60 %, preferably > 65 %, more preferably > 70 %, more preferably > 75 %, more preferably
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 16 is not identical to SEQ ID NO: 16.
  • a hydantoinase E 2 I 6 V has hydantoinase activity and preferably L-hydantoinase activity, determined as described under items 4.5.10.2 and 4.5.10.3.1.
  • a hydantoinase E 2 I 6 V preferably has hydantoinase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoinase activity of the hydantoinase E 2 I 6 S, wherein the hydantoinase activity of E 2 I 6 V, relative to the hydantoinase activity of E 2 i6s is determined by Assay F described under item 4.5.11 .2.
  • a hydantoinase E 2 iev has hydantoinase activity 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 % the hydantoinase activity of the hydantoinase E 2 i6s, wherein the hydantoinase activity of E 2 I 6 V, relative to the hydantoinase activity E 2 I 6 S is determined by Assay F described under item 4.5.11 .2.
  • step (p) of the method according to the present invention may be carried out under conditions known to the skilled person.
  • the reaction medium is preferably aqueous, more preferably an aqueous buffer.
  • Exemplary buffers commonly used in biotransformation reactions and advantageously used herein include Tris, phosphate, or any of Good's buffers, such as 2-(/V-morpholino)ethanesulfonic acid (“MES”), /V-(2-acetamido)iminodiacetic acid (“ADA”), piperazine-/V,/ ⁇ /'-bis(2-ethanesulfonic acid) (“PIPES”), /V-(2-acetamido)-2- aminoethanesulfonic acid (“ACES”), P-hydroxy-4- morpholinepropanesulfonic acid (“MOPSO”), cholamine chloride, 3-(/V-morpholino)propanesulfonic acid (“MOPS”), /V,/V-Bis(2-hydroxyethyl)- 2-aminoethanesulfonic acid (“BES”), 2-[[1 ,3-dihydroxy-MES”), /V-(2-acetamido)iminodiacetic acid
  • HEPES 1-piperazineethanesulfonic acid
  • DIPSO 3-(Bis(2-hydroxyethyl)amino)-2-hydroxypropane- 1 -sulfonic acid
  • acetamidoglycine
  • TAPSO 3-(/V-Tris(hydroxymethyl)methylamino(-2-hydroxypropane)sulfonic acid
  • POPSO piperazine- /V,/ ⁇ /'-bis(2-hydroxypropanesulfonic acid)
  • POPSO 4-(2- Hydroxyethyl)piperazine- 1-(2-hydroxypropanesulfonic acid)
  • HEPPSO 3-[4-(2-Hydroxyethyl)-1-piperazinyl]propanesulfonic acid
  • HPPS 3-[4-(2-Hydroxyethyl)-1-piperazinyl]propanesulfonic acid
  • TAPS 3-[[1 ,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1 -sulfonic acid
  • ammonium can act as a buffer.
  • One or more organic solvents can also be added to the reaction.
  • the buffer preferably contains metal salts, more preferably metal salts such as halogenides of metals, preferably halogenides of monovalent or bivalent or trivalent metals, preferably chlorides of monovalent or bivalent metals, preferably C0CI2 or MnCI 2 , preferably C0CI2.
  • metal salts more preferably metal salts such as halogenides of metals, preferably halogenides of monovalent or bivalent or trivalent metals, preferably chlorides of monovalent or bivalent metals, preferably C0CI2 or MnCI 2 , preferably C0CI2.
  • the concentration of these metal salts in the reaction medium is preferably in the range from 1 pM to 1 M, more preferably 1 mM to 100 mM, even more preferably 1 to 10 mM.
  • step (p) of the method according to the invention is carried out in a phosphate buffer.
  • the pH of the reaction medium in step (p) of the method according to the invention is preferably in the range of from 2 to 10, more preferably in the range of from 5 to 8, more preferably 7.2 to 7.5, most preferably 7.5.
  • step (p) of the method according to the invention is carried out at a temperature in the range of from 20 °C to 70 °C, more preferably in the range of from 30 °C to 55 °C, most preferably 50 °C.
  • the total concentration of all hydantoinases E 2 in the reaction solution in step (p) is in the range of from 1 pM to 10 mM, preferably 10 pM to 1 mM, more preferably 0.1 mM to 0.5 mM, most preferably 0.4 mM.
  • the total concentration of all hydantoinases E 2 in the reaction solution in step (p) is in the range of from 1 pg/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.
  • the initial concentration of all the compounds according to formula L-(lll) in the reaction medium in step (p) is in the range of from 1 pM to 1 M, preferably of from 10 pM to 0.5 M, more preferably of from 0.1 mM to 0.1 M, more preferably of from 1 mM to 10 mM, most preferably 1.25 mM.
  • the initial concentration of all the compounds according to formula D-(lll) in the reaction medium is preferably from 1 % to 100 % the concentration of all the compounds according to formula L-(lll), more preferably 10 % to 100 % the concentration, even more preferably 50 to 100 %, even more preferably 100 % the concentration of all the compounds according to formula L-(lll).
  • step (p) is carried out in the same reaction medium in which step (6) is carried out.
  • the initial concentration of all the compounds according to formula L-(lll) in the reaction medium in step (p) is in the range of from 1 pM to 1 M, preferably of from 10 pM to 0.5 M, more preferably of from 0.1 mM to 0.1 M, more preferably of from 1 mM to 10 mM, most preferably 1 .25 mM.
  • “Initial concentration of all the compounds according to formula L-(lll)/ D-(lll)” refers to the concentration of the respective compound L-(lll) or D-(lll), respectively, in the reaction medium when the respective compounds are employed in step (p).
  • the compound according to formula LL-(III) and/or, preferably and, the compound according to formula DL-(III) is obtained by a step (a) in which a compound according to formula LD-(III) is reacted to give a compound according to formula LL-(III) and/or, preferably and, a compound according to formula DD-(III) is reacted to give a compound according to formula DL-(III), wherein LD-(III) and DD-(III) have the following formulae:
  • Step (a) gives the starting material for step (p), and R in LD-(III) and DD-(III) has the same meaning as described for DL-(III) and LL-(III) [or DL-(II) and LL-(II) ].
  • the reaction according to step (a) may be carried out enzymatically or non-enzymatically, preferably enzymatically. More preferably, the reaction according to step (a) is catalyzed by a hydantoin racemase E 3 .
  • step (a) the compound according to formula D-(lll) is employed in step (a) as a mixture Mm** of LD-(III) and DD-(III) with the respective enantiomer DL-(III) and LL-(III) .
  • the mixture Mm** is a racemic mixture of enantiomer L-(lll) and enantiomer D-(lll), meaning that the molar ratio of all compounds according to L-(lll) to compounds according to D-(lll) is essentially 1 : 1.
  • the molar ratio of all compounds L-(lll) to all compounds D-(lll) in mixture Mm** is in the range of from 99 : 1 to 1 : 99, more preferably in the range of from 1.01 : 1 to 1 : 99, more preferably in the range of from 1 : 1 to 1 : 99, more preferably in the range of from 1 : 1.01 to 1 : 99, more preferably in the range of from 1 : 1.01 to 1 : 9, more preferably in the range of from 1 : 1.01 to 1 : 8, more preferably in the range of from 1 : 1.01 to 1 : 3.
  • Step (a) may be carried out non-enzymatically, i.e. without the use of an enzyme.
  • the reaction of compounds according to step D-(lll) to compounds according to L-(lll) proceeds in alkaline solution, as known to the skilled person and as described by Slomka et al., M. Bovarnick & H.T. Clarke, Journal of the American Chemical Society 1938, 60, 2426 - 2430, by R. A. Lazarus, J. Org. Chem. 1990, 55, 4755 - 4757, and by A. S. 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 acitivity” on page 164 and 167 in “Microbial Reagents in Organic Synthesis” Series C: Mathematical and Physical Sciences - Vol. 381 , S. Servi (Ed.), 1992, Springer Science+Business Media, B.V., Dordrecht.
  • step (a) is carried out non-enzymatically
  • the conditions that are preferably applied in the reaction medium in which non-enzymatic step (a) is carried out are preferably those that are described for the preferred conditions fpr step (p) (item 4.5.13), except that the pH is > 8, preferably in the range of 8 to 12, more preferably 8 to 11 , more preferably 8 to 10, even more preferably 8 to 9.
  • the preferred conditions in step (a) and (p) with respect to the pH ranges are different, it is preferable that the reaction media in steps (a) and (p) are different.
  • Step (a) is preferably carried out enzymatically, i.e. the reaction according to step (a) is preferably catalyzed by a hydantoin racemase E 3 .
  • hydantoin racemases catalyze the conversion of one of the two hydantoin enantiomers H L and HR into the other (see the following reaction ⁇ 3>): It was now surprisingly found that hydantoin racemases also accept substrates in which
  • Suitable hydantoin racemases are described e.g. in WO 01/23582 A1 and by U. Engel, J. Rudat, C. Syldatk in “The hydantoinase process: recent developments for the production of non-canonical amino acids” in the book “Industrial biocatalysis” by P. Grunwald (Ed.), Pan Stanford Series on Biocatalysis, 2015, pages 817 - 862, and by F. J. Las Heras-Vazquez, J. M. Clemente-Jimenez, S. Martinez-Rodriguez, F.
  • a hydantoin racemase E 3 that may be used in optional step (a) of the method according to the invention may originate from Agrobacterium sp., in particular Agrobacterium strain IPJ-671 ; Arthrobacter sp., in particular Arthrobacter aurescens, more in particular Arthrobacter aurescens DSM 3745 or Arthtrobacter sp. BT801 ; Flavobacterium sp., in particular Flavobacterium sp. AJ 11199; Microbacterium sp., in particular Microbacterium liquefaciens, preferably Microbacterium liquefaciens AJ 3912; Pasteurella sp., in particular Pasteurella sp.
  • Pseudomonas sp. in particular Pseudomonas sp. NS671
  • Pyrococcus sp. in particular Pyrococcus horikoshii OT3
  • Rhodococcus sp. in particular Rhodococcus R04
  • Sinorhizobium sp. in particular Sinorhizobium meliloti, more in particular Sinorhizobium meliloti CECT 4114, , most preferably from Arthrobacter aurescens DSM 3745.
  • a hydantoin racemase E 3 suitable for the method according to the present invention may be the enzyme HyuR, which originates from Arthrobacter aurescens DSM 3745. Another enzyme may be selected from HyuE, Hyu2, HRase, HyuA, PH1054.
  • the hydantoin racemase E 3 that may preferably be used in preferred step (a) of the method according to the invention may be categorized in the EC class 5.1 .99.5.
  • the following table 3 gives preferred examples for polypeptide sequences of hydantoin racemase E 3 that may be preferably used in step (a) of the method according to the invention.
  • the genes encoding the respective hydantoin racemase E 3 and the respective accession code are indicated as far as known.
  • the reaction according to step (a) is catalyzed by a hydantoin racemase E 3 , wherein the polypeptide sequence of E 3 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.
  • E 3 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
  • Assay G may be used to determine hydantoin racemase activity of a given enzyme E z and may advantageously be used according to the invention to determine carbamoylase and L-carbamoylase activity in variants of SEQ ID NO: 17, variants of 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 variants of SEQ ID NO: 23, variants of SEQ ID NO: 24, variants of SEQ ID NO: 25, variants of SEQ ID NO: 26.
  • the molar mass of the enzyme E z to be tested is calculated as the molar mass of the polypeptide sequence of E z .
  • the resulting solution is incubated at 25 °C, and the pH is held at pH 7.2 by addition of 0.5 M. After 300 minutes, the reaction is stopped by addition of 2 M HCI to achieve a pH of 2.5.
  • Whether a given enzyme E z may be considered a hydantoin racemase E 3 , may be determined in the context of the present invention by the following Assay H:
  • step H-1 is repeated, except that instead of the addition of 400 nmol E z in 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh), 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh) without E z is added.
  • E z is deemed to have hydantoin racemase activity, and hence may be considered a hydantoin racemase E 3 in the context of the invention.
  • An enzyme the polpypetide sequence of which is selected from the group consisting 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.
  • the polypeptide sequence of the hydantoin racemase E 3 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.
  • an enzyme the polypeptide sequence of which 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:
  • SEQ ID NO: 22 has hydantoin racemase activity.
  • the hydantoin racemase activity of a given hydantoin racemase E 3V may be quantified in the context of the present invention by the following Assay J.
  • hydantoin racemase E 3 the polypeptide sequence of which is selected from the group consisting 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, are generally denoted as “E 3S ”.
  • Hydantoin racemases E 3 the polypeptide sequence of which is selected from variants of a sequence selected from the group consisting of f 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, are generally denoted as “E 3V ”.
  • the reaction in step (a) is catalyzed by a hydantoin racemase E 3 , and the polypeptide sequence of the hydantoin racemase E 3 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.
  • step (a) is catalyzed by a hydantoin racemase E 3 , and the polypeptide sequence of the hydantoin racemase E 3 is selected from the group consisting of SEQ ID NO: 1 and variants of SEQ ID NO: 1 .
  • polypeptide sequence of the hydantoin racemase E 3 may also be a variant of SEQ ID NO: 17.
  • the hydantoin racemase E 3 the polypeptide sequence of which is SEQ ID NO: 17, is denoted as “E 3 i7s”.
  • Hydantoin racemases E 3 the polypeptide sequence of which is is selected from variants of SEQ ID NO: 17, are generally denoted as “E 3 I 7 V”.
  • a variant of the polypeptide sequence of SEQ ID NO: 17 is a polypeptide with sequence identity of 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
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 17 is not identical to SEQ ID NO: 17.
  • a hydantoin racemase E 3 i?v has hydantoin racemase activity, determined as described under item 4.5.14.4 and 4.5.14.5.
  • a hydantoin racemase E 3 i?v preferably has hydantoin racemase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoin racemase of the hydantoin racemase E 3i7 s, wherein the hydantoin racemase activity of E 3i7 v, relative to the hydantoin racemase activity E 3i7 s is determined by Assay J described under item 4.5.14.6.
  • a hydantoin racemase E 3i7 v has hydantoin racemase activity 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 % the hydantoin racemase activity of the hydantoin racemase E 3i7 s, wherein the hydantoin racemase activity of E 3i7 v, relative to the hydantoin racemase activity of E 3i7 s is determined by Assay J described under item 4.5.14.6.
  • polypeptide sequence of the hydantoin racemase E 3 may also be a variant of SEQ ID NO: 18.
  • the hydantoin racemase E 3 the polypeptide sequence of which is SEQ ID NO: 18, is denoted as “E 3 i8s”.
  • Hydantoin racemases E 3 the polypeptide sequence of which is is selected from variants of SEQ ID NO: 18, are generally denoted as “E 3i8 v”.
  • a variant of the polypeptide sequence of SEQ ID NO: 18 is a polypeptide with sequence identity of 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
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 18 is not identical to SEQ ID NO: 18.
  • a hydantoin racemase E 3i8V has hydantoin racemase activity, determined as described under item 4.5.14.4 and 4.5.14.5.
  • a hydantoin racemase E 3i8V preferably has hydantoin racemase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoin racemase of the hydantoin racemase E 3i8 s, wherein the hydantoin racemase activity of E 3i8 v, relative to the hydantoin racemase activity E 3i8S is determined by Assay J described under item 4.5.14.6.
  • a hydantoin racemase E 3i8V has hydantoin racemase activity 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 % the hydantoin racemase activity of the hydantoin racemase E 3i8S , wherein the hydantoin racemase activity of E 3i8V , relative to the hydantoin racemase activity of E 3i8S is determined by Assay J described under item 4.5.14.6.
  • polypeptide sequence of the hydantoin racemase E 3 may also be a variant of SEQ ID NO: 19.
  • the hydantoin racemase E 3 is denoted as “Ernes”.
  • Hydantoin racemases E 3 the polypeptide sequence of which is is selected from variants of SEQ ID NO: 19, are generally denoted as “E 3i9V ”.
  • a variant of the polypeptide sequence of SEQ ID NO: 19 is a polypeptide with sequence identity of 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
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 19 is not identical to SEQ ID NO: 19.
  • a hydantoin racemase E319V has hydantoin racemase activity, determined as described under item 4.5.14.4 and 4.5.14.5.
  • a hydantoin racemase E319V preferably has hydantoin racemase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoin racemase of the hydantoin racemase E 3i9S , wherein the hydantoin racemase activity of E 3i9V , relative to the hydantoin racemase activity E 3i9S is determined by Assay J described under item 4.5.14.6.
  • a hydantoin racemase E 3i9V has hydantoin racemase activity 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 % the hydantoin racemase activity of the hydantoin racemase E 3i9S , wherein the hydantoin racemase activity of E 3i9V , relative to the hydantoin racemase activity of E 3i9S is determined by Assay J described under item 4.5.14.6.
  • polypeptide sequence of the hydantoin racemase E 3 may also be a variant of SEQ ID NO: 20.
  • the hydantoin racemase E 3 the polypeptide sequence of which is SEQ ID NO: 20, is denoted as “E 32 OS”.
  • Hydantoin racemases E 3 the polypeptide sequence of which is is selected from variants of SEQ ID NO: 20, are generally denoted as “E 32 ov”.
  • a variant of the polypeptide sequence of SEQ ID NO: 20 is a polypeptide with sequence identity of 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
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 20 is not identical to SEQ ID NO: 20.
  • a hydantoin racemase E320V has hydantoin racemase activity, determined as described under item 4.5.14.4 and 4.5.14.5.
  • a hydantoin racemase E320V preferably has hydantoin racemase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoin racemase of the hydantoin racemase E 32 os, wherein the hydantoin racemase activity of E 32 ov, relative to the hydantoin racemase activity E 32 os is determined by Assay J described under item 4.5.14.6.
  • a hydantoin racemase E 32 ov has hydantoin racemase activity 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 % the hydantoin racemase activity of the hydantoin racemase E 32 os, wherein the hydantoin racemase activity of E 32 ov, relative to the hydantoin racemase activity of E 32 os is determined by Assay J described under item 4.5.14.6.
  • polypeptide sequence of the hydantoin racemase E 3 may also be a variant of SEQ ID NO: 21 .
  • the hydantoin racemase E 3 the polypeptide sequence of which is SEQ ID NO: 21 , is denoted as “E 32 IS”.
  • Hydantoin racemases E 3 the polypeptide sequence of which is is selected from variants of SEQ ID NO: 21 , are generally denoted as “E 32 iv”.
  • a variant of the polypeptide sequence of SEQ ID NO: 21 is a polypeptide with sequence identity of 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
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 21 is not identical to SEQ ID NO: 21.
  • a hydantoin racemase E321V has hydantoin racemase activity, determined as described under item 4.5.14.4 and 4.5.14.5.
  • a hydantoin racemase E321V preferably has hydantoin racemase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoin racemase of the hydantoin racemase E 32 is, wherein the hydantoin racemase activity of E 32 iv, relative to the hydantoin racemase activity E 32 is is determined by Assay J described under item 4.5.14.6.
  • a hydantoin racemase E 32 iv has hydantoin racemase activity 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 % the hydantoin racemase activity of the hydantoin racemase E 32 is, wherein the hydantoin racemase activity of E 32 iv, relative to the hydantoin racemase activity of E 32 is is determined by Assay J described under item 4.5.14.6.
  • polypeptide sequence of the hydantoin racemase E 3 may also be a variant of SEQ ID NO: 22.
  • the hydantoin racemase E 3 is denoted as “E 32 2S”.
  • Hydantoin racemases E 3 the polypeptide sequence of which is is selected from variants of SEQ ID NO: 22, are generally denoted as “E 32 2v”.
  • a variant of the polypeptide sequence of SEQ ID NO: 22 is a polypeptide with sequence identity of 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
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 22 is not identical to SEQ ID NO: 22.
  • a hydantoin racemase E322V has hydantoin racemase activity, determined as described under item 4.5.14.4 and 4.5.14.5.
  • a hydantoin racemase E322V preferably has hydantoin racemase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoin racemase of the hydantoin racemase E322S, wherein the hydantoin racemase activity of E322V, relative to the hydantoin racemase activity E322S is determined by Assay J described under item 4.5.14.6.
  • a hydantoin racemase E322V has hydantoin racemase activity 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 % the hydantoin racemase activity of the hydantoin racemase E322S, wherein the hydantoin racemase activity of E322V, relative to the hydantoin racemase activity of E322S is determined by Assay J described under item 4.5.14.6.
  • polypeptide sequence of the hydantoin racemase E 3 may also be a variant of SEQ ID NO: 23.
  • the hydantoin racemase E 3 is denoted as “E 3 23S”.
  • Hydantoin racemases E 3 the polypeptide sequence of which is is selected from variants of SEQ ID NO: 23, are generally denoted as “E 3 23v”.
  • a variant of the polypeptide sequence of SEQ ID NO: 23 is a polypeptide with sequence identity of 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
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 23 is not identical to SEQ ID NO: 23.
  • a hydantoin racemase E 3 23v has hydantoin racemase activity, determined as described under item 4.5.14.4 and 4.5.14.5.
  • a hydantoin racemase E 3 23v preferably has hydantoin racemase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoin racemase of the hydantoin racemase E 3 23s, wherein the hydantoin racemase activity of E 3 23v, relative to the hydantoin racemase activity E 3 23s is determined by Assay J described under item 4.5.14.6.
  • a hydantoin racemase E 3 23v has hydantoin racemase activity 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 % the hydantoin racemase activity of the hydantoin racemase E 3 23s, wherein the hydantoin racemase activity of E 3 23v, relative to the hydantoin racemase activity of E 3 23s is determined by Assay J described under item 4.5.14.6.
  • polypeptide sequence of the hydantoin racemase E 3 may also be a variant of SEQ ID NO: 24.
  • the hydantoin racemase E 3 the polypeptide sequence of which is SEQ ID NO: 24, is denoted as “E 3 24S”.
  • Hydantoin racemases E 3 the polypeptide sequence of which is is selected from variants of SEQ ID NO: 24, are generally denoted as “E 3 24v”.
  • a variant of the polypeptide sequence of SEQ ID NO: 24 is a polypeptide with sequence identity of 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
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 24 is not identical to SEQ ID NO: 24.
  • a hydantoin racemase E 3 24v has hydantoin racemase activity, determined as described under item 4.5.14.4 and 4.5.14.5.
  • a hydantoin racemase E 3 24v preferably has hydantoin racemase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoin racemase of the hydantoin racemase E 32 4s, wherein the hydantoin racemase activity of E 32 4v, relative to the hydantoin racemase activity E 32 4s is determined by Assay J described under item 4.5.14.6.
  • a hydantoin racemase E 32 4v has hydantoin racemase activity 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 % the hydantoin racemase activity of the hydantoin racemase E 32 4s, wherein the hydantoin racemase activity of E 32 4v, relative to the hydantoin racemase activity of E 32 4s is determined by Assay J described under item 4.5.14.6.
  • polypeptide sequence of the hydantoin racemase E 3 may also be a variant of SEQ ID NO: 25.
  • the hydantoin racemase E 3 is denoted as “E 32 5S”.
  • Hydantoin racemases E 3 the polypeptide sequence of which is is selected from variants of SEQ ID NO: 25, are generally denoted as “E 32 5v”.
  • a variant of the polypeptide sequence of SEQ ID NO: 25 is a polypeptide with sequence identity of 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
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 25 is not identical to SEQ ID NO: 25.
  • a hydantoin racemase E 3 25v has hydantoin racemase activity, determined as described under item 4.5.14.4 and 4.5.14.5.
  • a hydantoin racemase E 3 25v preferably has hydantoin racemase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoin racemase of the hydantoin racemase E 32 5s, wherein the hydantoin racemase activity of E 32 5v, relative to the hydantoin racemase activity E 32 5s is determined by Assay J described under item 4.5.14.6.
  • a hydantoin racemase E 32 5v has hydantoin racemase activity 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 % the hydantoin racemase activity of the hydantoin racemase E 32 5s, wherein the hydantoin racemase activity of E 32 5v, relative to the hydantoin racemase activity of E 32 5s is determined by Assay J described under item 4.5.14.6.
  • polypeptide sequence of the hydantoin racemase E 3 may also be a variant of SEQ ID NO: 26.
  • the hydantoin racemase E 3 the polypeptide sequence of which is SEQ ID NO: 26, is denoted as “E 32 6S”.
  • Hydantoin racemases E 3 the polypeptide sequence of which is is selected from variants of SEQ ID NO: 26, are generally denoted as “E 32 6v”.
  • a variant of the polypeptide sequence of SEQ ID NO: 26 is a polypeptide with sequence identity of 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
  • polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 26 is not identical to SEQ ID NO: 26.
  • a hydantoin racemase E 3 2ev has hydantoin racemase activity, determined as described under item 4.5.14.4 and 4.5.14.5.
  • a hydantoin racemase E 3 2ev preferably has hydantoin racemase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoin racemase of the hydantoin racemase E 32 6s, wherein the hydantoin racemase activity of E 32 ev, relative to the hydantoin racemase activity E 32 6s is determined by Assay J described under item 4.5.14.6.
  • a hydantoin racemase E 32 ev has hydantoin racemase activity 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 % the hydantoin racemase activity of the hydantoin racemase E 32 6s, wherein the hydantoin racemase activity of E 32 ev, relative to the hydantoin racemase activity of E 32 6s is determined by Assay J described under item 4.5.14.6.
  • step (a) in which step (a) is catalyzed by a hydantoin racemase E 3 , the reaction in step (a) may be carried out under conditions known to the skilled person.
  • the reaction medium is preferably aqueous, more preferably an aqueous buffer.
  • Exemplary buffers commonly used in biotransformation reactions and advantageously used herein include Tris, phosphate, or any of Good's buffers, such as 2-(/V-morpholino)ethanesulfonic acid (“MES”), /V-(2-acetamido)iminodiacetic acid (“ADA”), piperazine-/V,/ ⁇ /'-bis(2-ethanesulfonic acid) (“PIPES”), /V-(2-acetamido)-2- aminoethanesulfonic acid (“ACES”), P-hydroxy-4- morpholinepropanesulfonic acid (“MOPSO”), cholamine chloride, 3-(/V-morpholino)propanesulfonic acid (“MOPS”), /V,/V-Bis(2-hydroxyethyl)- 2-aminoethanesulfonic acid (“BES”), 2-[[1 ,3-dihydroxy-MES”), /V-(2-acetamido)iminodiacetic acid
  • TES 2-(hydroxymethyl)propan-2- yl]amino]ethanesulfonic acid
  • HPES 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid
  • DIPSO 3-(Bis(2-hydroxyethyl)amino)-2-hydroxypropane- 1-sulfonic acid
  • acetamidoglycine 2-(hydroxymethyl)propan-2- yl]amino]ethanesulfonic acid
  • HEPES 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid
  • DIPSO 3-(Bis(2-hydroxyethyl)amino)-2-hydroxypropane- 1-sulfonic acid
  • TAPSO 3-(/V-Tris(hydroxymethyl)methylamino(-2-hydroxypropane)sulfonic acid
  • POPSO piperazine- /V,/ ⁇ /'-bis(2-hydroxypropanesulfonic acid)
  • HEPPSO 4-(2- Hydroxyethyl)piperazine- 1-(2-hydroxypropanesulfonic acid)
  • HPPS 3-[4-(2-Hydroxyethyl)-1-piperazinyl]propanesulfonic acid
  • TAPS 3-[4-(2-Hydroxyethyl)-1-piperazinyl]propanesulfonic acid
  • HPPS 4-(2-Hydroxyethyl)-1-piperazinyl]propanesulfonic acid
  • tricine tricine
  • glycinamide glycinamide
  • bicine 3-[[1 ,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1 -sulfonic acid
  • ammonium can act as a buffer.
