Process for the production of (1r,2s)-2,6-dimethyl-1-indanamine

Abstract

Described is a process for producing nearly enantiomerically pure (1R,2S)-2,6-dimethyl-1-indanamine, said process being characterized by the reaction of a mixture of the four stereoisomers of 2,6-dimethyl-1-indanamine with an acylating or carboxylating agent in the presence of a protein with lipase activity.

Description

[0001] Process for the preparation of (lR,2S)-2,6-dimethyl-l-indanamine

[0002] Description

[0003] The present invention relates to a process for the preparation of virtually enantiomerically pure (lR,2S)-2,6-dimethyl-l-indanamine, which is a valuable building block for the synthesis of the herbicidal active ingredient indaziflam. Specifically, the present invention relates to a process for the preparation of virtually enantiomerically pure (lR,2S)-2,6-dimethyl-l-indanamine by enzyme-catalyzed, stereoselective acylation of racemic 2,6-dimethyl-l-indanamine.

[0004] To date, only processes for the production of almost enantiomerically pure (lR,2S)-2,6-dimethyl-l-indanamine are known. Due to the use of expensive reaction components and catalysts, these are only suitable for laboratory scale and not for industrial use. WO 2004 / 69814 A1 discloses a process characterized by the following five reaction steps:

[0005] 1. Preparation of a mixture of the four stereoisomers of 2,6-dimethyl-l-aminoindane by palladium-catalyzed reduction of the corresponding oxime.

[0006] 2. Column chromatographic separation of these four stereoisomers into the cis and trans isomers.

[0007] 3. Reaction of the trans isomer mixture with methyl 2-methoxyacetate in the presence of the enzyme Novozym 435® to give the corresponding acetylated (lR,2S)-2,6-dimethyl-l-indanamine.

[0008] 4. Isolation of the acetylated (1 R,2S)-2,6-dimethyl-l -indanamine.

[0009] 5. Acid hydrolysis of the acetylated (lR,2S)-2,6-dimethyl-l-indanamine to the free (lR,2S)-2,6-dimethyl-1-indanamine.

[0010] Tetrahedron 2007, 63 (29), 6755-6763 describes a process for the preparation of ( \ R.2S)-2.6-dimethyl-1-indanamine, which is characterized by the following two reaction steps:

[0011] 1. Racemic 1,6-dimethylindan-l-one is reduced diastereoselectively (96:4 dr) and enantioselectively (98:2 er) to the corresponding (1S,2S)-2,6-dimethylindan-l-ol in a yield of 80% using a chiral ruthenium catalyst.

[0012] 2. Reaction of the resulting (I,S',2,S')-2,6-dimethoxylindan-l-ol with diphenylphosphoryl azide and subsequent reduction with lithium aluminum hydride leads to (I,S',2,S')-2,6-dimethoxylindan-l-amine in a yield of 76%. A disadvantage of this process, in addition to the use of expensive reagents, is the long reaction time of eight days.

[0013] The object of the present invention was to provide a process for the preparation of almost enantiomerically pure (lR,2S)-2,6-dimethyl-l-indanamine which overcomes the disadvantages known from the prior art.

[0014] A process for the preparation of almost enantiomerically pure (lR,2S)-2,6-dimethyl-l-indanamine has been found, which is characterized by reacting a mixture of the four stereoisomers of 2,6-dimethyl-l-indanamine with an acylating or carboxylating agent in the presence of an enzyme.

[0015] The present invention relates to a process for the preparation of almost enantiomerically pure (lR,2S)-2,6-dimethyl-l-indanamine, characterized in that

[0016] 1. that in a first step, a mixture of the four stereoisomers of 2,6-dimethyl-l-indanamine (I) is reacted with an acylating or carboxylating agent RC(=O)R' in the presence of a protein with the activity of a lipase to give the corresponding amide or carbamate (II) and a mixture (III) of the unreacted stereoisomers of 2,6-dimethyl-l-indanamine: wherein the protein is encoded by an amino acid sequence selected from the group consisting of

[0017] I. Proteins which have 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 to the amino acid sequence shown under SEQ ID No. 1,

[0018] II. Proteins which have 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 to the amino acid sequence shown under SEQ ID No. 1, except that the amino acid sequence which 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 to the amino acid sequence shown under SEQ ID No. 1, has a modification selected from the group consisting of 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; that in a second step the amide or carbamate (II) is separated from the remaining 2,6-dimethyl-l-indanamines (III) and minor components by crystallization, and that in a third step the amide or carbamate (II) is converted into the almost enantiomerically pure (lÄ,2>S)-2,6-dimethyl-l-indanamine (IV) using an acid or a base. wherein R is a radical from the group CH2OCH3, CH2OCH2CH3, CH3, OCH3, OCH2CH3, OCH(CH3)2, OCH2CH2CH2CH3 , OCH2CHCH2 and OCH2(C6H5), and wherein R 1 a residue from the group OCH3, OCH2CH3, OCH(CH3)2, OCH2CH2CH2CH3, OCH2CHCH2 and OCH2(C6H5).

[0019] The mixture of the four stereoisomers of 2,6-dimethyl-l-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.

[0020] Preferably, R represents a radical from the group OCH3 and OCH2CH3, and

[0021] R 1 means a residue from the group OCH3 and OCH2CH3.

[0022] In the process according to the invention, proteins with the activity of a lipolytic enzyme or a lipase are used.

[0023] SEQ ID No. 1 represents the amino acid sequence of a wild-type lipolytic protein. The wild-type lipase is derived from an uncultured bacterium from an environmental sample, which can be obtained from GenPept (PDB) under accession no. QRD81023 (version ORD81023.1). In case of doubt 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.

[0024] Also described are proteins with the activity of a lipolytic enzyme or a lipase, wherein the amino acid sequences of these proteins represent variants of a known protein that exhibits the activity of a lipolytic enzyme or a lipase. In particular, the amino acid sequences of proteins with the activity of a lipase described herein represent variants of the amino acid sequence represented by the amino acid shown in SEQ ID No. 1, wherein in the sequence shown under SEQ ID No.1, at least the amino acid at position 186, the amino acid at position 280, the amino acid at position 312, the amino acid at position 3, the amino acid at position 29, the amino acid at position 17, the amino acid at position 4, the amino acid at position 18, the amino acid at position 202, the amino acid at position 301, the amino acid at position 309, the amino acid at position 31, the amino acid at position 111, the amino acid at position 85, the amino acid at position 8, the amino acid at position 79 or the amino acid at position 40 is different from the amino acid present at the corresponding amino acid position in the sequence shown under SEQ ID No. 1.

[0025] The term "variant," as used herein, means an object that differs from an object known in the prior art. With respect to nucleic acid molecules and proteins, variants are understood to mean a nucleic acid sequence or an amino acid sequence that differs from corresponding known sequences, but encodes a protein that has the same function or catalyzes the same reaction, e.g., the function of encoding a protein with the activity of an elipase. "Deviation" of nucleic acid molecule sequences and amino acid sequences from known nucleic acid sequences and protein sequences means that the sequences comprise substitutions (replacements) and / or deletions and / or insertions of nucleotides or amino acids, respectively, compared to the corresponding known nucleic acid sequences or amino acid sequences.

[0026] Typically, proteins with the activity of an eipase are used, wherein the proteins are encoded by an amino acid sequence which 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 to the amino acid sequence shown under SEQ ID No. 1.

[0027] Proteins with the activity of a lipase are also used, the proteins being encoded by an amino acid sequence which 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 to the amino acid sequence shown under SEQ ID No. 1, apart from the fact that the amino acid sequence which 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 to the amino acid sequence shown under SEQ ID No. 1, has a modification selected from the group consisting of 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, 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, 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.

[0028] The meaning of the amino acid abbreviations A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, TV, W, Y can be derived hereinafter in Table 2 in the section titled “Description of the sequences”.

[0029] A further embodiment of the invention relates to proteins with the activity of a lipase, wherein the proteins are selected from the group consisting of a) proteins which comprise the amino acid sequence shown in SEQ ID No. 1, except that the amino acid at position 186 is different from L; b) proteins with an amino acid sequence with 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 to the amino acid sequence shown under a), provided that the amino acid at position 186 is different from L.

[0030] Preferably, the amino acid in the proteins according to a) or b) 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. Most preferably, the amino acid at position 186 is W or Y. Most preferably, the amino acid at position 186 is Y.

[0031] An "amino acid corresponding to position X" in a first amino acid sequence (e.g., position 3 in SEQ ID No. 1) means herein that an amino acid of a second amino acid sequence, compared to the first amino acid sequence, appears at position x of the first amino acid sequence in a pairwise sequence alignment of the first amino acid sequence with the second amino acid sequence if the numbering of the amino acids of the second amino acid sequence differs from the amino acid numbering of the first amino acid sequence.

[0032] In the context of the present invention, the term "identity" in relation to sequence identity or identical sequences is to be understood as the number of identical amino acids or nucleotides that a first nucleic or amino acid sequence has in common with another (second) nucleic or amino acid sequence over the entire sequence length, expressed as a percentage.

