Preparation of (1R,2S)-2,6-dimethyl-1-indanamine

Abstract

A method for producing substantially enantiomerically pure (1R,25)-2,6-dimethyl-1-indanamine is described, which comprises reacting a mixture of four stereoisomers of 2,6-dimethyl-1-indanamine with an acylating or carboxylating agent in the presence of a protein having lipase activity.

Description

[Technical Field]

[0001] The present invention relates to a method for producing substantially enantiopure (1R,2S)-2,6-dimethyl-1-indanamine, a valuable building block for the synthesis of the herbicidal active ingredient indaziflam. Specifically, the present invention relates to a method for preparing substantially enantiopure (1R,2S)-2,6-dimethyl-1-indanamine by enzyme-catalyzed stereoselective acylation of racemic 2,6-dimethyl-1-indanamine. [Background technology]

[0002] The only method for preparing substantially enantiopure (1R,2S)-2,6-dimethyl-1-indanamine known to date from the prior art is only suitable for laboratory scale, but not for industrial use, due to the use of expensive reactants and catalysts. For example, WO 2004 / 69814 discloses a method characterized by the following five reaction steps: 1. Preparation of a mixture of four stereoisomers of 2,6-dimethyl-1-aminoindan by palladium-catalyzed reduction of the corresponding oxime.

[0003] 2. Column chromatographic separation of the four stereoisomers into cis and trans isomers.

[0004] 3. Reaction of the trans isomer with methyl 2-methoxyacetate in the presence of the enzyme Novozym 435® to form the corresponding acetylated (1R,2S)-2,6-dimethyl-1-indanamine.

[0005] 4. Isolation of acetylated (1R,2S)-2,6-dimethyl-1-indanamine.

[0006] 5. Acid hydrolysis of acetylated (1R,2S)-2,6-dimethyl-1-indanamine to form free (1R,2S)-2,6-dimethyl-1-indanamine.

[0007] Tetrahedron 2007, 63(29), 6755-6763 describes a method for the preparation of (1R,2S)-2,6-dimethyl-1-indanamine, which is characterized by the following two reaction steps: 1. Racemic 1,6-dimethylindan-1-one is diastereoselectively (96:4 d.r.) and enantioselectively (98:2 e.r.) reduced by a chiral ruthenium catalyst to form the corresponding (1S,2S)-2,6-dimethylindan-1-ol in 80% yield.

[0008] 2. The (1S,2S)-2,6-dimethylindan-1-ol thus obtained is reacted with diphenylphosphoryl azide and subsequent reduction with lithium aluminum hydride to give (1R,2S)-2,6-dimethyl-1-indanamine in 76% yield.

[0009] The drawbacks of this method are considered to be the use of expensive reagents as well as the long reaction time of 8 days in total. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2004 / 69814 Brochure [Non-patent literature]

[0011] [Non-Patent Document 1] Tetrahedron 2007, 63(29), 6755-6763 Summary of the Invention [Problem to be solved by the invention]

[0012] The object of the present invention is to provide a process for preparing substantially enantiopure (1R,2S)-2,6-dimethyl-1-indanamine, which overcomes the drawbacks known from the prior art. [Means for solving the problem]

[0013] The present inventors have discovered a method for preparing substantially enantiopure (1R,2S)-2,6-dimethyl-1-indanamine, characterized by the reaction of a mixture of four stereoisomers of 2,6-dimethyl-1-indanamine with an acylating or carboxylating agent in the presence of an enzyme.

[0014] The present invention provides a method for preparing substantially enantiopure (1R,2S)-2,6-dimethyl-1-indanamine, comprising the steps of: 1. In the first step, a mixture of four stereoisomers of 2,6-dimethyl-1-indanamine (I) is reacted with an acylating or carboxylating agent RC(=O)R 1 in the presence of a protein having lipase activity to form the corresponding amide or carbamate (II) and a mixture of unreacted stereoisomers of 2,6-dimethyl-1-indanamine (III): [ka] [wherein the protein is I. A protein having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and most preferably 99% identity with the amino acid sequence shown under SEQ ID NO: 1; II. An amino acid sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and most preferably 99% identity with the amino acid sequence shown under SEQ ID NO: 1, i. the amino acid at position 186 is different from L; ii. the amino acid at position 280 is different from L; iii. the amino acid at position 312 differs from that of P; iv. the amino acid at position 3 is different from M; v. The amino acid at position 29 is different from N; vi. the amino acid at position 17 is different from L; vii. the amino acid at position 4 is different from S; viii. The amino acid at position 18 is different from that of V; ix. the amino acid at position 202 is different from A; x. the amino acid at position 301 is different from D; xi. The amino acid at position 309 is different from that of P; xii. The amino acid at position 31 is different from Q; xiii. The amino acid at position 111 is different from Q; xiv. The amino acid at position 85 is different from that of W; xv. the amino acid at position 8 is different from K; xvi. The amino acid at position 79 is different from E; xvii. The amino acid at position 40 is different from K; A protein having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and most preferably 99% identity with the amino acid sequence shown under SEQ ID NO: 1, apart from the fact that it has a modification selected from the group consisting of: encoded by an amino acid sequence selected from the group consisting of 2. In a second step, the amide or carbamate (II) is separated from the remaining 2,6-dimethyl-1-indanamine (III) and secondary components by crystallization; and 3. In a third step, converting the amide or carbamate (II) to substantially enantiopure (1R,2S)-2,6-dimethyl-1-indanamine (IV) using an acid or base. [ka] wherein R represents a radical from the group consisting of CH2OCH3, CH2OCH2CH3, CH3, OCH3, OCH2CH3, OCH(CH3)2, OCH2CH2CH2CH3, OCH2CHCH2 and OCH2(C6H5), and In the formula, R 1 means a radical from the group consisting of OCH3, OCH2CH3, OCH(CH3)2, OCH2CH2CH2CH3, OCH2CHCH2 and OCH2(C6H5). The present invention provides a method characterized by:

[0015] The mixture of four stereoisomers of 2,6-dimethyl-1-indanamine (I) required as starting material for the process according to the invention is known and can be prepared, for example, as described in WO 2004 / 69814.

[0016] Preferably, R represents a radical from the group consisting of OCH3 and OCH2CH3, and R 1 means a radical from the group consisting of OCH3 and OCH2CH3.

[0017] In the method according to the invention, a protein having lipolytic enzyme or lipase activity is used.

[0018] SEQ ID NO: 1 is the amino acid sequence of the lipolytic wild-type protein. The wild-type lipase is derived from an uncultivated bacterium from an environmental sample from GenPept (PDB) under accession number QRD81023 (version ORD81023.1). In case of any dispute between the amino acid sequence shown in SEQ ID NO: 1 and the sequence shown in the above database entry, SEQ ID NO: 1 takes precedence.

[0019] Further described are proteins having lipolytic enzyme or lipase activity, wherein the amino acid sequences of these proteins are variants of known proteins having lipolytic enzyme or lipase activity. In particular, the amino acid sequences of the proteins having lipase activity described herein are variants of the amino acid sequence shown in SEQ ID NO: 1, wherein at least the amino acid at position 186, 280, 312, 3, 29, 17, 4, 18, 202, 301, 309, 31, 111, 85, 8, 79, or 40 in the amino acid sequence shown under SEQ ID NO: 1 is different from the amino acid at the corresponding amino acid position in the sequence shown under SEQ ID NO: 1.

[0020] The term "variant" as used herein refers to an entity distinct from that known in the prior art. With respect to nucleic acid molecules and proteins, variants are understood to mean nucleic acid or amino acid sequences that deviate from the corresponding known sequences but encode proteins with the same function or catalyze the same reaction, e.g., encoding proteins with lipase activity. "Deviation" of nucleic acid molecule and amino acid sequences from known nucleic acid and protein sequences means that the sequences contain base or amino acid substitutions (replacements) and / or deletions and / or insertions compared to the corresponding known nucleic acid or amino acid sequences.

[0021] Typically, a protein having lipase activity is used, wherein said protein is encoded by an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, most preferably 99% identity with the amino acid sequence shown under SEQ ID NO: 1.

[0022] Proteins having lipase activity are also used, wherein said proteins have an amino acid sequence that has at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, most preferably 99% identity with the amino acid sequence shown in SEQ ID NO: 1. i. the amino acid at position 186 is different from L, preferably the amino acid at position 186 is F, W, Y, E, D, Q, T, H, P, C, K, S, N, I or V, more preferably the amino acid at position 186 is F, W, Y, E, D or K, particularly preferably the amino acid at position 186 is W or Y, and most preferably the amino acid at position 186 is Y; ii. the amino acid at position 280 is different from L, preferably the amino acid at position 280 is E, S, K, D or A, more preferably the amino acid at position 280 is A; iii. the amino acid at position 312 is different from P, preferably the amino acid at position 312 is N, F, D, Q or K, more preferably the amino acid at position 312 is N; iv. the amino acid at position 3 is different from M, preferably the amino acid at position 3 is L, Q or C, more preferably the amino acid at position 3 is Q; v. the amino acid at position 29 is different from N, preferably the amino acid at position 29 is H, W or Y, more preferably the amino acid at position 29 is H or W, and most preferably the amino acid at position 29 is H; vi. the amino acid at position 17 is different from L, preferably the amino acid at position 17 is P or T, more preferably the amino acid at position 17 is P; vii. the amino acid at position 4 is different from S, preferably the amino acid at position 4 is P or L, more preferably the amino acid at position 4 is P; viii. the amino acid at position 18 is different from V, preferably the amino acid at position 18 is A, T, C, or S, more preferably the amino acid at position 18 is A or C; ix. the amino acid at position 202 is different from A, preferably the amino acid at position 202 is Q or N, more preferably the amino acid at position 202 is N; x. the amino acid at position 301 is different from D, preferably the amino acid at position 301 is A; xi. the amino acid at position 309 is different from P, preferably the amino acid at position 309 is C; xii. the amino acid at position 31 is different from Q, preferably the amino acid at position 31 is W; xiii. the amino acid at position 111 is different from Q, preferably the amino acid at position 111 is E; xiv. the amino acid at position 85 is different from W, preferably the amino acid at position 85 is H; xv. the amino acid at position 8 is different from K, preferably the amino acid at position 8 is E; xvi. the amino acid at position 79 is different from K, preferably the amino acid at position 79 is S, I, or W, more preferably the amino acid at position 79 is S; xvii. the amino acid at position 40 is different from K, preferably the amino acid at position 40 is M; Apart from the fact that the amino acid sequence has an alteration selected from the group consisting of:

[0023] The meanings of the amino acid abbreviations A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y can be found herein following Table 2 in the section entitled "Sequence Description."