  • One or more organic solvents can also be added to the reaction.
  • the buffer preferably contains metal salts, more preferably metal salts such as halogenides of monovalent or bivalent metals (e.g. C0CI2, MnCh).
  • the concentration of these metal salts in the reaction medium is preferably in the range from 1 pM to 1 M, more preferably 1 mM to 100 mM, even more preferably 1 to 10 mM.
  • step (a) of the method according to the invention is carried out in a phosphate buffer.
  • the pH of the reaction medium in step (a) of the method according to the invention is preferably in the range of from 2 to 10, more preferably in the range of from 5 to 8, more preferably 7.2 to 7.5, most preferably 7.5.
  • step (a) of the method according to the invention is carried out at a temperature in the range of from 20 °C to 70 °C, more preferably in the range of from 30 °C to 55 °C, most preferably 50 °C.
  • step (a) in which step (a) is catalyzed by a hydantoin racemase E 3 , the preferred reaction conditions in step (a) are the same as described for steps (p) and (6), confer items 4.5.7 and 4.5.13, respectively. It is even more preferred to carry out step (a) concomitantly with steps (p) and (6).
  • the total concentration of all hydantoin racemases E 3 in the reaction solution in step (a) is in the range of from 1 pM to 10 mM, preferably 10 pM to 1 mM, more preferably 0.1 mM to 0.5 mM, most preferably 0.4 mM.
  • the total concentration of all hydantoin racemases E 3 in the reaction solution in step (a) is in the range of from 1 pg/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.
  • step (a) is carried out in the same reaction medium in which steps (6) and (p) are carried out.
  • steps (6) and (p) are carried out.
  • the initial concentration of all the compounds according to formula D-(lll) in the reaction medium in step (a) is in the range of from 1 pM to 1 M, preferably of from 10 pM to 0.5 M, more preferably of from 0.1 mM to 0.1 M, more preferably of from 1 mM to 10 mM, most preferably 1.25 mM.
  • the initial concentration of all the compounds according to formula L-(lll) in the reaction medium is preferably from 1 to a 100 times the concentration of all the compounds according to formula D-(lll), more preferably 1 to 10 times the concentration, even more preferably 1 to 2 times even more preferably the same as the concentration of all the compounds according to formula D-(lll).
  • “Initial concentration of all the compounds according to formula L-(lll)/ D-(lll)” refers to the concentration of the respective compound L-(lll) or D-(lll), respectively, in the reaction medium when the respective compounds are employed in step (a).
  • step (6) a mixture Mu comprising the two diastereomers DL-(II), LL-(II) is reacted.
  • DL-(II) , LL-(II) may each also be denoted as “carbamoyl compounds”.
  • DL-(I), LL-(I) may also be denoted as “amino acid compounds”.
  • step (6) catalyzes the reaction of each carbamoylate compound DL-(II) to LL-(II) to the respective amino acid compounds DL-(I) and LL-(I) at a different rate.
  • This finding may advantageously be used in a method in which only of the two carbamoyl compounds DL-(II) and LL-(II) is reacted to the respective amino acid compound DL-(I) and LL-(I) or in which both carbamoyl compounds are reacted, but at different rates, so that in the resulting product mixture Mi comprising DL-(I) and LL-(I), one of DL-(I) and LL- (I) is present in excess to the other.
  • a mixture Mi comprising at least one amino acid compound of DL-(I), LL-(I).
  • rate at which DL-(II) is reacted to give DL-(I) is different from the rate at which LL-(II) is reacted to give LL-(I)
  • mixture Mi comprises both amino acid compounds DL-(I) and LL-(I)
  • the molar ratio of DL-(I) to LL-(I) in Mi is different from the molar ratio of DL-(II) to LL-(II) in Mu
  • DL-(II) reacts to DL-(I) and/or, preferably and, LL-(II) reacts to
  • Mi obtained after step (5) comprises LL-(I) as amino acid compound, but not DL-(I), and it comprises DL-(II) and, in case not all LL-(II) reacted to give LL-(I), optionally comprises LL-(II).
  • step (5) LL-(II) does not react to the respective amino acid compound LL-(I), but DL-(II) does react to the respective amino acid compound DL-(I).
  • Mi obtained after step (5) comprises DL-(I) as amino acid compound, but not LL-(I), and it comprises LL-(II) and, in case not all DL-(II) reacted to give DL-(I), optionally comprises DL-(II).
  • Mi obtained after step (5) comprises DL-(I) and LL-(I) as amino acid compound, and, in case not all DL-(II) reacted to give optionally DL-(I), it comprises DL-(II) and, in case not all LL-(II) reacted to give LL-(I), optionally comprises LL-(II).
  • Mi comprises DL-(II) and LL-(II).
  • the mixture Mi obtained in step (5) comprises at least one amino acid compound of DL-(I), LL-(I)
  • step (5) catalyzes the reaction of each carbamoylate compound DL-(II) to LL-(II) to the respective amino acid compounds DL-(I) and LL-(I) in a different rate, this means that in those cases in which mixture Mi comprises both amino acid compounds DL-(I) and LL-(I), the molar ratio of DL-(I) to LL-(I) in Mi is different from the molar ratio of DL-(II) to LL-(II) in Mu.
  • mixture Mi comprises both amino acid compounds DL-(I) and LL-(I)
  • the molar ratio of DL-(I) to LL-(I) in Mi is different from the molar ratio of DL-(II) to LL-(II) in Mu.
  • “Molar ratio of DL-(II) to LL-(II) in Mu” means the molar ratio of DL-(II) to LL-(II) in the mixture Mu that is initially employed in step (Y)-
  • mixture Mi comprises both carbamoyl compounds DL-(II) to LL-(II).
  • the molar ratio of DL-(I) to LL-(I) in Mi is different by a factor x from the molar ratio of DL-(II) to LL-(II) in Mu.
  • x is in the range of 1 .0001 to 10000, more preferably in the range of 1 .001 to 1000, more preferably in the range of 1 .01 to 100, more preferably in the range of 1 .10 to 100, more preferably in the range of, more preferably in the range of 1 .20 to 300, more preferably in the range of 1 .30 to 250, more preferably in the range of 1 .40 to 150, more preferably in the range of 1 .50 to 100, more preferably in the range of 1 .75 to 90, more preferably in the range of 2.50 to 80, more preferably in the range of 3.00 to 65, more preferably in the range of 5.00 to 50, more preferably in the range of 10.00 to 20.00.
  • one of the amino acid compounds selected from DL-(I), LL-(I) comprised by mixture Mi is at least partially separated from at least one of the carbamoyl compounds DL-(II), LL-(II) and/or from the other amino acid compound selected from DL-(I), LL-(I).
  • one of the amino acid compounds selected from DL-(I), LL-(I) comprised by mixture Mi is at least partially separated from at least one of the carbamoyl compounds DL-(II), LL-(II) and from the other amino acid compound selected from DL-(I), LL-(I).
  • This separation may be carried out by the person skilled in the art.
  • at least one of the following separation methods is used: chromatography, crystallization, wherein crystallization is preferred.
  • crystallization of the respective amino acid compound selected from DL-(I), LL-(I) is preferred.
  • the at least partial separation of one of the amino acid compounds selected from DL-(I), LL-(I) comprised by mixture Mi is performed by crystallizing the respective amino acid compound.
  • LL-(I) is then preferably at least partially separated from DL-(II) and, in case Mi comprises LL-(II), also from LL-(II).
  • DL-(I) is then preferably at least partially separated from LL-(II) and, in case Mi comprises DL-(II), also from DL-(II).
  • the L-glufosinate P- ester according to formula DL-(I) or LL-(I) obtained in purified form after step (£) with the method according to the invention may then be saponified to produce LGA, for example in an acidic aqueous medium, preferably at pH ⁇ 7, even more preferably at an pH between ⁇ 6, more preferably at an pH ⁇ 3, even more preferably at an pH of ⁇ 1 .
  • saponification conditions are known to the skilled person and described e.g by H.J. Zeiss, J. Org. Chem. 1991 , 56, 1783-1788.
  • hydantoinase dihydropyrimidinase, EC 3.5.2.2
  • L-carbamoylase /V-carbamoyl-L-amino-acid hydrolase, EC 3.5.1.87
  • hydantoin racemase EC 5.1 .99.5
  • Cloning of hydantoinase and L-carbamoylase into the rhamnose expression vector pJOE4036 was carried out in a plasmid derivative of the rhamnose expression vector pJOE4036.
  • Polynucleotides comprising the genes of the repective enzymes (SEQ ID NOs: 27, 28) were synthesized by GeneArt (ThermoFisher Scientific (Waltham, USA)). The polynucleotides carried additional sequences for EcoR1 and Hindlll restriction sites. Both enzymes were cloned into pJOE4036 using those restriction sites resulting in the plasmid pOM22c, under the control of a rhamnose promotor ( Figure 2). 5.2 Example 2: Production of strains positive for hydantoinase, L-carbamoylase and hydantoin racemase
  • the generated strain which was positive for hydantoinase- and carbamoylase was rendered chemically competent and transformed with 10 ng of the plasmid pOM21c.
  • An E. coll ET5 strain transformed with pOM21 c or pOM22c was incubated under shaking (250 U/min) at 30 °C for 18 hours in LB medium containing ampicillin (100 pg/l), chloramphenicol (50 pg/l), and 2 g/l rhamnose.
  • the biomass was separated by centrifugation, resuspended in 50 mM phosphate buffer (pH 7.2) and applied in biotransformation tests in the following examples.
  • the concentration of the biomass in the solution was 40-50 g/l.
  • the solution was used as catalyst (“catalyst 7”) in the following.
  • the concentration of the respective polypeptide carbamoylase, hydantoinase and racemase in the obtained solution may be determined by SDS page and analysis of the respective bands via the software GelQuant® (BiochemLabSolutions).
  • the mixture M Ci was dissolved in a stirring reactor with 25 ml water. 2.4 g of the catalyst 1 was added, followed by the addition of 50 pl C0CI2 solution. The suspension was set to pH 7.5 with 0.5 M NaOH. The total volume was replenished with water to 50 ml, so that the concentration of each enantiomer L-(IV) Ci and D-(IV) Ci was 0.025 mol/l and the concentration of C0CI2 in the final solution was 1 mM. The pH was held between 7.0 and 7.5 by HCI-titration or NaOH-titration. The temperature was maintained at 37 °C by a thermostat during the reaction.
  • the reaction was stopped after 120 hours by addition of 2 N HCI until pH 2.5 was reached.
  • the biomass was separated by centrifugation or filtration.
  • the enzyme reaction was monitored by ninhydrin test to determine the formation of amino acids. No formation of amino acids was detected by the ninhydrine test.
  • L-(V)C2 and D-(V) C 2 are carbamoylates with the following formulae, was used as substrate:
  • the mixture M C 2 was dissolved in a stirring reactor with 25 ml water. 2.4 g of the catalyst 1 was added, followed by the addition of 50 pl C0CI2 solution. The suspension was set to pH 7.5 with 0.5 M NaOH. The total volume was replenished with water to 50 ml, so that the concentration of each enantiomer l_-(V) C 2 and D-(V) C 2 was 0.025 mol/l and the concentration of C0CI2 in the final solution was 1 mM. The pH was held between 7.0 and 7.5 by HCI-titration or NaOH-titration. The temperature was maintained at 37 °C by a thermostat during the reaction.
  • the reaction was stopped after 120 hours by addition of 2 N HCI until pH 2.5 was reached.
  • the biomass was separated by centrifugation or filtration.
  • the enzyme reaction was monitored by ninhydrin test to determine the formation of amino acids. No formation of amino acids was detected by the ninhydrine test.
  • the reaction was stopped after 120 hours by addition of 2 N HCI until pH 2.5 was reached.
  • the biomass was separated by centrifugation or filtration.
  • the enzyme reaction was monitored by ninhydrin test to determine the formation of amino acids. Formation of amino acids was detected by the ninhydrine test.
  • reaction mixture was saponified at 100 °C for 10 hours by adding 6 M HCI to obtain L-glufosinate and, if present, D-glufosinate.
  • the final reaction mixture was analysed by LC-MS with a CR-I column as described under item 5.4 to determine the enantiomeric excess (“ee”) of either D- or L-glufosinate.
  • ee enantiomeric excess
  • An ee of LGA of 79 % over the D-enantiomer was detected.
  • the ee of the L-enantiomer (“eei”) is determined by the following formula in the context of the invention, wherein mi and mo are the detected molar masses of L- and D-glufosinate, respectively:
  • 2 was dissolved in a stirring reactor with 25 ml water. 2.4 g of the catalyst 1 was added, followed by the addition of 50 pl C0CI2 solution. The suspension was set to pH 7.5 with 0.5 M NaOH. The total volume was replenished with water to 50 ml, so that the concentration of each enantiomer L-(VII)I 2 and D-(VII)H was 0.025 mol/l and the concentration of C0CI2 in the final solution was 1 mM. The pH was held between 7.0 and 7.5 by HCI-titration or NaOH-titration. The temperature was maintained at 37 °C by a thermostat during the reaction.
  • the reaction was stopped after 120 hours by addition of 2 N HCI until pH 2.5 was reached.
  • the biomass was separated by centrifugation or filtration.
  • the enzyme reaction was monitored by ninhydrin test to determine the formation of amino acids. Formation of amino acids was detected by the ninhydrine test.
  • reaction mixture was saponified at 100 °C for 10 hours by adding 6 M HCI to obtain L-glufosinate and, if present, D-glufosinate.
  • the final reaction mixture was analysed by LC-MS with a CR-I column as described under item 5.4 to determine the enantiomeric excess (“ee”) of either D- or L-glufosinate. An ee of LGA of 77 % over the D-enantiomer was detected.
  • L-glufosinate and D-glufosinate were detected by LC-MS (“Liquid Chromatography - Mass Spectrometry”) with a chiral column [Daicel CROWNPAK CR-l-(-)] as follows.
  • LC-MS Liquid Chromatography - Mass Spectrometry
  • a chiral column [Daicel CROWNPAK CR-l-(-)] as follows.
  • a Daicel Chiralpak IA-U column may also be used.
  • Channel A H2O with trifluoroacetic acid (TFA) pH1.15;
  • Channel B ACN (acetonitrile); isocratic 80.0 % A 120.0 % B; stoptime 7.00 min
  • this analytical method is so specific that the four diastereomers of glufosinate-P-(n-butyl) ester, namely l_l_-(VIII)i 3 , DL-(VIII)
  • L-carbamoylases (/V-carbamoyl-L-amino-acid hydrolases, EC 3.5.1 .87) of different origins were tested for their ability to react with different carbamoyl substrates according to structure L-(ll) and D-(ll) to form the respective L-glufosinate/ D-glufosinate derivative according to structures L-(l) and D-(l), respectively.
  • Cloning and expression of the respective L-carbamoylase gene was essentially carried out as described by B. Wilms, A. Wiese, C. Syldatk, R. Mattes, J. Altenbuchner, M. Pietzsch, Journal of Biotechnology 1999, 68, 101 - 113 (hereinafter “Wilms et al.”), in particular, as set forth in the following.
  • Cloning of the respective L-carbamoylase gene into the rhamnose expression vector pJOE4036 was carried out in a plasmid derivative of the rhamnose expression vector pJOE4036.
  • Polynucleotides comprising the genes of the repective enzymes were synthesized by GeneArt (ThermoFisher Scientific (Waltham, USA)). The polynucleotides carried additional sequences for ndel and Hindlll restriction sites.
  • Each one of the genes encoding the enzymes was cloned into pJOE4036 using those restriction sites resulting in the respective plasmid , i.e pOM17c for expression of SEQ ID NO: 1 , pOM17c ⁇ Prha ⁇ [amaB_Gst] for expression of SEQ ID NO: 2, pOM17c ⁇ Prha ⁇ [atcC_Ps] for expression of SEQ ID NO: 3, pOM17c ⁇ Prha ⁇ [hyuc_Pau] for expression of SEQ ID NO: 5, pOM17c ⁇ Prha ⁇ [hyuC_Asp (co_Ec)] for expression of SEQ ID NO: 8, each under the control of a rhamnose promotor.
  • E. coll ET5 cells (as described in WO 2004/042047 A1) were transformed with 10 ng of the respective plasmid generated according to Example 3.
  • An E. coli ET5 strain transformed with the respective plasmid was incubated under shaking (250 U/min) at 30 °C for 18 hours in LB medium containing ampicillin (100 pg/l), and 2 g/l rhamnose.
  • the biomass was separated by centrifugation, resuspended in 50 mM phosphate buffer (pH 7.2) and applied in biotransformation tests in the following examples.
  • the concentration of the biomass in the solution was 12.2 g/l.
  • the solution was used as catalyst (“catalyst 2”) in the following.
  • the concentration of the respective polypeptide carbamoylase in the obtained solution may be determined by SDS page and analysis of the respective bands via the software GelQuant® (BiochemLabSolutions).
  • the carbamoylate compound according to formula L-(VII)I 3 was an equimolar mixture (1 : 1) of the two diastereoisomers LL-(VII)I 3 and DL-(VII)I 3
  • the carbamoylate compound according to formula D-(VII)I 3 was an equimolar mixture (1 : 1) of the two diastereoisomers LD-(VII)I 3 and DD-(VII)
  • 3 was dissolved in a stirring reactor with 25 ml water. 2.4 g of the catalyst 2 was added, followed by the addition of C0CI2 solution. The suspension was set to pH 7.4 with 0.5 M NaOH. The total volume was replenished with water to 50 ml, so that the concentration of each compound l_-(VII)i 3 and D-(VII)I 3 was 5 mmol/l and the concentration of C0CI2 in the final solution was 1 mM. The pH was held between 7.4 by HCI-titration or NaOH-titration. The temperature was maintained at 37 °C by a thermostat during the reaction.
  • the reaction was stopped after 48 hours by addition of 2 N HCI until pH 2.5 was reached.
  • the biomass was separated by centrifugation or filtration.
  • the enzyme reaction was monitored by ninhydrin test to determine the formation of amino acids L-(VIII)

Abstract

The present invention relates to a method for the production of an L-glufosinate P-ester from the corresponding L-glufosinate P-ester carbamoylate, which is enzymatically catalyzed by a carbamoylase. The L-glufosinate P-ester and the L-glufosinate P-ester carbamoylate have a chiral α-carbon atom with a L-configuration and a chiral phosphorous atom with a D- or L-configuration. Hence, both the L-glufosinate P-ester and the L-glufosinate P-ester carbamoylate each form two diastereoisomers. The method allows for selective reaction of one diastereoisomer of the carbamoylate to the respective L-glufosinate P-ester. One of the L-glufosinate P-ester diastereomer products is then at least partially separated from the other one and/or the L-glufosinate P-ester carbamoylate starting materials. The L-glufosinate P-ester carbamoylate may be obtained by reaction from the corresponding hydantoin compound through catalysis by a hydantoinase. Likewise, the L-glufosinate hydantoin P-ester may be obtained from the corresponding D-glufosinate hydantoin P-ester, which reaction is preferably catalyzed by a hydantoin racemase.

Description

Enzymatic method for the diastereoselective production of L-glufosinate P-esters
The present invention relates to a method for the production of an L-glufosinate P-ester from the corresponding L-glufosinate P-ester carbamoylate, which is enzymatically catalyzed by a carbamoylase.
The L-glufosinate P-ester and the L-glufosinate P-ester carbamoylate have a chiral a-carbon atom with a L-configuration and a chiral phosphorous atom with a D- or L-configuration. Hence, both the L-glufosinate P-ester and the L-glufosinate P-ester carbamoylate each form two diastereoisomers. The method allows for selective reaction of one diastereoisomer of the carbamoylate to the respective L-glufosinate P-ester. One of the L-glufosinate P-ester diastereomer products is then at least partially separated from the other one and/or the L-glufosinate P-ester carbamoylate starting materials.
The L-glufosinate P-ester carbamoylate may be obtained by reaction from the corresponding hydantoin compound through catalysis by a hydantoinase. Likewise, the L-glufosinate hydantoin P-ester may be obtained from the corresponding D-glufosinate hydantoin P-ester, which reaction is preferably catalyzed by a hydantoin racemase.
1. Background of the Invention
Organic phosphorous compounds, i.e. chemical agents comprising a carbon-phosphor bond, are widely applied as herbicides in the area of plant protection. Agents such as the herbicides glyphosate (Roundup®, Touchdown®) and glufosinate (Basta®, Liberty®) as well as the growth regulator glyphosine (Polaris®) are used for this purpose (as described for example by G. Horlein, Rev. Environ. Contam. Toxicol. 1994, 138, 73 - 145).
The esters of P-methyl phosphinic acid (for example, P-methyl phosphinic acid butyl ester; “MPBE”; CAS-No: 6172-80-1) have a key role as synthetic building blocks in the synthesis of the non-selective herbicide glufosinate. These esters are accessible via two fundamental synthetic pathways (summarized in Figures 3 a and 3 b, page 130, of the article of K. Haack, Chem. Unserer Zeit 2003, 37, 128 - 138): a. Reacting diethoxy chlorophosphite [CIP(OC2H5)2] with CHsMgCI provides methyl diethoxy phosphine [H3CP(OC2Hs)2; “DEMP”; CAS-No. 15715-41-0], which is partially hydrolyzed to give the corresponding methylphosphinic acid ethyl ester (MPEE; CAS-Nr: 16391-07-4). b. Alternatively, methane can be reacted with phosphor trichloride at 500 °C to give methyl dichloro phosphane H3CPCI2. The latter can then be solvolyzed in alcohols to give the corresponding methyl phosphinic acid esters. The esters of P-methyl phosphinic acid add to carbon-carbon double bonds regioselectively. This property is used in the synthesis of glufosinate for the formation of the second phosphor-carbon bond. For example, H3CPH(O)OR* (R* = Alkyl) reacts with 1 -cyano allyl acetate in an addition reaction to provide an intermediate. Subsequent exchange of the acetate substitent with ammonia and hydrolysis of the cyano group and the ester group of the phosphinic acid moiety give glufosinate.
Acrylic acid ester is a cheaper alternative starting material. It can react with the ester of P-methyl phosphinic acid to 3-[alkoxy(methyl)phosphinyl]propionic acid alkyl ester. Claisen reaction of this diester with diethyl oxalate, hydrolysis and decarboxylation provide the corresponding a-keto acid, which can be reductively aminated to give glufosinate.
These and further synthetic routes towards glufosinate are also described in the art, e.g. in WO 1999/009039 A1 , EP 0 508 296 A1 .
A general disadvantage of all synthetic routes to glufosinate is that the obtained glufosinate is a racemic mixture. However, as there is no herbicidal activity of the D-enantiomer, L-glufosinate [hereinafter “LGA”; CAS-Nr. 35597-44-5; other names “(S)-glufosinate”, “(-)-glufosinate”] is the enantiomer of economical interest.
Figure imgf000003_0001
LGA
For example, CN 111662325 A discloses a synthetic pathway in which L-homoserine is reacted to the respective hydantoin, followed by addition of methane phosphor dichloride, which results in a methane phosphane which is disubstituted with L-homoserine hydantoin. After a final Arbuzov reaction and hydrolysis, LGA is obtained. Although the disclosure describes a high enantiomeric excess (= “ee”), this ee could not be reproduced by the inventors of the present invention. A reason could be that during this synthesis pathway, strong acidic (HCI for saponification of the hydantoin phosphor bond) and alkaline conditions (NaOH, 100 °C for ring opening) are applied. Such conditions usually lead to racemization, as described by M. Bovarnick & H.T. Clarke, Journal of the American Chemical Society 1938, 60, 2426 - 2430, by R. A. Lazarus, J. Org. Chem. 1990, 55, 4755 - 4757, and by A. S. 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 acitivity” on page 164 and 167 in “Microbial Reagents in Organic Synthesis” Series C: Mathematical and Physical Sciences - Vol. 381 , S. Servi (Ed.), 1992, Springer Science+Business Media, B.V., Dordrecht.
For enantioselective syntheses of LGA, chemical and enzymatic pathways are described in the art. WO 2020/145513 A1 and WO 2020/145514 A1 describe a chemical route to LGA. In this route, an L-homoserine derivative such as O-acetyl L-homoserine or O-succinyl L-homoserine is used as starting material and L-glufosinate is obtained by a sequence of reactions including lactonization and halogenation.
WO 2020/145627 A1 describes a similar route, wherein during halogenation, a bromo derivative is obtained.
The route disclosed by CN 106083922 A resembles these synthetic pathways but starts off from L-methionine.
CN 108516991 A describes another synthetic pathway to LGA, starting from the azeotropic dehydration of L-homoserine to give L-3,6-bis(2-haloethyl)-2,5-diketopiperazine, followed by introduction of a methylphosphonate diester group, and hydrolysis.
WO 2017/151573 A1 discloses a two-step enzymatic synthesis of LGA from D-glufosinate. In the first step, D-glufosinate is oxidatively deaminated to give 2-oxo-4-[hydroxy(methyl)phosphinoyl]- butyric acid (“PPO”), followed by the specific amination of PPO to LGA as the second step. The first step is carried out by catalysis of a D-amino acid oxidase, the second step is catalyzed by a transaminase.
WO 2020/051188 A1 discloses a similar method of converting racemic glufosinate to the L-enantiomer. In addition, it discloses a step in which the a-ketoacid or ketone byproduct formed during amination of PPO with an amine donor is converted by ketoglutarate decarboxylase to further shift the equilibrium to LGA.
WO 2019/018406 A1 discloses a method of purifying LGA from a mixture comprising LGA and glutamate. Glutamate is converted to pyroglutamate enzymatically by glutaminyl-peptidyl cyclotransferase, and LGA is then purified from the resulting mixture with an ion-exchange resin.