[0033] Sequence identity can be determined by aligning two amino acid or two nucleotide sequences using global or local alignment algorithms, such as those included in popular software such as GAP or BESTFIT or the Emboss program "Needle." This software uses the global alignment algorithm of Needleman and Wunsch to align two sequences across their entire length, maximizing the number of matches and minimizing the number of gaps. Generally, the default parameters are used, with a gap creation penalty of 10 and a gap extension penalty of 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 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 used algorithms and output formats such as FASTA, BLAST, etc., but preferably, hits should be retrieved and pairwise aligned to conclusively determine sequence identity.

[0034] If sequences to be compared are of different lengths, the identity is to be determined by determining the identity as a percentage of the number of amino acids or nucleotides that the shorter sequence has in common with the longer sequence. Preferably, identity is determined using the well-known and 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, 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 from the mirrored websites of the EBI (European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK). Preferably, the computer program ClustalW version 1.8 is used to determine the identity between proteins described in the context of the present invention and other proteins. The parameters must 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.

[0035] Preferably, the ClustalW computer program version 1.8 is used to determine the identity, for example, between the nucleotide sequence of the nucleic acid molecules described in connection with the present invention and the nucleotide sequence of other nucleic acid molecules. The parameters must be set as follows:

[0036] KTUPLE=2, TOPDIAGS=4, PAIRGAP=5, DNAMATRIX :IUB, GAPOPEN=10, GAPEXT=5, MAXDIV=40, TRANSITIONS: unweighted.

[0037] "Identity" also means that there is functional and / or structural equivalence between the nucleic acid molecules in question or the proteins they encode. Functional equivalence means that the nucleic acid molecule sequences or the amino acid sequences encode a protein with lipase activity. Nucleic acid molecules that are homologous to the molecules described above and are derivatives of these molecules are generally variants of these molecules, representing modifications that have the same biological function or catalyze the same reaction, i.e., encode a protein with lipase activity. These can be either naturally occurring variants, for example, sequences from other species, or mutations, whereby these mutations may have occurred naturally or have been introduced by targeted mutagenesis.Furthermore, the variants can be synthetically produced sequences. The allelic variants can be either naturally occurring variants, synthetically produced variants, or variants generated by recombinant DNA techniques. However, crucial for the present invention is that these variants encode proteins with lipase activity and comprise the amino acid substitutions (replacements), deletions, or insertions described herein with respect to the proteins of the invention.

[0038] A special type of derivatives are, for example, nucleic acid molecules which differ from the nucleic acid molecules described in the present invention by the degeneracy of the genetic code.

[0039] According to the NC-IUBMB (Nomenclature Committee of the International Union of Biochemistry and Molecular Biology), lipases belong to the class of hydrolases (EC 3). Hydrolase is a class of enzymes that typically act as biochemical catalysts and use water to break a chemical bond, typically resulting in the division of a larger molecule into smaller molecules. The group of hydrolases includes enzymes that act on ester bonds (EC 3.1), such as carboxylic acid ester hydrolases (EC 3.1.1), and as a subgroup, lipases (EC 3.1.1.3). Lipases have been identified from plants, mammals, and microorganisms, e.g., in bacteria, fungi, and fungi. B. Pseudomonas, Vibrio, Acinetobacter, Burkholderia, Chromobacterium, cutinase from Fusarium solani (FSC), Candida antarctica A (CalA), Rhizopus oryzae (ROL), Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML), Aspergillus Niger, Fusarium heterosporum, Fusarium oxysporum or Fusarium culmorum.

[0040] If a protein exhibits lipase activity, this can be detected using methods known and described in the prior art. It is not critical which method is used to detect whether a protein according to the invention exhibits lipase activity. Preferably, in connection with the present invention, the method is described in the "Example" section.

[0041] Lipase variant proteins according to the invention may have further amino acid modifications (amino acid substitutions, deletions or insertions) compared to the amino acid sequences described above with respect to the amino acid sequence shown under SEQ ID No. 1.

[0042] In addition to the lipase variants described above under point a) with respect to the amino acid sequences shown under SEQ ID No. 1, the lipase variants may have at least one, two, three, four, five, six, seven further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. In other words, the protein according to the invention with the activity of a lipase is selected from the group consisting of a) proteins which comprise the amino acid sequence shown in SEQ ID No. 1, except that the amino acid at position 186 is different from L and that they have 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) proteins having an amino acid sequence with 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 to the amino acid sequence shown directly above under a), provided that the amino acid at position 186 is different from L and with at least one further amino acid substitution selected from the groups (i) to (ix) mentioned 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; preferably the amino acid at position 111 is E.;

[0043] Furthermore, lipase variant proteins according to the invention may, in addition to the further amino acid modifications, have additional amino acid substitutions compared to the amino acid sequences described above with respect to the amino acid sequence shown under SEQ ID No. 1. These additional amino acid substitutions refer to positions of the amino acid sequence that are different from position(s) 79, 202, 280, 301, 3, 11, 17, 40, or 111, which refer to the further amino acid modifications. The lipase variants described above under point a) with respect to the amino acid sequences shown under SEQ ID No. 1 may have at least one, two, three, four, five, six, seven, or more additional 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.

[0044] A further embodiment of the invention accordingly relates to proteins according to the invention which have further amino acid modifications, preferably these embodiments are proteins with the activity of a lipase, wherein the proteins are selected from the group consisting of:

[0045] Proteins having the amino acid sequence shown in SEQ ID No. 1, with the exception that

[0046] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E, wherein it is preferred that the amino acid at position 186 is preferably W or Y, more preferably Y, and that 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 it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N;

[0047] - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A;

[0048] - 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;

[0049] - 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;

[0050] - 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;

[0051] - the amino acid at position 186 is different from L and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 17 is preferably P;

[0052] - 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;

[0053] - 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;

[0054] - Proteins with 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%, most preferably 99% identical to the amino acid sequence shown under a), provided that the amino acid at position 186 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and wherein the proteins have at least one further amino acid substitution selected from the group shown under the listed symbols directly above. Preferably, the lipase variants described herein above under point a) with respect to the amino acid sequences shown under SEQ ID No. 1 can 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.

[0055] A further embodiment of the invention accordingly relates to proteins according to the invention which have further amino acid modifications, preferably these embodiments are proteins with the activity of a lipase, wherein the proteins are selected from the group consisting of proteins with the amino acid sequence shown in SEQ ID No. 1, with the exception that

[0056] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 202 is preferably N;

[0057] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 280 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 280 is preferably A;

[0058] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 301 is different from D, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 301 is preferably A;

[0059] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 3 is different from M, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that 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 79 is different from E and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 11 is preferably A;

[0060] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 17 is preferably P;

[0061] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 40 is preferably M;

[0062] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 111 is preferably E;

[0063] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 280 is preferably A;

[0064] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 301 is different from D, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that 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 202 is

[0065] A is different and the amino acid at position 3 is different from M, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 3 is preferably Q;

[0066] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 11 is preferably A;

[0067] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 17 is preferably P;

[0068] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 40 is preferably M;

[0069] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 111 is preferably E;

[0070] - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A, and that the amino acid at position 301 is preferably A;

[0071] - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 3 is different from M, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A, and that 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 280 is different from L and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A, and that the amino acid at position 11 is preferably A;

[0072] - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A, and that the amino acid at position 17 is preferably P;

[0073] - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A, and that the amino acid at position 40 is preferably M;

[0074] - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A, and that the amino acid at position 111 is preferably E;

[0075] - the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 301 is preferably A, and that the amino acid at position 3 is preferably Q;

[0076] - the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 301 is preferably A, and that the amino acid at position 11 is preferably A;

[0077] - the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 301 is preferably A, and that the amino acid at position 17 is preferably P;

[0078] - the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 301 is preferably A, and that the amino acid at position 40 is preferably M;

[0079] - the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 301 is preferably A, and that the amino acid at position 111 is preferably E;

[0080] - the amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 3 is preferably Q, and that the amino acid at position 11 is preferably A;

[0081] - the amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 3 is preferably Q, and that the amino acid at position 17 is preferably P;

[0082] - the amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 3 is preferably Q, and that the amino acid at position 40 is preferably M;

[0083] - the amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 3 is preferably Q, and that the amino acid at position 111 is preferably E;

[0084] - the amino acid at position 186 is different from L and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 11 is preferably A, and that the amino acid at position 17 is preferably P;

[0085] - the amino acid at position 186 is different from L and the amino acid at position 11 is different from C and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 11 is preferably A, and that the amino acid at position 40 is preferably M;

[0086] - the amino acid at position 186 is different from L and the amino acid at position 11 is different from C and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 11 is preferably A, and that the amino acid at position 111 is preferably E;

[0087] - the amino acid at position 186 is different from L and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 17 is preferably P, and that the amino acid at position 40 is preferably M;

[0088] - the amino acid at position 186 is different from L and the amino acid at position 17 is different from L and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 17 is preferably P, and that the amino acid at position 111 is preferably E;

[0089] - the amino acid at position 186 is different from L and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 40 is preferably M, and that the amino acid at position 111 is preferably E;

[0090] - 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), provided that the amino acid at position 186 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and wherein the proteins have at least two further amino acid substitutions selected from the group shown under the listed symbols directly above.