[0024] A further embodiment according to the present invention relates to a protein having the activity of a lipase, wherein said protein a) a protein comprising the amino acid sequence shown in SEQ ID NO: 1, apart from the fact that the amino acid at position 186 differs from L; b) a protein having an amino acid sequence that is at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and most preferably 99% identical to the amino acid sequence shown under a), with the proviso that the amino acid at position 186 is different from L. is selected from the group consisting of:

[0025] Preferably, the amino acid at position 186 in the protein according to a) or b) is F, W, Y, E, D, Q, T, H, P, C, K, S, N, I or V. More preferably, the amino acid at position 186 is F, W, Y, E, D or K. Particularly preferably, the amino acid at position 186 is W or Y. Most preferably, the amino acid at position 186 is Y.

[0026] As used herein, an "amino acid corresponding to position x" in a first amino acid sequence (e.g., position 3 in SEQ ID NO: 1) means that, when the amino acid numbering of the second amino acid sequence deviates from the amino acid numbering of the first amino acid sequence, compared to the first amino acid sequence, the amino acid of the second amino acid sequence appears at position x in the first amino acid sequence in a pairwise sequence alignment of the first and second amino acid sequences.

[0027] In the context of the present invention, the term "identity" with respect to sequence identity or identical sequences should be understood to mean the number of identical amino acids or nucleotides (expressed as a percentage) that a first nucleic acid or amino acid sequence has in common with another (second) nucleic acid or amino acid sequence over the entire sequence length.

[0028] "Sequence identity" can be determined by aligning two amino acid or two nucleotide sequences using a global or local alignment algorithm, such as that included in known software such as GAP or BESTFIT or the Emboss program "Needle." This software uses the global alignment algorithm by Needleman and Wunsch to align two sequences over their entire length, maximizing the number of matches and minimizing the number of gaps. Generally, the default parameters are used: gap creation penalty = 10 and gap extension penalty = 0.5 (for both nucleotide and protein alignment). For nucleotides, the default scoring matrix used is DNAFULL, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 10915-10919). Sequence alignments and percent sequence identity scores can be determined, for example, using software such as EMBOSS, available on the EBI website (ebi.ac.uk / Tools / emboss / ). Alternatively, sequence similarity or identity can be determined by searching databases (e.g., EMBL, GenBank) using commonly known algorithms and output formats such as FASTA, BLAST, etc., although it is preferred to search and pairwise align hits to ultimately determine sequence identity.

[0029] When the sequences to be compared are of different lengths, identity should be determined by determining the percentage of amino acids or nucleotides that the shorter sequence shares with the longer sequence.Preferably, identity is determined using the well-known, publicly available computer program ClustalW (Thompson et al., Nucleic Acids Research 22 (1994), 4673-4680).ClustalW is publicly available from Julie Thompson (Thompson@EMBL-Heidelberg.DE) and Toby Gibson (Gibson@EMBL-Heidelberg.DE), European Molecular Biology Laboratory, Meyerhofstrasse 1, D 69117 Heidelberg, Germany. ClustalW can also be downloaded from various websites, such as IGBMC (Institut de Genetique et de Biologie Moleculaire et Cellulaire, BP163, 67404 Illkirch Cedex, France; ftp: / / ftp-igbmc.u-strasbg.fr / pub / ) and EBI (ftp: / / ftp.ebi.ac.uk / pub / software / ) and mirror websites of EBI (European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK).

[0030] Preferably, the computer program ClustalW version 1.8 is used to determine identity between the proteins described in connection with the present invention and other proteins, where the parameters should be set as follows: KTUPLE=1, TOPDIAG=5, WINDOW=5, PAIRGAP=3, GAPOPEN=10, GAPEXTEND=0.05, GAPDIST=8, MAXDIV=40, MATRIX=GONNET, ENDGAPS(OFF), NOPGAP, NOHGAP.

[0031] Preferably, the computer program ClustalW version 1.8 is used to determine identity between, for example, the nucleotide sequences of the nucleic acid molecules described in connection with the present invention and the nucleotide sequences of other nucleic acid molecules, where the parameters should be set as follows: KTUPLE=2, TOPDIAGS=4, PAIRGAP=5, DNAMATRIX:IUB, GAPOPEN=10, GAPEXT=5, MAXDIV=40, TRANSITIONS: Unweighted.

[0032] "Identity" also means functional and / or structural equivalence between the nucleic acid molecules in question or the proteins encoded thereby. Functional equivalence means that the nucleic acid molecule sequence or amino acid sequence encodes a protein with lipase activity. Nucleic acid molecules that are homologous to and derivatives of the above molecules are usually variants of these molecules, having the same biological function or catalyzing the same reaction, i.e., encoding proteins with lipase activity. They can be naturally occurring variants, e.g., sequences from other species, or mutations, where the mutations can occur naturally or be introduced by targeted mutagenesis. Furthermore, variants can be synthetically produced sequences. Allelic variants can be naturally occurring or synthetically produced, or produced by recombinant DNA technology. However, what is important for the present invention is that these variants encode proteins with lipase activity and contain the amino acid substitutions, deletions, or insertions currently described for the proteins of the present invention.

[0033] A particular type of derivative is a nucleic acid molecule that differs from the nucleic acid molecules described in accordance with the present invention due, for example, to the degeneracy of the genetic code.

[0034] According to the NC-IUBMB (Commission on Nomenclature of the International Union of Biochemistry and Molecular Biology), lipases belong to the class of hydrolases (EC 3). Hydrolases are a class of enzymes that typically act as biochemical catalysts, using water to break chemical bonds, typically resulting in the splitting of larger molecules into smaller ones. The group of hydrolases includes enzymes that act on ester bonds (EC 3.1), such as carboxylic ester hydrolases (EC 3.1.1), and the subgroup lipases (EC 3.1.1.3). Lipases have been identified from plants, mammals, and microorganisms, such as cutinases from Pseudomonas, Vibrio, Acinetobacter, Burkholderia, Chromobacterium, Fusarium solani (FSC), Candida antarctica A (CalA), Rhizopus oryzae (ROL), Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML), Aspergillus Niger, and Fusarium heterosporum. heterosporum, Fusarium oxysporum or Fusarium culmorum.

[0035] If a protein has lipase activity, it can be detected using methods known and described in the prior art. It is not important which method is used to detect whether a protein according to the present invention has lipase activity. Preferably, the methods described in the "Examples" section in connection with the present invention.

[0036] The lipase variant proteins according to the present invention may have further amino acid modifications (amino acid substitutions, deletions or insertions) compared to the amino acid sequences described herein above in relation to the amino acid sequence shown under SEQ ID NO: 1.

[0037] With respect to the amino acid sequence shown under SEQ ID NO: 1, the lipase variants described herein under point a) may further have at least 1, 2, 3, 4, 5, 6 or 7 further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. In other words, a protein according to the present invention having lipase activity is a protein comprising the amino acid sequence set forth in SEQ ID NO: 1, apart from the fact that the amino acid at position 186 is different from L and that it has at least one, two, three, four, five, six, seven or more further amino acid substitutions selected from the group consisting of: (i) the amino acid at position 79 is different from E; (ii) the amino acid at position 202 is different from A; (iii) the amino acid at position 280 is different from L; (iv) the amino acid at position 301 is different from D; (v) the amino acid at position 3 is different from M; (vi) the amino acid at position 11 is different from C; (vii) the amino acid at position 17 is different from L; (viii) the amino acid at position 40 is different from K; (ix) the amino acid at position 111 is different from Q; and b) a) is selected from the group consisting of proteins having an amino acid sequence that is at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and most preferably 99% identical to the amino acid sequence shown directly under a), with the proviso that the amino acid at position 186 is different from L and has at least one further amino acid substitution selected from groups (i) to (ix) directly above. Preferably, the amino acid at position 79 is S, W, or I, more preferably S; preferably, the amino acid at position 202 is N; preferably, the amino acid at position 280 is A; preferably, the amino acid at position 301 is A; preferably, the amino acid at position 3 is Q; preferably, the amino acid at position 11 is A; preferably, the amino acid at position 17 is P; preferably, the amino acid at position 40 is M; and preferably, the amino acid at position 111 is E.

[0038] Furthermore, apart from the further amino acid modifications, the lipase variant proteins according to the present invention can have further amino acid substitutions compared to the amino acid sequence described herein above with respect to the amino acid sequence shown under SEQ ID NO: 1. These further amino acid substitutions relate to positions in the amino acid sequence that are different from positions 79, 202, 280, 301, 3, 11, 17, 40 or 111 associated with the further amino acid modifications. The lipase variants described above under point a) with respect to the amino acid sequence shown under SEQ ID NO: 1 can have at least one, two, three, four, five, six, seven or more further amino acid substitutions at positions 4, 8, 18, 29, 31, 42, 84, 85, 192, 217, 309 or 312. The amino acid at position 4 is different from S, preferably the amino acid at this position is P. The amino acid at position 8 is different from K, preferably the amino acid at this position is E. The amino acid at position 18 is different from V, preferably the amino acid at this position is C. The amino acid at position 29 is different from N, preferably the amino acid at this position is W or H. The amino acid at position 31 is different from Q, preferably the amino acid at this position is W. The amino acid at position 42 is different from L, preferably the amino acid at this position is D. The amino acid at position 84 is different from N, preferably the amino acid at this position is T. The amino acid at position 85 is different from W, preferably the amino acid at this position is H. The amino acid at position 192 is different from F, preferably the amino acid at this position is A or V. The amino acid at position 217 is different from Q, preferably the amino acid at this position is M. The amino acid at position 309 is different from P, preferably the amino acid at this position is C. The amino acid at position 312 is different from P, preferably the amino acid at this position is N.