WO 2013/072486 A1 disclose hydantoinase mutants which have a greater activity towards D-amino acids.
WO 00/58449 A1 disclose hydantoinase mutants which have a greater activity towards L-amino acids.
A first object of the present invention is to provide a further enzymatic process for producing glufosinate or glufosinate P-esters. This process should provide products with a high excess of the L-enantiomer over the D-enantiomer. Moreover, this process should allow to use new substrates which heretofore were not used in the enzymatic synthesis of glufosinate or glufosinate esters. Moreover, there is a need in the art for an enantioselective method for the production of L-glufosinate or L-glufosinate esters from starting materials comprising D- and L-enantiomers, such as racemic mixtures, in a minimal amount of synthetic steps.
In this context, it is noted that while glufosinate has only one chiral atom, i.e. the a-carbon, a further chiral atom is observed for glufosinate derivatives in which the OH-Group that is bound to the phosphorous atom is esterified. Hence, esters of LGA exist as two diastereomers, LL-(I) and DL-(I) with the following formulae (wherein R = alkyl or aryl):
Figure imgf000005_0001
LL-(I) DL-(I)
The stereoselective production of one diastereomer of a certain product compound as well as the separation of complex diastereomeric compounds from the respective mixture of diastereomers remains a challenge in modern chemistry. One common approach taken in order to separate one diastereomer from the other is kinetic racemate resolution. In this method, one of the diastereomers is reacted selectively to the respective product, while the corresponding reaction of the other diastereomer is kinetically inhibited.
Kinetic racemic resolutions are commonly used in chemical derivatisations using chiral auxiliaries, which, however, are in many cases costly and require further reaction steps. For example, asymmetric transition metal catalysts are described by D.A. Schichl, “Racemisierung und dynamische kinetische Racematspaltung von Aminosaurederivaten und sekundaren Alkoholen” Dissertation, TU Munchen 2001 ; online source: https://mediatum.ub.tum.de/doc/601184/601184.pdf, last retrieved May 12, 2022.
Alternatively, enzymes are used for racemic resolutions, e.g. as described by Schrittwieser et al., Angewandte Chemie 2014, 126, 3805-3809 or in case of selective acylations using lipases as described by A. Ghanem & H.Y. Aboul-Enein, Tetrahedron: Asymmetry 2004, 15, 3331-3351 .
While the above methods may only be used for certain class of products, no method is disclosed in the art that allows for stereoselective synthesis of either one of the compounds LL-(I) and DL-(I).
A further object of the present invention was hence the provision of such a method for synthesis of LL-(I) and DL-(I), wherein one of the diastereoisomers is preferably formed over the other. 2. Short Summary of the Invention
Namely, it was surprisingly found that the desired L-glufosinate P-esters can be produced by an enzymatically catalyzed reaction from the corresponding carbamoylates of formula L-(ll), wherein R is an alkyl group or an aryl group:
Figure imgf000006_0001
L-(ll)
Such carbamoylates were not used in the enzymatically catalyzed production of L-glufosinate or its P-esters before. This finding was even more surprising, as it turned out that the presence of an alkyl group or aryl group for R in formula L-(ll) is mandatory, as there is no corresponding reaction of compounds according to formula L-(ll) in which R = H. As R H, L-(ll) forms two diastereomers, LL-(II) and DL-(II) with the following formulae:
Figure imgf000006_0002
LL-(II) DL-(II)
It was surprisingly found that, when catalyzed by carbamoylases, one of the reactions of
LL-(II) LL-(I) and DL-(II) DL-(I) is kinetically favoured over the other, allowing for stereoselective synthesis of one of the two diastereoisomers LL-(I) and DL-(I) from a mixture comprising LL-(II) and DL-(II).
1) The present invention hence solves the problems mentioned above by providing a method for the production of a glufosinate P-ester according to formula LL-(I) and/or, preferably and, DL-(I), comprising steps (y), (6), and (e).
In step (y), a mixture Mu comprising carbamoyl compounds according to formulae LL-(II) and DL- (II) is provided, wherein LL-(II) and DL-(II) have the formulae shown above.
In step (6), in mixture Mu, a carbamoyl compound according to formula LL-(II) is reacted to give an amino acid compound according to formula LL-(I) and/or a carbamoyl compound according to formula DL-(II) is reacted to give an amino acid compound according to formula DL-(I), wherein LL-(I) and DL-(I) have the formulae shown above, wherein the reaction according step (6) is catalyzed by a carbamoylase Ei. In step (6), a mixture Mi is obtained comprising at least one amino acid compound of DL-(I), LL-(I), wherein, if mixture Mi comprises both amino acid compounds DL-(I) and LL-(I), the molar ratio of DL-(I) to LL-(I) in Mi is different from the molar ratio of DL-(II) to LL-(II) in Mu, and wherein mixture Mi comprises at least one of the carbamoyl compounds DL-(II), LL-(II).
“[...] the molar ratio of DL-(I) to LL-(I) in Mi is different from the molar ratio of DL-(II) to LL-(II) in Mu” refers to the the molar ratio of DL-(II) to LL-(II) in the mixture Mu provided in step (y).
In step (e), one of the amino acid compounds selected from DL-(I), LL-(I) comprised by mixture Mi is at least partially separated from at least one of the carbamoyl compounds DL-(II), LL-(II) and/or, more preferably and, from the other amino acid compound selected from DL-(I), LL-(I), wherein preferably the separation of at least one of the amino acid compounds selected from DL-(I), LL-(I) comprised by mixture Mi is performed by crystallizing the respective amino acid compound.
Preferably, the carbamoylase Ei is an L-enantioselective carbamoylase Ei (“L-enantioselective carbamoylase” = “L-carbamoylase”).
R is an alkyl group or aryl group, in particular an alkyl group or phenyl or benzyl, more preferably an alkyl group, more preferably an alkyl group with 1 to 10, even more preferably with 2 to 8, even more preferably with 2 to 4 carbon atoms, even more preferably R = ethyl or n-butyl, most preferably R = n-butyl.
The L-carbamoylase Ei is preferably categorized in the EC class 3.5.1.87.
The polypeptide sequence of the L-carbamoylase Ei 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.
2) In a preferred embodiment of the method according to the invention, the mixture Mu provided in step (y) is obtained by a step (p). In preferred step (p), in a mixture Mm comprising compounds according to LL-(III) and DL-(III), at least a part of the compounds according to formula LL-(III) are reacted to give compounds according to LL-(II), and at least a part of the compounds according to formula DL-(III) are reacted to give compounds according to DL-(II), wherein LL-(III) and DL-(III) have the following formulae:
Figure imgf000008_0001
wherein the reaction according to step (p) is catalyzed by a hydantoinase E2, and wherein R has the same meaning as described for LL-(I) and DL-(I).
The hydantoinase E2 is preferably an L-enantioselective hydantoinase E2 (“L-enantioselective hydantoinase” = “L-hydantoinase”).
The hydantoinase E2, in particular the L-hydantoinase E2, is preferably categorized in the EC class 3.5.2.2.
The polypeptide sequence of the hydantoinase E2, in particular the 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.
3) In an even more preferred embodiment of the method according to the invention, the compound according to formula L-(lll) and/or the compound according to formula DL-(III) is obtained by a step (a) in which a compound according to formula LD-(III) is reacted to give a compound according to formula LL-(III) and/or a compound according to formula DD-(III) is reacted to give a compound according to formula DL-(III), wherein LD-(III) and DD-(III) have the following formulae:
Figure imgf000008_0002
LD-(III) DD-(III) and wherein R has the same meaning as described for LL-(I) and DL-(I).
Preferably, the reaction according to step (a) is catalyzed by a hydantoin racemase E3, and the hydantoin racemase E3 is more preferably categorized in the EC class 5.1 .99.5.
The polypeptide sequence of the hydantoin racemase E3 in step (a) is even more 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. 3. Brief Description of the Drawings
Figure 1 shows the pOM21 c plasmid map.
Figure 2 shows the pOM22c plasmid map.
Figure 3 shows the pOM17c plasmid map.
Figure 4 shows the pOM17c {Prha}[amaB_Gst] plasmid map.
Figure 5 shows the pOM17c {Prha}[atc_Ps] plasmid map.
Figure 6 shows the pOM17c {Prha}[hyuC_Pau] plasmid map.
Figure 7 shows the pOM17c {Prha}[hyuC_Asp (co_Ec)] plasmid map.
Figure 8 shows the different formation of each one of the distereoisomers of LL-(I) and DL-(I) for R = n-butyl, wherein an L-carbamoylase with SEQ ID NO: 1 was used (plasmid name: pOM17c). The x-axis gives the reaction time in minutes. The y-axis gives the ratio ni/nu for each diastereomer LL-(I) and DL-(I) in percent. nu is the total amount of substance (in mole) of each diastereomers LL-(II) and DL-(II) initially employed. ni is the total amount of substance (in mole) of each one of the two diastereomers LL-(I) and DL-(I) obtained.
The broken graph gives the ratio ni/nu for one diastereomer, whereas the continuous graph gives the ratio ni/nu for the other diastereomer, wherein m is the molar amount of one of the two diastereoisomers LL-(I) or DL-(I).
Figure 9 shows the different formation of each one of the distereoisomers of LL-(I) and DL-(I) for R = n-butyl, wherein an L-carbamoylase with SEQ ID NO: 2 was used (plasmid name: pOM17c{Prha}[amaB_Gst]). The values of the x- and the y-axis are as described for Figure 8.
Figure 10 shows the different formation of each one of the distereoisomers of LL-(I) and DL-(I) for R = n-butyl, wherein an L-carbamoylase with SEQ ID NO: 3 was used (plasmid name: pOM17c{Prha}[atcC_Ps]). The values of the x- and the y-axis are as described for Figure 8.
Figure 11 shows the different formation of each one of the distereoisomers of LL-(I) and DL-(I) for R = n-butyl, wherein an L-carbamoylase with SEQ ID NO: 5 was used (plasmid name: pOM17c{Prha}[hyuc_Pau]). The values of the x- and the y-axis are as described for Figure 8. 4. Detailed Description of the Invention
4.1 Definitions
Any of the enzymes used according to any aspect of the present invention, may be an isolated enzyme. In particular, the enzymes 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 polypeptides or factors essential for its activity. Such factors may be metal ions such as Mn2+ or Co2+.
In particular, an enzyme according to the present application may be a carbamoylase Ei, a hydantoinase E2, or a hydantoin racemase E3.
“Enzymatically catalyzed” means that the respective reaction is catalyzed by an enzyme, which may be a carbamoylase Ei, a hydantoinase E2, or a hydantoin racemase E3.
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 is encoded by such a molecule, particularly a polypeptide or nucleic acid that, as such, does not occur naturally but is the result of genetic engineering or refers to a cell that comprises a recombinant molecule. For example, a nucleic acid molecule is recombinant if it comprises a promoter functionally linked to a sequence encoding a catalytically active polypeptide and the promoter has been engineered such that the catalytically active polypeptide is overexpressed relative to the level of the polypeptide in the corresponding wild type cell that comprises the original unaltered nucleic acid molecule.
A “polypeptide” (one or more peptides) is a chain of chemical building blocks called amino acids that are 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 native protein, respectively. These terms are sometimes used interchangeably. A polypeptide may or may not be glycosylated.
The term “overexpressed”, as used herein, means that the respective polypeptide encoded or expressed is expressed at a level higher or at higher activity than would normally be found in the cell under identical conditions in the absence of genetic modifications carried out to increase the expression, for example in the respective wild type cell.
4.2 Methods to obtain enzymes
The enzymes that can be used in the method according to the present invention can be synthesized by methods that are known to the skilled person. One approach, which is a preferred approach according to the invention, is to express the enzyme(s) in microorgan ism(s) such as Escherichia coli (= “E. coli), Saccharomyces cerevisiae, Pichia pastoris, and others, and to add the whole cells to the reactions as whole cell biocatalysts. Another approach is to express the enzyme(s), lyse the microorganisms, and add the cell lysate. Yet another approach is to purify, or partially purify, the enzyme(s) from a lysate and add pure or partially pure enzyme(s) to the reaction. If multiple enzymes are required for a reaction, the enzymes can be expressed in one or several microorganisms, including expressing all enzymes within a single microorganism.
For example, the skilled person can obtain the enzymes according to the invention by expression, in particular, overexpression, [hereinafter, “expression, in particular overexpression” is abbreviated as (over)expression”, and “express, in particular overexpress” is abbreviated as (over)express”] of these enzymes in a cell and subsequent isolation thereof, e.g. as described in DE 100 31 999 A1. Episomal plasmids, for example, are employed for increasing the expression of the respective genes. In such plasmids, the nucleic acid molecule to be (over)expressed or encoding the polypeptide or enzyme to be (over)expressed may be placed under the control of a strong inducible promoter such as the lac promoter, located upstream of the gene. A promoter is a DNA sequence consisting of about 40 to 50 base pairs which constitutes the binding site for an RNA polymerase holoenzyme and the transcriptional start point (M. Patek, J. Holatko, T. Busche, J. Kalinowski, J. Nesvera, Microbial Biotechnology 2013, 6, 103-117), whereby the strength of expression of the controlled polynucleotide or gene can be influenced. A “functional linkage” is obtained by the sequential arrangement of a promoter with a gene, which leads to a transcription of the gene.
Suitable strong promoters or methods of producing such promoters for increasing expression are known from the literature (e.g. S. Lisser & H. Margalit, Nucleic Acid Research 1993, 21, 1507-1516; M. Patek and J. Nesvera in H. Yukawa and M Inui (eds.), Corynebacterium glutamicum, Microbiology Monographs 23, Springer Verlag Berlin Heidelberg 2013, 51-88; B. J. Eikmanns, E. Kleinertz, W. Liebl, H. Sahm, Gene 1991 , 102, 93-98). For instance, native promoters may be optimized by altering the promoter sequence in the direction of known consensus sequences with respect to increasing the expression of the genes functionally linked to these promoters (M. Patek, B.J. Eikmanns, J. Patek, H. Sahm, Microbiology 1996, 142, 1297-1309; M. Patek, J. Holatko, T. Busche, J. Kalinowski, J. Nesvera, Microbial Biotechnology 2013, 6, 103-117).
Constitutive promoters are also suitable for the (over)expression, in which the gene encoding the enzyme activity is expressed continuously under the control of the promoter such as, for example, the glucose dependent dec promoter. Chemically induced promoters are also suitable, such as tac, lac, rha or trp. The most widespread system for the induction of promoters is the lac operon of E. coli. In this case, either lactose or isopropyl B-D-thiogalactopyranoside (IPTG) is used as inducer. Also, systems using arabinose (e.g. the pBAD system) or rhamnose (e.g. E. coli KRX) are common as inducers. A system for physical induction is, for example, the temperature-induced cold shock promoter system based on the E. coli cspA promoter from Takara or Lambda PL and also osmotically inducible promoters, for example, osmB (e.g. WO 95/25785 A1).
Suitable plasmids or vectors are in principle all embodiments available for this purpose to the person skilled in the art. The state of the art describes standard plasmids that may be used for this purpose, for example the pET system of vectors exemplified by pET-3a or pET-26b(+) (commercially available from Novagen). Further plasmids and vectors can be taken, for example, pOM21 described in WO 2004/111227 A2, pOM22 described in WO 00/058449 A1 or pOM18 described in WO 2013/072486 A1 or from the brochures of the companies Novagen, Promega, New England Biolabs, Clontech or Gibco BRL. Further preferred plasmids and vectors can be found in: Glover, D.M. (1985) DNA cloning: a practical approach, Vol. I-III, IRL Press Ltd., Oxford; Rodriguez, R.L. and Denhardt, D. T (eds) (1988) Vectors: a survey of molecular cloning vectors and their uses, 179-204, Butterworth, Stoneham; Goeddel, D. V. (1990) Systems for heterologous gene expression, Methods Enzymol. 185, 3-7; Sambrook, J.; Fritsch, E. F. and Maniatis, T. (1989), Molecular cloning: a laboratory manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York. Of these plasmids, pOM21 and pOM22 are preferred.
The plasmid vector, which contains the gene to be amplified, is then converted to the desired strain, e.g. by conjugation or transformation. The method of conjugation is described, for example, by A. Schafer, J. Kalinowski, A. Piihler, Applied and Environmental Microbiology 1994, 60, 756 - 759. Methods for transformation are described, for example, in G. Thierbach, A. Schwarzer, A. Piihler, Applied Microbiology and Biotechnology 1988, 29, 356 - 362, L.K. Dunican & E. Shivnan, Bio/Technology 1989, 7, 1067-1070 and A. Tauch, O. Kirchner, L. Wehmeier, J. Kalinowski, A. Piihler, FEMS Microbiology Letters 1994, 123, 343-347. After homologous recombination by means of a “cross-over” event, the resulting strain contains at least two copies of the gene concerned.
The desired enzyme can be isolated by disrupting cells which contain the desired activity in a manner known to the person skilled in the art, for example with the aid of a ball mill, a French press or of an ultrasonic disintegrator and subsequently separating off cells, cell debris and disruption aids, such as, for example, glass beads, by centrifugation for 10 minutes at 13,000 rpm and 4 °C. Using the resulting cell-free crude extract, enzyme assays with subsequent LC-ESI-MS detection of the products can then be carried out. Alternatively, the enzyme can be enriched in the manner known to the person skilled in the art by chromatographic methods (such as nickel-nitrilotriacetic acid affinity chromatography, streptavidin affinity chromatography, gel filtration chromatography or ion-exchange chromatography) or else purified to homogeneity. Quantification of the enzyme can be performed by methods known to the person skilled in the art, for example by determination of the concentration of the respective polypeptide of the enzyme (e.g. carbamoylase, hydantoinase and racemase) in the obtained solution by SDS page and analysis of the respective bands via the software GelQuant® (BiochemLabSolutions).
Moreover, whether or not a nucleic acid or polypeptide is (over)expressed, may be determined by way of quantitative PCR reaction in the case of a nucleic acid molecule, SDS polyacrylamide electrophoreses, Western blotting or comparative activity assays in the case of a polypeptide. Genetic modifications may be directed to transcriptional, translational, and/or post-translational modifications that result in a change of enzyme activity and/or selectivity under selected and/or identified culture conditions.
4.3 Variants
In the context of the present invention, the term “variant” with respect to polypeptide sequences refers to a polypeptide sequence with a degree of identity to the reference sequence (“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 %. In still further particular 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 %.
It goes without saying that a “variant” of a certain polypeptide sequence is not identical to the polypeptide sequence.
Such variants may be prepared by introducing deletions, insertions, substitutions, or combinations thereof, in particular in amino acid sequences, as well as fusions comprising such macromolecules or variants thereof.
Modifications of amino acid residues of a given polypeptide sequence which lead to no significant modifications of the properties and function of the given polypeptide are known to those skilled in the art. Thus for example many amino acids can often be exchanged for one another without problems; examples of such suitable amino acid substitutions are: Ala by Ser; Arg by Lys; Asn by Gin or His; Asp by Glu; Cys by Ser; Gin by Asn; Glu by Asp; Gly by Pro; His by Asn or Gin; He by Leu or Vai; Leu by Met or Vai; Lys by Arg or Gin or Glu; Met by Leu or He; Phe by Met or Leu or Tyr; Ser by Thr; Thr by Ser; Trp by Tyr; Tyr by Trp or Phe; Vai by lie 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, often exert no significant influence on the function of the polypeptide.
In line with this, preferable 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 that comprises the complete polypeptide sequence 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, or at least the amino acids of the respective sequence 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, for example the catalytic activity of a protein, or the fold or structure of the protein. The other amino acids may be deleted, substituted or replaced by insertions or essential amino acids are replaced in a conservative manner to the effect that the activity of the enzyme, in particular the 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), is preserved.
4.4 Sequence Identity
The person skilled in the art is aware that various computer programs are available for the calculation of similarity or identity between two nucleotide or polypeptide sequences. Preferred methods for determining the sequence identity initially generate the greatest alignment between the sequences to be compared. Computer programs for determining the sequence identity include, but are not limited to, the GCG program package including
GAP [J. Deveroy et al., Nucleic Acid Research 1984, 12, page 387, Genetics Computer Group University of Wisconsin, Medicine (Wl)], and
BLASTP, BLASTN and FASTA (S. Altschul et al., Journal of Molecular Biology 1990, 215, 403 - 410). The BLAST program can be obtained from the National Center for Biotechnology Information (NCBI) and from other sources (BLAST Handbook, S. Altschul et al., NCBI NLM NIH Bethesda ND 22894; S. Altschul et al., above).
For instance, the percentage identity between two polypeptide sequences can be determined by the algorithm developed by S. B. 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 either a BLOSUM62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1 , 2, 3, 4, 5 or 6. The person skilled in the art will recognize that the use of different parameters will lead to slightly different results, but that the percentage identity between two polypeptide overall will not be significantly different. The BLOSUM62 matrix is typically used applying the default settings (gap weight: 12, length weight: 1).
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.
Most preferably, the degree of identity between sequences is determined in the context of the present invention by the programme “Needle” using the substitution matrix BLOSUM62, the gap opening penalty of 10, and the gap extension penalty of 0.5. The Needle program implements the global alignment algorithm described in S. B. Needleman & C. D. Wunsch, J. Mol. Biol. 1970, 48, 443-453. The substitution matrix used according to the present invention is BLOSUM62, gap opening penalty is 10, and gap extension penalty is 0.5. The preferred version used in the context of this invention is the one presented by F. Madeira, Y.M. Park, J. Lee, N. Buso, T. Gur, N. Madhusoodanan, P. Basutkar, A.R.N. Tivey, S.C. Potter, R.D. Finn, Nucleic Acids Research 2019, 47, W636-W641 , Web Server issue (preferred version accessible online on May 14, 2022 via https://www.ebi.ac.uk/Tools/psa/emboss_needle/).
In a particular embodiment, the percentage of identity of an amino acid sequence of a polypeptide with, or to, a reference polypeptide sequence is determined by i) aligning the two amino acid sequences using the Needle program, with the BLOSUM62 substitution matrix, a gap opening penalty of 10, and a gap extension penalty of 0.5; ii) counting the number of exact matches in the alignment; iii) dividing the number of exact matches by the length of the longest of the two amino acid sequences, and iv) converting the result of the division of iii) into percentage. 4.5 Method for production of an L-glufosinate P-ester
The present invention relates to a method for the production of an L-glufosinate P-ester according to formula LL-(I) and/or DL-(I):
Figure imgf000016_0001
LL-(I) DL-(I)
The method according to the invention comprises steps (y), (5), and (e).
In step (y) a mixture Mu comprising carbamoyl compounds according to formulae LL-(II) and DL-(II) is provided, wherein LL-(II) and DL-(II) have the following formulae:
Figure imgf000016_0002
LL-(II) DL-(II)
The molar ratio of all carbamoyl compounds according to formula LL-(II) in mixture Mu provided in step (Y) to all carbamoyl compounds according to formula DL-(II) in mixture Mu provided in step (y) is not specifically limited.
In a preferred embodiment, the molar ratio of all carbamoyl compounds according to formula LL-(II) in mixture Mu provided in step (y) to all carbamoyl compounds according to formula DL-(II) provided in step (Y) in mixture Mu is in the range of 999 : 1 to 1 : 999, preferably 99 : 1 to 1 : 99, more preferably 9 : 1 to 1 : 9, more preferably 4 : 1 to 1 : 4, more preferably 7 : 3 to 3 : 7, more preferably 3 : 2 to 2 : 3, most preferably 1 : 1.
In a further preferred embodiment, mixture Mu provided in step (y) further comprises the diastereoisomers LD-(II) and DD-(II), wherein LD-(II) and DD-(II) have the following formulae:
Figure imgf000017_0001
LD-(II) DD-(II)
In case mixture Mu provided in step (y) further comprises the diastereoisomers LD-(II) and DD-(II), the molar ratio of all carbamoyl compounds according to formula LD-(II) in mixture Mu provided in step (y) to all carbamoyl compounds according to formula DD-(II) in mixture Mu provided in step (y) is not specifically limited.
In a preferred embodiment, the molar ratio of all carbamoyl compounds according to formula LD-(II) in mixture Mu provided in step (y) to all carbamoyl compounds according to formula DD-(II) in mixture Mu provided in step (y) is in this case in the range of 999 : 1 to 1 : 999, preferably 99 : 1 to 1 : 99, more preferably 9 : 1 to 1 : 9, more preferably 4 : 1 to 1 : 4, more preferably 7 : 3 to 3 : 7, more preferably 3 : 2 to 2 : 3, most preferably 1 : 1.
In case mixture Mu provided in step (y) further comprises the diastereoisomers LD-(II) and DD-(II), the molar ratio of all carbamoyl compounds LL-(II) and DL-(II) to all carbamoyl compounds LD-(II) and DD-(II) in mixture Mu provided in step (y) is not specifically limited.
In a preferred embodiment, the molar ratio of all carbamoyl compounds according to formulae LL-(II) and DL-(II) in mixture Mu provided in step (y) to all carbamoyl compounds according to formulae LD-(II) and DD-(II) in mixture Mu provided in step (y) is in this case in the range of 999 : 1 to 1 : 999, preferably 99 : 1 to 1 : 99, more preferably 9 : 1 to 1 : 9, more preferably 4 : 1 to 1 : 4, more preferably 7 : 3 to 3 : 7, more preferably 3 : 2 to 2 : 3, most preferably 1 : 1.
R is an alkyl group or an aryl group. R is the same for LL-(II) and DL-(II), and, in those embodiments in which mixture Mu provided in step (y) comprises compounds according to formulae LL-(H), DL-(II) and at least one, preferably both of LD-(II) and DD-(II), is, in particular, the same in all the compounds according to these formulae in mixture Mu provided in step (y).
In particular, R is an alkyl group or phenyl or benzyl, more preferably an alkyl group, more preferably an alkyl group with 1 to 10, even more preferably with 2 to 8, even more preferably with 2 to 4 carbon atoms, even more preferably R = ethyl or n-butyl, most preferably R = n-butyl. 4.5.1 Step (5)
4.5. 1. 1 Enzymatic catalysis of step (5)
The method according to the invention comprises a step (5).
In step (5), in mixture Mu, a carbamoyl compound according to formula LL-(II) is reacted to give an amino acid compound according to formula LL-(I) and/or a carbamoyl compound according to formula DL-(II) is reacted to give an amino acid compound according to formula DL-(I):
Figure imgf000018_0001
LL-(I) DL-(I)
In a preferred embodiment of step (6), in mixture Mu, a carbamoyl compound according to formula LL-(II) is reacted to give an amino acid compound according to formula LL-(I) and a carbamoyl compound according to formula DL-(II) is reacted to give an amino acid compound according to formula DL-(I).