[0091] Preferably, the lipase variants described hereinabove under point a) with respect to the amino acid sequences shown under SEQ ID No. 1 may have at least three further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111.

[0092] 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.

[0093] A further embodiment of the invention accordingly relates to proteins according to the invention which have further amino acid modifications, preferably these embodiments are proteins with the activity of a lipase, wherein the proteins are selected from the group consisting of proteins with the amino acid sequence shown in SEQ ID No. 1, with the exception that

[0094] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 202 is preferably N, and that the amino acid at position 280 is preferably A;

[0095] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A and the amino acid at position 301 is different from D, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 202 is preferably N, and that the amino acid at position 301 is preferably A;

[0096] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A and the amino acid at position 3 is different from M, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 202 is preferably N, and that 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 79 is different from E and the amino acid at position 202 is different from A and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 202 is preferably N, and that the amino acid at position 11 is preferably A;

[0097] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 202 is preferably N, and that the amino acid at position 17 is preferably P;

[0098] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 202 is preferably N, and that the amino acid at position 40 is preferably M;

[0099] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 202 is preferably N, and that the amino acid at position 111 is preferably E;

[0100] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 280 is preferably A, and that 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 79 is different from E and the amino acid at position 280 is different from L and the amino acid at position 3 is different from M, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 280 is preferably A, and that the amino acid at position 3 is preferably Q;

[0101] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 280 is different from L and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 280 is preferably A, and that the amino acid at position 11 is preferably A;

[0102] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 280 is different from L and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 280 is preferably A, and that the amino acid at position 17 is preferably P;

[0103] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 280 is different from L and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 280 is preferably A, and that the amino acid at position 40 is preferably M;

[0104] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 280 is different from L and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 280 is preferably A, and that the amino acid at position 111 is preferably E;

[0105] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 301 is preferably A, and that 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 79 is different from E and the amino acid at position 301 is different from D and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 301 is preferably A, and that the amino acid at position 11 is preferably A;

[0106] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 301 is different from D and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 301 is preferably A, and that the amino acid at position 17 is preferably P;

[0107] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 301 is different from D and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, more preferably Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 301 is preferably A, and that the amino acid at position 40 is preferably M, wherein it is particularly preferred that the amino acid at position 186 is Y and that the amino acid at position 79 is S and that the amino acid at position 301 is A and that the amino acid at position 40 is M;

[0108] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 301 is different from D and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 301 is preferably A, and that the amino acid at position 111 is preferably E;

[0109] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 3 is preferably Q, and that 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 79 is different from E and the amino acid at position 3 is different from M and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 3 is preferably Q, and that the amino acid at position 17 is preferably P;

[0110] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 3 is different from M and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 3 is preferably Q, and that the amino acid at position 40 is preferably M;

[0111] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 3 is different from M and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 3 is preferably Q, and that the amino acid at position 111 is preferably E;

[0112] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 11 is preferably A, and that the amino acid at position 17 is preferably P;

[0113] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 11 is different from C and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 11 is preferably A, and that 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 79 is different from E and the amino acid at position 11 is different from C and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 11 is preferably A, and that the amino acid at position 111 is preferably E;

[0114] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 17 is preferably P, and that the amino acid at position 40 is preferably M;

[0115] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 17 is different from L and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 17 is preferably P, and that the amino acid at position 111 is preferably E;

[0116] - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 79 is preferably S, W or I, more preferably S, and that the amino acid at position 40 is preferably M, and that the amino acid at position 111 is preferably E;

[0117] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 280 is preferably A, and that the amino acid at position 301 is preferably A;

[0118] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L and the amino acid at position 3 is different from M, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 280 is preferably A, and that 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 202 is different from A and the amino acid at position 280 is different from L and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 280 is preferably A, and that the amino acid at position 11 is preferably A;

[0119] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 280 is preferably A, and that the amino acid at position 17 is preferably P;

[0120] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 280 is preferably A, and that the amino acid at position 40 is preferably M;

[0121] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 280 is preferably A, and that the amino acid at position 111 is preferably E;

[0122] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 301 is preferably A, and that 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 202 is

[0123] A is different and the amino acid at position 301 is different from D and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 301 is preferably A, and that the amino acid at position 11 is preferably A;

[0124] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 301 is different from D and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 301 is preferably A, and that the amino acid at position 17 is preferably P;

[0125] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 301 is different from D and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 301 is preferably A, and that the amino acid at position 40 is preferably M;

[0126] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 301 is different from D and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 301 is preferably A, and that the amino acid at position 111 is preferably E;

[0127] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 3 is preferably Q, and that the amino acid at position 11 is preferably A;

[0128] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 3 is different from M and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 3 is preferably Q, and that 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 202 is different from A and the amino acid at position 3 is different from M and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 3 is preferably Q, and that the amino acid at position 40 is preferably M;

[0129] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 3 is different from M and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 3 is preferably Q, and that the amino acid at position 111 is preferably E;

[0130] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 11 is preferably A, and that the amino acid at position 17 is preferably P;

[0131] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 11 is different from C and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 11 is preferably A, and that the amino acid at position 40 is preferably M;

[0132] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 11 is different from C and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 11 is preferably A, and that the amino acid at position 111 is preferably E; the amino acid at position 186 is different from L and the amino acid at position 202 is

[0133] A is different and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 17 is preferably P, and that the amino acid at position 40 is preferably M;

[0134] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 17 is different from L and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 17 is preferably P, and that the amino acid at position 111 is preferably E;

[0135] - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 202 is preferably N, and that the amino acid at position 40 is preferably M, and that the amino acid at position 111 is preferably E;

[0136] - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A, and that the amino acid at position 301 is preferably A, and that the amino acid at position 3 is preferably Q;

[0137] - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A, and that the amino acid at position 301 is preferably A, and that the amino acid at position 11 is preferably A;

[0138] - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A, and that the amino acid at position 301 is preferably A, and that 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 280 is different from L and the amino acid at position 301 is different from D and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A, and that the amino acid at position 301 is preferably A, and that the amino acid at position 40 is preferably M;

[0139] - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A, and that the amino acid at position 301 is preferably A, and that the amino acid at position 111 is preferably E;

[0140] - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A, and that the amino acid at position 3 is preferably Q, and that the amino acid at position 11 is preferably A;

[0141] - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 3 is different from M and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A, and that the amino acid at position 3 is preferably Q, and that the amino acid at position 17 is preferably P;

[0142] - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 3 is different from M and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A, and that the amino acid at position 3 is preferably Q, and that 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 280 is different from L and the amino acid at position 3 is different from M and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A, and that the amino acid at position 3 is preferably Q, and that the amino acid at position 111 is preferably E;

[0143] - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A, and that the amino acid at position 11 is preferably A, and that the amino acid at position 17 is preferably P;

[0144] - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 11 is different from C and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A, and that the amino acid at position 11 is preferably A, and that the amino acid at position 40 is preferably M;

[0145] - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 11 is different from C and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A, and that the amino acid at position 11 is preferably A, and that the amino acid at position 111 is preferably E;

[0146] - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A, and that the amino acid at position 17 is preferably P, and that the amino acid at position 40 is preferably M;

[0147] - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 17 is different from L and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A, and that the amino acid at position 17 is preferably P, and that the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 280 is preferably A, and that the amino acid at position 40 is preferably M, and that the amino acid at position 111 is preferably E;

[0148] - the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 301 is preferably A, and that the amino acid at position 3 is preferably Q, and that the amino acid at position

[0149] II is preferably A;

[0150] - the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 301 is preferably A, and that the amino acid at position 3 is preferably Q, and that the amino acid at position 17 is preferably P;

[0151] - the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 301 is preferably A, and that the amino acid at position 3 is preferably Q, and that the amino acid at position 40 is preferably M;

[0152] - the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 301 is preferably A, and that the amino acid at position 3 is preferably Q, and that the amino acid at position

[0153] III is preferably E; the amino acid at position 186 is different from L and the amino acid at position 301 is different from

[0154] D is different and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 301 is preferably A, and that the amino acid at position 11 is preferably A, and that the amino acid at position 17 is preferably P;

[0155] - the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 11 is different from C and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 301 is preferably A, and that the amino acid at position 11 is preferably A, and that the amino acid at position 40 is preferably M;

[0156] - the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 11 is different from C and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 301 is preferably A, and that the amino acid at position 11 is preferably A, and that the amino acid at position 111 is preferably E;

[0157] - the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 301 is preferably A, and that the amino acid at position 17 is preferably P, and that the amino acid at position 40 is preferably M;

[0158] - the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 17 is different from L and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 301 is preferably A, and that the amino acid at position 17 is preferably P, and that the amino acid at position 111 is preferably E;

[0159] - the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 301 is preferably A, and that the amino acid at position 40 is preferably M, and that the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 3 is preferably Q, and that the amino acid at position 11 is preferably A, and that the amino acid at position 17 is preferably P;