[0039] Therefore, a further embodiment according to the invention relates to proteins according to the invention with further amino acid modifications; preferably these embodiments are proteins with the activity of a lipase, wherein said protein comprises: - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E, wherein the amino acid at position 186 is preferably W or Y, more preferably Y, and the amino acid at position 79 is preferably S, W or I, more preferably S; - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N; - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A; - the amino acid at position 186 is different from L and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 301 is preferably A; - the amino acid at position 186 is different from L and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 111 is preferably E; Apart from that, a protein having the amino acid sequence set forth in SEQ ID NO: 1, - proteins having an amino acid sequence which is at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, most preferably 99% identical to the amino acid sequence shown under a), with the proviso that the amino acid at position 186 is different from L, wherein the amino acid at position 186 is preferably W or Y, and said protein has at least one further amino acid substitution selected from the group shown immediately above under the recited symbol. is selected from the group consisting of:

[0040] Preferably, with respect to the amino acid sequence shown under SEQ ID NO: 1, the lipase variants described herein above under point a) may have at least two further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. Preferably, the amino acid at position 79 is S, W or I, more preferably S; preferably, the amino acid at position 202 is N; preferably, the amino acid at position 280 is A; preferably, the amino acid at position 301 is A; preferably, the amino acid at position 3 is Q; preferably, the amino acid at position 11 is A; preferably, the amino acid at position 17 is P; preferably, the amino acid at position 40 is M; preferably, the amino acid at position 111 is E.

[0041] Therefore, a further embodiment according to the invention relates to proteins according to the invention with further amino acid modifications, preferably these embodiments are proteins with the activity of a lipase, wherein said proteins - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, and the amino acid at position 202 is different from A, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 202 is preferably N; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, and the amino acid at position 280 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 280 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 301 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 11 is more preferably A; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, and the amino acid at position 280 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, and the amino acid at position 280 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, and the amino acid at position 301 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A and the amino acid at position 301 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 11 is different from C, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 11 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 11 is different from C, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 11 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 11 is different from C, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 11 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 17 is preferably P, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 17 is preferably P, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E; Apart from that, a protein having the amino acid sequence set forth in SEQ ID NO: 1, - proteins having an amino acid sequence which is at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, most preferably 99% identical to the amino acid sequence shown under a), with the proviso that the amino acid at position 186 is different from L, wherein the amino acid at position 186 is preferably W or Y, and wherein said protein has at least two further amino acid substitutions selected from the group shown immediately above under the recited symbol. is selected from the group consisting of:

[0042] Preferably, with respect to the amino acid sequence shown under SEQ ID NO: 1, the lipase variants described herein under point a) may have at least three further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. Preferably, the amino acid at position 79 is S, W or I, more preferably S; preferably, the amino acid at position 202 is N; preferably, the amino acid at position 280 is A; preferably, the amino acid at position 301 is A; preferably, the amino acid at position 3 is Q; preferably, the amino acid at position 11 is A; preferably, the amino acid at position 17 is P; preferably, the amino acid at position 40 is M; preferably, the amino acid at position 111 is E.

[0043] Therefore, a further embodiment according to the invention relates to proteins according to the invention with further amino acid modifications, preferably these embodiments are proteins with the activity of a lipase, wherein said protein - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 202 is different from A, and the amino acid at position 280 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 202 is preferably N, and the amino acid at position 280 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 202 is different from A, and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 202 is preferably N, and the amino acid at position 301 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 202 is different from A, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 202 is preferably N, and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 202 is different from A, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 202 is preferably N, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 202 is different from A, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 202 is preferably N, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 202 is different from A, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 202 is preferably N, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 202 is different from A, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 202 is preferably N, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 280 is different from L, and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 280 is preferably A, and the amino acid at position 301 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 280 is different from L, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 280 is preferably A, and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 280 is different from L, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 280 is preferably A, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 280 is different from L, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 280 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 280 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 280 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 280 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 280 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 301 is different from D, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 301 is preferably A, and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 301 is different from D, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 301 is preferably A, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 301 is different from D, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 301 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 301 is different from D, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, more preferably Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 301 is preferably A, and the amino acid at position 40 is preferably M, wherein the amino acid at position 186 is particularly preferably Y, the amino acid at position 79 is particularly preferably S, the amino acid at position 301 is particularly preferably A, and the amino acid at position 40 is particularly preferably M; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 301 is different from D, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 301 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 3 is different from M, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 3 is preferably Q, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 3 is different from M, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 3 is preferably Q, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 3 is different from M, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 3 is preferably Q, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 3 is different from M, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 3 is preferably Q, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 11 is different from C, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 11 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 11 is different from C, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 11 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 11 is different from C, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 11 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 17 is preferably P, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 17 is preferably P, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 280 is different from L, and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 280 is preferably A, and the amino acid at position 301 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 280 is different from L, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 280 is preferably A, and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 280 is different from L, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 280 is preferably A, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 280 is different from L, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 280 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 280 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 280 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 280 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 280 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 301 is different from D, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 301 is preferably A, and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 301 is different from D, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 301 is preferably A, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 301 is different from D, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 301 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 301 is different from D, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 301 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 301 is different from D, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 301 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 3 is different from M, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 3 is preferably Q, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 3 is different from M, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 3 is preferably Q, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 3 is different from M, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 3 is preferably Q, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 3 is different from M, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 3 is preferably Q, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 11 is different from C, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 11 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 11 is different from C, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 11 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 11 is different from C, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 11 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 17 is preferably P, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 17 is preferably P, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 301 is preferably A, and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 301 is preferably A, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 301 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 301 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 301 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 11 is different from C, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 11 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 11 is different from C, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 11 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 11 is different from C, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 11 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 17 is preferably P, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 17 is preferably P, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 3 is different from M, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 3 is different from M, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 3 is different from M, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 3 is different from M, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 11 is different from C, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 11 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 11 is different from C, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 11 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 11 is different from C, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 11 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 17 is preferably P, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 17 is preferably P, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, the amino acid at position 11 is different from C, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, the amino acid at position 11 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, the amino acid at position 11 is different from C, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, the amino acid at position 11 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, the amino acid at position 11 is different from C, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, the amino acid at position 11 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, the amino acid at position 17 is preferably P, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, the amino acid at position 17 is preferably P, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 11 is different from C, the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 11 is preferably A, the amino acid at position 17 is preferably P, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 11 is different from C, the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 11 is preferably A, the amino acid at position 17 is preferably P, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 11 is different from C, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 11 is preferably A, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 17 is different from L, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 17 is preferably P, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E; Apart from that, a protein having the amino acid sequence set forth in SEQ ID NO: 1, - proteins having an amino acid sequence which is at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, most preferably 99% identical to the amino acid sequence shown under a), with the proviso that the amino acid at position 186 is different from L, wherein the amino acid at position 186 is preferably W or Y, more preferably Y, and wherein said protein has at least three further amino acid substitutions selected from the group shown immediately above under the recited symbol. is selected from the group consisting of:

[0044] The lipase variants described herein above under point a) in relation to the amino acid sequence shown under SEQ ID NO: 1 may have at least four further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. Preferably, the amino acid at position 79 is S, W or I, more preferably S; preferably, the amino acid at position 202 is N; preferably, the amino acid at position 280 is A; preferably, the amino acid at position 301 is A; preferably, the amino acid at position 3 is Q; preferably, the amino acid at position 11 is A; preferably, the amino acid at position 17 is P; preferably, the amino acid at position 40 is M; preferably, the amino acid at position 111 is E.

[0045] The lipase variants described herein under point a) with reference to the amino acid sequence shown under SEQ ID NO: 1 may have at least five further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. Preferably, the amino acid at position 79 is S, W or I, more preferably S; preferably, the amino acid at position 202 is N; preferably, the amino acid at position 280 is A; preferably, the amino acid at position 301 is A; preferably, the amino acid at position 3 is Q; preferably, the amino acid at position 11 is A; preferably, the amino acid at position 17 is P; preferably, the amino acid at position 40 is M; preferably, the amino acid at position 111 is E.

[0046] The lipase variants described herein under point a) with reference to the amino acid sequence shown under SEQ ID NO: 1 may have at least six further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. Preferably, the amino acid at position 79 is S, W or I, more preferably S; preferably, the amino acid at position 202 is N; preferably, the amino acid at position 280 is A; preferably, the amino acid at position 301 is A; preferably, the amino acid at position 3 is Q; preferably, the amino acid at position 11 is A; preferably, the amino acid at position 17 is P; preferably, the amino acid at position 40 is M; preferably, the amino acid at position 111 is E.

[0047] The lipase variants described herein under point a) with reference to the amino acid sequence shown under SEQ ID NO: 1 may have at least seven further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. Preferably, the amino acid at position 79 is S, W or I, more preferably S; preferably, the amino acid at position 202 is N; preferably, the amino acid at position 280 is A; preferably, the amino acid at position 301 is A; preferably, the amino acid at position 3 is Q; preferably, the amino acid at position 11 is A; preferably, the amino acid at position 17 is P; preferably, the amino acid at position 40 is M; preferably, the amino acid at position 111 is E.

[0048] Preferred embodiments according to the present invention are those represented by SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 19, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 31 9, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401.

[0049] Particularly preferred embodiments according to the present invention are proteins encoding lipases having the amino acid sequences shown under SEQ ID NOs: 233 and 399.