The reaction according step (6) is enzymatically catalyzed, namely it is catalyzed by a carbamoylase Ei.
4.5. 1.2 Enantioselective and enantiospecific catalysis of step (5)
Step (6) of the method according to the invention is preferably L-enantioselective, even more preferably L-enantiospecific.
In such a preferred embodiment, a mixture Mu is in particular employed in step (6), wherein the mixture Mu comprises, besides compounds according to formulae LL-(II) and DL-(II), the respective enantiomers according to formulae LD-(II) and DD-(II). Such mixtures comprising all four diastereomers LL-(II), DL-(II), LD-(II), and DD-(II) are referred to as “mixture Mu* hereinafter.
For such mixtures Mu*, a parallel reaction according to step (6)* may be observed. Namely, in the reaction according to step (6)*, LD-(II), i.e. the enantiomer of DL-(II), in mixture Mu* is reacted to give a compound according to formula LD-(I), and/or, preferably and, DD-(II), i.e. the enantiomer of LL-(II), in mixture Mu* is reacted to give a compound according to formula DD-(I), wherein
Figure imgf000019_0001
LD-(I) DD-(I)
In case that step (6) is “L-enantioselective”, this means that in case a mixture Mu* is employed in step (5), then there is either no reaction according to step (5)* or, in case there is a reaction according to step (5)*, then the rate of reaction according to step (5)* is lesser than the rate of the reaction according to step (5).
Step (5) is “L-enantiospecific”, if the rate of reaction according to step (5)* is essentially zero, i.e. there is no reaction according to step (5)*.
Step (6) is in particular L-enantioselective, if it is catalyzed by an L-carbamoylase Ei, which may be determined by the skilled person as set forth under 4.5.4.3.
In case step (6) is L-enantioselective, the reaction according to step (6) proceeds preferably at a reaction rate that is at least 2 times greater, preferably at least 10 times greater, more preferably at least 100 times greater, even more preferably at least 103 times greater, even more preferably at least 104 times greater, even more preferably at least 105 times greater than the reaction rate at which step (6)* proceeds.
To quantify the factor at which the reaction rate of step (6) proceeds compared to the reaction rate of step (6)*, the following test may be carried out:
(1) An equimolar mixture of compounds according to L-(ll) and D-(ll) [i.e. the ratio of the sum of the molar masses of the diastereosiomers [LL-(II) and DL-(II)] to the sum of the molar masses of the diastereosiomers [LD-(II) and DD-(II)] is 1 : 1] is provided. Likewise, the molar ratio of LL-(II) to DL-(II) is 1 : 1 , and the molar ratio of LD-(II) to DD-(II) is 1 : 1 .
Figure imgf000019_0002
This mixture subjected to the respective reactions conditions and the development of the products
L-(l) [LL-(I) and DL-(I)] versus the development of the products D-(l) [LD-(I) and DD-(I)] is monitored overtime (e.g. by LC-MS as set forth under item 5.4):
Figure imgf000020_0001
(2) When nLno = 10 mol-% of the initially employed L-(ll) have reacted to the respective product L-(l), the amount of D-(l) that was formed by reaction from D-(ll) [in mol-% relative to the initially employed D-(ll)] is measured (= nDno).
(3) The ratio of nLno I nDno = 10 / nDno gives the factor at which the reaction rate of step (5) proceeds compared to the reaction rate of step (5)*.
If nLno I nDno > 1 , the reaction rate of step (5) is greater than the reaction rate of step (5)*. If nLno I nDno < 1 , the reaction rate of step (5) is lesser than the reaction rate of step (5)*.
If nLno I nDno = 1 , the reaction rates of steps (5) and (5)* are the same.
4.5.2 Carbamoylases
The reaction according to step (5) of the method according to the invention is catalyzed by a carbamoylase Ei.
Namely, it was surprisingly found that carbamoylases accept compounds of formulae LL-(II) and DL-(II) as substrates and convert them to the respective product LL-(I) and DL-(I), and hence can be used to catalyze the reaction according to step (6). This finding is of high scientific and economic value, as it opens new synthetic routes based on new starting materials for the production of L-glufosinate P-esters and L-gluofsinate. Even more surprisingly, it was found that L-glufosinate carbamoylate, i.e. the compound according to LL-(II) and DL-(II), in which R = H, does not undergo reaction by carbamoylases to give L-glufosinate.
Even further surprisingly, it was found that the reaction rate at which these carbamolyases catalyze the conversion of compounds of formulae LL-(II) and DL-(II) to the respective product LL-(I) and DL-(I) are different, so that a specific conversion of one of the substrates LL-(II) and DL-(II) to the respective product LL-(I) and DL-(I) is favored over the other.
In nature, carbamoylases generally catalyze the following reaction <1>, wherein Rx may be an organic residue, e.g. a side chain of one of the naturally occurring amino acids.
Figure imgf000021_0001
It was now surprisingly found that carbamoylases also accept substrates in which
Figure imgf000021_0005
Surprisingly, they do not accept substrates in which Rx = RY =
Figure imgf000021_0002
wherein R = H.
In the context of the present invention, a “carbamoylase Ei” is a carbamoylase that catalyzes the following reaction <1A> of a carbamoyl substrate SL to the respective amino acid product PL, wherein Rx = RY and preferably Rx = Rz.
Figure imgf000021_0003
In particular, the carbamoylase Ei is a “L-carbamolyase”, i.e. it has a greater catalytic activity for reaction <1A> than for reaction <1 B>, wherein the substrate SD in the reaction <1 B> is the enantiomer of the substrate SL in the reaction <1A>:
Figure imgf000021_0004
<1 B>: SD PD
As an L-carbamoylase has a higher catalytic activity for reaction <1A> than for reaction <1 B>, it is “L-enantioselective”. An L-carbamoylase that has no catalytic activity for reaction <1 B> and thus only has catalytic activity for reaction <1A> is “L-enantiospecific”. A “D-carbamoylase” is defined as a carbamoylase which is “D-enantioselective”, 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 thus only has catalytic activity for reaction <1B> is “D-enantiospecific”.
A carbamoylase which has the same catalytic activity for reaction <1B> as for reaction <1A>, is referred to as “non-enantioselective carbamoylase”.
For determination whether a carbamoylase may be denoted as “L-carbamoylase”, “D-carbamoylase” or “non-enantioselective carbamoylase” in the context of the present invention, the procedure according to Assay B (item 4.5.4) may preferably be used.
The carbamoylase Ei, in particular the L-carbamoylase Ei, that may be used in step (6) of the method according to invention may originate 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 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.; Pseudomonas sp.; Ralstonia sp., in particular Ralstonia pickettii; Sinorhizobium sp., in particular Sinorhizobium meliloti.
An L-carbamoylase Ei suitable for the method according to the present invention may be the enzyme HyuC, which originates from Arthrobacter. Other enzymes are AmaB, AtcC, Inc, SinmeB_2280.
WO 01/23582 A1 discloses an example of an enzyme having carbamoylase activity according to the invention.
The carbamoylase Ei that may be used in step (6) of the method according to the present invention may be an L-carbamoylase categorized in the EC class EC 3.5.1 .87.
L-carbamoylase enzymes are for example described by J. Ogawa, H. Miyake, S. Shimizu, Appl. Microbiol. Biotechnol. 1995 43, 1039 - 1043 and in WO 01/23582 A1 .
A L-carbamoylase Ei that may preferably be used in step (6) according to the first aspect of the invention may originate from Arthrobacter sp., in particular Arthrobacter crystallopoietes, Arthrobacter aurescens, Arthrobacter sp. BT801 , Arthrobacter aurescens DSM 3747; Bacillus sp. , in particular Bacillus fordii; Geobacillus sp., in particular Geobacillus stearothermophilus, Geobacillus kaustophilus', Microbacterium sp., in particular Microbacterium liquefaciens strain AJ3912; Paenarthrobacter sp., in particular Paenarthrobacter aurescens Pseudomonas sp. , in particular Pseudomonas sp. QR-101 Sinorhizobium sp., in particular Sinorhizobium meliloti. Even more preferably, the L-carbamoylase Ei that may preferably be used in step (6) according to the first aspect of the invention may originate from Arthrobacter sp., in particular Arthrobacter crystallopoietes, Arthrobacter aurescens, Arthrobacter sp. BT801 , Arthrobacter aurescens DSM 3747, most preferably from Arthrobacter aurescens DSM 3747.
The respective sequences can be derived from databases such as the Braunschweig Enzyme Database (BRENDA, Germany, available underwww.brenda-enzymes.org/index.php), the National Center for Biotechnological Information (NCBI, available under https://www.ncbi.nlm.nih.gov/) or the Kyoto Encyclopedia of Genes and Genomes (KEGG, Japan, available under www. https://www.genome.jp/kegg/).
The following table 1 gives preferred examples for polypeptide sequences of L-carbamoylases Ei that may be preferably used in step (6) of the method according to the invention. The genes encoding the respective L-carbamoylase Ei and the respective accession code are indicated as far as known.
Table 1
Figure imgf000023_0001
In a preferred embodiment of the method according to the present invention, the reaction according to step (5) is catalyzed by an L-carbamoylases Ei, wherein the polypeptide sequence of Ei is selected from the group consisting of 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.
4.5.3 Assays AL and AD for determining carbamoylase activity
The skilled person is aware of carbamoylases, in particular L-carbamoylases, that may be used in step (6) of the method according to the invention.
In particular, Assay AL, described in the following, may be used to determine carbamolyase and L-carbamoylase activity of a given enzyme Ex and may advantageously be used according to the invention to determine carbamoylase and L-carbamoylase activity 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, variants of SEQ ID NO: 9.
For comparative reasons, Assay AD may be used to determine D-carbamoylase activity of a given enzyme Ex.
For the purpose of Assay AL and Assay AD, the molar mass of the enzyme Ex to be tested is calculated as the molar mass of the polypeptide sequence of Ex.
4.5.3.1 Assay AL:
To 0.9 ml of an aqueous reaction solution (phosphate buffer, pH 7.2, 10 mM MnCh), containing 50 mM of an n-butyl P-ester of carbamoyl glufosinate according to formula L-(ll), wherein R = n-butyl, and wherein LL-(II) and DL-(II) are equimolar, are added 400 nmol of Ex in 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh). The resulting solution is incubated at 25 °C, and the pH is held at pH 7.2 by addition of 0.5 M NaOH. After 300 minutes, the reaction is stopped by addition of 2 M HCI to achieve a pH of 2.5, and the molar amount of the respective LGA P-(n-butyl) ester according to formula L-(l), wherein R = n-butyl, is determined. L-(l), i.e. wherein R = n-butyl, may be detected by the LC-MS method as described in the example section (item 5.4) for detection of LGA.
4.5.3.2 Assay AD:
To 0.9 ml of an aqueous reaction solution (phosphate buffer, pH 7.2, 10 mM MnCh), containing
50 mM of an n-butyl P-ester of carbamoyl glufosinate according to formula D-(ll), wherein
R = n-butyl, and wherein LD-(II) and DD-(II) are equimolar, are added 400 nmol of Ex in 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh). The resulting solution is incubated at 25 °C, and the pH is held at pH 7.2 by addition of 0.5 M NaOH. After 300 minutes, the reaction is stopped by addition of 2 M HCI to achieve a pH of 2.5, and the molar amount of the respective D-glufosinate P-(n-butyl) ester D-(l), wherein R = n-butyl, is determined. D-(l), wherein R = n-butyl, may be detected by the LC-MS method described in the example section (item 5.4) for detection of LGA.
4.5.4 Assay B for identifiying carbamoylases, L-carbamoylases, D-carbamoylases, L- and D-enantiospecificity
The carbamoylase Ei according to the invention is preferably an L-carbamoylase, more preferably L-enantiospecific.
Whether a given enzyme Ex may be considered a carbamoylase Ei, in particular an L-carbamoylase, may be determined in the context of the present invention by the following Assay B.
4.5.4.1 Assay B:
B-1 . Firstly, Assay AL as set forth under item 4.5.3.1 is conducted, and the obtained molar amount of the compound of the formula L-(l), wherein R = n-butyl, is determined according to Assay AL.
B-2. Secondly, Assay AD as set forth under item 4.5.3.2 is conducted, and the obtained molar amount of the compound of the formula D-(l), wherein R = n-butyl, is determined according to Assay AD.
B-3. Then, step B-1 is repeated, except that instead of the addition of 400 nmol Ex in 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh), 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh) without Ex is added.
B-4. Then, step B-2 is repeated, except that instead of the addition of 400 nmol Ex in 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh), 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh) without Ex is added.
4.5.4.2 Carbamoylase activity
If the molar amount of the compound of the formula L-(l), wherein R = n-butyl, that is determined in step B-1 , is greater than the molar amount of the compound of the formula L-(l), wherein
R = n-butyl, that is determined in step B-3, then Ex is deemed to have carbamoylase activity, and hence may be considered a carbamoylase Ei in the context of the invention. 4.5.4.3 L-Carbamoylase activity
4.5.4.3.1 L-Carbamoylases
(i) If the molar amount of the compound of formula L-(l), wherein R = n-butyl, that is determined in step B-1 , is greater than the molar amount of the compound of formula L-(l), wherein R = n-butyl, that is determined in step B-3, and
(ii) if the molar amount of the compound of formula D-(l), wherein R = n-butyl, that is determined in step B-2, is greater than or the same as the molar amount of the compound of formula D-(l), wherein R = n-butyl, that is determined in step B-4, and
(iii) if, in addition, the molar amount of the compound of formula of L-(l), wherein R = n-butyl, that is determined in step B-1 , is greater than the molar amount of the compound of formula of D-(l), wherein R = n-butyl, that is determined in step B-2, then Ex is deemed to have L-carbamoylase activity, and hence may be considered an L-carbamoylase in the context of the invention. In this case, Ex is deemed to be “L-enantioselective” in the context of the invention.
For the sake of clarity, it is pointed out that condition (iii) is automatically fulfilled in those cases in which the molar amount of the compound of formula D-(l), wherein R = n-butyl, that is determined in step B-2, is the same as the molar amount of the compound of formula D-(l), wherein R = n-butyl, that is determined in step B-4.
(i) If the molar amount of the compound of formula L-(l), wherein R = n-butyl, that is determined in step B-1 , is greater than the molar amount of the compound of formula L-(l), wherein R = n-butyl, that is determined in step B-3, and
(ii) if the molar amount of the compound of formula D-(l), wherein R = n-butyl, that is determined in step B-2 is the same as the molar amount of the compound of formula D-(l), wherein R = n-butyl, that is determined in step B-4, then Ex is deemed to have L-carbamoylase activity, and hence may be considered to be an L- carbamoylase in the context of the invention. In this case, Ex is not only “L-enantioselective”, but also “L-enantiospecific” in the context of the invention.
For L-carbamyolases Ex that are not L-enantiospecific, the L-enantioselectivity may then be quantified by dividing the molar amount of the compound of formula L-(l), wherein R = n-butyl, that is determined in step B-1 , by the molar amount of the compound of formula D-( I) , wherein R = n-butyl, that is determined in step B-2, and then multiplying the obtained value by 100, giving the L-enantioselectivity of Ex in %. 4.5.4.3.2 D-carbamoylases
(i) If the molar amount of the compound of formula L-(l), wherein R = n-butyl, that is determined in step B-1 , is greater than or the same as the molar amount of the compound of formula L-(l), wherein R = n-butyl, that is determined in step B-3, and
(ii) if the molar amount of the compound of formula D-(l), wherein R = n-butyl, that is determined in step B-2, is greater than the molar amount of the compound of formula D-(l), wherein R = n-butyl, that is determined in step B-4, and
(iii) if, in addition, the molar amount of the compound of formula D-(l), wherein R = n-butyl, that is determined in step B-2 is greater than the molar amount of the compound of formula L-(l), wherein R = n-butyl, that is determined in step B-1 , then Ex is deemed to have D-carbamoylase activity, and hence may be considered a D-carbamoylase. In this case, Ex is “D-enantioselective” in the context of the invention.
For the sake of clarity, it is pointed out that condition (iii) is automatically fulfilled in those cases in which the molar amount of the compound of formula L-(l), wherein R = n-butyl, that is determined in step B-1 is the same as the molar amount of the compound of formula L-(l), wherein R = n-butyl, that is determined in step B-3.
(i) If the molar amount of the compound of formula L-(l), wherein R = n-butyl, that is determined in step B-1 , is the same as the molar amount of the compound of formula L-( I) , wherein R = n-butyl, that is determined in step B-3, and
(ii) if the molar amount of the compound of formula D-(l), wherein R = n-butyl, that is determined in step B-2, is greater than the molar amount of the compound of formula D-(l), wherein R = n-butyl, that is determined in step B-4, then Ex is deemed to have D-carbamoylase activity, and hence may be considered to be a D-carbamoylase. In this case, Ex is not only “D-enantioselective”, but also “D-enantiospecific” in the context of the invention.
For D-carbamoylases Ex, that are not D-enantiospecific, the D-enantioselectivity may then be quantified by dividing the molar amount of the compound of formula D-(l), wherein R = n-butyl, that is determined in step B-2 by the molar amount of the compound of formula L-(l), wherein R = n-butyl, that is determined in step B-1 and then multiplying the obtained value by 100, giving the D-enantioselectivity of Ex in %.
4.5.4.3.3 Non-enantioselective carbamoylases
(i) If the molar amount of the compound of formula L-(l), wherein R = n-butyl, that is determined in step B-1 , is greater than the molar amount of the compound of formula L-(l), wherein R = n-butyl, that is determined in step B-3, and (ii) if the molar amount of the compound of formula D-(l), wherein R = n-butyl, that is determined in step B-2 is greater than the molar amount of the compound of formula D-(l), wherein R = n-butyl, that is determined in step B-4, and
(iii) if, in addition, the molar amount of the compound of formula D-(l), wherein R = n-butyl, that is determined in step B-2 is the same as the molar amount of the compound of formula L-(l), wherein R = n-butyl, that is determined in step B-1 , then Ex is deemed to be a non-enantioselective carbamoylase in the context of the invention. In this case, Ex is “non-enantioselective”.
4.5.5 Assay C for identifiying preferred carbamoylase variants of SEQ ID NOs: 1 - 9
4.5.5.1 L- and D-enantioselective carbamoylase variants
An enzyme, the polpypetide sequence of which is 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 has carbamoylase, and L-carbamoylase activity.
In a preferred embodiment of the method according to the invention, the polypeptide sequence of the L-carbamoylase Ei is selected from the group consisting of 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.
In an even more preferred embodiment of the method according to the invention, the polypeptide sequence of the L-carbamoylase Ei is selected from the group consisting of SEQ ID NO: 1 and variants thereof, SEQ ID NO: 2 and variants thereof, SEQ ID NO: 3 and variants thereof, SEQ ID NO: 5 and variants thereof, SEQ ID NO: 8 and variants thereof, more preferably, the polypeptide sequence of the L-carbamoylase Ei is selected from the group consisting of SEQ ID NO: 1 and variants thereof, SEQ ID NO: 2 and variants thereof, SEQ ID NO: 3 and variants thereof, SEQ ID NO: 5 and variants thereof, more preferably the polypeptide sequence of the L-carbamoylase Ei is selected from SEQ ID NO: 1 and variants thereof.
The term “variant” is defined under item 4.3.
In the context of the invention, an enzyme Ei, the polypeptide sequence of which 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 has carbamoylase activity, preferably L-carbamoylase activity, more preferably is L-enantiospecific. Whether a given enzyme Ex, the polypeptide sequence of which 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, has carbamoylase activity, L carbamoylase activity and/or is L-enantiospecific may be determined as set forth under items 4.5.4.2 and 4.5.4.3.1 , respectively.
The carbamoylase activity of a given L-carbamoylase EiV, the polypeptide sequence of which is a variant of one 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, relative to the carbamoylase activity of an L-carbamoylase Eis, wherein the polypeptide sequence of Eis is selected from 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, may be quantified in the context of the present invention by the following Assay C:
4.5.5.2 Assay C
C-1 Assay AL as set forth under item 4.5.3.1 is conducted, wherein Eis is the enzyme to be tested. The obtained molar amount of the compound according to formula L-(l), wherein R = n-butyl, is determined according to Assay AL.
C-2 Step C-1 is repeated, except that, instead of Eis, Eiv is used as the enzyme to be tested.
C-3. Then, the molar amount of the compound according to formula L-(l), wherein R = n-butyl, that is determined in step C-2, is divided by the molar amount of the compound according to formula L-(l), wherein R = n-butyl, that is determined in step C-1 , and the obtained ratio is multiplied by 100, giving the carbamoylase activity of L-carbamoylase EiV, relative to the carbamoylase activity of the L-carbamoylase Eis, in %.
4.5.6 Preferred carbamoylase variants of SEQ ID NOs: 1 - 9
In the context of the invention, L-carbamoylases Ei, the polypeptide sequence of which is 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, are generally denoted as tis ■
L-carbamoylases Ei, the polypeptide sequence of which is selected from variants of 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, are generally denoted as “Eiv”.
In a preferred embodiment of the method according to the present invention, the reaction in step (6) is catalyzed by an L-carbamoylase Ei, and the polypeptide sequence of the L-carbamoylase Ei is selected from the group consisting of 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 (5) is catalyzed by an L-carbamoylase Ei, and the polypeptide sequence of the L-carbamoylase Ei is selected from the group consisting of SEQ ID NO: 1 and variants of SEQ ID NO: 1.
4.5.6.1 Preferred variants of SEQ ID NO: 1
According to the invention, the polypeptide sequence of the L-carbamoylase Ei may also be a variant of SEQ ID NO: 1 .
The L-carbamoylase Ei, the polypeptide sequence of which is SEQ ID NO: 1 , is denoted as “Eiois”. L-carbamoylases Ei, the polypeptide sequence of which is selected from variants of SEQ ID NO: 1 , are generally denoted as “Eioiv”.
A variant of the polypeptide sequence of SEQ ID NO: 1 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 1 .
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 1 is not identical to SEQ ID NO: 1.
According to the invention, an L-carbamoylase Eioiv has carbamoylase activity and L-carbamoylase activity, determined as described under items 4.5.4.2 and 4.5.4.3.1 .
According to the invention, an L-carbamoylase Eioiv preferably has carbamoylase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least
60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least
90 %, more preferably of at least 99 %, more preferably of at least 100 % the carbamoylase activity of the L-carbamoylase Eiois, wherein the carbamoylase activity of Eioiv, relative to the carbamoylase activity of Eiois, is determined by Assay C described under item 4.5.5.2.
It is even more preferable according to the invention, that an L-carbamoylase Eioiv has carbamoylase activity 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 % the carbamoylase activity of the
L-carbamoylase Eiois, wherein the carbamoylase activity of Eioiv, relative to the carbamoylase activity of Eiois, is determined by Assay C described under item 4.5.5.2.
4.5.6.2 Preferred variants of SEQ ID NO: 2
According to the invention, the polypeptide sequence of the L-carbamoylase Ei may also be a variant of SEQ ID NO: 2.
The L-carbamoylase Ei, the polypeptide sequence of which is SEQ ID NO: 2, is denoted as “EI02S”. L-carbamoylases Ei, the polypeptide sequence of which is selected from variants of SEQ ID NO: 2, are generally denoted as “EI02V”.
A variant of the polypeptide sequence of SEQ ID NO: 2 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 2.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 2 is not identical to SEQ ID NO: 2.
According to the invention, an L-carbamoylase EI02V has carbamoylase activity and L-carbamoylase activity, determined as described under items 4.5.4.2 and 4.5.4.3.1 .
According to the invention, an L-carbamoylase EI02V preferably has carbamoylase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least
60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least
90 %, more preferably of at least 99 %, more preferably of at least 100 % the carbamoylase activity of the L-carbamoylase EI02S, wherein the carbamoylase activity of EI02V, relative to the carbamoylase activity of EI02S, is determined by Assay C described under item 4.5.5.2.
It is even more preferable according to the invention, that an L-carbamoylase EI02V has carbamoylase activity 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 % the carbamoylase activity of the L-carbamoylase EI02S, wherein the carbamoylase activity of EI02V, relative to the carbamoylase activity of EI02S, is determined by Assay C described under item 4.5.5.2.
4.5.6.3 Preferred variants of SEQ ID NO: 3
According to the invention, the polypeptide sequence of the L-carbamoylase Ei may also be a variant of SEQ ID NO: 3.
The L-carbamoylase Ei, the polypeptide sequence of which is SEQ ID NO: 3, is denoted as “Eioss”. L-carbamoylases Ei, the polypeptide sequence of which is selected from variants of SEQ ID NO: 3, are generally denoted as “Eiosv”.
A variant of the polypeptide sequence of SEQ ID NO: 3 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 3.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 3 is not identical to SEQ ID NO: 3.
According to the invention, an L-carbamoylase Ewsv has carbamoylase activity and L-carbamoylase activity, determined as described under items 4.5.4.2 and 4.5.4.3.1 .
According to the invention, an L-carbamoylase Ewsv preferably has carbamoylase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least
60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least
90 %, more preferably of at least 99 %, more preferably of at least 100 % the carbamoylase activity of the L-carbamoylase Ewss, wherein the carbamoylase activity of Ewsv, relative to the carbamoylase activity of Ewss, is determined by Assay C described under item 4.5.5.2.
It is even more preferable according to the invention, that an L-carbamoylase Ewsv has carbamoylase activity 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 % the carbamoylase activity of the L-carbamoylase Ewss, wherein the carbamoylase activity of Ewsv, relative to the carbamoylase activity of Ewss, is determined by Assay C described under item 4.5.5.2.
4.5.6.4 Preferred variants of SEQ ID NO: 4
According to the invention, the polypeptide sequence of the L-carbamoylase Ei may also be a variant of SEQ ID NO: 4.
The L-carbamoylase Ei, the polypeptide sequence of which is SEQ ID NO: 4, is denoted as “Ems”. L-carbamoylases Ei, the polypeptide sequence of which is selected from variants of SEQ ID NO: 4, are generally denoted as “Emv”.
A variant of the polypeptide sequence of SEQ ID NO: 4 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 4.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 4 is not identical to SEQ ID NO: 4.
According to the invention, an L-carbamoylase Emv has carbamoylase activity and L-carbamoylase activity, determined as described under items 4.5.4.2 and 4.5.4.3.1 .
According to the invention, an L-carbamoylase Emv preferably has carbamoylase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least
60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least
90 %, more preferably of at least 99 %, more preferably of at least 100 % the carbamoylase activity of the L-carbamoylase Ems, wherein the carbamoylase activity of Emv, relative to the carbamoylase activity of Ems, is determined by Assay C described under item 4.5.5.2.