[0160] - the amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 3 is preferably Q, and that the amino acid at position 11 is preferably A, and that the amino acid at position 40 is preferably M;

[0161] - the amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 3 is preferably Q, and that the amino acid at position 11 is preferably A, and that the amino acid at position 111 is preferably E;

[0162] - the amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 3 is preferably Q, and that the amino acid at position 17 is preferably P, and that the amino acid at position 40 is preferably M;

[0163] - the amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 17 is different from L and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 3 is preferably Q, and that the amino acid at position 17 is preferably P, and that the amino acid at position 111 is preferably E; the amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 3 is preferably Q, and that the amino acid at position 40 is preferably M, and that the amino acid at position 111 is preferably E;

[0164] - the amino acid at position 186 is different from L and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 11 is preferably A, and that the amino acid at position 17 is preferably P, and that the amino acid at position 40 is preferably M;

[0165] - the amino acid at position 186 is different from L and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 11 is preferably A, and that the amino acid at position 17 is preferably P, and that the amino acid at position 111 is preferably E;

[0166] - the amino acid at position 186 is different from L and the amino acid at position 11 is different from C and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 11 is preferably A, and that the amino acid at position 40 is preferably M, and that the amino acid at position 111 is preferably E;

[0167] - the amino acid at position 186 is different from L and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and that the amino acid at position 17 is preferably P, and that the amino acid at position 40 is preferably M, and that the amino acid at position 111 is preferably E;

[0168] - 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), provided that the amino acid at position 186 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, more preferably Y, and wherein the proteins have at least three further amino acid substitutions selected from the group shown under the listed symbols directly above.

[0169] The lipase variants described hereinabove under point a) with respect to the amino acid sequences shown under SEQ ID No. 1 can 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.

[0170] The lipase variants described hereinabove under point a) with respect to the amino acid sequences 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.

[0171] The lipase variants described hereinabove under point a) with respect to the amino acid sequences 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.

[0172] The lipase variants described above under point a) with regard to the amino acid sequences shown under SEQ ID No. 1 can 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. Preferred embodiments of the invention are proteins which encode lipases with the amino acid sequences shown under 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,.

[0173] 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191,

[0174] 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233,

[0175] 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275,

[0176] 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317,

[0177] 319, 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.

[0178] Particularly preferred embodiments of the invention are proteins which encode lipases having the amino acid sequences shown under SEQ ID Nos. 233 and 399, respectively.

[0179] Additional proteins with lipolytic enzyme or lipase activity have been tested. The amino acid sequences of these additional proteins represent variants of the amino acid sequence represented by the amino acid in SEQ ID No. 1, where:

[0180] - in 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 indicated at the corresponding amino acid positions in the sequence shown under SEQ ID No. 1. In a particular variant tested with respect 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.

[0181] - in the amino acid sequence shown under SEQ ID No. 1, the two amino acids at positions 40 and 186 are different from the amino acids indicated at the corresponding amino acid positions in the sequence shown under SEQ ID No. 1. In a particular variant tested with respect 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;

[0182] - in 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 indicated at the corresponding amino acid positions in the sequence shown under SEQ ID No. 1. In a particular variant tested with respect 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;

[0183] - in the amino acid sequence shown under SEQ ID No. 1, the two amino acids at positions 79 and 186 are different from the amino acids indicated at the corresponding amino acid positions in the sequence shown under SEQ ID No. 1. In a particular variant tested with respect 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;

[0184] - in the amino acid sequence shown under SEQ ID No. 1, the two amino acids at positions 79 and 301 are different from the amino acids indicated at the corresponding amino acid positions in the sequence shown under SEQ ID No. 1. In a particular variant tested with respect 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;

[0185] - In the amino acid sequence shown under SEQ ID No. 1, the two amino acids at positions 186 and 301 are different from the amino acids indicated at the corresponding amino acid positions in the sequence shown under SEQ ID No. 1. In a particular variant tested with respect 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.

[0186] Further proteins with lipolytic enzyme or lipase activity have been tested. The amino acid sequences of these additional proteins represent variants of the amino acid sequence represented by the amino acid in SEQ ID No. 1, where:

[0187] - in the amino acid sequence shown under SEQ ID No. 1, the three amino acids at positions 40, 79, and 186 are different from the amino acids indicated at the corresponding amino acid positions in the sequence shown under SEQ ID No. 1. In a particular variant tested with respect 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;

[0188] - in the amino acid sequence shown under SEQ ID No. 1, the three amino acids at positions 40, 79, and 301 are different from the amino acids indicated at the corresponding amino acid positions in the sequence shown under SEQ ID No. 1. In a particular variant tested with respect 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 301 is A;

[0189] - in the amino acid sequence shown under SEQ ID No. 1, the three amino acids at positions 40, 186, and 301 are different from the amino acids indicated at the corresponding amino acid positions in the sequence shown under SEQ ID No. 1. In a particular variant tested with respect 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;

[0190] - in the amino acid sequence shown under SEQ ID No. 1, the three amino acids at positions 79, 186, and 301 are different from the amino acids indicated at the corresponding amino acid positions in the sequence shown under SEQ ID No. 1. In a particular variant tested with respect 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;

[0191] The lipases and lipase variants according to the invention exhibit high selectivity and / or high specific activity with respect to the stereoselective acylation or carboxylation of 2,6-dimethyl-l-indanamine (DMAI) and are capable of producing enantiomerically enriched or nearly pure methyl [(lR,2S)-2,6-dimethyl-2,3-dihydro-lH-inden-l-yl] carbamate. Methyl [(lR,2S)-2,6-dimethyl-2,3-dihydro-lH-inden-l-yl] carbamate is an important intermediate for the synthesis of the herbicidal compound indaziflam.

[0192] “Enantiomerically enriched” means herein that one of two enantiomers is present in a composition in greater amounts than the other enantiomer, preferably one enantiomer is present in the composition to at least 60%, more preferably one enantiomer is present in the composition to at least 65%, even more preferably one enantiomer is present in the composition to at least 70%, even more preferably one enantiomer is present in the composition to at least 75%, even more preferably one enantiomer is present in the composition to at least 80%, more preferably one enantiomer is present in the composition to at least 85%, most preferably one enantiomer is present in the composition to at least 90%, or most preferably one enantiomer is present in the composition to at least 94%.

[0193] “Enantiomerically nearly pure” means herein that one of two enantiomers is present in a composition in amounts of at least 95.0%, preferably one of two enantiomers is present in a composition in amounts of at least 95.5%, more preferably one of two enantiomers is present in a composition in amounts of at least 96.0%, even more preferably one of two enantiomers is present in a composition in amounts of at least 96.5%, even more preferably one of two enantiomers is present in a composition in amounts of at least 97.0%, even more preferably one of two enantiomers is present in a composition in amounts of at least 98.0%, particularly preferably one of two enantiomers is present in a composition in amounts of at least 98.5%, most preferably one of two enantiomers is present in a composition in amounts of at least 99.0% or most preferably one of two enantiomers is present in a composition in amounts of at least 99.5%.

[0194] A further embodiment of the invention relates to nucleic acid molecules encoding a protein of the invention.

[0195] Nucleic acid molecules according to the invention can be any type of nucleic acid, provided the nucleic acid encodes a protein according to the invention. The nucleic acids can be ribonucleic acid molecules (e.g., RNA, mRNA) or deoxyribonucleic acid molecules (DNA, including genomic DNA, which may or may not include introns and coding DNA).

[0196] Of particular interest for the invention are nucleic acid molecules encoding proteins having the activity of a lipase, comprising those listed under 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,

[0197] 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,

[0198] 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245,

[0199] 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287,

[0200] 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 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.

[0201] The invention further relates to nucleic acid molecules which encode a protein with the activity of a lipase, selected from the group consisting of a) nucleic acid molecules which have the amino acid sequences shown under 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, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,

[0202] 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,

[0203] 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234,

[0204] 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272,

[0205] 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310,

[0206] 312, 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; b) nucleic acid molecules which have 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 most preferably 99% identity to the nucleic acid sequences shown under a).

[0207] In the context of the present invention, "hybridizing with" means hybridization under conventional hybridization conditions, preferably under stringent conditions, as described, for example, in Sambrook et al. (Molecular Cloning, A Laboratory Manual, 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ISBN: 0879695773) or in 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:

[0208] Hybridization buffer:

[0209] 2xSSC; 10xDenhardt solution (Fikoll 400+PEG+BSA; ratio 1:1:1); 0.1% SDS; 5 mM EDTA; 50 mM Na2HPO4; 250 pg / ml herring sperm DNA; 50 pg / ml tRNA; or

[0210] 25 M sodium phosphate buffer, pH 7.2; 1 mM EDTA; 7% SDS

[0211] Hybridization temperature: T = 65 to 68 °C

[0212] Wash buffer: 0.1 l x SSC; 0.1% SDS

[0213] Washing temperature: T = 65 to 68 °C.

[0214] Nucleic acid molecules that hybridize with nucleic acid molecules encoding a protein with lipase activity can originate from any organism; accordingly, they can originate from bacteria, fungi, animals, humans, plants, or viruses.

