Process for producing (1R, 2S)-2, 6-dimethyl-1-aminoindan

Abstract

A process for the production of substantially enantiomerically pure (1R, 2S)-2, 6-dimethyl-1-aminoindan is described, said process being characterized in that a mixture of four stereoisomers of 2, 6-dimethyl-1-aminoindan is reacted with an acylating or carboxylating agent in the presence of a protein having lipase activity.

Description

[0001] The present invention relates to a method for preparing substantially enantiopure (1R,2S)-2,6-dimethyl-1-aminoindan, which is a valuable building block for the synthesis of the herbicidal active ingredient indaziflam. In particular, the present invention relates to a method for preparing substantially enantiopure (1R,2S)-2,6-dimethyl-1-aminoindan by enzymatic stereoselective acylation of racemic 2,6-dimethyl-1-aminoindan.

[0002] From the prior art, the only methods known to date for preparing substantially enantiopure (1R,2S)-2,6-dimethyl-1-aminoindan are those which are only suitable for laboratory scale but not for industrial use due to the use of expensive reaction components and catalysts. For example, WO 2004 / 69814 A1 discloses a method which is characterized by the following five reaction steps:

[0003] 1. A mixture of four stereoisomers of 2,6-dimethyl-1-aminoindan was prepared by palladium-catalyzed reduction of the corresponding oxime.

[0004] 2. The four stereoisomers were separated into cis and trans isomers by column chromatography.

[0005] 3. Trans isomer mixture and 2-methoxyacetic acid methyl ester in Novozyme The reaction is carried out in the presence of an enzyme to form the corresponding acetylated (1R,2S)-2,6-dimethyl-1-aminoindan.

[0006] 4. Separation of acetylated (1R,2S)-2,6-dimethyl-1-aminoindan.

[0007] 5. Acid hydrolysis acetylates (1R,2S)-2,6-dimethyl-1-aminoindan to form free (1R,2S)-2,6-dimethyl-1-aminoindan.

[0008] Tetrahedron 2007, 63 (29), 6755-6763 describes a process for preparing (1R, 2S)-2,6-dimethyl-1-aminoindan, characterized by the following two reaction steps:

[0009] 1. Racemic 1,6-dimethylindan-1-one was reduced diastereoselectively (96:4 d.r.) and enantioselectively (98:2 e.r.) over a chiral ruthenium catalyst to form the corresponding (1S,2S)-2,6-dimethylindan-1-ol in 80% yield.

[0010] 2. The (1S,2S)-2,6-dimethylindan-1-ol thus obtained was reacted with diphenylphosphoryl azide and subsequently reduced with lithium aluminum hydride to give (1R,2S)-2,6-dimethyl-1-aminoindan in a yield of 76%.

[0011] The disadvantages of this method are considered to be not only the use of expensive reagents but also the total reaction time of up to eight days.

[0012] It is an object of the present invention to provide a process for preparing substantially enantiopure (1R,2S)-2,6-dimethyl-1-aminoindan which overcomes the known disadvantages of the prior art.

[0013] We have found a process for preparing substantially enantiopure (1R,2S)-2,6-dimethyl-1-aminoindan, characterized in that a mixture of four stereoisomers of 2,6-dimethyl-1-aminoindan is reacted with an acylating or carboxylating agent in the presence of an enzyme.

[0014] The present invention provides a method for preparing substantially enantiopure (1R,2S)-2,6-dimethyl-1-aminoindan, characterized in that

[0015] 1. In the first step, a mixture of four stereoisomers of 2,6-dimethyl-1-aminoindan (I) is reacted with an acylating or carboxylating agent RC(=O)R in the presence of a protein having lipase activity. 1 Reaction to form the corresponding amide or carbamate (II) and a mixture of stereoisomers of unreacted 2,6-dimethyl-1-aminoindan (III):

[0016]

[0017] wherein the protein is encoded by an amino acid sequence selected from the group consisting of:

[0018] I. A protein having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and most preferably 99% identity with the amino acid sequence shown in SEQ ID No. 1,

[0019] II. A protein having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and most preferably 99% identity to the amino acid sequence shown in SEQ ID No. 1, except that the amino acid sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and most preferably 99% identity to the amino acid sequence shown in SEQ ID No. 1 has a modification selected from the group consisting of:

[0020] i. The amino acid at position 186 is different from L;

[0021] ii. The amino acid at position 280 is different from L;

[0022] iii. The amino acid at position 312 is different from P;