It is even more preferable according to the invention, that an L-carbamoylase Emv has carbamoylase activity 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 % the carbamoylase activity of the L-carbamoylase Ems, wherein the carbamoylase activity of E , relative to the carbamoylase activity of Ems, is determined by Assay C described under item 4.5.5.2.
4.5.6.5 Preferred variants of SEQ ID NO: 5
According to the invention, the polypeptide sequence of the L-carbamoylase Ei may also be a variant of SEQ ID NO: 5.
The L-carbamoylase Ei, the polypeptide sequence of which is SEQ ID NO: 5, is denoted as “Ems”. L-carbamoylases Ei, the polypeptide sequence of which is selected from variants of SEQ ID NO: 5, are generally denoted as “Emv”.
A variant of the polypeptide sequence of SEQ ID NO: 5 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 5.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 5 is not identical to SEQ ID NO: 5.
According to the invention, an L-carbamoylase E v has carbamoylase activity and L-carbamoylase activity, determined as described under items 4.5.4.2 and 4.5.4.3.1 .
According to the invention, an L-carbamoylase E v preferably has carbamoylase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least
60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least
90 %, more preferably of at least 99 %, more preferably of at least 100 % the carbamoylase activity of the L-carbamoylase Ems, wherein the carbamoylase activity of E v, relative to the carbamoylase activity of Ems, is determined by Assay C described under item 4.5.5.2.
It is even more preferable according to the invention, that an L-carbamoylase E v has carbamoylase activity 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 % the carbamoylase activity of the
L-carbamoylase Ems, wherein the carbamoylase activity of E v, relative to the carbamoylase activity of Ems, is determined by Assay C described under item 4.5.5.2. 4.5.6.6 Preferred variants of SEQ ID NO: 6
According to the invention, the polypeptide sequence of the L-carbamoylase Ei may also be a variant of SEQ ID NO: 6.
The L-carbamoylase Ei, the polypeptide sequence of which is SEQ ID NO: 6, is denoted as “Ewes”. L-carbamoylases Ei, the polypeptide sequence of which is selected from variants of SEQ ID NO: 6, are generally denoted as “Eioev”.
A variant of the polypeptide sequence of SEQ ID NO: 6 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 6.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 6 is not identical to SEQ ID NO: 6.
According to the invention, an L-carbamoylase Eioev has carbamoylase activity and L-carbamoylase activity, determined as described under items 4.5.4.2 and 4.5.4.3.1 .
According to the invention, an L-carbamoylase Eioev preferably has carbamoylase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least
60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least
90 %, more preferably of at least 99 %, more preferably of at least 100 % the carbamoylase activity of the L-carbamoylase Ewes, wherein the carbamoylase activity of Eioev, relative to the carbamoylase activity of Ewes, is determined by Assay C described under item 4.5.5.2.
It is even more preferable according to the invention, that an L-carbamoylase Eioev has carbamoylase activity 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 % the carbamoylase activity of the
L-carbamoylase Ewes, wherein the carbamoylase activity of Eioev, relative to the carbamoylase activity of Ewes, is determined by Assay C described under item 4.5.5.2. 4.5.6.7 Preferred variants of SEQ ID NO: 7
According to the invention, the polypeptide sequence of the L-carbamoylase Ei may also be a variant of SEQ ID NO: 7.
The L-carbamoylase Ei, the polypeptide sequence of which is SEQ ID NO: 7, is denoted as “Eiozs”. L-carbamoylases Ei, the polypeptide sequence of which is selected from variants of SEQ ID NO: 7, are generally denoted as “Eiozv”.
A variant of the polypeptide sequence of SEQ ID NO: 7 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 7.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 7 is not identical to SEQ ID NO: 7.
According to the invention, an L-carbamoylase EI07V has carbamoylase activity and L-carbamoylase activity, determined as described under items 4.5.4.2 and 4.5.4.3.1 .
According to the invention, an L-carbamoylase EI07V preferably has carbamoylase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least
60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least
90 %, more preferably of at least 99 %, more preferably of at least 100 % the carbamoylase activity of the L-carbamoylase EI07S, wherein the carbamoylase activity of EI07V, relative to the carbamoylase activity of EI07S, is determined by Assay C described under item 4.5.5.2.
It is even more preferable according to the invention, that an L-carbamoylase EI07V has carbamoylase activity 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 % the carbamoylase activity of the
L-carbamoylase EI07S, wherein the carbamoylase activity of EI07V, relative to the carbamoylase activity of EI07S, is determined by Assay C described under item 4.5.5.2. 4.5.6.8 Preferred variants of SEQ ID NO: 8
According to the invention, the polypeptide sequence of the L-carbamoylase Ei may also be a variant of SEQ ID NO: 8.
The L-carbamoylase Ei, the polypeptide sequence of which is SEQ ID NO: 8, is denoted as “Eioss”. L-carbamoylases Ei, the polypeptide sequence of which is selected from variants of SEQ ID NO: 8, are generally denoted as “Eiosv”.
A variant of the polypeptide sequence of SEQ ID NO: 8 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 8.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 8 is not identical to SEQ ID NO: 8.
According to the invention, an L-carbamoylase Eiosv has carbamoylase activity and L-carbamoylase activity, determined as described under items 4.5.4.2 and 4.5.4.3.1 .
According to the invention, an L-carbamoylase Eiosv preferably has carbamoylase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least
60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least
90 %, more preferably of at least 99 %, more preferably of at least 100 % the carbamoylase activity of the L-carbamoylase Eioss, wherein the carbamoylase activity of Eiosv, relative to the carbamoylase activity of Eioss, is determined by Assay C described under item 4.5.5.2.
It is even more preferable according to the invention, that an L-carbamoylase Eiosv has carbamoylase activity 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 % the carbamoylase activity of the
L-carbamoylase Eioss, wherein the carbamoylase activity of Ews, relative to the carbamoylase activity of Eioss, is determined by Assay C described under item 4.5.5.2. 4.5.G.9 Preferred variants of SEQ ID NO: 9
According to the invention, the polypeptide sequence of the L-carbamoylase Ei may also be a variant of SEQ ID NO: 9.
The L-carbamoylase Ei, the polypeptide sequence of which is SEQ ID NO: 9, is denoted as “Eioss”. L-carbamoylases Ei, the polypeptide sequence of which is selected from variants of SEQ ID NO: 9, are generally denoted as “Eiosv”.
A variant of the polypeptide sequence of SEQ ID NO: 9 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 9.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 9 is not identical to SEQ ID NO: 9.
According to the invention, an L-carbamoylase EiOgv has carbamoylase activity and L-carbamoylase activity, determined as described under items 4.5.4.2 and 4.5.4.3.1 .
According to the invention, an L-carbamoylase EiOgv preferably has carbamoylase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least
60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least
90 %, more preferably of at least 99 %, more preferably of at least 100 % the carbamoylase activity of the L-carbamoylase EI09S, wherein the carbamoylase activity of EI09V, relative to the carbamoylase activity of EI09S, is determined by Assay C described under item 4.5.5.2.
It is even more preferable according to the invention, that an L-carbamoylase EiOgv has carbamoylase activity 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 % the carbamoylase activity of the
L-carbamoylase EI09S, wherein the carbamoylase activity of EI09V, relative to the carbamoylase activity of EI09S, is determined by Assay C described under item 4.5.5.2. 4.5.7 Preferred method conditions in step (5)
The reaction in step (6) of the method according to the present invention may be carried out under conditions known to the skilled person.
The reaction medium is preferably aqueous, more preferably an aqueous buffer.
Exemplary buffers commonly used in biotransformation reactions and advantageously used herein include Tris, phosphate, or any of Good's buffers, such as 2-(/V-morpholino)ethanesulfonic acid (“MES”), /V-(2-acetamido)iminodiacetic acid (“ADA”), piperazine-/V,/\/'-bis(2-ethanesulfonic acid) (“PIPES”), /V-(2-acetamido)-2- aminoethanesulfonic acid (“ACES”), P-hydroxy-4- morpholinepropanesulfonic acid (“MOPSO”), cholamine chloride, 3-(/V-morpholino)propanesulfonic acid (“MOPS”), /V,/V-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”), acetamidoglycine,
3-(/V-Tris(hydroxymethyl)methylamino(-2-hydroxypropane)sulfonic acid (“TAPSO”), piperazine- /V,/\/'-bis(2-hydroxypropanesulfonic acid) (“POPSO”), 4-(2- Hydroxyethyl)piperazine- 1-(2-hydroxypropanesulfonic acid) (“HEPPSO”), 3-[4-(2-Hydroxyethyl)-1-piperazinyl]propanesulfonic acid (“HEPPS”), tricine, glycinamide, bicine, or 3-[[1 ,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1 -sulfonic acid (“TAPS”).
In some embodiments, ammonium can act as a buffer. One or more organic solvents can also be added to the reaction.
The buffer preferably contains metal salts, more preferably metal salts such as halogenides of metals, preferably halogenides of monovalent or bivalent or trivalent metals, preferably chlorides of monovalent or bivalent metals, preferably C0CI2 or MnCh, preferably C0CI2.
The concentration of these metal salts in the reaction medium is preferably in the range from 1 pM to 1 M, more preferably 1 mM to 100 mM, even more preferably 1 to 10 mM.
Preferably, step (6) of the method according to the invention is carried out in a phosphate buffer.
The pH of the reaction medium in step (6) of the method according to the invention is preferably in the range of from 2 to 10, more preferably in the range of from 5 to 8, more preferably 7.2 to 7.5, most preferably 7.5.
Preferably, step (6) of the method according to the invention is carried out at a temperature in the range of from 20 °C to 70 °C, more preferably in the range of from 30 °C to 55 °C, most preferably 50 °C. Preferably, the total concentration of all carbamoylases Ei in the reaction solution in step (6) is in the range of from 1 pM to 10 mM, preferably 10 pM to 1 mM, more preferably 0.1 mM to 0.5 mM, most preferably 0.4 mM.
In alternative preferred embodiments, the total concentration of all carbamoylases Ei in the reaction solution in step (6) is in the range of from 1 pg/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.
Preferably, the initial concentration of all the compounds according to formula L-(ll) in the reaction medium in step (6) is in the range of from 1 pM to 1 M, preferably of from 10 pM to 0.5 M, more preferably of from 0.1 mM to 0.1 M, more preferably of from 1 mM to 10 mM, most preferably 1.25 mM.
If compounds according to formula D-(ll) are present in the reaction medium in step (6), the initial concentration of all the compounds according to formula D-(ll) in the reaction medium is preferably from 1 % to 100 % the concentration of all the compounds according to formula L-(ll), more preferably 10 % to 100 % the concentration, even more preferably 50 to 100 %, even more preferably 100 % the concentration of all the compounds according to formula L-(ll).
“Initial concentration of all the compounds according to formula L-(ll)/ D-(ll)”” refers to the concentration of the respective compound L-(ll) or D-(ll) respectively, in the reaction medium when the respective compounds are employed in step (6).
4.5.8 Step (fi) [optional]
4.5.8.1 Enzymatic catalysis of step (fi)
In a preferred embodiment of the method according to the invention, the mixture Mu provided in step (y) is obtained by a step (p) wherein in a mixture Mm comprising compounds according to LL- (III) and DL-(III), at least a part of the compounds according to formula LL-(III) are reacted to give compounds according to LL-(II), and at least a part of the compounds according to formula DL-(III) are reacted to give compounds according to DL-(II), wherein LL-(III) and DL-(III) have the following formulae:
Figure imgf000041_0001
The reaction according to step (p) is catalyzed by a hydantoinase E2.
R in LL-(IH) and DL-(III) has the same meaning as described for LL-(I) and DL-(I).
Step (p) gives the starting material for step (6)
The reaction according step (p) is enzymatically catalyzed, namely it is catalyzed by a hydantoinase E2.
4.5.8.2 Enantioselective and enantiospecific catalysis of step (/3)
Step (p) of the method according to the present invention is preferably L-enantioselective, even more preferably L-enantiospecific.
In such a preferred embodiment, Mixture Mm comprises, besides LL-(III) and DL-(III), the respective enantiomer of LL-(III) and DL-(III), which is DD-(III) and LD-(III):
Figure imgf000041_0002
LD-(III) DD-(III)
Such mixtures comprising all four diastereomers LL-(III), DL-(III), LD-(III), and DD-(III) are referred to as “mixture Mm* hereinafter.
For such mixtures Mm*, a parallel reaction according to step (p)* may be observed. Namely, in the reaction according to step (p)*, LD-(III), i.e. the enantiomer of DL-(III), in mixture M * is reacted to give a compound according to formula LD-(II), and/or, preferably and, DD-(III), i.e. the enantiomer of LL-(III), in mixture Mm* is reacted to give a compound according to formula DD-(II).
In case that step (p) is “L-enantioselective”, this means that in case a mixture M * is employed in step (p), then there is either no reaction according to step (p)* or, in case there is a reaction according to step (p)*, then the rate of reaction according to step (p)* is lesser than the rate of the reaction according to step (p). Step (p) is “L-enantiospecific”, if the rate of reaction according to step (p)* is essentially zero, i.e. there is no reaction according to step (p)*.
In a preferred embodiment, the mixture Mm* is a racemic mixture of enantiomer L-(lll) and enantiomer D-(lll), meaning that the molar ratio of all compounds according to L-(lll) to compounds according to D-(lll) is essentially 1 : 1 , wherein:
Figure imgf000042_0001
In other preferred embodiments, the molar ratio of all compounds L-(lll) to all compounds D-(lll) in mixture Mm* is in the range of from 99 : 1 to 1 : 99, more preferably in the range of from 1.01 : 1 to 1 : 99, more preferably in the range of from 1 : 1 to 1 : 99, more preferably in the range of from 1 : 1.01 to 1 : 99, more preferably in the range of from 1 : 1.01 to 1 : 9, more preferably in the range of from 1 : 1.01 to 1 : 8, more preferably in the range of from 1 : 1.01 to 1 : 3.
Step (p) is in particular L-enantioselective, if it is preferably catalyzed by an L-hydantoinase E2, which may be determined by the skilled person as set forth under 4.5.10.3.
In case step (p) is L-enantioselective, the reaction according to step (p) proceeds preferably at a reaction rate that is at least 2 times greater, preferably at least 10 times greater, more preferably at least 100 times greater, even more preferably at least 103 times greater, even more preferably at least 104 times greater, even more preferably at least 105 times greater than the reaction rate at which step (p)* proceeds.
To quantify the factor at which the reaction rate of step (p) proceeds compared to the reaction rate of step (p)*, the following test may be carried out:
(1) An equimolar mixture of compounds L-(lll) and D-(lll) [i.e. the ratio of the sum of the molar masses of the diastereosiomers [LL-(III) and DL-(III)] to the sum of the molar masses of the diastereosiomers [LD-(III) and DD-(III)] is 1 : 1] is provided. Likewise, the molar ratio of LL-(III) to DL-(III) is 1 : 1 , and the molar ratio of LD-(III) to DD-(III) is 1 : 1 .
The mixture is subjected to the respective reactions conditions and the development of the two products L-(ll) [LL-(II) and DL-(II)] versus the development of the products D-(ll) [LD-(II) and DD-(II)] is monitored overtime (e.g. by LC-MS as set forth under item 5.4). (2) When nLmo = 10 mol-% of the initially employed L-(lll) has reacted to the respective product L-(ll), the molar amount of D-(ll) that was formed by reaction from D-(lll) [in mol-% relative to the initially employed D-(ll)] is measured (= nDino).
(3) The ratio of nLino / nDmo = 10 / nDino gives the factor by which the reaction rate of step (p) is higher than the reaction rate of step (p)*.
-> If nLin o I nDm o > 1 , the reaction rate of step (p) is greater than the reaction rate of step (p)*.
-> If nLin o I nDm o < 1 , the reaction rate of step (p) is lesser than the reaction rate of step (p)*.
-> If nLin o / nDm o = 1 , the reaction rates of steps (b and (p)* are the same.
The reaction according to step (p) of the preferred embodiment of the invention is catalyzed by a hydantoinase (“dihydropyrimidinase”) E2.
Namely, it was surprisingly found that hydantoinases accept compounds of formula L-(lll) as substrates and convert them to products according to formulae L-(ll), and hence catalyze the reaction according to step (p). This finding is of high scientific and economic value, as it further broadens the scope of synthetic routes based on new starting materials for the production of L-glufosinate P-esters. Even more surprisingly, it is suggested that L-glufosinate hydantoin, i.e. the compound according to formula L-(lll), in which R = H, does not undergo reaction by hydantoinases to give the respective LGA carbamoylate.
In nature, hydantoinases (“dihydropyrimidinases”) generally catalyze the reaction of 5,6-dihydrouracil to produce ureidopropionate (see the following reaction <2A>):
Figure imgf000043_0001
They also catalyze the ring opening of monosubstituted hydantoins to give the respective carbamoyl amino acid according to the following reaction <2B>, wherein R* may be an organic residue, e.g. a side chain of one of the naturally occurring amino acids.
<2B>:
Figure imgf000043_0002
Chapter 1 .3 (pages 7 to 11) of the dissertation “ Untersuchungen zur Substratspezifitat und Enantioselektivitat mikrobieller Hydantoinasen/ Investigations of substrate specificity and enantioselectivity of microbial hydantoinases" by T. Waniek, University of Stuttgart, 2000 (available under: https://elib.uni-stuttgart.de/bitstream/11682/1511/1/Diss. pdf) gives an overview over hydantoinases. It was now surprisingly found that hydantoinases also accept substrates in which
O ii
Figure imgf000044_0004
Surprisingly, they supposedly do not accept substrates in which R* = RY =
Figure imgf000044_0001
wherein R = H.
In the context of the present invention, a “hydantoinase E2” is a hydantoinase that catalyzes the following reaction <20 of a carbamoyl substrate S’L to the respective amino acid product P’L, wherein R* = RY and preferably R* = Rz:
Figure imgf000044_0002
In particular, the hydantoinase E2 is a “L-hydantoinase”, i.e. it has a greater catalytic activity for reaction <20 than for reaction <20, wherein the substrate S’D in the reaction <20 is the enantiomer of the substrate SL in the reaction <20:
Figure imgf000044_0003
As an L-hydantoinase has a higher catalytic activity for reaction <20 than for reaction <2D>, it is “L-enantioselective”. An L-hydantoinase that has no catalytic activity for reaction <20 and thus only has catalytic activity for reaction <20 is “L-enantiospecific”.
A “D-hydantoinase” is defined as a hydantoinase which is “D-enantioselective”, i.e. it has a higher catalytic activity for reaction <20 than for reaction <20. A D-carbamoylase that does not catalyze reaction <20 and thus only has catalytic activity for reaction <20 is “D-enantiospecific”. A hydantoinase which has the same catalytic activity for reaction <20 as for reaction <2D>, is referred to as “non enantioselective hydantoinase”.
For determination whether a hydantoinase may be denoted as “L-hydantoinase”, “D-hydantoinase” or “non-enantioselective hydantoinase” in the context of the present invention, the procedure according to Assay E (item 4.5.10) may preferably be used.
The hydantoinase E2, in particular the L-hydantoinase E2, that may be used in step (p) of the preferred embodiment of the method according to the invention may originate from Arthrobacter sp., in particular Arthrobacter crystallopoietes, Arthrobacter aurescens, Arthrobacter sp. BT801 ; Alcaligenes sp., in particular Alcaligenes faecalis subsp. faecalis; Bacillus sp., in particular Bacillus fordii; Microbacterium sp., in particular Microbacterium liquefaciens strain AJ3912; Pseudomonas sp., in particular Pseudomonas fluorescens, Pseudomonas aeruginosa.
A hydantoinase E2, in particular an L-hydantoinase E2 suitable for the method according to the present invention may be the enzyme HyuH, which originates from Arthrobacter. Another enzyme may be Dht.
Even more preferably, the hydantoinase E2, in particular the L-hydantoinase E2, that may preferably be used in step (p) according to the first aspect of the invention may originate from Arthrobacter sp., in particular Arthrobacter crystallopoietes, Arthrobacter aurescens, Arthrobacter sp. BT801 , Arthrobacter aurescens DSM 9771 , most preferably from Arthrobacter aurescens DSM 9771.
A hydantoinase suitable for the method according to the present invention is described e.g. in WO 01/23582 A1 and by J.M. Clemente-Jimenez, S. Martinez-Rodriguez, F. Rod rig uez- Vico, F.J.L. Heras-Vazquez, Recent Pat. Biotechnology 2008, 2, 35 - 46; G. Latacz, E. Pekala, K. Kiec- Kononowicz, Biotechnologia 2006, 2, 189 - 205.
Further suitable hydantoinases are described by K. Yokozeki, H. Yoshiteru, K. Kubota, Agric. Biol. Chem. 1987, 51 , 737 - 746.
The hydantoinase E2 that may be used in preferred step (p) of the method according to the present invention may be a hydantoinase categorized in the EC class EC 3.5.2.2.
The following table 2 gives preferred examples for polypeptide sequences of hydantoinases E2 that may be preferably used in step (p) of the preferred embodiment of the method according to the invention. The genes encoding the respective hydantoinase E2 and the respective accession code are indicated as far as known. Table 2
Figure imgf000046_0001
In a preferred embodiment of the method according to the present invention, the reaction according to step (p) is catalyzed by an hydantoinase E2, wherein the polypeptide sequence of 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, preferably SEQ ID NO: 10 and variants thereof.
4.5.9 Assays DL and DD for determining hydantoinase activity
The skilled person is aware of hydantoinases, in particular L-hydantoinases, that may be used in step (p) of the preferred method according to the invention.
In particular, Assay DL, described in the following, may be used to determine hydantoinase and L-hydantoinase activity of a given enzyme EY and may advantageously be used according to the invention to determine hydantoinase and L-hydantoinase activity 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, variants of SEQ ID NO: 16.
For comparative reasons, Assay DD may be used to determine D-hydantoinase activity of a given enzyme EY.
For the purpose of Assay DL and Assay DD, the molar mass of the enzyme EY to be tested is calculated as the molar mass of the polypeptide sequence of EY. 4.5.9.1 Assay DL:
To 0.9 ml of an aqueous reaction solution (phosphate buffer, pH 7.2, 10 mM MnCh), containing 50 mM of an n-butyl P-ester of hydantoin glufosinate of the formula L-(lll), wherein R = n-butyl, and wherein LL-(III) and DL-(III) are equimolar, are added 400 nmol of EY in 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh). The resulting solution is incubated at 25 °C, and the pH is held at pH
7.2 by addition of 1 M NaOH. After 300 minutes, the reaction is stopped by addition of 2 M HCI to achieve a pH of 2.5, and the molar amount of the n-butyl P-ester of carbamoyl glufosinate according to formula L-(ll), wherein R = n-butyl, is determined. L-(ll), wherein R = n-butyl, may be detected by the LC-MS method as described in the example section (item 5.4) for detection of LGA.
4.5.9.2 Assay DD:
To 0.9 ml of an aqueous reaction solution (phosphate buffer, pH 7.2, 10 mM MnCh), containing 50 mM of an n-butyl P-ester of hydantoin glufosinate of the formula D-(lll), and wherein LD-(III) and DD-(III) are equimolar, wherein R = n-butyl, are added 400 nmol of EY in 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh). The resulting solution is incubated at 25 °C, and the pH is held at pH
7.2 by addition of 1 M NaOH. After 300 minutes, the reaction is stopped by addition of 2 M HCI to achieve a pH of 2.5, and the molar amount of the n-butyl P-ester of carbamoyl glufosinate according to formula D-(ll), wherein R = n-butyl, is determined. D-(ll), wherein R = n-butyl, may be detected by the LC-MS method as described in the example section (item 5.4) for detection of LGA.
4.5.10 Assay E for identifying hydantoinases, L-hydantoinases, D-hydantoinases, L- and D-enantiospecificity
The hydantoinase E2 according to the invention is preferably an L-hydantoinase, more preferably L-enantiospecific.
Whether a given enzyme EY may be considered a hydantoinase E2, in particular an L-hydantoinase, may be determined in the context of the present invention by the following Assay E:
4.5.10.1 Assay E:
E-1 . Firstly, Assay DL as set forth under item 4.5.9.1 is conducted, and the obtained molar amount of the compound of the formula L-(ll), wherein R = n-butyl, is determined according to Assay DL. E-2 Secondly, Assay DD as set forth under item 4.5.9.2 is conducted, and the obtained molar amount of the compound of the formula D-(ll), wherein R = n-butyl, is determined according to Assay DD.
E-3. Then, step E-1 is repeated, except that instead of the addition of 400 nmol EY in 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh), 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh) without EY is added.
E-4 Then, step E-2 is repeated, except that instead of the addition of 400 nmol EY in 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh), 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh) without EY is added.
4.5.10.2 Hydantoinase activity:
If the molar amount of the compound of the formula L-(ll), wherein R = n-butyl, that is determined in step E-1 , is greater than the molar amount of the compound of formula L-(ll), wherein R = n-butyl, that is determined in step E-3, then EY is deemed to have hydantoinase activity, and hence may be considered a hydantoinase E2 in the context of the invention.
4.5.10.3 L-Hydantoinase activity
4.5.10.3.1 L-hydantoinases
(i) If the molar amount of the compound of the formula L-(ll), wherein R = n-butyl, that is determined in step E-1 , is greater than the molar amount of the compound of the formula L-(ll), wherein R = n-butyl, that is determined in step E-3, and
(ii) if the molar amount of the compound of the formula D-(ll), wherein R = n-butyl, that is determined in step E-2, is greater than or the same as the molar amount of the compound of the formula D-(ll), wherein R = n-butyl, that is determined in step E-4, and
(iii) if, in addition, the molar amount of the compound of the formula L-(ll), wherein R = n-butyl, that is determined in step E-1 is greater than the molar amount of the compound of the formula D-(ll), wherein R = n-butyl, that is determined in step E-2, then EY is deemed to have L-hydantoinase activity, and hence may be considered an L-hydantoinase in the context of the invention. In this case, EY is deemed to be “L-enantioselective” in the context of the invention.
For the sake of clarity, it is pointed out that condition (iii) is automatically fulfilled in those cases in which the molar amount of the compound of formula D-(ll), wherein R = n-butyl, that is determined in step E-2 is the same as the molar amount of the compound of formula D-(ll), wherein R = n-butyl, that is determined in step E-4. (i) If the molar amount of the compound of formula L-(ll), wherein R = n-butyl, that is determined in step E-1 , is greater than the molar amount of the compound of formula L-(ll), wherein R = n-butyl, that is determined in step E-3, and
(ii) if the molar amount of the compound of the formula D-(ll), wherein R = n-butyl, that is determined in step E-2, is the same as the molar amount of the compound of the formula D-(ll), wherein R = n-butyl, that is determined in step E-4, and then EY is deemed to have L-hydantoinase activity, and hence may be considered a L-hydantoinase in the context of the invention. In this case, EY is deemed to be not only “L-enantioselective”, but also “L-enantiospecific” in the context of the invention.