[0050] Additional proteins with lipolytic enzyme or lipase activity have been tested. The amino acid sequences of these additional proteins are variants of the amino acid sequence described by amino acid sequence SEQ ID NO: 1, wherein: - in the case of the amino acid sequence shown under SEQ ID NO: 1, the two amino acids at positions 40 and 79 are different from the amino acids shown at the corresponding amino acid positions in the sequence shown under SEQ ID NO: 1. In the particular variant tested in relation to the amino acid sequence shown under SEQ ID NO: 1, the amino acid at position 40 is M and the amino acid at position 79 is S; - in the case of the amino acid sequence shown under SEQ ID NO: 1, the two amino acids at positions 40 and 186 differ from the amino acids shown at the corresponding amino acid positions in the sequence shown under SEQ ID NO: 1. In the particular variant tested in relation to the amino acid sequence shown under SEQ ID NO: 1, the amino acid at position 40 is M and the amino acid at position 186 is Y; - in the case of the amino acid sequence shown under SEQ ID NO: 1, the two amino acids at positions 40 and 301 are different from the amino acids shown at the corresponding amino acid positions in the sequence shown under SEQ ID NO: 1. In the particular variant tested in relation to the amino acid sequence shown under SEQ ID NO: 1, the amino acid at position 40 is M and the amino acid at position 301 is A; - in the case of the amino acid sequence shown under SEQ ID NO: 1, the two amino acids at positions 79 and 186 differ from the amino acids shown at the corresponding amino acid positions in the sequence shown under SEQ ID NO: 1. In the particular variant tested in relation to the amino acid sequence shown under SEQ ID NO: 1, the amino acid at position 79 is S and the amino acid at position 186 is Y; - in the case of the amino acid sequence shown under SEQ ID NO: 1, the two amino acids at positions 79 and 301 differ from the amino acids shown at the corresponding amino acid positions in the sequence shown under SEQ ID NO: 1. In the particular variant tested in relation to the amino acid sequence shown under SEQ ID NO: 1, the amino acid at position 79 is S and the amino acid at position 301 is A; - In the case of the amino acid sequence shown under SEQ ID NO: 1, the two amino acids at positions 186 and 301 differ from the amino acids shown at the corresponding amino acid positions in the sequence shown under SEQ ID NO: 1. In the particular variant tested in relation to the amino acid sequence shown under SEQ ID NO: 1, the amino acid at position 186 is Y and the amino acid at position 301 is A.

[0051] Additional proteins with lipolytic enzyme or lipase activity have been tested. The amino acid sequences of these additional proteins are variants of the amino acid sequence described by amino acid sequence SEQ ID NO: 1, wherein: - in the case of the amino acid sequence shown under SEQ ID NO: 1, the three amino acids at positions 40, 79 and 186 differ from the amino acids shown at the corresponding amino acid positions in the sequence shown under SEQ ID NO: 1. In the particular variant tested in relation to the amino acid sequence shown under SEQ ID NO: 1, the amino acid at position 40 is M, the amino acid at position 79 is S, and the amino acid at position 186 is Y; - in the case of the amino acid sequence shown under SEQ ID NO: 1, the three amino acids at positions 40, 79 and 301 differ from the amino acids shown at the corresponding amino acid positions in the sequence shown under SEQ ID NO: 1. In the particular variant tested in relation to the amino acid sequence shown in SEQ ID NO: 1, the amino acid at position 40 is M, the amino acid at position 79 is S, and the amino acid at position 301 is A; - in the case of the amino acid sequence shown under SEQ ID NO: 1, the three amino acids at positions 40, 186 and 301 differ from the amino acids shown at the corresponding amino acid positions in the sequence shown under SEQ ID NO: 1. In the particular variant tested in relation to the amino acid sequence shown under SEQ ID NO: 1, the amino acid at position 40 is M, the amino acid at position 186 is Y, and the amino acid at position 301 is A; - In the case of the amino acid sequence shown under SEQ ID NO: 1, the three amino acids at positions 79, 186 and 301 differ from the amino acids shown at the corresponding amino acid positions in the sequence shown under SEQ ID NO: 1. In the particular variant tested in relation to the amino acid sequence shown under SEQ ID NO: 1, the amino acid at position 79 is S, the amino acid at position 186 is Y, and the amino acid at position 301 is A.

[0052] The lipases and lipase variants of the present invention have high selectivity and / or high specific activity for the stereoselective acylation or carboxylation of 2,6-dimethyl-1-indanamine (DMAI) to produce enantiomerically enriched or substantially pure methyl[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate, which is a key intermediate for the synthesis of the herbicidal compound indaziflam.

[0053] As used herein, "enantiomerically enriched" means that one of the two enantiomers in the composition is present in greater amount than the other enantiomer, preferably, one enantiomer is present in the composition to an extent of at least 60%, more preferably, one enantiomer is present in the composition to an extent of at least 65%, even more preferably, one enantiomer is present in the composition to an extent of at least 70%, even more preferably, one enantiomer is present in the composition to an extent of at least 75%, even more preferably, one enantiomer is present in the composition to an extent of at least 80%, particularly preferably, one enantiomer is present in the composition to an extent of at least 85%, most preferably, one enantiomer is present in the composition to an extent of at least 90%, or very particularly preferably, one enantiomer is present in the composition to an extent of at least 94%.

[0054] As used herein, "enantiomerically substantially pure" means that one of the two enantiomers in the composition is present in an amount of at least 95.0%, preferably that one of the two enantiomers in the composition is present in an amount of at least 95.5%, more preferably that one of the two enantiomers in the composition is present in an amount of at least 96.0%, even more preferably that one of the two enantiomers in the composition is present in an amount of at least 96.5%, even more preferably that one of the two enantiomers in the composition is present in an amount of at least 97.0%, even more preferably that one of the two enantiomers in the composition is present in an amount of at least 98.0%, particularly preferably that one of the two enantiomers in the composition is present in an amount of at least 98.5%, most preferably that one of the two enantiomers in the composition is present in an amount of at least 99.0%, or very particularly preferably that one of the two enantiomers in the composition is present in an amount of at least 99.5%.

[0055] A further embodiment according to the invention relates to a nucleic acid molecule encoding a protein according to the invention.

[0056] The nucleic acid molecule according to the present invention can be any type of nucleic acid, provided that it encodes a protein according to the present invention. The nucleic acid can be a ribonucleic acid molecule (e.g., RNA, mRNA) or a deoxyribonucleic acid molecule (DNA, including genomic DNA, which may or may not contain introns and coding DNA).

[0057] Of particular interest for the present invention are SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 1 9, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 22 1, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 322 3, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401.

[0058] The present invention further comprises: a) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 2, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218 , 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, a nucleic acid molecule comprising a nucleic acid sequence set forth under 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402; b) a nucleic acid molecule having at least 60%, preferably 70%, more preferably 80%, even more preferably 90%, even more preferably 95%, even more preferably 96%, particularly preferably 97%, most preferably 98% or very particularly preferably 99% identity with the nucleic acid sequence shown under a). The present invention relates to a nucleic acid molecule encoding a protein having lipase activity selected from the group consisting of:

[0059] In the context of the present invention, "hybridizing" means hybridization under conventional hybridization conditions, preferably under stringent conditions, such as those described, for example, by Sambrook et al. (Molecular Cloning, A Laboratory Manual, 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ISBN: 0879695773) or Ausubel et al. (Short Protocols in Molecular Biology, John Wiley & Sons, 5th Edition (2002), ISBN: 0471250929). Particularly preferably, "hybridization" means hybridization under the following conditions: Hybridization buffer: 2xSSC; 10x Denhardt's solution (Fikoll 400 + PEG + BSA; 1:1:1 ratio); 0.1% SDS; 5mM EDTA; 50mM Na2HPO4; 250μg / ml herring sperm DNA; 50μg / ml tRNA; or 25M sodium phosphate buffer, pH 7.2; 1mM EDTA; 7% SDS Hybridization temperature: T=65℃~68℃ Wash buffer: 0.1xSSC; 0.1% SDS Washing temperature: T=65℃~68℃.

[0060] Nucleic acid molecules that hybridize to nucleic acid molecules encoding proteins with the activity of a lipase can be derived from any organism; therefore, they can be derived from bacteria, fungi, animals, humans, plants or viruses.

[0061] The nucleic acid molecule that hybridizes to the nucleic acid molecule encoding a protein having lipase activity is preferably derived from a microorganism, more preferably from a fungus or bacterium, and most preferably from a bacterium.

[0062] The nucleic acid molecules that hybridize with the aforementioned molecules can be isolated from, for example, genomic DNA or cDNA library.These nucleic acid molecules can be identified and isolated by using the nucleic acid molecules described herein, or by using the parts of these molecules or the reverse complements of these molecules, for example, by standard hybridization (for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ISBN: 0879695773; Ausubel et al., Short Protocols in Molecular Biology, John Wiley & Sons; 5th Edition (2002), ISBN: 0471250929) or PCR amplification.

[0063] The fragments used as hybridization probes may also be synthetic fragments or oligonucleotides produced using conventional synthesis techniques, the sequences of which are substantially identical to the nucleic acid molecules described in connection with the present invention. When a gene that hybridizes to the nucleic acid sequence described in connection with the present invention is identified and isolated, the sequence should be determined, and the properties of the protein encoded by the sequence should be analyzed to determine whether it is a protein with lipase activity. Methods for determining whether a protein has the activity of a protein with lipase activity are known to those skilled in the art.

[0064] Molecules that hybridize to the nucleic acid molecules described in accordance with the present invention include in particular fragments, derivatives, and allelic variants of the aforementioned nucleic acid molecules. In the context of the present invention, the term "derivative" means that the sequences of these molecules differ from the sequences of the above-mentioned nucleic acid molecules at one or more positions and are highly identical to these sequences. Differences with respect to the above-mentioned nucleic acid molecules can be due, for example, to deletions, additions, substitutions, insertions, or recombinations.

[0065] Preferred nucleic acid molecules according to the present invention are those set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 16, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 2 14, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 311, 312 12, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402.

[0066] The meanings of the nucleotide abbreviations a, c, g, t and degenerate nucleotide abbreviations r, y, s, w, k, m, b, d, h, v, n can be found herein in the section entitled "Sequence Description" following Table 1. The amino acids encoded by degenerate nucleotides can be found herein in the section entitled "Sequence Description" following Table 3.

[0067] Recombinant nucleic acid molecules comprising nucleic acid molecules suitable for the methods according to the invention are also disclosed.