[0215] Nucleic acid molecules that hybridize with nucleic acid molecules encoding a protein with lipase activity preferably originate from microorganisms, more preferably from fungi or bacteria, most preferably from bacteria.

[0216] Nucleic acid molecules that hybridize with the aforementioned molecules can be isolated, for example, from genomic or cDNA libraries. These nucleic acid molecules can be identified and isolated using the nucleic acid molecules described herein, or they can be identified and isolated using portions of these molecules or the reverse complements of these molecules, for example, by hybridization according to standard procedures (see, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ISBN: 0879695773; Ausubel et al., Short Protocols in Molecular Biology, John Wiley & Sons; 5th ed. (2002), ISBN: 0471250929) or by amplification using PCR.

[0217] The fragments used as hybridization probes may also be synthetic fragments or oligonucleotides prepared using conventional synthesis techniques, the sequence of which is substantially identical to the nucleic acid molecule described in the context of the present invention. If genes that hybridize with the nucleic acid sequences described in the context of the present invention are identified and isolated, the sequence should be determined, and the properties of the proteins encoded by this sequence should be analyzed to determine whether they are proteins with lipase activity. Methods for determining whether a protein has lipase activity are known to those of ordinary skill in the art.

[0218] The molecules that hybridize with the nucleic acid molecules described in the present invention include, in particular, fragments, derivatives, and allelic variants of said nucleic acid molecules. In the context of the present invention, the term "derivative" means that the sequences of these molecules differ in one or more positions from the sequences of the nucleic acid molecules described above and are highly identical to these sequences. The differences from the nucleic acid molecules described above can be due, for example, to deletion, addition, substitution, insertion, or recombination.

[0219] Preferred nucleic acid molecules according to the invention are those under 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, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,

[0220] 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202,

[0221] 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244,

[0222] 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286,

[0223] 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328,

[0224] 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370,

[0225] 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402. The meaning of the nucleotide abbreviations a, c, g, t as well as the abbreviations for degenerate nucleotides r, y, s, w, k, m, b, d, h, v, n can be derived hereinafter from Table 1 in the section titled "Description of the Sequences". The amino acids encoded by degenerate nucleotides can be derived hereinafter from Table 3 in the section titled "Description of the Sequences".

[0226] Also disclosed are recombinant nucleic acid molecules comprising a nucleic acid molecule suitable for the method according to the invention.

[0227] The term "recombinant nucleic acid molecule" is understood to mean a nucleic acid molecule that, in addition to the nucleic acid molecule suitable for the process according to the invention, contains further sequences that do not occur naturally in the combination in which they occur in the nucleic acids recombinant for the process according to the invention. The above-mentioned additional sequences can be any sequences, preferably functional or regulatory sequences (promoters, termination signals, enhancers, ribosome binding sites (RBS), leader sequences that increase transcription, translation, or RNA stability, subcellular targeting sequences, etc.). More preferably, they are functional or regulatory sequences that are active in microorganisms, and most preferably, they are regulatory sequences that are active in fungi, in particular yeasts, or bacteria.Methods for producing the recombinant nucleic acid molecules suitable for the method according to the invention are known to those of ordinary skill in the art. These include genetic methods, such as binding nucleic acid molecules by ligation, genetic recombination, or de novo synthesis of nucleic acid molecules. 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 in Ausubel et al. (Short Protocols in Molecular Biology, John Wiley & Sons; 5th edition (2002), ISBN: 0471250929).

[0228] The recombinant nucleic acid molecule usable for the method according to the invention comprises a suitable nucleic acid molecule linked to regulatory sequences that initiate transcription in prokaryotic or eukaryotic cells.

[0229] Regulatory sequences that initiate transcription in a cell are also known as promoters. Information regarding regulatory sequences and plasmids is well known to those of ordinary skill in the art and is described, for example, in the Registry of Standard Biological Parts, 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).

[0230] Regulatory sequences that initiate transcription in prokaryotic organisms, such as E. coli, as well as in eukaryotic organisms, have been extensively described in the literature, particularly those related to expression in yeast, e.g., Saccharomyces cerevisiae. An overview of various systems for protein expression in different host organisms can be found, for example, in Methods in Enzymology 153 (1987), 383-516, as well as in Bitter et al. (Methods in Enzymology 153 (1987), 516-544), or in 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 are pAOX1, pHIS4, pGAL, and pScADH2 (Baghban et al., 2018, see above). Common bacterial promoters are T5, T7, rhamnose-inducible, arabinose-inducible, PhoA, and the 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).

[0231] A further embodiment of recombinant nucleic acid molecules usable for the method according to the invention are vectors of plasmids comprising the suitable nucleic acid molecules.

[0232] "Vectors" are well known in the field of molecular biology and, as used herein, represent a nucleic acid sequence or a vehicle comprising a nucleic acid sequence used to transfer genetic material (DNA or RNA) into a target cell. Vectors can be plasmids, e.g., T-DNA or binary vectors for generating transgenic plants, expression vectors for expressing nucleic acid sequences in a host cell, shuttle vectors capable of replicating in different hosts, or vectors can be virus particles or bacteriophages modified to deliver foreign genetic material into a host.

[0233] “Plasmids” are well known in the field of molecular biology and represent an autonomously self-replicating, often circular DNA molecule which, when present in a host cell, is separated from the chromosomal DNA.

[0234] Suitable nucleic acid molecules, recombinant nucleic acid molecules, vectors or plasmids can be used to produce proteins for the method according to the invention, e.g. by expressing the suitable nucleic acid molecule in host cells.

[0235] Also disclosed are hosts or host cells which comprise or express a nucleic acid molecule suitable for the method according to the invention or which comprise suitable proteins having the activity of a lipase or which comprise a recombinant nucleic acid molecule suitable for the method according to the invention or which comprise a vector suitable for the method according to the invention or which comprise a plasmid suitable for the method according to the invention.

[0236] Suitable nucleic acid molecules encoding a protein with lipase activity can be expressed in host cells, e.g., for their replication or to produce proteins with lipase activity. For expression in host cells, suitable nucleic acid molecules can be contained on vectors or plasmids or can be stably integrated into the genome of a respective host cell. The suitable nucleic acid molecules can also be contained in vectors that support their introduction into host cells.

[0237] Also disclosed is a host or host cell suitable for the method according to the invention, comprising a nucleic acid molecule suitable for the method according to the invention or comprising a recombinant nucleic acid molecule suitable for the method according to the invention or comprising a vector suitable for the method according to the invention or comprising a plasmid suitable for the method according to the invention and in each case a protein suitable for the method according to the invention.Also disclosed is a host or host cell suitable for the method according to the invention, comprising a nucleic acid molecule suitable for the method according to the invention or comprising a recombinant nucleic acid molecule suitable for the method according to the invention or comprising a vector suitable for the method according to the invention or comprising a plasmid suitable for the method according to the invention and each expressing a protein suitable for the method according to the invention, wherein the protein preferably has the activity of a lipase.

[0238] "Expressing a nucleic acid molecule" is to be understood herein as meaning that, if the nucleic acid molecule is RNA or mRNA, the nucleic acid molecule is translated into a protein, preferably into a protein having the activity of a lipase, or if the nucleic acid molecule is DNA or cDNA, it is transcribed (and processed in the case of genomic DNA containing introns) into mRNA, preferably into an mRNA encoding a protein having the activity of a lipase, and then translated into a protein, preferably into a protein having the activity of a lipase.

[0239] The transcription of a particular nucleic acid molecule in a host can be detected by methods known to those of ordinary skill in the art, for example by detecting specific transcripts (mRNA) of foreign nucleic acid molecules by Northern blot analysis or by RT-PCR.

[0240] Whether hosts or host cells comprise a particular protein or comprise a protein derived from the expression of a nucleic acid molecule can be determined by methods known to those of ordinary skill in the art, for example, by immunological methods such as Western blot analysis, ELISA (Enzyme-Linked Immunosorbent Assay), or RIA (Radio Immune Assay). Those of ordinary skill in the art are familiar with methods for producing antibodies that specifically react with a particular protein, i.e., that specifically bind to a particular protein (see, for example, Lottspeich and Zorbas (eds.), 1998, Bioanalytik, Spektrum akad, Verlag, Heidelberg, Berlin, ISBN 3-8274-0041-4). Some companies (Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA USA 0245; GenScript, 60 Centennial Ave., Piscataway, NJ 08854, USA) offer the production of these antibodies as a service on demand.

[0241] Furthermore, those of ordinary skill in the art can check whether a host or host cell comprises a protein suitable for the method according to the invention by detecting the (additional) activity of proteins with the activity of a lipase in a corresponding host cell. Preferably, the activity of proteins with additional lipase activity in a corresponding host cell is detected by comparing the activities of lipases in a host cell used for the method according to the invention with the corresponding activity of host cells that do not comprise a protein suitable for the method according to the invention. Checking whether a protein has lipase activity can be carried out using methods known in the art.

[0242] Hosts or host cells used for the method of the invention can be prepared by those of ordinary skill in the art using known methods for genetically modifying or transforming organisms.