For L-hydantoinases EY that are not L-enantiospecific, the L-enantioselectivity may then be quantified by dividing the molar amount of the compound of formula L-(ll), wherein R = n-butyl, that is determined in E-1 , by the molar amount of the compound of formula D-(ll), wherein R = n-butyl, that is determined in E-2, and then multiplying the obtained value by 100, giving the L-enantioselectivity of EY in %.
4.5.10.3.2 D-hydantoinases
(i) If the molar amount of the compound of formula L-(ll), wherein R = n-butyl, that is determined in step E-1 , is greater than or the same as the molar amount of the compound of formula L-(ll), wherein R = n-butyl, that is determined in step E-3, and
(ii) if the molar amount of the compound of formula D-(ll), wherein R = n-butyl, that is determined in step E-2 is greater than the molar amount of the compound of formula D-(ll), wherein R = n-butyl, that is determined in step E-4, and
(iii) if, in addition, the molar amount of the compound of formula D-(ll), wherein R = n-butyl, that is determined in step E-2, is greater than the molar amount of the compound of formula L-(ll), wherein R = n-butyl, that is determined in step E-1 , then EY is deemed to have D-hydantoinase activity, and hence may be considered a D-hydantoinase. In this case, EY is “D-enantioselective” in the context of the invention.
For the sake of clarity, it is pointed out that condition (iii) is automatically fulfilled in those cases in which the molar amount of the compound of formula L-(ll), wherein R = n-butyl, that is determined in step E-1 is the same as the molar amount of the compound of formula L-(ll), wherein R = n-butyl, that is determined in step E-3. (i) If the molar amount of the compound of formula L-(ll), wherein R = n-butyl, that is determined in step E-1 , is the same as the molar amount of the compound of formula L-(ll), wherein R = n-butyl, that is determined in step E-3, and
(ii) if the molar amount of the compound of formula D-(ll), wherein R = n-butyl, that is determined in step E-2, is the greater than the molar amount of the compound of formula D-(ll), wherein
R = n-butyl, that is determined in step E-4, then EY is deemed to have D-hydantoinase activity, and hence may be considered a D-hydantoinase in the context of the invention. In this case, EY is deemed to be not only “D-enantioselective”, but also “D-enantiospecific” in the context of the invention.
For D-hydantoinases EY that are not D-enantiospecific, the D-enantioselectivity may then be quantified by dividing the molar amount of the compound of formula D-(ll), wherein R = n-butyl, that is determined in step E-2, by the molar amount of the compound of formula L-(ll), wherein
R = n-butyl, that is determined in step E-1 and then multiplying the obtained value by 100, giving the D-enantioselectivity of EY in %.
4.5.10.3.3 Non-enantioselective hydantoinases
(i) If the molar amount of the compound of formula L-(ll), wherein R = n-butyl, that is determined in step E-1 , is greater than the molar amount of the compound of formula L-(ll), wherein R = n-butyl, that is determined in step E-3, and
(ii) if the molar amount of the compound of formula D-(ll), wherein R = n-butyl, that is determined in step E-2, is greater than the molar amount of the compound of formula D-(ll), wherein R = n-butyl, that is determined in step E-4, and
(iii) if, in addition, the molar amount of the compound of formula D-(ll), wherein R = n-butyl, that is determined in step E-2 is the same as the molar amount of the compound of formula L-(ll), wherein R = n-butyl, that is determined in step E-1 , then EY is deemed to be a non-enantioselective hydantoinase in the context of the invention. In this case, EY is “non-enantioselective”.
4.5.11 Assay F for identifying preferred hydantoinases variants of SEQ ID NOs: 10 - 16
4.5.11.1 L- and D-enantioselective hydantoinase variants
An enzyme, the polpypetide sequence of which 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 has hydantoinase, in particular L-hydantoinase activity.
In a preferred embodiment of the method according to the invention, the polypeptide sequence of the hydantoinase, in particular the L-hydantoinase E2 is selected from the group consisting 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.
The term variant is defined under item 4.3.
In the context of the invention, an enzyme, the polypeptide sequence of which 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-enantiospecific.
Whether a given enzyme EY, the polypeptide sequence of which 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, L-hydantoinase activity and is L-enantiospecific may be determined as set forth under items 4.5.10.2 and 4.5.10.3.1 , respectively.
The hydantoinase activity of a given hydantoinase E2V, the polypeptide sequence of which 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, relative to the hydantoinase activity of an hydantoinase E2S, wherein the polypeptide sequence of E2S 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, may be determined in the context of the present invention by the following Assay F:
4.5.11.2 Assay F
F-1 Assay DL as set forth under item 4.5.9.1 is conducted, wherein E2S is the enzyme to be tested. The obtained molar amount of the compound according to formula L-(ll), wherein R = n-butyl, is determined according to Assay DL.
F-2 Step F-1 is repeated, except that, instead of E2S, E2V is used as the enzyme to be tested.
F-3. Then, the molar amount of the compound according to formula L-(ll), wherein R = n-butyl, that is determined in step F-2, is divided by the molar amount of the compound according to formula L-(ll), wherein R = n-butyl, that is determined in step F-1 , and the obtained ratio is multiplied by 100, giving the hydantoinase activity of hydantoinase E2V relative to the hydantoinase activity of the hydantoinase E2S, in %.
4.5.12 Preferred hydantoinase variants of SEQ ID NOs: 10 - 16
In the context of the invention, hydantoinases E2, the polypeptide sequence of which 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, are generally denoted as “E2S”. Hydantoinases E2, the polypeptide sequence of which is selected from variants of a sequence 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, are generally denoted as “E2V”.
In a preferred embodiment of the method according to the present invention, the reaction in step (p) is catalyzed by a hydantoinase E2, preferably an L-hydantoinase E2, and the polypeptide sequence of the hydantoinase E2, preferably the L-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.
More preferably, the reaction in step (p) is catalyzed by a hydantoinase E2, preferably an L-hydantoinase E2, and the polypeptide sequence of the hydantoinase E2, preferably the L-hydantoinase E2, is selected from the group consisting of SEQ ID NO: 10 and variants of SEQ ID NO: 10.
4.5.12.1 Preferred variants of SEQ ID NO: 10
According to the invention, the polypeptide sequence of the hydantoinase E2, preferably the polypeptide sequence of the L-hydantoinase E2, may also be a variant of SEQ ID NO: 10.
The hydantoinase E2, the polypeptide sequence of which is SEQ ID NO: 10, is denoted as “E2IOS”. The hydantoinase E2, the polypeptide sequence of which is selected from variants of SEQ ID NO: 10, are generally denoted as “E2IOV”.
A variant of the polypeptide sequence of SEQ ID NO: 10 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 10.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 10 is not identical to SEQ ID NO: 10.
According to the invention, a hydantoinase E2IOV has hydantoinase activity and preferably L-hydantoinase activity, determined as described under items 4.5.10.2 and 4.5.10.3.1.
According to the invention, a hydantoinase E2IOV preferably has hydantoinase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoinase activity of the hydantoinase E2IOS, wherein the hydantoinase activity of E2IOV, relative to the hydantoinase activity of E2IOS is determined by Assay F described under item 4.5.11 .2.
It is even more preferably according to the invention, that a hydantoinase E2IOV has hydantoinase activity 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 % the hydantoinase activity of the hydantoinase E2IOS, wherein the hydantoinase activity of E2IOV, relative to the hydantoinase activity E2IOS is determined by Assay F described under item 4.5.11 .2.
4.5.12.2 Preferred variants of SEQ ID NO: 11
According to the invention, the polypeptide sequence of the hydantoinase E2, preferably the polypeptide sequence of the L-hydantoinase E2, may also be a variant of SEQ ID NO: 11 .
The hydantoinase E2, the polypeptide sequence of which is SEQ ID NO: 11 , is denoted as “E2nS”.
The hydantoinase E2, the polypeptide sequence of which is selected from variants of SEQ ID NO: 1 1 , are generally denoted as “E2nV”.
A variant of the polypeptide sequence of SEQ ID NO: 11 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 11 .
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 11 is not identical to SEQ ID NO: 11.
According to the invention, a hydantoinase E2nV has hydantoinase activity and preferably L-hydantoinase activity, determined as described under items 4.5.10.2 and 4.5.10.3.1.
According to the invention, a hydantoinase E2nV preferably has hydantoinase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoinase activity of the hydantoinase E2ns, wherein the hydantoinase activity of E2nv, relative to the hydantoinase activity of E2HS is determined by Assay F described under item 4.5.11 .2.
It is even more preferably according to the invention, that a hydantoinase E2nV has hydantoinase activity 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 % the hydantoinase activity of the hydantoinase E2nS, wherein the hydantoinase activity of E2nV, relative to the hydantoinase activity E2nS is determined by Assay F described under item 4.5.11 .2.
4.5.12.3 Preferred variants of SEQ ID NO: 12
According to the invention, the polypeptide sequence of the hydantoinase E2, preferably the polypeptide sequence of the L-hydantoinase E2, may also be a variant of SEQ ID NO: 12.
The hydantoinase E2, the polypeptide sequence of which is SEQ ID NO: 12, is denoted as “E2I2S”. The hydantoinase E2, the polypeptide sequence of which is selected from variants of SEQ ID NO: 12, are generally denoted as “E2I2V”.
A variant of the polypeptide sequence of SEQ ID NO: 12 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 12.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 12 is not identical to SEQ ID NO: 12.
According to the invention, a hydantoinase E2I2V has hydantoinase activity and preferably L-hydantoinase activity, determined as described under items 4.5.10.2 and 4.5.10.3.1.
According to the invention, a hydantoinase E2I2V preferably has hydantoinase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoinase activity of the hydantoinase E212S, wherein the hydantoinase activity of E212V, relative to the hydantoinase activity of E212S is determined by Assay F described under item 4.5.11 .2.
It is even more preferably according to the invention, that a hydantoinase E212V has hydantoinase activity 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 % the hydantoinase activity of the hydantoinase E212S, wherein the hydantoinase activity of E212V, relative to the hydantoinase activity E212S is determined by Assay F described under item 4.5.11 .2.
4.5.12.4 Preferred variants of SEQ ID NO: 13
According to the invention, the polypeptide sequence of the hydantoinase E2, preferably the polypeptide sequence of the L-hydantoinase E2, may also be a variant of SEQ ID NO: 13.
The hydantoinase E2, the polypeptide sequence of which is SEQ ID NO: 13, is denoted as “E2i3s”.
The hydantoinase E2, the polypeptide sequence of which is selected from variants of SEQ ID NO: 13, are generally denoted as “E2i3v”.
A variant of the polypeptide sequence of SEQ ID NO: 13 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 13.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 13 is not identical to SEQ ID NO: 13.
According to the invention, a hydantoinase E213V has hydantoinase activity and preferably L-hydantoinase activity, determined as described under items 4.5.10.2 and 4.5.10.3.1.
According to the invention, a hydantoinase E213V preferably has hydantoinase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoinase activity of the hydantoinase E213S, wherein the hydantoinase activity of E2isv, relative to the hydantoinase activity of E2i3s is determined by Assay F described under item 4.5.11 .2.
It is even more preferably according to the invention, that a hydantoinase E2I3V has hydantoinase activity 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 % the hydantoinase activity of the hydantoinase E2I3S, wherein the hydantoinase activity of E2I3V, relative to the hydantoinase activity E2I3S is determined by Assay F described under item 4.5.11 .2.
4.5.12.5 Preferred variants of SEQ ID NO: 14
According to the invention, the polypeptide sequence of the hydantoinase E2, preferably the polypeptide sequence of the L-hydantoinase E2, may also be a variant of SEQ ID NO: 14.
The hydantoinase E2, the polypeptide sequence of which is SEQ ID NO: 14, is denoted as “E2I4S”. The hydantoinase E2, the polypeptide sequence of which is selected from variants of SEQ ID NO: 14, are generally denoted as “E2I4V”.
A variant of the polypeptide sequence of SEQ ID NO: 14 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 14.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 14 is not identical to SEQ ID NO: 14.
According to the invention, a hydantoinase E2I4V has hydantoinase activity and preferably L-hydantoinase activity, determined as described under items 4.5.10.2 and 4.5.10.3.1.
According to the invention, a hydantoinase E2I4V preferably has hydantoinase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoinase activity of the hydantoinase E2I4S, wherein the hydantoinase activity of E2I4V, relative to the hydantoinase activity of E2I4S is determined by Assay F described under item 4.5.11 .2. It is even more preferably according to the invention, that a hydantoinase E214V has hydantoinase activity 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 % the hydantoinase activity of the hydantoinase E2i4s, wherein the hydantoinase activity of E2I4V, relative to the hydantoinase activity E2I4S is determined by Assay F described under item 4.5.11 .2.
4.5.12.6 Preferred variants of SEQ ID NO: 15
According to the invention, the polypeptide sequence of the hydantoinase E2, preferably the polypeptide sequence of the L-hydantoinase E2, may also be a variant of SEQ ID NO: 15.
The hydantoinase E2, the polypeptide sequence of which is SEQ ID NO: 15, is denoted as “E2I5S”. The hydantoinase E2, the polypeptide sequence of which is selected from variants of SEQ ID NO: 15, are generally denoted as “E2I5V”. A variant of the polypeptide sequence of SEQ ID NO: 15 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 15.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 15 is not identical to SEQ ID NO: 15. According to the invention, a hydantoinase E2I5V has hydantoinase activity and preferably L-hydantoinase activity, determined as described under items 4.5.10.2 and 4.5.10.3.1.
According to the invention, a hydantoinase E2I5V preferably has hydantoinase activity of at least
1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoinase activity of the hydantoinase E2I5S, wherein the hydantoinase activity of E2I5V, relative to the hydantoinase activity of E2i5s is determined by Assay F described under item 4.5.11 .2. It is even more preferably according to the invention, that a hydantoinase E2I5V has hydantoinase activity 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 % the hydantoinase activity of the hydantoinase E2I5S, wherein the hydantoinase activity of E2I5V, relative to the hydantoinase activity E2I5S is determined by Assay F described under item 4.5.11 .2.
4.5.12.7 Preferred variants of SEQ ID NO: 16
According to the invention, the polypeptide sequence of the hydantoinase E2, preferably the polypeptide sequence of the L-hydantoinase E2, may also be a variant of SEQ ID NO: 16.
The hydantoinase E2, the polypeptide sequence of which is SEQ ID NO: 16, is denoted as “E2I6S”.
The hydantoinase E2, the polypeptide sequence of which is selected from variants of SEQ ID NO: 16, are generally denoted as “E2I6V”.
A variant of the polypeptide sequence of SEQ ID NO: 16 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 16.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 16 is not identical to SEQ ID NO: 16.
According to the invention, a hydantoinase E2I6V has hydantoinase activity and preferably L-hydantoinase activity, determined as described under items 4.5.10.2 and 4.5.10.3.1.
According to the invention, a hydantoinase E2I6V preferably has hydantoinase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoinase activity of the hydantoinase E2I6S, wherein the hydantoinase activity of E2I6V, relative to the hydantoinase activity of E2i6s is determined by Assay F described under item 4.5.11 .2. It is even more preferably according to the invention, that a hydantoinase E2iev has hydantoinase activity 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 % the hydantoinase activity of the hydantoinase E2i6s, wherein the hydantoinase activity of E2I6V, relative to the hydantoinase activity E2I6S is determined by Assay F described under item 4.5.11 .2.
4.5.13 Preferred method conditions in step (/3)
The reaction in step (p) of the method according to the present invention may be carried out under conditions known to the skilled person.
The reaction medium is preferably aqueous, more preferably an aqueous buffer.
Exemplary buffers commonly used in biotransformation reactions and advantageously used herein include Tris, phosphate, or any of Good's buffers, such as 2-(/V-morpholino)ethanesulfonic acid (“MES”), /V-(2-acetamido)iminodiacetic acid (“ADA”), piperazine-/V,/\/'-bis(2-ethanesulfonic acid) (“PIPES”), /V-(2-acetamido)-2- aminoethanesulfonic acid (“ACES”), P-hydroxy-4- morpholinepropanesulfonic acid (“MOPSO”), cholamine chloride, 3-(/V-morpholino)propanesulfonic acid (“MOPS”), /V,/V-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”), acetamidoglycine,
3-(/V-Tris(hydroxymethyl)methylamino(-2-hydroxypropane)sulfonic acid (“TAPSO”), piperazine- /V,/\/'-bis(2-hydroxypropanesulfonic acid) (“POPSO”), 4-(2- Hydroxyethyl)piperazine- 1-(2-hydroxypropanesulfonic acid) (“HEPPSO”), 3-[4-(2-Hydroxyethyl)-1-piperazinyl]propanesulfonic acid (“HEPPS”), tricine, glycinamide, bicine, or 3-[[1 ,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1 -sulfonic acid (“TAPS”).
In some embodiments, ammonium can act as a buffer. One or more organic solvents can also be added to the reaction.
The buffer preferably contains metal salts, more preferably metal salts such as halogenides of metals, preferably halogenides of monovalent or bivalent or trivalent metals, preferably chlorides of monovalent or bivalent metals, preferably C0CI2 or MnCI2, preferably C0CI2.
The concentration of these metal salts in the reaction medium is preferably in the range from 1 pM to 1 M, more preferably 1 mM to 100 mM, even more preferably 1 to 10 mM. Preferably, step (p) of the method according to the invention is carried out in a phosphate buffer.
The pH of the reaction medium in step (p) of the method according to the invention is preferably in the range of from 2 to 10, more preferably in the range of from 5 to 8, more preferably 7.2 to 7.5, most preferably 7.5.
Preferably, step (p) of the method according to the invention is carried out at a temperature in the range of from 20 °C to 70 °C, more preferably in the range of from 30 °C to 55 °C, most preferably 50 °C.
Preferably, the total concentration of all hydantoinases E2 in the reaction solution in step (p) is in the range of from 1 pM to 10 mM, preferably 10 pM to 1 mM, more preferably 0.1 mM to 0.5 mM, most preferably 0.4 mM.
In alternative preferred embodiments, the total concentration of all hydantoinases E2 in the reaction solution in step (p) is in the range of from 1 pg/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.
Preferably, the initial concentration of all the compounds according to formula L-(lll) in the reaction medium in step (p) is in the range of from 1 pM to 1 M, preferably of from 10 pM to 0.5 M, more preferably of from 0.1 mM to 0.1 M, more preferably of from 1 mM to 10 mM, most preferably 1.25 mM.
If compounds according to formula D-(lll) are present in the reaction medium in step (p), the initial concentration of all the compounds according to formula D-(lll) in the reaction medium is preferably from 1 % to 100 % the concentration of all the compounds according to formula L-(lll), more preferably 10 % to 100 % the concentration, even more preferably 50 to 100 %, even more preferably 100 % the concentration of all the compounds according to formula L-(lll).
Preferably, step (p) is carried out in the same reaction medium in which step (6) is carried out.
In this case, preferably, the initial concentration of all the compounds according to formula L-(lll) in the reaction medium in step (p) is in the range of from 1 pM to 1 M, preferably of from 10 pM to 0.5 M, more preferably of from 0.1 mM to 0.1 M, more preferably of from 1 mM to 10 mM, most preferably 1 .25 mM.
“Initial concentration of all the compounds according to formula L-(lll)/ D-(lll)” refers to the concentration of the respective compound L-(lll) or D-(lll), respectively, in the reaction medium when the respective compounds are employed in step (p).
4.5.14 Step (a) [optional] In a preferred embodiment of the method according to the invention, the compound according to formula LL-(III) and/or, preferably and, the compound according to formula DL-(III) is obtained by a step (a) in which a compound according to formula LD-(III) is reacted to give a compound according to formula LL-(III) and/or, preferably and, a compound according to formula DD-(III) is reacted to give a compound according to formula DL-(III), wherein LD-(III) and DD-(III) have the following formulae:
Figure imgf000061_0001
LD-(III) DD-(III) and wherein R has the same meaning as described for LL-(I) and DL-(I).
Step (a) gives the starting material for step (p), and R in LD-(III) and DD-(III) has the same meaning as described for DL-(III) and LL-(III) [or DL-(II) and LL-(II) ].
The reaction according to step (a) may be carried out enzymatically or non-enzymatically, preferably enzymatically. More preferably, the reaction according to step (a) is catalyzed by a hydantoin racemase E3.
In preferred step (a), the compound according to formula D-(lll) is employed in step (a) as a mixture Mm** of LD-(III) and DD-(III) with the respective enantiomer DL-(III) and LL-(III) .
In a preferred embodiment, the mixture Mm** is a racemic mixture of enantiomer L-(lll) and enantiomer D-(lll), meaning that the molar ratio of all compounds according to L-(lll) to compounds according to D-(lll) is essentially 1 : 1.
In other preferred embodiments, the molar ratio of all compounds L-(lll) to all compounds D-(lll) in mixture Mm** is in the range of from 99 : 1 to 1 : 99, more preferably in the range of from 1.01 : 1 to 1 : 99, more preferably in the range of from 1 : 1 to 1 : 99, more preferably in the range of from 1 : 1.01 to 1 : 99, more preferably in the range of from 1 : 1.01 to 1 : 9, more preferably in the range of from 1 : 1.01 to 1 : 8, more preferably in the range of from 1 : 1.01 to 1 : 3.
4.5.14.1 Step (a) without enzymatic catalysis
Step (a) may be carried out non-enzymatically, i.e. without the use of an enzyme. The reaction of compounds according to step D-(lll) to compounds according to L-(lll) proceeds in alkaline solution, as known to the skilled person and as described by Slomka et al., M. Bovarnick & H.T. Clarke, Journal of the American Chemical Society 1938, 60, 2426 - 2430, by R. A. Lazarus, J. Org. Chem. 1990, 55, 4755 - 4757, and by A. S. 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 acitivity” on page 164 and 167 in “Microbial Reagents in Organic Synthesis” Series C: Mathematical and Physical Sciences - Vol. 381 , S. Servi (Ed.), 1992, Springer Science+Business Media, B.V., Dordrecht.
Therefore, if step (a) is carried out non-enzymatically, the conditions that are preferably applied in the reaction medium in which non-enzymatic step (a) is carried out are preferably those that are described for the preferred conditions fpr step (p) (item 4.5.13), except that the pH is > 8, preferably in the range of 8 to 12, more preferably 8 to 11 , more preferably 8 to 10, even more preferably 8 to 9. As in these embodiments, the preferred conditions in step (a) and (p) with respect to the pH ranges are different, it is preferable that the reaction media in steps (a) and (p) are different.
4.5.14.2 Enzymatic conversion of D-(lll) into L-(lll)
Step (a) is preferably carried out enzymatically, i.e. the reaction according to step (a) is preferably catalyzed by a hydantoin racemase E3.
Namely, it was surprisingly found that hydantoin racemases accept compounds of formula D-(lll) as substrates and convert them to products according to formulae L-(lll), and hence catalyze the reaction according to step (a), while there is no catalysis of the corresponding compound according to formula D-(lll), in which R = H.
This finding is of high scientific and economic value, as it further expands the scope of new starting materials for the production of L-glufosinate P-esters via new synthetic routes. Moreover, this finding also opens new possibilities of enantioselective production of LGA from racemic mixtures of L-(lll) and D-(lll).
In nature, hydantoin racemases catalyze the conversion of one of the two hydantoin enantiomers HL and HR into the other (see the following reaction <3>):
Figure imgf000062_0001
It was now surprisingly found that hydantoin racemases also accept substrates in which
O ii
Figure imgf000063_0002
O II Surprisingly, they do not accept substrates in which R* = RY =
Figure imgf000063_0001
, wherein R = H.
Suitable hydantoin racemases are described e.g. in WO 01/23582 A1 and by U. Engel, J. Rudat, C. Syldatk in “The hydantoinase process: recent developments for the production of non-canonical amino acids” in the book “Industrial biocatalysis” by P. Grunwald (Ed.), Pan Stanford Series on Biocatalysis, 2015, pages 817 - 862, and by F. J. Las Heras-Vazquez, J. M. Clemente-Jimenez, S. Martinez-Rodriguez, F. Rodriguez-Vico in “Hydantoin racemase: the key enzyme for the production of optically pure a-amino acids” in chapter 12 of the book “Modern Biocatalysis: Stereoselective and environmentally friendly reactions” by W. Fessner, T. Anthonsen (Eds), Weinheim: WILEY- VCH Verlag GmbH & Co, 2009, pages 173 - 193.
A hydantoin racemase E3 that may be used in optional step (a) of the method according to the invention may originate from Agrobacterium sp., in particular Agrobacterium strain IPJ-671 ; Arthrobacter sp., in particular Arthrobacter aurescens, more in particular Arthrobacter aurescens DSM 3745 or Arthtrobacter sp. BT801 ; Flavobacterium sp., in particular Flavobacterium sp. AJ 11199; Microbacterium sp., in particular Microbacterium liquefaciens, preferably Microbacterium liquefaciens AJ 3912; Pasteurella sp., in particular Pasteurella sp. AJ11221 ; Pseudomonas sp., in particular Pseudomonas sp. NS671 ; Pyrococcus sp., in particular Pyrococcus horikoshii OT3; Rhodococcus sp., in particular Rhodococcus R04; Sinorhizobium sp., in particular Sinorhizobium meliloti, more in particular Sinorhizobium meliloti CECT 4114, , most preferably from Arthrobacter aurescens DSM 3745.
A hydantoin racemase E3 suitable for the method according to the present invention may be the enzyme HyuR, which originates from Arthrobacter aurescens DSM 3745. Another enzyme may be selected from HyuE, Hyu2, HRase, HyuA, PH1054.
The hydantoin racemase E3 that may preferably be used in preferred step (a) of the method according to the invention may be categorized in the EC class 5.1 .99.5.
The following table 3 gives preferred examples for polypeptide sequences of hydantoin racemase E3 that may be preferably used in step (a) of the method according to the invention. The genes encoding the respective hydantoin racemase E3 and the respective accession code are indicated as far as known.