[0068] The term "recombinant nucleic acid molecule" should be understood to mean not only a nucleic acid molecule suitable for the method according to the invention, but also a nucleic acid molecule containing additional sequences that do not naturally occur in the resulting combination in the recombinant nucleic acid for the method according to the invention. The aforementioned additional sequences can be any sequences; preferably, they are functional or regulatory sequences (promoters, termination signals, enhancers, ribosome binding sites (rbs), leader sequences that increase transcription, translation, or RNA stability, intracellular targeting sequences, etc.), particularly preferably, they are functional or regulatory sequences that are active in microorganisms, and very particularly preferably, they are regulatory sequences that are active in fungi, especially yeast, or bacteria. Methods for producing recombinant nucleic acid molecules suitable for the method according to the invention are known to those skilled in the art. These include genetic methods such as ligation of nucleic acid molecules, combining nucleic acid molecules by genetic recombination, or de novo synthesis. These methods are described, for example, in Sambrok et al. (Molecular Cloning, A Laboratory Manual, 3rd Edition (2001) Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY. ISBN: 0879695773) or Ausubel et al. (Short Protocols in Molecular Biology, John Wiley & Sons; 5th Edition (2002), ISBN: 0471250929).

[0069] Recombinant nucleic acid molecules that can be used in the methods according to the invention include suitable nucleic acid molecules linked to regulatory sequences that initiate transcription in prokaryotic or eukaryotic cells.

[0070] The regulatory sequence that initiates transcription in cells is also known as promoter.Regulatory sequence and plasmid information are well known to those skilled in the art, and can be found, for example, in the standard biological parts registry supported by The International Genetically Engineered Machine (iGEM) Foundation (One Kendall Square, Suite B6104, Cambridge, MA 02139, USA) on the Internet (http: / / parts.igem.org / Catalog).

[0071] Regulatory sequences for initiating transcription in prokaryotes such as Escherichia coli and eukaryotes are widely described in the literature, particularly those relevant for expression in yeasts such as Saccharomyces cerevisiae. Overviews of different systems for protein expression in different host organisms are found, for example, in Methods in Enzymology 153 (1987), 383-516 and Bitter et al. (Methods in Enzymology 153 (1987), 516-544) or Gomes et al. (2016, Advances in Animal and Veterinary Sciences, 4(4), 346) and Baghban et al. (2018, Current Pharmaceutical Biotechnology, 19(6)). Common yeast promoters include pAOX1, pHIS4, pGAL, and pScADH2 (Baghban et al., 2018, see above). Conventional bacterial promoters are the T5 promoter, T7 promoter, rhamnose-inducible promoter, arabinose-inducible promoter, PhoA promoter, and artificial trc (trp-lac) promoter, as described by Marschall et al. (2017, Appl Microbiol Biotechnol 101, 501-512) and Tegel et al. (2011, FEBS Journal 278, 729-739).

[0072] A further embodiment of a recombinant nucleic acid molecule that can be used in the method according to the invention is a plasmid vector that contains a suitable nucleic acid molecule.

[0073] The term "vector" is well known in the field of molecular biology, and as used herein, refers to a nucleic acid sequence or a vehicle containing a nucleic acid sequence that is used to introduce genetic material (DNA or RNA) into a target cell. A vector can be a plasmid, such as a T-DNA or binary vector for producing a transgenic plant, an expression vector for expressing a nucleic acid sequence in a host cell, a shuttle vector that can propagate in different hosts, or a vector can be a virus particle or bacteriophage that has been modified to deliver foreign genetic material into a host.

[0074] "Plasmid" is well known in the field of molecular biology and, as used herein, refers to an autonomously self-replicating, usually circular, DNA molecule that, when present in a host cell, is separate from chromosomal DNA.

[0075] Any suitable nucleic acid molecule, recombinant nucleic acid molecule, vector or plasmid can be used to produce a protein for the method according to the invention, for example by expression of the suitable nucleic acid molecule in a host cell.

[0076] Also disclosed are hosts or host cells which contain or express a nucleic acid molecule suitable for the method according to the invention, or which contain a suitable protein having the activity of a lipase, or which contain a recombinant nucleic acid molecule suitable for the method according to the invention, or which contain a vector suitable for the method according to the invention, or which contain a plasmid suitable for the method according to the invention.

[0077] Suitable nucleic acid molecules encoding proteins having lipase activity can be expressed in host cells, for example, for their propagation or for the production of proteins having lipase activity. For expression in host cells, suitable nucleic acid molecules can be present on vectors or plasmids, or can be stably integrated into the genome of a particular host cell. Suitable nucleic acid molecules can also be present in vectors that facilitate their introduction into host cells. Also disclosed are hosts or host cells suitable for the methods of the present invention that contain nucleic acid molecules suitable for the methods of the present invention, or that contain recombinant nucleic acid molecules suitable for the methods of the present invention, or that contain vectors suitable for the methods of the present invention, or that contain plasmids suitable for the methods of the present invention, each containing a protein suitable for the methods of the present invention. Also disclosed are hosts or host cells suitable for the methods of the present invention that contain nucleic acid molecules suitable for the methods of the present invention, or that contain recombinant nucleic acid molecules suitable for the methods of the present invention, or that contain vectors suitable for the methods of the present invention, or that contain plasmids suitable for the methods of the present invention, each expressing a protein suitable for the methods of the present invention, wherein said protein preferably has lipase activity.

[0078] "Expression of a nucleic acid molecule" as used herein should be understood to mean that, if the nucleic acid molecule is RNA or mRNA, the nucleic acid molecule is translated into a protein, preferably into a protein having lipase activity, or, if the nucleic acid molecule is DNA or cDNA, is transcribed into mRNA (and in the case of genomic DNA containing introns is processed), preferably into an mRNA encoding a protein having lipase activity, which is subsequently translated into a protein, preferably into a protein having lipase activity.

[0079] Transcription of a specific nucleic acid molecule in the host can be detected by methods known to those skilled in the art, such as detecting specific transcripts (mRNA) of the foreign nucleic acid molecule by Northern blot analysis or RT-PCR.

[0080] Determining whether a host or host cell contains a specific protein or a protein derived from the expression of a nucleic acid molecule can be performed by methods known to those skilled in the art, for example, immunological methods such as Western blot analysis, ELISA (enzyme-linked immunosorbent assay), or RIA (radioimmunoassay). Those skilled in the art are familiar with methods for producing antibodies that specifically react with, i.e., specifically bind to, a specific protein (see, for example, Lottspeich and Zorbas (eds.), 1998, Bioanalytik, Spektrum akad, Verlag, Heidelberg, Berlin, ISBN 3-8274-0041-4). Several companies (Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA USA 0245; GenScript, 60 Centennial Ave., Piscataway, NJ 08854, USA) offer custom-made services for producing such antibodies.

[0081] Furthermore, a person skilled in the art can check whether a host or host cell contains a protein suitable for the method according to the invention by detecting the (additional) activity of a protein having lipase activity in the corresponding host cell. Preferably, the activity of a protein having lipase activity in the corresponding host cell is detected by comparing the lipase activity in the host cell used in the method according to the invention with the corresponding activity in a host cell that does not contain a protein suitable for the method according to the invention. Checking whether a protein has lipase activity can be carried out using known methods according to the prior art.

[0082] The hosts or host cells used for the method according to the invention can be produced by those skilled in the art with the aid of known methods for the genetic modification or transformation of organisms.

[0083] Thus, also disclosed are hosts or host cells, particularly prokaryotic or eukaryotic hosts or host cells, suitable for the method according to the invention, which have been genetically modified (or transformed) using a suitable nucleic acid molecule, or using a suitable recombinant nucleic acid molecule, or using a suitable vector, or using a suitable plasmid. Preferably, the genetically modified (transformed) host or host cell used in the method according to the invention expresses a protein having lipase activity; more preferably, the genetically modified (transformed) host or host cell expresses a protein suitable for the method according to the invention. "Genetically modified with a nucleic acid molecule" or "transformed with a nucleic acid molecule" as used herein should be understood to mean that the nucleic acid molecule is or has been introduced into the host or host cell by technical and / or non-natural means, preferably by technical methods from the fields of molecular biology, biotechnology or gene technology.

[0084] Also disclosed are the descendants or progeny of hosts or host cells used in a method according to the invention; preferably, said descendants or progeny comprise a suitable nucleic acid molecule, or comprise a suitable recombinant nucleic acid molecule, or comprise a suitable plasmid, or comprise a suitable protein for a method according to the invention, more preferably, said descendants or progeny comprise a suitable nucleic acid molecule, or comprise a suitable recombinant nucleic acid molecule, or comprise a suitable vector, or comprise a suitable plasmid, which in each case express a protein having the activity of a lipase, and even more preferably, said descendants or progeny comprise a suitable nucleic acid molecule, or comprise a suitable recombinant nucleic acid molecule, or comprise a suitable vector, or comprise a suitable plasmid, which in each case express a protein having the activity of a lipase that can be used in a method according to the invention.

[0085] The host or host cell for the method according to the invention may be of prokaryotic or eukaryotic origin. Host cells or host cells are bacteria or bacterial cells (e.g. Escherichia coli, in particular bacteria of the genus Bacillus, in particular Bacillus subtilis, in particular Agrobacterium subtilis, in particular Agrobacterium tumefaciens or Agrobacterium rhizogenes, Pseudomonas, in particular Pseudomonas fluorescens, Streptomyces spp., Rhodococcus spp., in particular Rhodococcus rhodochrous, Vibrio natrigens, natrigens, Corynebacterium, in particular Corynebacterium glutamicum) or fungi or fungal cells (e.g., Agaricus, in particular Agaricus bisporus, Aspergillus, Trichoderma or yeasts, in particular S. cerevisiae, Pichia ssp., e.g., P. pastoris), and also plants or plant cells, or they may be plants or plant cells or animal cells. Preferred host cells are microbial cells. For the purposes of this patent application, it is assumed that all bacteria and protists (e.g. fungi, especially yeasts and algae) as defined, for example, in Schlegel "General Microbiology" (Georg Thieme Verlag (1985), 1-2) are included.With regard to microorganisms, the host or host cell used in the method according to the invention is preferably a bacterium / bacterial cell or a yeast / yeast cell, more preferably a bacterium / bacterial cell, even more preferably a Bacillus / Bacillus cell or an E. coli / E. coli cell, most preferably an E. coli / E. coli cell. Alternatively, Pseudomonas, particularly Pseudomonas fluorescens, Streptomyces, Rhodococcus, particularly Rhodococcus rhodochrous, Vibrio, particularly Vibrio natrigens, Corynebacterium, particularly Corynebacterium glutamicum, or other host or host cell can be used for the method according to the invention.