[0243] Furthermore, a host or host cell suitable for the method according to the invention, in particular a prokaryotic or eukaryotic host or host cell, genetically modified (or transformed) with a suitable nucleic acid molecule or with a suitable recombinant nucleic acid molecule or with a suitable vector or with a suitable plasmid, is disclosed. Preferably, the genetically modified (transformed) host or host cell used for the method according to the invention expresses a protein with the activity of a lipase; 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” is to be understood herein as meaning that a nucleic acid molecule is or has been introduced into a host or host cell by technical and / or non-naturally occurring means, preferably by technical processes from the field of molecular biology, biotechnology or genetic engineering.

[0244] Descendants or progeny of hosts or host cells used for the method according to the invention are also disclosed, preferably these descendants or progeny comprise a nucleic acid molecule suitable for the method according to the invention or comprise a suitable recombinant nucleic acid molecule or comprise a suitable vector or comprise a suitable plasmid or comprise a suitable protein, more preferably these 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 and in each case they express a protein, wherein the protein has the activity of a lipase,Even more preferably, these progeny 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 and in each case they express a protein, which protein has the activity of a lipase which can be used in the process according to the invention.

[0245] The host or host cell for the method according to the invention may be a host or host cell of a prokaryotic or a eukaryotic organism. The host or host cells can be bacteria or bacterial cells (e.g. E. coli, bacteria of the genus Bacillus, in particular Bacillus subtilis, Agrobacterium, in particular Agrobacterium tumefaciens or Agrobacterium rhizogenes, Pseudomonas, in particular Pseudomonas fluorescens, Streptomyces spp, Rhodococcus spp, in particular Rhodococcus rhodochrous, Vibrio natrigens, Corynebacterium, in particular Corynebacterium glutamicum) or fungi or fungal cells (e.g. Agaricus, in particular Agaricus bisporus, Aspergillus, Trichoderma or yeasts, in particular Aspergillus cerevisiae, Pichia ssp. such as P. pastoris), as well as plants or plant cells, or they can be animals or animal cells. Preferred host cells are cells of microorganisms.In the context of the present patent application, it is assumed that all bacteria and protists (e.g., fungi, especially yeasts and algae), as defined, for example, in Schlegel's "General Microbiology" (Georg Thieme Verlag (1985), 1-2), are included. With regard to microorganisms, the hosts or host cells used for the process according to the invention are preferably bacteria / bacterial cells or yeasts / yeast cells, more preferably bacteria / bacterial cells, even more preferably Bacillus species / BacZZZus species cells or Escherichia coli / Escherichia coli cells, most preferably Escherichia coli / Escherichia coli cells. Alternatively, Pseudomonas, in particular Pseudomonas fluorescens, Streptomyces spp, Rhodococcus spp, in particular Rhodococcus rhodochrous, Vibrio spp, in particular Vibrio natrigens, Corynebacterium, in particular Corynebacterium glutamicum or other hosts or host cells can be used for the method according to the invention.

[0246] Preferred hosts or host cells for the method according to the invention comprise a suitable nucleic acid molecule, wherein the suitable nucleic acid molecule is characterized in that the codons of the nucleic acid molecule are modified such that they are adapted to the codon usage frequency of the host or a host cell. Description of the sequences

[0247] Throughout the application, abbreviations for nucleotides and amino acids are used according to the following lUPAC codes:

[0248] Table 1

[0249] To distinguish between amino acids and nucleotides, the abbreviated nucleotide codes written in capital letters in the table above are written in lower case here.

[0250] Table 2

[0251] The use of codons herein follows the so-called “general genetic code” as shown in the following table, where “t” in ribonucleotide acid (RNA) sequences is to be replaced by “u”.

[0252] Table 3

[0253] Table 4

[0254] The sequence listing associated with this application is submitted in electronic format and is hereby incorporated by reference in its entirety into this patent specification. "PRT" stands for "protein" and "NUC" for "nucleic acid."

[0255]

[0256] The protein with lipase activity is preferably used in an amount of 0.1-50 wt.% based on the mixture (I). An amount of 0.5-10 wt.% is preferred. An amount of 1-5 wt.% is particularly preferred.

[0257] Step 1 of the process according to the invention can be carried out in the absence of a solvent or in the presence of one. Preferred solvents are those selected from the group consisting of methyl t-butyl ether, heptane, toluene, xylenes, mesitylene, anisole, chlorobenzene, n-butanol, z-propanol, n-propanol, and ethanol, as well as mixtures thereof. Particular preference is given to solvents selected from the group consisting of toluene, xylenes, mesitylene, n-butanol, and ethanol, as well as mixtures thereof. The reaction is also particularly preferably carried out in the absence of a solvent.

[0258] Based on the amount of substance used in the mixture (I), the acylating or carboxylating agent RC(=O)R' is preferably used in an amount of 0.4-25 equivalents. It is particularly preferably used in an amount of 0.6-10 equivalents. It is especially preferably used in an amount of 1-5 equivalents.

[0259] The reaction according to step 1 is typically carried out at temperatures of 20-130 °C. Preferably, the reaction is carried out at 70-130 °C. Particularly preferably, the reaction is carried out at 80-120 °C.

[0260] The separation of component (II) mentioned in step 2 of the process according to the invention is carried out by a crystallization known to the person skilled in the art.

[0261] The bases mentioned in step 3 of the process according to the invention typically come 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 preferred bases are lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium ethoxide, sodium ethoxide, and potassium ethoxide.

[0262] The bases lithium hydroxide, sodium hydroxide and potassium hydroxide are particularly preferred.

[0263] Typically, the base is used in a stoichiometry of 1.00-3.00 equivalents based on the amount of component (II) used. Preferably, the base is used in an amount of 1.50-2.50 equivalents. Particularly preferably, the base is used in an amount of 1.75 to 2.25 equivalents.

[0264] Preferred solvents are ethanol, z-propanol, z-propanol, n-butanol, i-butanol, sec-butanol, tert-butanol, and l-methoxypropan-2-ol, as well as toluene, xylenes, and veratrole. Particular preference is given to ethanol, n-butanol, z-propanol, l-methoxypropan-2-ol, toluene, xylenes, and veratrole. Ethanol, n-butanol, and xylenes are particularly preferred.

[0265] The reaction according to step 3 is typically carried out at temperatures of 60-140 °C. Preferably, the reaction is carried out at 80-120 °C. Particularly preferably, the reaction is carried out at 90-110 °C.

[0266] A further advantage of the process according to the invention is that the mixture of 2,6-dimethyl-l-indanamines (III) remaining in step 1 or step 2 can be isomerized again in a metal-catalyzed, resource-saving manner to a mixture of the four stereoisomers of 2,6-dimethyl-l-indanamine (I).

[0267] This isomerization can be carried out as follows:

[0268] The preferred metal catalyst is a palladium-on-carbon (Pd / C), a palladium-on-calcium carbonate (Pd / CaCCL), or a palladium-on-aluminum (Pd / AhOi) catalyst with a palladium loading of 1-20 wt.%. Also preferred is the Shvo catalyst with the IUPAC name 1-hydroxytetraphenylcyclopentadienyl-(tetraphenyl-2,4-cyclopentadien-1-one)-p-hydrotetracarbonyldiruthenium(II) in a stoichiometry of 1-10 mol%. Particular preference is given to using a palladium-on-carbon (Pd / C) or a palladium-on-aluminum (Pd / AhOi) catalyst with a palladium loading of 1-20 wt.%. Particularly preferred is a palladium-on-alumina (Pd / AhCl) catalyst with a palladium loading of 1-20 wt.%. The supported catalysts are used in an amount of 0.1-10 wt.% relative to the compounds of formula (III); preferably, 0.5-5 wt.%.The amount of catalysts used is calculated based on the dry mass of the catalysts.

[0269] Preferred bases are pyridine, iV-methylmorpholine, morpholine, cyclohexylamine, di- / z-butylamine, tri-n-butylamine, triethylamine, diisopropylethylamine, piperidine, potassium carbonate, sodium carbonate, lithium carbonate, potassium ethoxide, sodium ethoxide, and lithium ethoxide, or no base at all. Particular preference is given to using piperidine, morpholine, diisopropylethylamine, tri-n-butylamine, potassium carbonate, and sodium ethoxide, or no base at all. Piperidine and sodium ethoxide, or no base at all, are particularly preferred.

[0270] Toluene, xylenes, mesitylene, anisole, chlorobenzene, n-butanol, i-propanol, n-propanol, and ethanol are preferred solvents, or the reaction is carried out in the absence of a solvent. Toluene, xylenes, mesitylene, n-butanol, and ethanol are particularly preferred solvents, or the reaction is carried out in the absence of a solvent. The reaction is particularly preferably carried out in the absence of a solvent.

[0271] The reaction is preferably carried out at a pressure between 0 and 20 bar. The reaction is particularly preferably carried out at a pressure between 0 and 10 bar. The reaction is particularly preferably carried out at a pressure between 1 and 6 bar.