Table 3
Figure imgf000064_0001
In a preferred embodiment of the preferred method according to the present invention, the reaction according to 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, SEQ ID NO: 26 and variants thereof. 4.5.14.3 Assay G for determining hydantoin racemase activity
The skilled person is aware of hydantoin racemases, that may be used in preferred step (a) of the method according to the invention. In particular, Assay G, described in the following, may be used to determine hydantoin racemase activity of a given enzyme Ez and may advantageously be used according to the invention to determine carbamoylase and L-carbamoylase activity in variants of SEQ ID NO: 17, variants of 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 variants of SEQ ID NO: 23, variants of SEQ ID NO: 24, variants of SEQ ID NO: 25, variants of SEQ ID NO: 26.
For the purpose of Assay G, the molar mass of the enzyme Ez to be tested is calculated as the molar mass of the polypeptide sequence of Ez.
Assay G:
To 0.9 ml of an aqueous reaction solution (phosphate buffer, pH 7.2, 10 mM Mg2CI), containing 50 mM of the pure D-enantiomer of an n-butyl ester of hydantoin glufosinate of the formula D-(lll), wherein LD-(III) and DD-(III) are equimolar, wherein R = n-butyl, are added 400 nmol of Ez in 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh).
The resulting solution is incubated at 25 °C, and the pH is held at pH 7.2 by addition of 0.5 M. After 300 minutes, the reaction is stopped by addition of 2 M HCI to achieve a pH of 2.5. The molar amount of the L-enantiomer of formula L-(lll), wherein R = n-butyl, is measured, at least every 3 minutes (determination by LC-MS, e.g. by the LC-MS method described in the example section, item 5.4, for the detection of LGA).
4.5.14.4 Assay H for identif lying hydantoin racemases
Whether a given enzyme Ez may be considered a hydantoin racemase E3, may be determined in the context of the present invention by the following Assay H:
H-1 Firstly, Assay G as set forth under item 4.5.14.3 is conducted, and the obtained molar amount of L-(lll) is determined according to Assay G.
H-2 Then, step H-1 is repeated, except that instead of the addition of 400 nmol Ez in 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh), 0.1 ml aqueous phosphate buffer (pH 7.2, 10 mM MnCh) without Ez is added.
4.5.14.5 Hydantoin racemase activity
If the molar amount of the compound of formula L-(lll), wherein R = n-butyl, that is determined in step H-1 is greater than the molar amount of the compound of formula L-(lll), wherein R = n-butyl, that is determined in step H-2, then Ez is deemed to have hydantoin racemase activity, and hence may be considered a hydantoin racemase E3 in the context of the invention.
4.5.14.6 Assay J for identifying preferred hydantoin racemase variants of SEQ ID NO: 17 - 26
An enzyme, the polpypetide sequence of which is selected from the group consisting 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.
In a preferred embodiment of the method according to the invention, 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.
The term “variant” is defined under item 4.3.
In the context of the invention, an enzyme, the polypeptide sequence of which 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.
Whether a given enzyme Ez, the polypeptide sequence of which is a variant of one 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 may be determined as set forth under items 4.5.14.4 and.4.5.14.5.
The hydantoin racemase activity of a given hydantoin racemase E3V, the polypeptide sequence of which 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, relative to the hydantoin racemase activity of a hydantoin racemase E3S, wherein the polypeptide sequence of E3S 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, may be quantified in the context of the present invention by the following Assay J.
Assay J:
J-1 Firstly, Assay G as set forth under item 4.5.14.3 is conducted, wherein E3S is the enzyme to be tested. The molar amount of the compound according to formula L-(lll), wherein R = n-butyl, is determined according to Assay G. J-2 Step J-1 is repeated, except that, instead of E3s, E3v is used as the enzyme to be tested.
J-3. Then, the molar amount of the compound according to formula L-(lll), wherein R = n-butyl, is determined in step J-2, is divided by the molar amount of the compound according to formula L-(lll), wherein R = n-butyl, is determined in step J-1 , and the obtained ratio is multiplied by 100, giving the hydantoin racemase activity of hydantoin racemase E3V, relative to the hydantoin racemase activity of the hydantoin racemase E3S, in %.
4.5.14.7 Preferred hydantoin racemase variants SEQ ID NO: 17 - 26
In the context of the present invention, hydantoin racemase E3, the polypeptide sequence of which is selected from the group consisting 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, are generally denoted as “E3S”.
Hydantoin racemases E3, the polypeptide sequence of which is selected from variants of a sequence selected from the group consisting of f 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, are generally denoted as “E3V”.
In a preferred embodiment of the method according to 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 of SEQ ID NO: 1 .
4.5.14.7.1 Preferred variants of SEQ ID NO: 17
According to the invention, the polypeptide sequence of the hydantoin racemase E3 may also be a variant of SEQ ID NO: 17.
The hydantoin racemase E3, the polypeptide sequence of which is SEQ ID NO: 17, is denoted as “E3i7s”. Hydantoin racemases E3, the polypeptide sequence of which is is selected from variants of SEQ ID NO: 17, are generally denoted as “E3I7V”.
A variant of the polypeptide sequence of SEQ ID NO: 17 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 17.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 17 is not identical to SEQ ID NO: 17.
According to the invention, a hydantoin racemase E3i?v has hydantoin racemase activity, determined as described under item 4.5.14.4 and 4.5.14.5.
According to the invention, a hydantoin racemase E3i?v preferably has hydantoin racemase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoin racemase of the hydantoin racemase E3i7s, wherein the hydantoin racemase activity of E3i7v, relative to the hydantoin racemase activity E3i7s is determined by Assay J described under item 4.5.14.6.
It is even more preferably according to the invention, that a hydantoin racemase E3i7v has hydantoin racemase activity 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 % the hydantoin racemase activity of the hydantoin racemase E3i7s, wherein the hydantoin racemase activity of E3i7v, relative to the hydantoin racemase activity of E3i7s is determined by Assay J described under item 4.5.14.6.
4.5.14.7.2 Preferred variants of SEQ ID NO: 18
According to the invention, the polypeptide sequence of the hydantoin racemase E3 may also be a variant of SEQ ID NO: 18.
The hydantoin racemase E3, the polypeptide sequence of which is SEQ ID NO: 18, is denoted as “E3i8s”. Hydantoin racemases E3, the polypeptide sequence of which is is selected from variants of SEQ ID NO: 18, are generally denoted as “E3i8v”.
A variant of the polypeptide sequence of SEQ ID NO: 18 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 18.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 18 is not identical to SEQ ID NO: 18.
According to the invention, a hydantoin racemase E3i8V has hydantoin racemase activity, determined as described under item 4.5.14.4 and 4.5.14.5.
According to the invention, a hydantoin racemase E3i8V preferably has hydantoin racemase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoin racemase of the hydantoin racemase E3i8s, wherein the hydantoin racemase activity of E3i8v, relative to the hydantoin racemase activity E3i8S is determined by Assay J described under item 4.5.14.6.
It is even more preferably according to the invention, that a hydantoin racemase E3i8V has hydantoin racemase activity 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 % the hydantoin racemase activity of the hydantoin racemase E3i8S, wherein the hydantoin racemase activity of E3i8V, relative to the hydantoin racemase activity of E3i8S is determined by Assay J described under item 4.5.14.6.
4.5.14.7.3 Preferred variants of SEQ ID NO: 19
According to the invention, the polypeptide sequence of the hydantoin racemase E3 may also be a variant of SEQ ID NO: 19.
The hydantoin racemase E3, the polypeptide sequence of which is SEQ ID NO: 19, is denoted as “Ernes”. Hydantoin racemases E3, the polypeptide sequence of which is is selected from variants of SEQ ID NO: 19, are generally denoted as “E3i9V”.
A variant of the polypeptide sequence of SEQ ID NO: 19 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 19.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 19 is not identical to SEQ ID NO: 19.
According to the invention, a hydantoin racemase E319V has hydantoin racemase activity, determined as described under item 4.5.14.4 and 4.5.14.5.
According to the invention, a hydantoin racemase E319V preferably has hydantoin racemase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoin racemase of the hydantoin racemase E3i9S, wherein the hydantoin racemase activity of E3i9V, relative to the hydantoin racemase activity E3i9S is determined by Assay J described under item 4.5.14.6.
It is even more preferably according to the invention, that a hydantoin racemase E3i9V has hydantoin racemase activity 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 % the hydantoin racemase activity of the hydantoin racemase E3i9S, wherein the hydantoin racemase activity of E3i9V, relative to the hydantoin racemase activity of E3i9S is determined by Assay J described under item 4.5.14.6.
4.5.14.7.4 Preferred variants of SEQ ID NO: 20
According to the invention, the polypeptide sequence of the hydantoin racemase E3 may also be a variant of SEQ ID NO: 20.
The hydantoin racemase E3, the polypeptide sequence of which is SEQ ID NO: 20, is denoted as “E32OS”. Hydantoin racemases E3, the polypeptide sequence of which is is selected from variants of SEQ ID NO: 20, are generally denoted as “E32ov”.
A variant of the polypeptide sequence of SEQ ID NO: 20 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 20.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 20 is not identical to SEQ ID NO: 20.
According to the invention, a hydantoin racemase E320V has hydantoin racemase activity, determined as described under item 4.5.14.4 and 4.5.14.5.
According to the invention, a hydantoin racemase E320V preferably has hydantoin racemase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoin racemase of the hydantoin racemase E32os, wherein the hydantoin racemase activity of E32ov, relative to the hydantoin racemase activity E32os is determined by Assay J described under item 4.5.14.6.
It is even more preferably according to the invention, that a hydantoin racemase E32ov has hydantoin racemase activity 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 % the hydantoin racemase activity of the hydantoin racemase E32os, wherein the hydantoin racemase activity of E32ov, relative to the hydantoin racemase activity of E32os is determined by Assay J described under item 4.5.14.6.
4.5.14.7.5 Preferred variants of SEQ ID NO: 21
According to the invention, the polypeptide sequence of the hydantoin racemase E3 may also be a variant of SEQ ID NO: 21 .
The hydantoin racemase E3, the polypeptide sequence of which is SEQ ID NO: 21 , is denoted as “E32IS”. Hydantoin racemases E3, the polypeptide sequence of which is is selected from variants of SEQ ID NO: 21 , are generally denoted as “E32iv”.
A variant of the polypeptide sequence of SEQ ID NO: 21 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 21 .
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 21 is not identical to SEQ ID NO: 21.
According to the invention, a hydantoin racemase E321V has hydantoin racemase activity, determined as described under item 4.5.14.4 and 4.5.14.5.
According to the invention, a hydantoin racemase E321V preferably has hydantoin racemase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoin racemase of the hydantoin racemase E32is, wherein the hydantoin racemase activity of E32iv, relative to the hydantoin racemase activity E32is is determined by Assay J described under item 4.5.14.6.
It is even more preferably according to the invention, that a hydantoin racemase E32iv has hydantoin racemase activity 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 % the hydantoin racemase activity of the hydantoin racemase E32is, wherein the hydantoin racemase activity of E32iv, relative to the hydantoin racemase activity of E32is is determined by Assay J described under item 4.5.14.6.
4.5.14.7.6 Preferred variants of SEQ ID NO: 22
According to the invention, the polypeptide sequence of the hydantoin racemase E3 may also be a variant of SEQ ID NO: 22.
The hydantoin racemase E3, the polypeptide sequence of which is SEQ ID NO: 22, is denoted as “E322S”. Hydantoin racemases E3, the polypeptide sequence of which is is selected from variants of SEQ ID NO: 22, are generally denoted as “E322v”.
A variant of the polypeptide sequence of SEQ ID NO: 22 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 22.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 22 is not identical to SEQ ID NO: 22.
According to the invention, a hydantoin racemase E322V has hydantoin racemase activity, determined as described under item 4.5.14.4 and 4.5.14.5.
According to the invention, a hydantoin racemase E322V preferably has hydantoin racemase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoin racemase of the hydantoin racemase E322S, wherein the hydantoin racemase activity of E322V, relative to the hydantoin racemase activity E322S is determined by Assay J described under item 4.5.14.6.
It is even more preferably according to the invention, that a hydantoin racemase E322V has hydantoin racemase activity 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 % the hydantoin racemase activity of the hydantoin racemase E322S, wherein the hydantoin racemase activity of E322V, relative to the hydantoin racemase activity of E322S is determined by Assay J described under item 4.5.14.6.
4.5. 14.7.7 Preferred variants of SEQ ID NO: 23
According to the invention, the polypeptide sequence of the hydantoin racemase E3 may also be a variant of SEQ ID NO: 23.
The hydantoin racemase E3, the polypeptide sequence of which is SEQ ID NO: 23, is denoted as “E323S”. Hydantoin racemases E3, the polypeptide sequence of which is is selected from variants of SEQ ID NO: 23, are generally denoted as “E323v”.
A variant of the polypeptide sequence of SEQ ID NO: 23 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 23.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 23 is not identical to SEQ ID NO: 23.
According to the invention, a hydantoin racemase E323v has hydantoin racemase activity, determined as described under item 4.5.14.4 and 4.5.14.5.
According to the invention, a hydantoin racemase E323v preferably has hydantoin racemase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoin racemase of the hydantoin racemase E323s, wherein the hydantoin racemase activity of E323v, relative to the hydantoin racemase activity E323s is determined by Assay J described under item 4.5.14.6.
It is even more preferably according to the invention, that a hydantoin racemase E323v has hydantoin racemase activity 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 % the hydantoin racemase activity of the hydantoin racemase E323s, wherein the hydantoin racemase activity of E323v, relative to the hydantoin racemase activity of E323s is determined by Assay J described under item 4.5.14.6.
4.5.14.7.8 Preferred variants of SEQ ID NO: 24
According to the invention, the polypeptide sequence of the hydantoin racemase E3 may also be a variant of SEQ ID NO: 24.
The hydantoin racemase E3, the polypeptide sequence of which is SEQ ID NO: 24, is denoted as “E324S”. Hydantoin racemases E3, the polypeptide sequence of which is is selected from variants of SEQ ID NO: 24, are generally denoted as “E324v”.
A variant of the polypeptide sequence of SEQ ID NO: 24 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 24.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 24 is not identical to SEQ ID NO: 24.
According to the invention, a hydantoin racemase E324v has hydantoin racemase activity, determined as described under item 4.5.14.4 and 4.5.14.5.
According to the invention, a hydantoin racemase E324v preferably has hydantoin racemase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoin racemase of the hydantoin racemase E324s, wherein the hydantoin racemase activity of E324v, relative to the hydantoin racemase activity E324s is determined by Assay J described under item 4.5.14.6.
It is even more preferably according to the invention, that a hydantoin racemase E324v has hydantoin racemase activity 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 % the hydantoin racemase activity of the hydantoin racemase E324s, wherein the hydantoin racemase activity of E324v, relative to the hydantoin racemase activity of E324s is determined by Assay J described under item 4.5.14.6.
4.5.14.7.9 Preferred variants of SEQ ID NO: 25
According to the invention, the polypeptide sequence of the hydantoin racemase E3 may also be a variant of SEQ ID NO: 25.
The hydantoin racemase E3, the polypeptide sequence of which is SEQ ID NO: 25, is denoted as “E325S”. Hydantoin racemases E3, the polypeptide sequence of which is is selected from variants of SEQ ID NO: 25, are generally denoted as “E325v”.
A variant of the polypeptide sequence of SEQ ID NO: 25 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 25.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 25 is not identical to SEQ ID NO: 25.
According to the invention, a hydantoin racemase E325v has hydantoin racemase activity, determined as described under item 4.5.14.4 and 4.5.14.5.
According to the invention, a hydantoin racemase E325v preferably has hydantoin racemase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoin racemase of the hydantoin racemase E325s, wherein the hydantoin racemase activity of E325v, relative to the hydantoin racemase activity E325s is determined by Assay J described under item 4.5.14.6.
It is even more preferably according to the invention, that a hydantoin racemase E325v has hydantoin racemase activity 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 % the hydantoin racemase activity of the hydantoin racemase E325s, wherein the hydantoin racemase activity of E325v, relative to the hydantoin racemase activity of E325s is determined by Assay J described under item 4.5.14.6.
4.5.14.7.10 Preferred variants of SEQ ID NO: 26
According to the invention, the polypeptide sequence of the hydantoin racemase E3 may also be a variant of SEQ ID NO: 26.
The hydantoin racemase E3, the polypeptide sequence of which is SEQ ID NO: 26, is denoted as “E326S”. Hydantoin racemases E3, the polypeptide sequence of which is is selected from variants of SEQ ID NO: 26, are generally denoted as “E326v”.
A variant of the polypeptide sequence of SEQ ID NO: 26 is a polypeptide with sequence identity of 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 polypeptide sequence SEQ ID NO: 26.
The polypeptide sequence of a variant of the polypeptide sequence SEQ ID NO: 26 is not identical to SEQ ID NO: 26.
According to the invention, a hydantoin racemase E32ev has hydantoin racemase activity, determined as described under item 4.5.14.4 and 4.5.14.5.
According to the invention, a hydantoin racemase E32ev preferably has hydantoin racemase activity of at least 1 %, preferably of at least 10 %, more preferably of at least 20 %, more preferably of at least 30 %, more preferably of at least 40 %, more preferably of at least 50 %, more preferably of at least 60 %, more preferably of at least 70 %, more preferably of at least 80 %, more preferably of at least 90 %, more preferably of at least 99 %, more preferably of at least 100 % the hydantoin racemase of the hydantoin racemase E326s, wherein the hydantoin racemase activity of E32ev, relative to the hydantoin racemase activity E326s is determined by Assay J described under item 4.5.14.6.
It is even more preferably according to the invention, that a hydantoin racemase E32ev has hydantoin racemase activity 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 % the hydantoin racemase activity of the hydantoin racemase E326s, wherein the hydantoin racemase activity of E32ev, relative to the hydantoin racemase activity of E326s is determined by Assay J described under item 4.5.14.6.
4.5.15 Preferred method conditions in step (a)
In this preferred embodiment, in which step (a) is catalyzed by a hydantoin racemase E3, the reaction in step (a) may be carried out under conditions known to the skilled person.
The reaction medium is preferably aqueous, more preferably an aqueous buffer.
Exemplary buffers commonly used in biotransformation reactions and advantageously used herein include Tris, phosphate, or any of Good's buffers, such as 2-(/V-morpholino)ethanesulfonic acid (“MES”), /V-(2-acetamido)iminodiacetic acid (“ADA”), piperazine-/V,/\/'-bis(2-ethanesulfonic acid) (“PIPES”), /V-(2-acetamido)-2- aminoethanesulfonic acid (“ACES”), P-hydroxy-4- morpholinepropanesulfonic acid (“MOPSO”), cholamine chloride, 3-(/V-morpholino)propanesulfonic acid (“MOPS”), /V,/V-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”), acetamidoglycine,
3-(/V-Tris(hydroxymethyl)methylamino(-2-hydroxypropane)sulfonic acid (“TAPSO”), piperazine- /V,/\/'-bis(2-hydroxypropanesulfonic acid) (“POPSO”), 4-(2- Hydroxyethyl)piperazine- 1-(2-hydroxypropanesulfonic acid) (“HEPPSO”),
3-[4-(2-Hydroxyethyl)-1-piperazinyl]propanesulfonic acid (“HEPPS”), tricine, glycinamide, bicine, or 3-[[1 ,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1 -sulfonic acid (“TAPS”).
In some embodiments, ammonium can act as a buffer. One or more organic solvents can also be added to the reaction.
The buffer preferably contains metal salts, more preferably metal salts such as halogenides of monovalent or bivalent metals (e.g. C0CI2, MnCh).
The concentration of these metal salts in the reaction medium is preferably in the range from 1 pM to 1 M, more preferably 1 mM to 100 mM, even more preferably 1 to 10 mM.
Preferably, step (a) of the method according to the invention is carried out in a phosphate buffer.
The pH of the reaction medium in step (a) of the method according to the invention is preferably in the range of from 2 to 10, more preferably in the range of from 5 to 8, more preferably 7.2 to 7.5, most preferably 7.5.
Preferably, step (a) of the method according to the invention is carried out at a temperature in the range of from 20 °C to 70 °C, more preferably in the range of from 30 °C to 55 °C, most preferably 50 °C.
In this preferred embodiment, in which step (a) is catalyzed by a hydantoin racemase E3, the preferred reaction conditions in step (a) are the same as described for steps (p) and (6), confer items 4.5.7 and 4.5.13, respectively. It is even more preferred to carry out step (a) concomitantly with steps (p) and (6).
Preferably, the total concentration of all hydantoin racemases E3 in the reaction solution in step (a) is in the range of from 1 pM to 10 mM, preferably 10 pM to 1 mM, more preferably 0.1 mM to 0.5 mM, most preferably 0.4 mM. In alternative preferred embodiments, the total concentration of all hydantoin racemases E3 in the reaction solution in step (a) is in the range of from 1 pg/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.
Preferably, step (a) is carried out in the same reaction medium in which steps (6) and (p) are carried out. The advantage is that this allows for a one-pot synthesis in which all the steps (a), (p), and (6) are carried out.
Preferably, the initial concentration of all the compounds according to formula D-(lll) in the reaction medium in step (a) is in the range of from 1 pM to 1 M, preferably of from 10 pM to 0.5 M, more preferably of from 0.1 mM to 0.1 M, more preferably of from 1 mM to 10 mM, most preferably 1.25 mM.
If compounds according to formula L-(lll) are present in the reaction medium in step (a), the initial concentration of all the compounds according to formula L-(lll) in the reaction medium is preferably from 1 to a 100 times the concentration of all the compounds according to formula D-(lll), more preferably 1 to 10 times the concentration, even more preferably 1 to 2 times even more preferably the same as the concentration of all the compounds according to formula D-(lll).
“Initial concentration of all the compounds according to formula L-(lll)/ D-(lll)”” refers to the concentration of the respective compound L-(lll) or D-(lll), respectively, in the reaction medium when the respective compounds are employed in step (a).
In step (6), a mixture Mu comprising the two diastereomers DL-(II), LL-(II) is reacted. In this application “DL-(II) , LL-(II)” may each also be denoted as “carbamoyl compounds”. Furthermore, in this application “DL-(I), LL-(I)” may also be denoted as “amino acid compounds”.
It has now been surprisingly found that the carbamoylase Ei employed in step (6) catalyzes the reaction of each carbamoylate compound DL-(II) to LL-(II) to the respective amino acid compounds DL-(I) and LL-(I) at a different rate. This finding may advantageously be used in a method in which only of the two carbamoyl compounds DL-(II) and LL-(II) is reacted to the respective amino acid compound DL-(I) and LL-(I) or in which both carbamoyl compounds are reacted, but at different rates, so that in the resulting product mixture Mi comprising DL-(I) and LL-(I), one of DL-(I) and LL- (I) is present in excess to the other.
Therefore, after step (6), a mixture Mi is obtained comprising at least one amino acid compound of DL-(I), LL-(I). Moreover, as the rate at which DL-(II) is reacted to give DL-(I) is different from the rate at which LL-(II) is reacted to give LL-(I), if mixture Mi comprises both amino acid compounds DL-(I) and LL-(I), the molar ratio of DL-(I) to LL-(I) in Mi is different from the molar ratio of DL-(II) to LL-(II) in Mu, Hence, in step (6) of Method I, DL-(II) reacts to DL-(I) and/or, preferably and, LL-(II) reacts to
LL-(I)
Therefore, in performing step (5), the following scenarios (5-1), (5-2), and (5-3) are possible:
(5-1) in the reaction in step (5), only LL-(II) reacts to the respective amino acid compound LL-(I), but DL-(II) does not react to the respective amino acid compound DL-(I). In this case, Mi obtained after step (5) comprises LL-(I) as amino acid compound, but not DL-(I), and it comprises DL-(II) and, in case not all LL-(II) reacted to give LL-(I), optionally comprises LL-(II).
(5-2) in the reaction in step (5), LL-(II) does not react to the respective amino acid compound LL-(I), but DL-(II) does react to the respective amino acid compound DL-(I). In this case, Mi obtained after step (5) comprises DL-(I) as amino acid compound, but not LL-(I), and it comprises LL-(II) and, in case not all DL-(II) reacted to give DL-(I), optionally comprises DL-(II).
(5-3) in the reaction in step (5), LL-(II) and DL-(II) react to the respective amino acid compound LL-(I) and DL-(II). In this case, In this case, Mi obtained after step (5) comprises DL-(I) and LL-(I) as amino acid compound, and, in case not all DL-(II) reacted to give optionally DL-(I), it comprises DL-(II) and, in case not all LL-(II) reacted to give LL-(I), optionally comprises LL-(II). In case (5-3), it is preferred that Mi comprises DL-(II) and LL-(II).
Hence, the mixture Mi obtained in step (5) comprises at least one amino acid compound of DL-(I), LL-(I)
As the carbamoylase Ei employed in step (5) catalyzes the reaction of each carbamoylate compound DL-(II) to LL-(II) to the respective amino acid compounds DL-(I) and LL-(I) in a different rate, this means that in those cases in which mixture Mi comprises both amino acid compounds DL-(I) and LL-(I), the molar ratio of DL-(I) to LL-(I) in Mi is different from the molar ratio of DL-(II) to LL-(II) in Mu.
If mixture Mi comprises both amino acid compounds DL-(I) and LL-(I), the molar ratio of DL-(I) to LL-(I) in Mi is different from the molar ratio of DL-(II) to LL-(II) in Mu. “Molar ratio of DL-(II) to LL-(II) in Mu” means the molar ratio of DL-(II) to LL-(II) in the mixture Mu that is initially employed in step (Y)-
In this embodiment, it is even more preferred that mixture Mi comprises both carbamoyl compounds DL-(II) to LL-(II).
In this embodiment, it is further preferred, that the molar ratio of DL-(I) to LL-(I) in Mi is different by a factor x from the molar ratio of DL-(II) to LL-(II) in Mu. “Different by a factor x” means that oil/ con = x or UJII/ oil = 1/x ; wherein x 4 1 wherein uii is the molar ratio of DL-(I) to LL-(I) in Mi, and wherein a>u is the molar ratio of DL-(II) to LL-(II) in Mu.
Preferably x is in the range of 1 .0001 to 10000, more preferably in the range of 1 .001 to 1000, more preferably in the range of 1 .01 to 100, more preferably in the range of 1 .10 to 100, more preferably in the range of, more preferably in the range of 1 .20 to 300, more preferably in the range of 1 .30 to 250, more preferably in the range of 1 .40 to 150, more preferably in the range of 1 .50 to 100, more preferably in the range of 1 .75 to 90, more preferably in the range of 2.50 to 80, more preferably in the range of 3.00 to 65, more preferably in the range of 5.00 to 50, more preferably in the range of 10.00 to 20.00.
4.5.16 Step (£)
In step (e), one of the amino acid compounds selected from DL-(I), LL-(I) comprised by mixture Mi is at least partially separated from at least one of the carbamoyl compounds DL-(II), LL-(II) and/or from the other amino acid compound selected from DL-(I), LL-(I).