[0086] Preferred hosts or host cells for the methods according to the invention comprise suitable nucleic acid molecules characterized in that the codons of the nucleic acid molecule have been modified to match the codon usage in the host or host cell.

[0087] Array Description Throughout this application, the following abbreviations for nucleotides and amino acids according to the IUPAC code are used: Table 1 [Table 1] To distinguish between amino acids and nucleotides, the abbreviated nucleotide codes capitalized in the table above are not capitalized herein.

[0088] Table 2 [Table 2] The codon usage herein follows the so-called "universal genetic code" with "t" substituted for "u" in ribonucleic acid (RNA) sequences according to the following table:

[0089] Table 3 [Table 3] TIFF2025527617000006.tif251168TIFF2025527617000007.tif202168 Table 4 The Sequence Listing associated with this application has been filed in electronic format and is incorporated herein by reference in its entirety. "PRT" means "protein" and "NUC" means "nucleic acid." [Table 4] TIFF2025527617000009.tif252167TIFF2025527617000010.tif252169TIFF2025527617 000011.tif253166TIFF2025527617000012.tif251168TIFF2025527617000013.tif2521 68TIFF2025527617000014.tif251168TIFF2025527617000015.tif252168TIFF20255276 17000016.tif247168TIFF2025527617000017.tif255168TIFF2025527617000018.tif25 3168TIFF2025527617000019.tif253169TIFF2025527617000020.tif251169TIFF202552 7617000021.tif255166TIFF2025527617000022.tif255164TIFF2025527617000023.tif The protein having lipase activity is preferably used in an amount of 0.1 to 50% by weight, more preferably 0.5 to 10% by weight, and particularly preferably 1 to 5% by weight, based on the weight of mixture (I).

[0090] Step 1 of the process according to the present invention can be carried out in the absence of a solvent or in such a solvent. Preferred are solvents from the group consisting of methyl tert-butyl ether, heptane, toluene, xylene, mesitylene, anisole, chlorobenzene, n-butanol, isopropanol, n-propanol, ethanol, and mixtures thereof. Particularly preferred are solvents from the group consisting of toluene, xylene, mesitylene, n-butanol, ethanol, and mixtures thereof. Also particularly preferred is carrying out the reaction in the absence of a solvent.

[0091] Relative to the molar amount of mixture (I) used, the acylating or carboxylating agent RC(=O)R 1 is preferably used in an amount of 0.4 to 25 equivalents, particularly preferably 0.6 to 10 equivalents, and particularly preferably 1 to 5 equivalents.

[0092] The reaction in step 1 is usually carried out at a temperature of 20 to 130°C. Preferably, the reaction is carried out at a temperature of 70 to 130°C. Particularly preferably, the reaction is carried out at a temperature of 80 to 120°C.

[0093] The isolation of component (II) specified in step 2 of the process according to the invention is carried out by crystallization as known to those skilled in the art.

[0094] The base specified in step 3 of the process according to the invention is usually taken from the group consisting of lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, lithium methoxide, sodium methoxide, potassium methoxide, lithium ethoxide, sodium ethoxide and potassium ethoxide. Particularly preferably, the bases lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium ethoxide, sodium ethoxide and potassium ethoxide are used.

[0095] Very particularly preferably, the bases lithium hydroxide, sodium hydroxide and potassium hydroxide are used.

[0096] Typically, the base is used in a stoichiometric amount of 1.00 to 3.00 equivalents relative to the molar amount of component (II) used. Preferably, the base is used in an amount of 1.50 to 2.50 equivalents. Particularly preferably, the base is used in an amount of 1.75 to 2.25 equivalents.

[0097] Preferably, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, tert-butanol and 1-methoxypropan-2-ol, as well as toluene, xylene and veratrole are used as solvents. Particularly preferably, ethanol, n-butanol, isopropanol, 1-methoxypropan-2-ol, toluene, xylene and veratrole are used. Particularly preferably, ethanol, n-butanol and xylene are used.

[0098] The reaction in step 3 is usually carried out at a temperature of 60 to 140°C. Preferably, the reaction is carried out at 80 to 120°C. Particularly preferably, the reaction is carried out at 90 to 110°C.

[0099] A further advantage of the process according to the present invention is that the mixture of 2,6-dimethyl-1-indanamine (III) remaining in steps 1 and 2 can be re-isomerized under a metal catalyst to form a mixture of four stereoisomers of 2,6-dimethyl-1-indanamine (I) in a resource-saving manner.

[0100] This isomerization can be carried out as follows: Preferably, palladium on carbon (Pd / C), palladium on calcium carbonate (Pd / CaCO3), or palladium on aluminum oxide (Pd / Al2O3) catalysts with a palladium loading of 1 to 20% by weight are used as the metal catalyst. Similarly, Shvo's catalyst, with the IUPAC name 1-hydroxytetraphenylcyclopentadienyl(tetraphenyl-2,4-cyclopentadien-1-one)-μ-hydrotetracarbonyldiruthenium(II), is used in a stoichiometry of 1 to 10 mol%. Palladium on carbon (Pd / C) or palladium on aluminum oxide (Pd / Al2O3) with a palladium loading of 1 to 20% by weight is particularly preferred. Palladium on aluminum oxide (Pd / Al2O3) catalysts with a palladium loading of 1 to 20% by weight are especially preferred. The supported catalyst is used in an amount of 0.1 to 10% by weight, preferably 0.5 to 5% by weight, based on the compound of formula (III). The amount of catalyst used is calculated based on the dry weight of the catalyst.

[0101] Preferably, pyridine, N-methylmorpholine, morpholine, cyclohexylamine, di-n-butylamine, tri-n-butylamine, triethylamine, diisopropylethylamine, piperidine, potassium carbonate, sodium carbonate, lithium carbonate, potassium ethoxide, sodium ethoxide and lithium ethoxide are used as bases, or no base is used. Particularly preferably, piperidine, morpholine, diisopropylethylamine, tri-n-butylamine, potassium carbonate and sodium ethoxide are used, or no base is used. Especially preferably, piperidine and sodium ethoxide are used, or no base is used.

[0102] Preferably, toluene, xylene, mesitylene, anisole, chlorobenzene, n-butanol, isopropanol, n-propanol, and ethanol are used as solvents, or the reaction is carried out in the absence of a solvent. Particularly preferably, toluene, xylene, mesitylene, n-butanol, and ethanol are used as solvents, or the reaction is carried out in the absence of a solvent. Especially preferably, the reaction is carried out in the absence of a solvent.

[0103] The reaction is preferably carried out at a gauge pressure of 0 to 20 bar. Particularly preferably, the reaction is carried out at a gauge pressure of 0 to 10 bar. Especially preferably, the reaction is carried out at a gauge pressure of 1 to 6 bar.

[0104] The reaction pressure is substantially achieved by injecting a gas / gas mixture. Preferably, hydrogen, nitrogen, or argon, or a mixture of hydrogen and nitrogen, or a mixture of hydrogen and argon, is injected. Particularly preferably, hydrogen or a mixture of nitrogen and hydrogen is injected. Especially preferably, pure hydrogen is injected.

[0105] The reaction is preferably carried out at a temperature of 80 to 150°C. Particularly preferably, the reaction is carried out at a temperature of 100 to 130°C. Particularly preferably, the reaction is carried out at a temperature of 110 to 125°C.

[0106] Thus, the present invention further provides a process for the preparation of a mixture of all four stereoisomers of 2,6-dimethyl-1-indanamine (I) by metal-catalyzed isomerization of a mixture of the three stereoisomers of (1R,2R)-, (1S,2R)-, and (1S,2S)-2,6-dimethyl-1-indanamine: [ka] [Example]

[0107] The following examples more particularly illustrate the present invention: Terrific Broth (TB) culture medium was prepared in demineralized water with 47.6 g / l granulation medium and 4 ml / l glycerol and sterilized at 121°C for 20 minutes.

[0108] Lipase cloning Nucleotide sequences encoding the lipases and lipase variants described herein can be synthesized as known in the art, for example, by relevant service providers such as Eurofins Genomics GmbH (Eurofins Genomics GmbH, Anzinger Str. 7a, 85560 Ebersberg, Germany). Briefly, the nucleic acid sequence of the wild-type lipase (SEQ ID NO: 2) or related variants described herein was cloned into an expression vector based on the vector pKA81a. Genetic elements were introduced into the modified pKA81a vector by means of commonly known methods. The wild-type lipase and lipase variants were expressed by introducing the expression vector into electrocompetent E. coli MG1655 cells.

[0109] Generation of enzyme mutants Nucleotide substitutions (replacements) were introduced into the parent nucleic acid sequence, for example, to exchange an amino acid for another amino acid. Many molecular biology methods can be used to achieve these substitutions. One useful method for producing mutant nucleic acids and corresponding mutant proteins according to the present invention is site-directed mutagenesis at codons encoding one or more preselected amino acids. Methods for achieving these site-directed mutations are well known to those skilled in the art and are fully described in the literature (in particular: Directed Mutagenesis: A Practical Approach, 1991, edited by MJ McPHERSON, IRL PRESS), or commercially available kits (e.g., QUIKCHANGE™ Lightning Mutagenesis Kit from Qiagen or Stratagene) can be used. After site-directed mutagenesis, the nucleic acid was transformed into E. coli MG1655 cells.