[0272] The reaction pressure is primarily achieved by injecting a gas / gas mixture. Hydrogen, nitrogen, or argon, or a mixture of hydrogen and nitrogen, or hydrogen and argon, is preferably injected. Hydrogen or a mixture of nitrogen and hydrogen is particularly preferably injected. Pure hydrogen is especially preferred. The reaction is preferably carried out at temperatures of 80-150 °C. The reaction is particularly preferably carried out at 100-130 °C. The reaction is particularly preferably carried out at 110-125 °C.

[0273] A further subject of the present invention is therefore also a process for the preparation of a mixture of all four stereoisomers of 2,6-dimethyl-l-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:

[0274] The following examples further illustrate the invention: Terrific Broth (TB) culture media were prepared in demineralized water using 47.6 g / l granulated medium and 4 ml / l glycerol and sterilized at 121 °C for 20 minutes.

[0275] Cloning of lipases

[0276] Nucleotide sequences encoding lipases and lipase variants as described herein can be synthesized as known in the art, e.g., as offered by appropriate service providers such as Eurofins Genomics GmbH (Eurofins Genomics GmbH, Anzinger Str. 7a, 85560 Ebersberg, Germany). Briefly, nucleic acid sequences of wild-type lipase (SEQ ID No. 2) or related variants as described herein were cloned into an expression vector based on the pKA81a vector. Genetic elements were introduced into the modified pKA81a vector using well-known methods. For expression of wild-type lipase or lipase variants, the expression vectors were introduced into electrocompetent MG1655 cells of Escherichia coli.

[0277] Generation of enzyme variants

[0278] Nucleotide substitutions (replacements) have been introduced into the nucleic acid parent sequences, e.g., to achieve an amino acid exchange with other amino acids. Several molecular biological methods can be used to achieve these replacements. One useful method for producing a mutated nucleic acid and the corresponding mutated protein of the invention is site-directed mutagenesis at codons encoding one or more amino acids that are selected in advance. The methods for achieving these site-directed mutations are well known to those of ordinary skill in the art and are adequately described in the literature (in particular: Directed Mutagenesis: A Practical Approach, 1991, edited by MJ McPHERSON, IRL PRESS) or are methods for which commercial kits (e.g., the QUIKCHANGE™ Lightning Mutagenesis Kit from Qiagen or Stratagene) can be used.After site-directed mutagenesis, nucleic acids were transformed into Escherichia coli MG1655 cells.

[0279] Transformed cells were tested in appropriate biotransformation reactions to determine product yield and selectivity. Suitable biotransformation reactions are described below. Sequence verification was performed as known in the art.

[0280] Glycerol stocks of E. coZZ cultures transformed with the respective expression plasmids were prepared by adding one volume of a 40% glycerol solution to one volume of an E. coZZ culture. To isolate individual bacterial colonies, appropriate dilutions of E. coZZ cultures were plated on LB agar plates containing appropriate concentrations of kanamycin and incubated at 37°C until single colonies were obtained.

[0281] Synthesis of ethyl [( 1 R,2S)-2,6-dimethyl-2,3-dihydro- 1 H-inden- 1 -yl] carbamate

[0282] Example 1:

[0283] In a comparative experiment, three different proteins with lipase activity were investigated for their suitability: the wild-type lipase (SEQ ID No. 1) and the lipase variants with SEQ ID No. 233 and SEQ ID No. 399. For this purpose, 9.25 g of 2,6-dimethylindan-l-amine (racemic mixture of 82% trans- and 18% cis-2,6-dimethylindan-l-amine) were placed in a 25 mL three-neck flask equipped with a magnetic stirrer under argon and diluted with 13.6 g of diethyl carbonate. This solution was heated to an internal temperature of 110 °C, and then 250 mg of protein with lipase activity was added in one portion. The reaction progress was monitored using HPLC measurements. After complete conversion of the (lR,2S)-2,6-dimethylindan-l-amine isomer, the reaction was terminated by adding 5 mL of diethyl carbonate, cooling to 100 °C and filtering this reaction mixture through a glass frit (porosity 3).An additional 5 mL of diethyl carbonate was used to wash the filter cake. The combined, orange filtrate was then distilled at 50 °C and 35-10 mbar vacuum. 50 mL of n-heptane was added to the distillation residue, and the solution was stirred at 40 °C for 20 minutes. During cooling to 2 °C, a suspension 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 vacuum. The resulting white solid was then analyzed for chemical purity by quantitative 'H NMR measurement and for stereoisomeric purity by chiral HPLC measurement. The results are compared in Table 5 below:

[0284] Table 5

[0285] The yields here refer to the content of pure ethyl [(lR,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl] carbamate. Example 2:

[0286] In a 500 mL four-neck round-bottom flask equipped with an internal thermometer, a magnetic stirrer, an inert gas inlet, and a bubble counter, 80.0 g of 2,6-dimethylindan-l-amine (racemic mixture of 82% trans- and 18% cis-2,6-dimethylindan-l-amine) was dissolved in 130.0 g of diethyl carbonate under argon. This solution was heated to an internal temperature of 110 °C with stirring. Subsequently, 1.2 g of E. coli culture transformed with SEQ ID No. 234 was added in one portion, and the reaction was continued at 110 °C with stirring and argon. After a reaction time of 7 hours, HPLC measurement indicated almost complete conversion of (lR,2S)-2,6-dimethylindan-l-amine. The reaction was stopped by cooling to 80 °C and subsequent filtration through a glass frit (porosity 3). The filter cake was then washed with 2 x 40 mL of diethyl carbonate, and the combined filtrate was subjected to distillation at 50 °C and a vacuum of 35-10 mbar.A total of 157.4 g of distillate was separated 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 form a suspension. The solid was separated 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 were obtained. A quantitative 'H NMR measurement confirmed ethyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate with a chemical purity of 85.4%. Using a chiral HPEC measurement, the stereoisomeric purity was determined to be 98.5%. This corresponds to a yield of 27% pure ethyl [(lR,2S)-2,6-dimethyl-2,3-dihydro-lH-inden-l-yl]carbamate. 'H NMR (400 MHz; CDCl) 5 = 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).

[0287] The derivative methyl [(lR,2S)-2,6-dimethyl-2,3-dihydro-lH-inden-l-yl]carbamate was prepared analogously using dimethyl carbonate and was obtained as a white solid. 'H NMR (400 MHz; CDCl) δ = 7.08-7.01 (m, 3H), 4.79-4.78 (m, 2H), 3.74 (s, 3H), 3.00 (dd, δ = 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).

[0288] Example 3:

[0289] In a 25 mE 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. Subsequently, 10.00 g of 2,6-dimethylindan-l-amine (racemic mixture of 82% trans- and 18% cis-2,6-dimethylindan-l-amine) and 0.25 g of E. coli culture transformed with SEQ ID No. 234 were added in one portion, and the reaction was continued under argon for 10 hours at 110 °C with stirring until an HPEC measurement indicated almost complete conversion of the (lR,2S)-2,6-dimethylindan-l-amine. The reaction mixture was then diluted with an additional 5 mL of diethyl carbonate and then filtered at 100 °C through a glass frit (porosity 3). The filter cake was then washed with 5 mL of diethyl carbonate heated to 80 °C. The combined filtrate was then subjected to distillation at 50 °C and a vacuum of 35-10 mbar.Subsequently, 50 mL of n-heptane was 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 over a period of 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. A quantitative 'H NMR measurement confirmed ethyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate with a chemical purity of 99%. Using chiral HPLC, the stereoisomeric purity was determined to be 98.5%. This corresponds to a yield of 34% pure ethyl [(lR,2S)-2,6-dimethyl-2,3-dihydro-lH-inden-l-yl]carbamate.

[0290] Example 4:

[0291] In a 600 mL autoclave, 100.0 g of 2,6-dimethylindan-l-amine (racemic mixture of 82% trans- and 18% cis-2,6-dimethylindan-l-amine), 139.2 g of diethyl carbonate, and 2 g of biomass GÖ171-7-032 were stirred under argon at 110 °C for 7 h. After cooling to 80 °C, the mixture was filtered 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 by adding 150 mL of methylcyclohexane in an ice bath. The solid was filtered through a glass frit and washed with a little methylcyclohexane. After drying under vacuum, 47.6 g of a white solid were obtained. A quantitative 'H NMR measurement confirmed ethyl [(lR,2S)-2,6-dimethyl-2,3-dihydro-lH-inden-l-yl]carbamate with a chemical purity of 91.9%. A chiral HPLC measurement determined the stereoisomeric purity to be 96.1%.This corresponds to a yield of 31.9% of pure ethyl [(lR,2S)-2,6-dimethyl-2,3-dihydro-lH-inden-l-yl]carbamate. The filtrate was concentrated under reduced pressure at 50 °C, and the content of the 2,6-dimethylindan-l-amine isomers was determined by chiral GC to have an isomer ratio of R,R : S,S : S,R : R,S = 6.0 : 16.2 : 77.2 : 0.