In a preferred step (£), one of the amino acid compounds selected from DL-(I), LL-(I) comprised by mixture Mi is at least partially separated from at least one of the carbamoyl compounds DL-(II), LL-(II) and from the other amino acid compound selected from DL-(I), LL-(I).
This separation may be carried out by the person skilled in the art. Preferably, at least one of the following separation methods is used: chromatography, crystallization, wherein crystallization is preferred. In particular, crystallization of the respective amino acid compound selected from DL-(I), LL-(I) is preferred. Hence, in a preferred step (£), the at least partial separation of one of the amino acid compounds selected from DL-(I), LL-(I) comprised by mixture Mi is performed by crystallizing the respective amino acid compound.
“[...] is separated from at least one of the carbamoyl compounds DL-(II), LL-(II) and/or the other amino acid compound selected from DL-(I), LL-(I) comprised by mixture Mi [...]” means in particular, that one of the following steps (d), (e2), (e3) is carried out:
(e1) in the above-described case (6-1) in which Mi comprises LL-(I), but not DL-(I), and Mi further comprises DL-(II) and optionally LL-(II): LL-(I) is then preferably at least partially separated from DL-(II) and, in case Mi comprises LL-(II), also from LL-(II). (e2) in the above-described case (6-2) in which Mi comprises DL-(I), but not LL-(I), and Mi further comprises LL-(II) and optionally DL-(II): DL-(I) is then preferably at least partially separated from LL-(II) and, in case Mi comprises DL-(II), also from DL-(II). (£3) in the above-described case (6-3) in which Mi comprises DL-(I) and LL-(I) and optionally at least one of, preferably both of DL-(II) and LL-(II), DL-(I) and LL-(I) are at least partially separated from at least one of, preferably both of DL-(II), LL-(II), and even more preferably DL-(I) and LL-(I) are further at least partially separated from each other. 4.5.18 Saponification
The L-glufosinate P- ester according to formula DL-(I) or LL-(I) obtained in purified form after step (£) with the method according to the invention may then be saponified to produce LGA, for example in an acidic aqueous medium, preferably at pH < 7, even more preferably at an pH between < 6, more preferably at an pH < 3, even more preferably at an pH of < 1 . These saponification conditions are known to the skilled person and described e.g by H.J. Zeiss, J. Org. Chem. 1991 , 56, 1783-1788.
5. Examples
5.1 Example 1 Identification of suitable enzymes and construction of plasmids
Genes of different origins encoding a hydantoinase (dihydropyrimidinase, EC 3.5.2.2), L-carbamoylase (/V-carbamoyl-L-amino-acid 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 structure L-(lll) and D-(lll) to form the respective enantioselective L-glufosinate derivative according to structure L-(l).
5.1.1 Examined enzymes
Details of the strains and genes of the respective enzymes that were used in the examples are summarized in table 4.
Table 4
Figure imgf000083_0001
5.1.2 Cloning of the enzymes
Cloning of the hydantoin racemase and generation of the plasmid pOM21c (Figure 1) was carried out as described in example 1 of WO 2004/111227 A2. In particular, a polynucleotide was used which comprised the respective gene (SEQ ID NO: 29) and additional sequences for Ndel and Pstl restriction sites.
Cloning of hydantoinase and L-carbamoylase into the rhamnose expression vector pJOE4036 was carried out in a plasmid derivative of the rhamnose expression vector pJOE4036.
Polynucleotides comprising the genes of the repective enzymes (SEQ ID NOs: 27, 28) were synthesized by GeneArt (ThermoFisher Scientific (Waltham, USA)). The polynucleotides carried additional sequences for EcoR1 and Hindlll restriction sites. Both enzymes were cloned into pJOE4036 using those restriction sites resulting in the plasmid pOM22c, under the control of a rhamnose promotor (Figure 2). 5.2 Example 2: Production of strains positive for hydantoinase, L-carbamoylase and hydantoin racemase
Chemically competent E. coll ET5 cells (as described in WO 2004/042047 A1) were transformed with 10 ng of the plasmid pOM22c generated according to Example 1.
The generated strain which was positive for hydantoinase- and carbamoylase was rendered chemically competent and transformed with 10 ng of the plasmid pOM21c.
An E. coll ET5 strain transformed with pOM21 c or pOM22c was incubated under shaking (250 U/min) at 30 °C for 18 hours in LB medium containing ampicillin (100 pg/l), chloramphenicol (50 pg/l), and 2 g/l rhamnose.
The biomass was separated by centrifugation, resuspended in 50 mM phosphate buffer (pH 7.2) and applied in biotransformation tests in the following examples. The concentration of the biomass in the solution was 40-50 g/l. The solution was used as catalyst (“catalyst 7”) in the following.
The concentration of the respective polypeptide carbamoylase, hydantoinase and racemase in the obtained solution may be determined by SDS page and analysis of the respective bands via the software GelQuant® (BiochemLabSolutions).
5.3 Comparative Examples 11 , 12, C1 , C2: Production of P-Alkyl phosphinothricin ester using the strains produced in Examples 1 and 2
In the following examples, different hydantoin substrates were tested to determine whether the respective polpypetide catalyzed the reaction of the respective substrate enantioselectively to give the corresponding L amino acid.
5.3.1 Example C1
In example C1 , a racemic mixture MCi of 1.25 mmol L-(IV)Ci and 1.25 mmol D-( I V)Ci , wherein L- (IV)ci and D-(IV)ci are hydantoins with the following formulae, was used as substrate:
Figure imgf000084_0001
The mixture MCi was dissolved in a stirring reactor with 25 ml water. 2.4 g of the catalyst 1 was added, followed by the addition of 50 pl C0CI2 solution. The suspension was set to pH 7.5 with 0.5 M NaOH. The total volume was replenished with water to 50 ml, so that the concentration of each enantiomer L-(IV)Ci and D-(IV)Ci was 0.025 mol/l and the concentration of C0CI2 in the final solution was 1 mM. The pH was held between 7.0 and 7.5 by HCI-titration or NaOH-titration. The temperature was maintained at 37 °C by a thermostat during the reaction.
The reaction was stopped after 120 hours by addition of 2 N HCI until pH 2.5 was reached. The biomass was separated by centrifugation or filtration.
The enzyme reaction was monitored by ninhydrin test to determine the formation of amino acids. No formation of amino acids was detected by the ninhydrine test.
5.3.2 Example C2
In example C2, a racemic mixture MC2 of 1 .25 mmol l_-(V)C2 and 1 .25 mmol D-(V)C2, wherein
L-(V)C2 and D-(V)C2 are carbamoylates with the following formulae, was used as substrate:
Figure imgf000085_0002
L-(V)C2 O-(V)C2
The mixture MC2 was dissolved in a stirring reactor with 25 ml water. 2.4 g of the catalyst 1 was added, followed by the addition of 50 pl C0CI2 solution. The suspension was set to pH 7.5 with 0.5 M NaOH. The total volume was replenished with water to 50 ml, so that the concentration of each enantiomer l_-(V)C2 and D-(V)C2 was 0.025 mol/l and the concentration of C0CI2 in the final solution was 1 mM. The pH was held between 7.0 and 7.5 by HCI-titration or NaOH-titration. The temperature was maintained at 37 °C by a thermostat during the reaction.
The reaction was stopped after 120 hours by addition of 2 N HCI until pH 2.5 was reached. The biomass was separated by centrifugation or filtration.
The enzyme reaction was monitored by ninhydrin test to determine the formation of amino acids. No formation of amino acids was detected by the ninhydrine test.
5.3.3 Example 11
In example 11 , a racemic mixture Mu of 1 .25 mmol l_-(VI)n and 1 .25 mmol D-(VI)H , wherein l_-(VI)n and D-(VI)H were hydantoins with the following formulae, was used as substrate:
Figure imgf000085_0001
L-(VI)h D-(VI)M The mixture Mu was dissolved in a stirring reactor with 25 ml water. 2.4 g of the catalyst 1 was added, followed by the addition of 50 pl C0CI2 solution. The suspension was set to pH 7.5 with 0.5 M NaOH. The total volume was replenished with water to 50 ml, so that the concentration of each enantiomer l_-(VI)n and D-(VI)H was 0.025 mol/l and the concentration of C0CI2 in the final solution was 1 mM. The pH was held between 7.0 and 7.5 by HCI-titration or NaOH-titration. The temperature was maintained at 37 °C by a thermostat during the reaction.
The reaction was stopped after 120 hours by addition of 2 N HCI until pH 2.5 was reached. The biomass was separated by centrifugation or filtration.
The enzyme reaction was monitored by ninhydrin test to determine the formation of amino acids. Formation of amino acids was detected by the ninhydrine test.
In a final step the reaction mixture was saponified at 100 °C for 10 hours by adding 6 M HCI to obtain L-glufosinate and, if present, D-glufosinate. The final reaction mixture was analysed by LC-MS with a CR-I column as described under item 5.4 to determine the enantiomeric excess (“ee”) of either D- or L-glufosinate. An ee of LGA of 79 % over the D-enantiomer was detected. The ee of the L-enantiomer (“eei”) is determined by the following formula in the context of the invention, wherein mi and mo are the detected molar masses of L- and D-glufosinate, respectively:
Figure imgf000086_0001
5.3.4 Example 12
In example 12, a racemic mixture M|2 of 1 .25 mmol l_-(VII)i2 and 1.25 mmol D-(VII)I2, wherein l_-(VII)i2 and D-(VII)I2 were hydantoins with the following formulae, was used as substrate:
Figure imgf000086_0002
L-(VII),2 D-(VII)I2
The mixture M|2 was dissolved in a stirring reactor with 25 ml water. 2.4 g of the catalyst 1 was added, followed by the addition of 50 pl C0CI2 solution. The suspension was set to pH 7.5 with 0.5 M NaOH. The total volume was replenished with water to 50 ml, so that the concentration of each enantiomer L-(VII)I2 and D-(VII)H was 0.025 mol/l and the concentration of C0CI2 in the final solution was 1 mM. The pH was held between 7.0 and 7.5 by HCI-titration or NaOH-titration. The temperature was maintained at 37 °C by a thermostat during the reaction.
The reaction was stopped after 120 hours by addition of 2 N HCI until pH 2.5 was reached. The biomass was separated by centrifugation or filtration.
The enzyme reaction was monitored by ninhydrin test to determine the formation of amino acids. Formation of amino acids was detected by the ninhydrine test.
In a final step the reaction mixture was saponified at 100 °C for 10 hours by adding 6 M HCI to obtain L-glufosinate and, if present, D-glufosinate. The final reaction mixture was analysed by LC-MS with a CR-I column as described under item 5.4 to determine the enantiomeric excess (“ee”) of either D- or L-glufosinate. An ee of LGA of 77 % over the D-enantiomer was detected.
5.3.5 Results
The results of the examples C1 , C2, 11 , 12 are summarized in table 5.
Table 5
Figure imgf000087_0001
5.3.6 Conclusion 1
The results as summarized in table 5 show that certain hydantoin esters such as the alkylated hydantoin esters can be used as substrates for the enzymatic enantioselective synthesis of L-glufosinate (11 , 12). In contrast, substrates in which the phosphinic acid group is not protected are not accepted by the catalytic system (C1 , C2). In this regard, C2 suggests that at least one reason for this is that the L-carbamoylase enzyme does not accept the respective substrate in which the phosphinic acid function is not protected by an ester group.
Moreover, in contrast to the process for enantioselective production according to the prior art (ON 111662325 A), the ee excess could be maintained throughout the process, because saponification is carried out not at the hydantoin stage, but at the stage of the amino acid ester.
5.4 Analytical Methods
Analytical Method 1
L-glufosinate and D-glufosinate were detected by LC-MS (“Liquid Chromatography - Mass Spectrometry”) with a chiral column [Daicel CROWNPAK CR-l-(-)] as follows. For hydantoins, a Daicel Chiralpak IA-U column may also be used.
This detection method may also be used for detection and quantification of the the LGA P-(n-butyl) ester according to formula L-(l), wherein R = n-butyl, according to Assay AL (item 4.5.3.1); the D-glufosinate P-(n-butyl) ester according to formula D-(l), wherein R = n-butyl, according to Assay AD (item 4.5.3.2); the carbamoyl LGA P-(n-butyl) ester according to formula L-(ll), wherein R = n-butyl, according to Assay DL (item 4.5.9.1); the carbamoyl D-glufosinate P-(n-butyl) ester according to formula D-(ll), wherein
R = n-butyl, according to Assay DD (item 4.5.9.2); the L-enantiomer of a n-butyl ester of hydantoin glufosinate of the formula L-(lll), wherein R = n-butyl, according to Assay G (item 4.5.14.3). the D-enantiomer of a n-butyl ester of hydantoin glufosinate of the formula D-(lll), wherein R = n-butyl, according to Assay G (item 4.5.14.3).
5.4.1 Aquisition Method Details
CR-l-(-), 3.0 mm I.D. x 150 mm, 5 pm, Crownpak, Part. No. 54784
MS QQQ Mass Spectrometer 6420, Agilent
ESI+
MS2 SIM, Glufosinat m/z 182.06 Unit Positive
Centroid
5.4.2 Source Parameter
Parameter Value (+); Gas Temp (°C): 350; Gas Flow (l/min): 12; Nebulizer (psi): 25; Capillary (V): 3000; Sampler Module: G1329B; Injection Volume 1.00 pL; 5°C
5.4.3 Binary Pump Module
G1312B
Flow: 0.200 mL/min;
Channel A: H2O with trifluoroacetic acid (TFA) pH1.15;
Channel B: ACN (acetonitrile); isocratic 80.0 % A 120.0 % B; stoptime 7.00 min
Column Comp. Module: G1316A
Temperature 5.0 °C
Analytical Method 2
A further analytical method was used to determine the yield of the LGA P-(n-butyl) ester according to formula L-(l), wherein R = n-butyl, and the D-glufosinate P-(n-butyl) ester according to formula D-(l), wherein R = n-butyl. In particular this method was used to determine the yield of L-(VII l)i3 and D-(VIII)i3, as summarized in table 6 hereinafter. Moreover, this analytical method is so specific that the four diastereomers of glufosinate-P-(n-butyl) ester, namely l_l_-(VIII)i3, DL-(VIII)|3, LD-(VIII)|3 and DD-(VIII)i3, may be separated from each other:
Figure imgf000089_0001
l_L-(VIII),3 DL-(VIII)I3
Hence, for this separation task, a LC-MS (“Liquid Chromatography - Mass Spectrometry”) with a chiral column [Chiralpak 3pm ZWIX (+), 150 x 3 mm, DAICEL] was used as follows. The observed error of measurement of this Method 2 was approximately ± 5 %.
5.4.4 Aquisition Method Details
ZWIX (+), 150 x 3 mm; 3pm
MS QQQ Mass Spectrometer 6420, Agilent
ESI+
MS2 SIM, Glufosinat-carbamoyl-butyl ester m/z 238.1 Unit Positive
Centroid
5.4.5 Source Parameter
Parameter Value (+); Gas Temp (°C): 350; Gas Flow (l/min): 12; Nebulizer (psi): 50; Capillary (V): 4000; Sampler Module: G1329B; Injection Volume 1.00 pL; 5°C
5.4.6 Binary Pump Module
G1312B
Flow: 0.300 mL/min; isocratic 49.0 % acetonitrile / 49.0 % methanol, 2.0 % H2O; stoptime 15.00 min
Column Comp. Module: G1316A
Temperature 5.0 °C
5.5 Example 3 Identification of further carbamoylase enzymes and construction of plasmids
In a further test series, genes encoding L-carbamoylases (/V-carbamoyl-L-amino-acid hydrolases, EC 3.5.1 .87) of different origins were tested for their ability to react with different carbamoyl substrates according to structure L-(ll) and D-(ll) to form the respective L-glufosinate/ D-glufosinate derivative according to structures L-(l) and D-(l), respectively. Cloning and expression of the respective L-carbamoylase gene was essentially carried out as described by B. Wilms, A. Wiese, C. Syldatk, R. Mattes, J. Altenbuchner, M. Pietzsch, Journal of Biotechnology 1999, 68, 101 - 113 (hereinafter “Wilms et al."), in particular, as set forth in the following.
5.5.1 Examined enzymes
Details of the strains and genes of the respective L-carbamoylase that were used in the examples are summarized in table 5.
Table 5
Figure imgf000090_0001
5.5.2 Cloning of the enzymes
Cloning of the respective L-carbamoylase gene into the rhamnose expression vector pJOE4036 was carried out in a plasmid derivative of the rhamnose expression vector pJOE4036.
Polynucleotides comprising the genes of the repective enzymes were synthesized by GeneArt (ThermoFisher Scientific (Waltham, USA)). The polynucleotides carried additional sequences for ndel and Hindlll restriction sites. Each one of the genes encoding the enzymes was cloned into pJOE4036 using those restriction sites resulting in the respective plasmid , i.e pOM17c for expression of SEQ ID NO: 1 , pOM17c{Prha}[amaB_Gst] for expression of SEQ ID NO: 2, pOM17c{Prha}[atcC_Ps] for expression of SEQ ID NO: 3, pOM17c{Prha}[hyuc_Pau] for expression of SEQ ID NO: 5, pOM17c {Prha}[hyuC_Asp (co_Ec)] for expression of SEQ ID NO: 8, each under the control of a rhamnose promotor.
5.6 Example 4: Production of strains positive for L-carbamoylase
Chemically competent E. coll ET5 cells (as described in WO 2004/042047 A1) were transformed with 10 ng of the respective plasmid generated according to Example 3. An E. coli ET5 strain transformed with the respective plasmid was incubated under shaking (250 U/min) at 30 °C for 18 hours in LB medium containing ampicillin (100 pg/l), and 2 g/l rhamnose.
The biomass was separated by centrifugation, resuspended in 50 mM phosphate buffer (pH 7.2) and applied in biotransformation tests in the following examples. The concentration of the biomass in the solution was 12.2 g/l. The solution was used as catalyst (“catalyst 2") in the following.
The concentration of the respective polypeptide carbamoylase in the obtained solution may be determined by SDS page and analysis of the respective bands via the software GelQuant® (BiochemLabSolutions).
In the following examples, the different carbamolyases were tested to determine whether the respective polpypetide catalyzed the reaction of the carbamoyl substrate enantioselectively to give the corresponding L amino acid.
5.6.1 Example 13 according to the inventive method
In inventive example I3, a racemic mixture M|3 of 0.25 mmol L-(VII)I3 and 0.25 mmol D-(VII)I3, wherein L-(VII)I3 and D-(VII)I3 were carbamoyles with the following formulae, was used as substrate:
Figure imgf000091_0001
L-(VII)|3 D-(VII)I3
The carbamoylate compound according to formula L-(VII)I3 was an equimolar mixture (1 : 1) of the two diastereoisomers LL-(VII)I3 and DL-(VII)I3, while the carbamoylate compound according to formula D-(VII)I3 was an equimolar mixture (1 : 1) of the two diastereoisomers LD-(VII)I3 and DD-(VII)|3.
Figure imgf000091_0002
LD-(VII)I3 DD-(VII)I3
Figure imgf000092_0001
LL-(VII)I3 DL-(VII)I3
The mixture M|3 was dissolved in a stirring reactor with 25 ml water. 2.4 g of the catalyst 2 was added, followed by the addition of C0CI2 solution. The suspension was set to pH 7.4 with 0.5 M NaOH. The total volume was replenished with water to 50 ml, so that the concentration of each compound l_-(VII)i3 and D-(VII)I3 was 5 mmol/l and the concentration of C0CI2 in the final solution was 1 mM. The pH was held between 7.4 by HCI-titration or NaOH-titration. The temperature was maintained at 37 °C by a thermostat during the reaction.
The reaction was stopped after 48 hours by addition of 2 N HCI until pH 2.5 was reached. The biomass was separated by centrifugation or filtration.
The enzyme reaction was monitored by ninhydrin test to determine the formation of amino acids L-(VIII)|3 and/or D-(VIII)|3.
Figure imgf000092_0002
L-(VIII)I3 D-(VIII)I3
The overall conversion of l_-(VII)i3 to the respective product l_-(VIII)i3 and of D-(VII)I3 to the respective product D-(VIII)I3 after 48 hours was quantified and is shown in table 6. For this analysis, the column: Chiralpak 3pm ZWIX (+), 150 x 3 mm, DAICEL was used (“Analytical Method 2”). Table 6 gives the yield of l_-(VIII)i3 relative to the compound l_-(VII)i3 employed and the yield of D-(VIII)I3 relative to the compound D-(VII)I3 employed.
Table 6
Figure imgf000092_0003
n.d. = not detectable .
Furthermore, the formation of the two diastereomers LL-(VIII)I3 and DL-(VIII)I3 was specifically investigated in case of the reaction with four of the five plasmids, i.e pOM17c for expression of SEQ ID NO: 1 , pOM17c{Prha}[amaB_Gst] for expression of SEQ ID NO: 2, pOM17c{Prha}[atcC_Ps] for expression of SEQ ID NO: 3, pOM17c{Prha}[hyuc_Pau] for expression of SEQ ID NO: 5.
Figure imgf000093_0001
l_L-(VIII)l3 DL-(VIII)I3
The results are shown in Figures 8 to 11 .
These results show that by the Analytical Method 2, the two two diastereomers LL-(VIII)I3 and DL-(VIII)i3 are separated from each other. Alternatively, a crystallization/ precipitation of each amino acid diastereomer LL-(VIII)I3 and DL-(VIII)I3 to separate it from the respective starting material LL-(VII)I3 and DL-(VII)I3, or LD-(VII)I3 and DD-(VII)I3 may also be carried out.
5.6.2 Conclusion 2
The results as summarized in table 6 are further evidence that certain carbamoylate esters such as the alkylated carbamoylate esters can be used as substrates for the enzymatic enantioselective synthesis of L-glufosinate. It further shows that these carbamoylases specifically catalyse the conversion of the L-glufosinate carbamoylate, but not the D-glufosinate carbamoylate.
5.6.3 Conclusion 3
The diagrams in Figures 8 to 10 clearly show that the carbamoylases do not only have a specificity for the chiral center at the a-carbon atom at the glufosinate carbamoyl substrate, but also display a different reactivity depending on the chirality at the phosphorous atom. Thereby, the method according to the present invention allows for diastereoselective synthesis and separation of specific diastereomers of glufosinate P-ester from the respective carbamoyls. In particular, SEQ ID NO:1 as shown in Figure 8 is especially selective in the catalysis of the conversion of only one of the diastereomers LL-(VII)I3 and DL-(VII)I3 into the respective amino acid compound.

Claims

Claims
1. A method for the production of an L-glufosinate P-ester according to formula LL-(I) and/or DL-(I), comprising steps (y), (5), and (e), wherein:
(y) a mixture Mu comprising carbamoyl compounds according to formulae LL-(II) and DL-(II) is provided, wherein LL-(II) and DL-(II) have the following formulae:
Figure imgf000094_0001
LL-(II) DL-(II)
(5) in mixture Mu, a carbamoyl compound according to formula LL-(II) is reacted to give an amino acid compound according to formula LL-(I) and/or a carbamoyl compound according to formula DL-(II) is reacted to give an amino acid compound according to formula DL-(I):
Figure imgf000094_0002
LL-(I) DL-(I) wherein the reaction according to step (6) is catalyzed by a carbamoylase Ei, and wherein R is an alkyl group or an aryl group, so that a mixture Mi is obtained comprising at least one amino acid compound of DL-(I), LL-(I), wherein, if mixture Mi comprises both amino acid compounds DL-(I) and LL-(I), the molar ratio of DL-(I) to LL-(I) in Mi is different from the molar ratio of DL-(II) to LL-(II) in Mu, and wherein mixture Mi comprises at least one of the carbamoyl compounds DL-(II), LL-(II),
(e) and wherein one of the amino acid compounds selected from DL-(I), LL-(I) comprised by mixture Mi is at least partially separated from at least one of the carbamoyl compounds DL-(II), LL-(II) and/or from the other amino acid compound selected from DL-(I), LL-(I).
2. The method according to Claim 1 , wherein R is an alkyl group.
3. The method according to Claim 1 or 2, wherein the reaction according to step (5) is catalyzed by an L-carbamoylase Ei.
4. The method according to Claim 3, wherein the L-carbamoylase Ei is categorized in the EC class 3.5.1 .87.
5. The method according to Claim 3 or 4, wherein the polypeptide sequence of the L-carbamoylase Ei is selected from the group consisting of 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 one of Claims 1 to 5, wherein the mixture Mu provided in step (y) is obtained by a step (p) wherein in a mixture Mm comprising compounds according to LL-(III) and DL-(III), at least a part of the compounds according to formula LL-(III) are reacted to give compounds according to LL-(II), and at least a part of the compounds according to formula DL-(III) are reacted to give compounds according to DL-(II), wherein LL-(III) and DL-(III) have the following formulae:
Figure imgf000095_0001
LL-(III) DL-(III) wherein the reaction according to step (p) is catalyzed by a hydantoinase E2, and wherein R has the same meaning as described for LL-(I) and DL-(I).
7. The method according to Claim 6, wherein the hydantoinase E2 is categorized in the EC class 3.5.2.2.
8. The method according to Claim 6 or 7, 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.
9. The method according to one of Claims 6 to 8, wherein the hydantoinase E2 is an L-hydantoinase E2.
10. The method according to one of Claims 6 to 9, wherein the compound according to formula LL-(III) and/or the compound according to formula DL-(III) is obtained by a step (a) in which a compound according to formula LD-(III) is reacted to give a compound according to formula LL-(III) and/or a compound according to formula DD-(III) is reacted to give a compound according to formula DL-(III), wherein LD-(III) and DD-(III) have the following formulae:
Figure imgf000096_0001
LD-(III) DD-(III) and wherein R has the same meaning as described for LL-(I) and DL-(I).
11. The method according to Claim 10, wherein the reaction according to step (a) is catalyzed by a hydantoin racemase E3.
12. The method according to Claim 11 , wherein the hydantoin racemase E3 is categorized in the EC class 5.1 .99.5.
13. The method according to Claim 11 or 12, wherein 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.
14. The method according to one of Claims 1 to 13, wherein in step (e) the at least partial separation of one of the amino acid compounds selected from DL-(I), LL-(I) comprised by mixture Mi is performed by crystallizing the respective amino acid compound.
15. The method according to one of Claims 1 to 14, wherein the molar ratio of all carbamoyl compounds according to formula LL-(II) in mixture Mu provided in step (y) to all carbamoyl compounds according to formula DL-(II) in mixture Mu provided in step (y) is in the range of 999 : 1 to 1 : 999.
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