[0110] The transformed cells were tested in suitable biotransformation reactions to determine product yield and product selectivity. Suitable biotransformation reactions are described below. Sequence verification was performed as known in the art.

[0111] Glycerol stocks of E. coli cultures transformed with each expression plasmid were prepared by adding 1 volume of 40% glycerol solution to 1 volume of E. coli culture.

[0112] To isolate single bacterial colonies, appropriate dilutions of E. coli cultures were plated onto LB agar plates containing an appropriate concentration of kanamycin and incubated at 37°C until single colonies were obtained.

[0113] Synthesis of ethyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate Example 1: In a comparative experiment, the suitability of three different proteins with lipase activity was tested: wild-type lipase (SEQ ID NO: 1) and lipase variants with SEQ ID NO: 233 and SEQ ID NO: 399. For this purpose, 9.25 g of 2,6-dimethylindan-1-amine (a racemic mixture of 82% trans- and 18% cis-2,6-dimethylindan-1-amine) was initially placed in a 25 mL three-necked flask equipped with a magnetic stirrer under argon and diluted with 13.6 g of diethyl carbonate in each case. After heating the solution to an internal temperature of 110 ° C, 250 mg of the proteins with lipase activity were added in each batch. The reaction progress was monitored by HPLC measurement for its conversion. After complete conversion of the (1R,2S)-2,6-dimethylindan-1-amine isomer, the reaction was terminated by adding 5 mL of diethyl carbonate, cooled to 100 °C, and the reaction mixture was filtered through a glass frit (porosity 3). Another 5 mL of diethyl carbonate was used to wash the filter cake. The combined orange filtrate was then subjected to distillation at 50 °C under a vacuum of 35-10 mbar. 50 mL of n-heptane was added to the distillation residue and stirred at 40 °C for 20 min. Upon cooling to 2 °C, a suspension was formed, which was filtered through a glass frit (porosity 3). The filter cake was washed with 5 mL of n-heptane and then dried under reduced pressure. The resulting white solid was then quantitatively separated. 1 The compounds were tested for chemical purity by H-NMR and for stereoisomeric purity by chiral HPLC, and the results are compared in Table 5 below.

[0114] Table 5 [Table 5] The yield is based on the content of pure ethyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate.

[0115] Example 2: In a 500 mL four-neck round-bottom flask equipped with an internal thermometer, magnetic stirrer, inert gas inlet, and bubble counter, 80.0 g of 2,6-dimethylindan-1-amine (a racemic mixture of 82% trans- and 18% cis-2,6-dimethylindan-1-amine) was dissolved in 130.0 g of diethyl carbonate under argon. The solution was heated to an internal temperature of 110°C with stirring. Then, 1.2 g of the E. coli culture transformed with SEQ ID NO:234 ​​was added in one portion, and the reaction was continued at 110°C with stirring under argon. After 7 hours of reaction time, essentially complete conversion of (1R,2S)-2,6-dimethylindan-1-amine was determined by high-performance liquid chromatography, and the reaction was stopped by cooling to 80°C followed by filtration through a glass frit (porosity 3). The filter cake was then washed with 2 x 40 mL of diethyl carbonate, and the combined filtrates were subjected to distillation at 50 °C under a reduced pressure of 35-10 mbar. This resulted in a total of 157.4 g of distillate being removed from the initial 264.2 g of filtrate. 120 mL of methylcyclohexane was then added to the distillation residue, and the resulting mixture was cooled to 2 °C to obtain a suspension. The solid was removed by filtration through a glass frit (porosity 3) and washed with a small amount of methylcyclohexane. After drying under vacuum, 37.0 g of a white solid was obtained. Quantitative 1 H-NMR analysis confirmed that the chemical purity of ethyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate was 85.4%. Chiral HPLC analysis determined the stereoisomeric purity to be 98.5%. This corresponds to a 27% yield of pure ethyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate.

[0116] 1H-NMR (400 MHz; CDCl3) δ = 7.08-7.01 (m, 3H), 4.81-4.71 (m, 2H), 4.19 (q, J = 8.0 Hz, 2H), 3.00 (dd, J = 8.0, 14.0 Hz, 1H), 2.51-2.45 (m, 1H), 2.33 (s, 3H), 2.16 (dt, J = 8.0, 14.0 Hz, 1H), 1.31-1.26 (m, 6H). The derivative methyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate was prepared analogously using dimethyl carbonate and was obtained in the form of a white solid. 1 H-NMR (400 MHz; CDCl3) δ = 7.08-7.01 (m, 3H), 4.79-4.78 (m, 2H), 3.74 (s, 3H), 3.00 (dd, J = 8.0, 16.0 Hz, 1H), 2.51-2.45 (m, 1H), 2.32 (s, 3H), 2.20-2.10 (m, 1H), 1.27 (d, J = 8.0 Hz, 3H). Example 3: In a 25 mL three-neck flask equipped with an internal thermometer, an inert gas inlet, a bubble counter, and a magnetic stirrer, 14.65 g of diethyl carbonate was heated to an internal temperature of 110°C under argon. Then, 10.00 g of 2,6-dimethylindan-1-amine (a racemic mixture of 82% trans- and 18% cis-2,6-dimethylindan-1-amine) and 0.25 g of an E. coli culture transformed with SEQ ID NO: 234 were both added at once, and the reaction was continued at 110°C for 10 hours with stirring under argon until HPLC analysis showed essentially complete conversion of (1R,2S)-2,6-dimethylindan-1-amine. The mixture was then diluted with another 5 mL of diethyl carbonate and filtered through a glass frit (porosity 3) at 100°C. The filter cake was then washed with 5 mL of diethyl carbonate adjusted to a temperature of 80°C. The combined filtrate was then subjected to distillation at 50°C under a reduced pressure of 35-10 mbar. 50 mL of n-heptane was then added to the distillation residue, resulting in a thick suspension. The suspension was heated to 40°C and stirred at this temperature for 20 minutes. The suspension was then cooled to 21°C within 30 minutes. The suspension was then further cooled to 2°C and filtered through a glass frit (porosity 3). The filter cake was washed with 20 mL of n-heptane cooled to 5°C and then dried at 40°C and 10 mbar vacuum. 5.1 g of a white solid was obtained. Quantitative 1 H-NMR analysis confirmed that the chemical purity of ethyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate was 99%. The stereoisomeric purity was determined to be 98.5% by chiral HPLC, corresponding to a 34% yield of pure ethyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate.

[0117] Example 4: In a 600 mL autoclave, 100.0 g of 2,6-dimethylindan-1-amine (a racemic mixture of 82% trans- and 18% cis-2,6-dimethylindan-1-amine), 139.2 g of diethyl carbonate, and 2 g of biomass Goe171-7-032 were stirred under argon at 110 °C for 7 hours. After cooling to 80 °C, filtration was carried out through a glass frit, and the filter cake was washed with diethyl carbonate. The combined filtrate was concentrated under reduced pressure at 50 °C, and the distillation residue was crystallized in an ice bath by the addition of 150 mL of methylcyclohexane. The solid was removed by filtration through a glass frit and washed with a small amount of methylcyclohexane. After drying under vacuum, 47.6 g of a white solid was obtained. Quantitative 1 H-NMR analysis confirmed that the chemical purity of ethyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate was 91.9%. Chiral HPLC analysis determined the stereoisomeric purity to be 96.1%. This corresponds to a 31.9% yield of pure ethyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate. The filtrate was concentrated under reduced pressure at 50 °C, and the content of 2,6-dimethylindan-1-amine isomers was determined by chiral GC in an isomeric ratio of R,R:S,S:S,R:R,S = 6.0:16.2:77.2:0.

[0118] Synthesis of (1R,2S)-2,6-dimethylindan-1-amine from methyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate Example 5: At 21 °C, 25 g of methyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate (98% stereoisomeric purity, 98% chemical purity) was suspended in 80 mL of n-butanol in a 250 mL jacketed reactor inerted with argon. The suspension was heated to an internal temperature of 65 °C, resulting in a clear solution. Then, 15 g of potassium hydroxide (85% chemical purity) was added in one portion, and the solution was further heated to an internal temperature of 100 °C. The solution was further stirred at this temperature for 1 h while an insoluble solid precipitated. Complete conversion was indicated by HPLC measurement. Subsequently, a vacuum of 360 mbar was applied, and 55 mL of solvent was distilled off. Then, 100 mL of p-xylene was added, and the reaction mixture was cooled to 20 °C. The mixture was washed with 1 × 50 mL of deionized water. The organic phase was then distilled at 40° C. to a vacuum of 5 mbar to quantitatively obtain 17.8 g of (1R,2S)-2,6-dimethylindan-1-amine in the form of a pale yellow oil. 1 It was obtained in 95% chemical purity as determined by H-NMR, corresponding to a yield of 95%. The stereoisomeric purity was determined to be 98% by chiral GC.

[0119] 1 H NMR (500 MHz, CDCl3): δ 7.12 (s, 1H), 7.05 (d, J = 7.5 Hz, 1H), 6.99 (d, J = 7.5 Hz, 1H), 3.74 (d, J = 8.5 Hz, 1H), 2.98 (dd, J = 7.5, 15.5 Hz, 1H), 2.42 (dd, J = 9.5, 15.5 Hz, 1H), 2.34 (s, 3H), 1.98 (m, 1H), 1.72 (bs, 2H), 1.24 (d, J = 6.5 Hz, 3H). Example 6: Synthesis of (1R,2S)-2,6-dimethylindan-1-amine hydrochloride from methyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate At 21 °C, 50 g of methyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate (98% stereoisomeric purity, 98% chemical purity) was suspended in 160 mL of n-butanol in a 250 mL jacketed reactor inerted with argon. The suspension was heated to an internal temperature of 65 °C, resulting in a clear solution. 31.1 g of potassium hydroxide (85% chemical purity) was then added in one portion, and the solution was further heated to an internal temperature of 100 °C. The solution was further stirred at this temperature for 1 h while an insoluble solid precipitated. Complete conversion was indicated by HPLC measurement. Subsequently, a vacuum of 360 mbar was applied, and 110 mL of solvent was distilled off. 200 mL of p-xylene was then added, and the reaction mixture was cooled to 20 °C. The mixture was washed with 1 × 100 mL of deionized water. The organic phase was then cooled to 10° C. and mixed with 22.8 g of concentrated hydrochloric acid (37% by weight in water) without allowing the temperature to rise above 25° C. 100 mL of solvent were distilled off from the resulting suspension at 50° C. and filtered off through a glass frit (porosity 3) at 20° C. The filter cake was washed with 1×100 mL of toluene and then dried at 40° C. under a reduced pressure of 10 mbar to obtain a quantitative yield in the form of a white solid. 1 40 g of (1R,2S)-2,6-dimethylindan-1-amine hydrochloride were obtained, corresponding to a yield of 95%, with a chemical purity of 99% as determined by H-NMR. The stereoisomeric purity was determined to be 98% by weight by chiral GC.