[0292] Synthesis of (lR,2S)-2,6-dimethylindan-l-amine from methyl [(lR,2S)-2,6-dimethyl-2,3-dihydro-lH-inden-1-yl]carbamate

[0293] Example 5:

[0294] In a 250 mL jacketed reactor inertized with argon at 21 °C, 25 g of methyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-lH-inden-l-yl]carbamate (98% stereomeric purity, 98% chemical purity) was suspended in 80 mL of n-butanol. The suspension was warmed to an internal temperature of 65 °C, resulting in a clear solution. Subsequently, 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 stirred at this temperature for one hour, during which insoluble solids precipitated. An HPLC measurement indicated complete conversion. A vacuum of 360 mbar was then applied, and 55 mE of solvent were distilled off. Subsequently, 100 mL of p-xylene was added and the reaction mixture was cooled to 20 °C. The mixture was washed with 1 x 50 mL of deionized water.The organic phase was then distilled at 40 °C to a vacuum of 5 mbar, yielding 17.8 g of (1R,2S)-2,6-dimethylindan-1-amine as a pale yellow oil with a chemical purity of 95%, determined by quantitative 'H NMR analysis, corresponding to a yield of 95%. The stereoisomeric purity was determined to be 98% by chiral GC measurement. >H NMR (500 MHz, CDC13): 5 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, 7 = 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).

[0295] Example 6:

[0296] Synthesis of (lR,2S)-2,6-dimethylindan-l-amine hydrochloride from ethyl [(lR,2S)-2,6-dimethyl-2,3-dihydro- IH-inden- 1 -yl]carbamate

[0297] In a 250 mL jacketed reactor inertized with argon at 21 °C, 50 g of methyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-lH-inden-l-yl]carbamate (98% stereomeric purity, 98% chemical purity) was suspended in 160 mL of / z-butanol. The suspension was warmed to an internal temperature of 65 °C, resulting in a clear solution. Subsequently, 31.1 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 stirred at this temperature for one hour, during which insoluble solids precipitated. An HPLC measurement indicated complete conversion. A vacuum of 360 mbar was then applied, and 110 mL of solvent was distilled off. Subsequently, 200 mL of p-xylene was added and the reaction mixture was cooled to 20 °C. The mixture was washed with 1 x 100 mL of deionized water.The organic phase was then cooled to 10 °C and treated with 22.8 g of concentrated hydrochloric acid (37 wt.% in water) at a temperature such that the temperature did not rise above 25 °C. 100 mL of solvent was then distilled from the resulting suspension at 50 °C, and the solid was filtered off at 20 °C using a glass frit (porosity 3). The filter cake was washed with 1 x 100 mL of toluene and then dried at 40 °C and a vacuum of 10 mbar. 40 g of (1R,2S)-2,6-dimethylindan-1-amine hydrochloride were obtained as a white solid with a chemical purity of 99%, determined by quantitative 'H-NMR measurement, corresponding to a yield of 95%. The stereoisomeric purity was determined to be 98% by chiral GC measurement. 'H-NMR (400 MHz; DMSO-d6) 5 = 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, 7 = 8.0 Hz, 3H).

[0298] Isomerization of 2,6-dimethylindan-l-amine mixture (III) to 2,6-dimethylindan-l-amine mixture (I) Example 7:

[0299] In a 25 ml autoclave, 10 g of the resulting 2,6-dimethylindan-l-amine mixture (III) (concentrated filtrate from Example 4) were mixed with 396 mg of palladium catalyst (5% Pd / AhOi). The autoclave was purged three times with 5 bar argon, then pressurized with 3 bar hydrogen and stirred for 10 h at 120°C under 3 bar hydrogen pressure. After cooling to room temperature and depressurizing the autoclave, the content of 2,6-dimethylindan-l-amine isomers in the resulting mixture was determined by chiral GC to an isomer ratio of R,R : S,S : S,R : R,S = 9.2 : 8.9 : 43.0 : 39.0.

[0300] Isomerization of (lR,2S)-2,6-dimethylindan-l-amine to 2,6-dimethylindan-l-amine (racemic mixture of 82% trans and 18% cis isomers)

[0301] Examples 8-27:

[0302] A 6 mL Wheaton screw-capped vial was filled with 1.0 g of (lR,2S)-2,6-dimethylindan-l-amine, 39.6 mg of palladium catalyst (5% Pd / AhCl), 1.47 g of diethyl carbonate, and 10-20 mol% of a base. The vial was then sealed with a screw cap and shaken at 90-110 °C for 6 hours at 400 rpm. The mixture was then cooled to 21 °C and analyzed by chiral GC and achiral HPLC. The results are tabulated. The activity determines the isomerization progress from an isomer ratio of 0:0:0:100 to an equilibrium ratio of 9:9:41:41 and is primarily measured by the degradation progress of the 1R,2S isomer from 100% to 41%. The activity is therefore determined from the measured maximum degradation rate of 59%.Chemoselectivity describes 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 minor components. The corresponding data are shown in Table 6 below.

[0303] Table 6

Claims

Patent claims:

1. A process for the preparation of almost enantiomerically pure (lR,25)-2,6-dimethyl-l-indanamine, characterized in that 1. that in a first step, a mixture of the four stereoisomers of 2,6-dimethyl-l-indanamine (I) is reacted with an acylating or carboxylating agent RC(=O)R' in the presence of a protein with the activity of a lipase to give the corresponding amide or carbamate (II) and a mixture (III) of the unreacted stereoisomers of 2,6-dimethyl-l-indanamine: wherein the protein is encoded by an amino acid sequence selected from the group consisting of I. Proteins that have at least 80% identity to the amino acid sequence shown under SEQ ID No. 1, II. Proteins which have at least 80% identity to the amino acid sequence shown under SEQ ID No. 1, except that the amino acid sequence has a modification selected from the group consisting of 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; 2. that in a second step the amide or carbamate (II) is separated from the remaining 2,6-dimethyl-l-indanamines (III) and minor components by crystallization, and 3. that in a third step the amide or carbamate (II) is converted using an acid or a base to the almost enantiomerically pure (lR,2S)-2,6-dimethyl-l-indanamine (IV), wherein R is a radical from the group CH2OCH3, CH2OCH2CH3, CH3, OCH3, OCH2CH3, OCH(CH3)2, OCH2CH2CH2CH3, OCH2CHCH2and OCH2(C6H5), and wherein R 1 a residue from the group OCH3, OCH2CH3, OCH(CH3)2, OCH2CH2CH2CH3, OCH2CHCH2 and OCH2(CeH5).

2. A process according to claim 1, wherein R represents a residue from the group OCH3 and OCH2CH3, and R 1 a residue from the group OCH3 and OCH2CH3.

3. The method according to claim 1 or 2, characterized in that the protein is selected from the group consisting of a) proteins which comprise the amino acid sequence shown in SEQ ID No. 1, apart from the fact that the amino acid at position 186 is different from L; b) proteins with an amino acid sequence with at least 80% identity to the amino acid sequence shown under a), provided that the amino acid at position 186 is different from L, wherein the amino acid in the proteins according to a) or b) at position 186 is preferably F, W, Y, E, D, Q, T, H, P, C, K, S, N, I or V.

4. The method according to claim 3, characterized in that the protein has at least one, preferably at least two, more preferably at least three further amino acid substitutions selected from the group consisting of (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.

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

6. Process according to one of claims 1 to 5, characterized in that the protein is used in an amount of 0.1-50 wt.% based on the mixture (I).

7. Process according to one of claims 1 to 6, characterized in that the protein is used in an amount of 0.5-10 wt.% based on the mixture (I).

8. Process according to one of claims 1 to 7, characterized in that the protein is used in an amount of 1-5 wt.% based on the mixture (I).

9. Process according to one of claims 1 to 8, characterized in that step 1 is carried out without solvent or in the presence of a solvent selected from the group consisting of toluene, xylenes, mesitylene and n-butanol.

10. Process according to one of claims 1 to 9, characterized in that in step 1 the acylating or carboxylating agent RC(=O)R' is preferably used in an amount of 0.4-25 equivalents based on the amount of substance used in the mixture (I).

11. Process according to one of claims 1 to 10, characterized in that in step 1 the acylating or carbocarboxylating agent RC(=O)R' is preferably used in an amount of 1.0-5.0 equivalents based on the amount of substance used in the mixture (I).

12. Process according to one of claims 1 to 11, characterized in that in step 1 the reaction is carried out at temperatures of 20-130 °C.

13. Process according to one of claims 1 to 12, characterized in that in step 1 the reaction is carried out at temperatures of 80-120 °C.

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

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 1.00-3.00 equivalents based on the amount of component (II).

16. Process for the preparation of a mixture of all four stereoisomers of 2,6-dimethyl-l-indanamine (I) by metal-catalyzed isomerization of a mixture of the three stereoisomers of (1R,2R)-, (1S,2R)- and (1S,2S)-2,6-dimethyl-l-indanamine:

17. The process according to claim 16, characterized in that a palladium on carbon (Pd / C) or a palladium on aluminum oxide (Pd / AhCh) catalyst with a palladium loading of 0.5-10 wt. % is used.

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

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