[0120] 1 H-NMR (400 MHz; DMSO-d6) δ = 8.66 (bs, 3H), 7.45 (s, 1H), 7.14 (dd, J = 8.0, 16.0 Hz, 2H), 4.20 (d, J = 8.0 Hz, 1H), 3.18-3.12 (m, 1H), 2.50-2.40 (m, 1H), 2.30 (s, 3H), 1.22 (d, J = 8.0 Hz, 3H). Isomerization of 2,6-dimethylindan-1-amine mixture (III) to form 2,6-dimethylindan-1-amine mixture (I) Example 7: In a 25 ml autoclave, 10 g of the resulting 2,6-dimethylindan-1-amine mixture (III) (concentrated filtrate from Example 4) was mixed with 396 mg of palladium catalyst (5% Pd / Al2O3). The autoclave was purged three times with 5 bar of argon, then filled with 3 bar of hydrogen, and stirred under 3 bar of hydrogen at 120 °C for 10 hours. After cooling to room temperature and venting the autoclave, the content of 2,6-dimethylindan-1-amine isomers in the resulting mixture was determined by chiral GC in an isomer ratio of R,R:S,S:S,R:R,S = 9.2:8.9:43.0:39.0.

[0121] Isomerization of (1R,2S)-2,6-dimethylindan-1-amine to 2,6-dimethylindan-1-amine (racemic mixture of 82% trans and 18% cis isomers) Examples 8 to 27: A 6 mL Wheaton screw-cap vial was charged with 1.0 g of (1R,2S)-2,6-dimethylindan-1-amine, 39.6 mg of palladium catalyst (5% Pd / Al2O3), 1.47 g of diethyl carbonate, and 10–20 mol% base. The vial was then closed with a screw cap and shaken at 400 rpm at 90–110 °C for 6 h. The mixture was then cooled to 21 °C and analyzed by chiral GC analysis and achiral HPLC. The results are tabulated. Activity is defined as the isomerization progression from an isomer ratio of 0:0:0:100 to an equilibrium ratio of 9:9:41:41, primarily measured by the degradation of the 1R,2S isomer from 100% to 41%. Therefore, activity is determined from the measured percentage of degradation below 59%. Chemoselectivity represents the degree of desired components (all DMAI reactant isomers and all DMAI carbamate product isomers) relative to the sum of all components formed in the isomerization, including secondary components. Relevant data are shown in Table 6 below: Table 6 [Table 6]

Claims

1. 1. A process for preparing substantially enantiopure (1R,2S)-2,6-dimethyl-1-indanamine, comprising:

1. In the first step, a mixture of four stereoisomers of 2,6-dimethyl-1-indanamine (I) is reacted with an acylating or carboxylating agent R—C(═O)R 1 in the presence of a protein having lipase activity to form the corresponding amide or carbamate (II) and a mixture of unreacted stereoisomers of 2,6-dimethyl-1-indanamine (III): 【Chemical 1】 [wherein the protein is I. A protein having at least 80% identity with the amino acid sequence shown under SEQ ID NO: 1; II. The amino acid sequence is i. the amino acid at position 186 is different from L; ii. the amino acid at position 280 is different from L; iii. the amino acid at position 312 is different from P; iv. the amino acid at position 3 is different from M; v. the amino acid at position 29 is different from N; vi. the amino acid at position 17 is different from L; vii. the amino acid at position 4 is different from S; viii. the amino acid at position 18 is different from V; ix. the amino acid at position 202 is different from A; x. the amino acid at position 301 is different from D; xi. the amino acid at position 309 is different from P; xii. the amino acid at position 31 is different from Q; xiii. the amino acid at position 111 is different from Q; xiv. the amino acid at position 85 is different from W; xv. the amino acid at position 8 is different from K; xvi. the amino acid at position 79 is different from E; xvii. the amino acid at position 40 is different from K; A protein having at least 80% identity with the amino acid sequence shown under SEQ ID NO: 1, except for the fact that it has a modification selected from the group consisting of: encoded by an amino acid sequence selected from the group consisting of 2. In a second step, the amide or carbamate (II) is separated from the remaining 2,6-dimethyl-1-indanamine (III) and secondary components by crystallization; and 3. In a third step, converting the amide or carbamate (II) to substantially enantiopure (1R,2S)-2,6-dimethyl-1-indanamine (IV) using an acid or base. 【Chemistry 2】 wherein R is CH ２ OCH ３ , C.H. ２ OCH ２ CH ３ , C.H. ３ , OCH ３ , OCH ２ CH ３ , OCH(CH ３ ) ２ , OCH ２ CH ２ CH ２ CH ３ , OCH ２ CHCH ２ and OCH ２ (C ６ H ５ ) means a radical from the group consisting of and In the formula, R １ is OCH ３ , OCH ２ CH ３ , OCH(CH ３ ) ２ , OCH ２ CH ２ CH ２ CH ３ , OCH ２ CHCH ２ and OCH ２ (C ６ H ５ ) means a group selected from the group consisting of A method characterized by:

2. R is OCH ３ and OCH ２ CH ３ means a radical from the group consisting of and R １ But OCH ３ and OCH ２ CH ３ The method of claim 1, wherein the group is selected from the group consisting of:

3. The protein is a) a protein comprising the amino acid sequence shown in SEQ ID NO: 1, apart from the fact that the amino acid at position 186 differs from L; b) Proteins having an amino acid sequence with at least 80% identity to the amino acid sequence shown under a), with the proviso that the amino acid at position 186 is different from L, wherein the amino acid at position 186 in the protein according to a) or b) is preferably F, W, Y, E, D, Q, T, H, P, C, K, S, N, I or V.

3. The method according to claim 1 or 2, characterized in that the compound is selected from the group consisting of:

4. The protein is (i) the amino acid at position 79 is different from E, preferably the amino acid at position 79 is S, W or I, more preferably the amino acid at position 79 is S; (ii) the amino acid at position 202 is different from A, preferably the amino acid at position 202 is N; (iii) the amino acid at position 280 is different from L, preferably the amino acid at position 280 is A; (iv) the amino acid at position 301 is different from D, preferably the amino acid at position 301 is A; (v) the amino acid at position 3 is different from M, preferably the amino acid at position 3 is Q; (vi) the amino acid at position 11 is different from C, preferably the amino acid at position 11 is A; (vii) the amino acid at position 17 is different from L, preferably the amino acid at position 17 is P; (viii) the amino acid at position 40 is different from K, preferably the amino acid at position 40 is M; (ix) the amino acid at position 111 is different from Q, preferably the amino acid at position 111 is E; 4. The method of claim 3, characterized in that the amino acid sequence has at least one, preferably at least two, more preferably at least three additional amino acid substitutions selected from the group consisting of:

5. 5. The method of claim 3 or 4, wherein the protein has amino acid substitutions at positions 186, 79, 301 and 40, wherein preferably the amino acid at position 186 is Y, the amino acid at position 79 is S, the amino acid at position 301 is A and the amino acid at position 40 is M.

6. 6. The method according to claim 1, wherein the protein is used in an amount of 0.1 to 50% by weight, based on the mixture (I).

7. 7. The method according to claim 1, wherein the protein is used in an amount of 0.5 to 10% by weight, based on the mixture (I).

8. 8. The method according to claim 1, wherein the protein is used in an amount of 1 to 5% by weight, based on the mixture (I).

9. 9. The process according to claim 1, wherein step 1 is carried out without a solvent or in the presence of a solvent from the group consisting of toluene, xylene, mesitylene and n-butanol.

10. In step 1, an acylating or carboxylating agent R—C(═O)R １ is preferably used in an amount of 0.4 to 25 equivalents relative to the molar amount of mixture (I) used.

11. In step 1, an acylating or carboxylating agent R—C(═O)R １ is preferably used in an amount of 1.0 to 5.0 equivalents relative to the molar amount of mixture (I) used.

12. 12. The method according to any one of claims 1 to 11, characterized in that in step 1, the reaction is carried out at a temperature of 20 to 130°C.

13. 13. The method according to any one of claims 1 to 12, characterized in that in step 1, the reaction is carried out at a temperature of 80 to 120°C.

14. 14. The method according to any one of claims 1 to 13, characterized in that in step 3 a base from the group consisting of lithium hydroxide, sodium hydroxide and potassium hydroxide is used.

15. 15. The process according to any one of claims 1 to 14, characterized in that in step 3, the base is used in a stoichiometry of 1.00 to 3.00 equivalents relative to the molar amount of component (II).

16. A method for the preparation of a mixture of all four stereoisomers of 2,6-dimethyl-1-indanamine (I) by metal-catalyzed isomerization of a mixture of the three stereoisomers of (1R,2R)-, (1S,2R)-, and (1S,2S)-2,6-dimethyl-1-indanamine. 【Chemistry 3】

17. Palladium-carbon (Pd / C) or palladium-aluminum oxide (Pd / Al) having a palladium loading of 0.5 to 10 mass % ２ O ３ 17. The method of claim 16, wherein a

18. 18. The process according to claim 16 or 17, characterized in that the reaction is carried out under injection of a gas mixture consisting of nitrogen and hydrogen at a gauge pressure of 1 to 6 bar.

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