[0023] iv. The amino acid at position 3 is different from M;

[0024] v. The amino acid at position 29 is different from N;

[0025] vi. The amino acid at position 17 is different from L;

[0026] vii. The amino acid at position 4 is different from S;

[0027] viii. The amino acid at position 18 is different from V;

[0028] ix. The amino acid at position 202 is different from A;

[0029] x. The amino acid at position 301 is different from D;

[0030] xi. The amino acid at position 309 is different from P;

[0031] xii. The amino acid at position 31 is different from Q;

[0032] xiii. The amino acid at position 111 is different from Q;

[0033] xiv. The amino acid at position 85 is different from W;

[0034] xv. The amino acid at position 8 is different from K;

[0035] xvi. The amino acid at position 79 is different from E;

[0036] xvii. The amino acid at position 40 is different from K;

[0037] 2. In a second step, the amide or carbamate (II) is separated from the remaining 2,6-dimethyl-1-aminoindan (III) and secondary components by crystallization, and

[0038] 3. In the third step, the amide or carbamate (II) is converted into substantially enantiomerically pure (1R,2S)-2,6-dimethyl-1-aminoindan (IV) using an acid or a base,

[0039]

[0040] wherein R represents a group selected from the group consisting of CH2OCH3, CH2OCH2CH3, CH3, OCH3, OCH2CH3, OCH(CH3)2, OCH2CH2CH2CH3, OCH2CHCH2 and OCH2(C6H5),

[0041] and

[0042] Where R 1 It represents a group selected from the group consisting of OCH3, OCH2CH3, OCH(CH3)2, OCH2CH2CH2CH3, OCH2CHCH2 and OCH2(C6H5).

[0043] The mixture of four stereoisomers of 2,6-dimethyl-1-aminoindan (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.

[0044] Preferably, R represents a group selected from the group consisting of: OCH3 and OCH2CH3,

[0045] and

[0046] R 1 represents a group selected from the group consisting of OCH3 and OCH2CH3.

[0047] In the process according to the invention, proteins having lipolytic enzyme or lipase activity are used.

[0048] SEQ ID No. 1 is an amino acid sequence of a wild-type protein of lipolysis. The wild-type lipase is derived from uncultured bacteria in environmental samples, which are available from GenPept (PDB) under accession number QRD81023 (version number 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 shall prevail.

[0049] Further described are proteins having lipolytic or lipase activity, wherein the amino acid sequences of these proteins are variants of known proteins having lipolytic or lipase activity. In particular, the amino acid sequences of the proteins having lipase activity described herein are variants of the amino acid sequences described by the amino acids set forth in SEQ ID No. 1, wherein in the amino acid sequence set forth in 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 at the corresponding amino acid position in the sequence set forth in SEQ ID No. 1.

[0050] The term "variant" as used herein refers to an entity that is different from an entity known according to the prior art. With respect to nucleic acid molecules and proteins, variants are understood to refer to nucleic acid sequences or amino acid sequences that are different from the corresponding known sequences but encode the same function or catalyze the same reaction (e.g., the function of encoding a protein with lipase activity). The "deviation" of nucleic acid molecule sequences and amino acid sequences from known nucleic acid sequences and protein sequences refers to a sequence that comprises a substitution (replacement) and / or a deletion and / or an insertion of nucleotides or amino acids compared to the corresponding known nucleic acid sequence or amino acid sequence.

[0051] Generally, a protein having lipase activity is used, wherein the protein is encoded by an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and most preferably 99% identity with the amino acid sequence shown in SEQ ID No. 1.

[0052] Also used is a protein having lipase activity, wherein the amino acid sequence is encoded by an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, most preferably 99% identity to the amino acid sequence shown in SEQ ID No. 1, except that the amino acid sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, most preferably 99% identity to the amino acid sequence shown in SEQ ID No. 1 has a modification selected from the group consisting of:

[0053] i. The amino acid at position 186 is different from L, preferably the amino acid at position 186 is F, W, Y, E, D, Q, T, H, P, C, K, S, N, I or V, more preferably the amino acid at position 186 is F, W, Y, E, D or K, particularly preferably the amino acid at position 186 is W or Y, and most preferably the amino acid at position 186 is Y;

[0054] 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;

[0055] 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;

[0056] 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;

[0057] v. The amino acid at position 29 is different from N, preferably the amino acid at position 29 is H, W or Y, more preferably the amino acid at position 29 is H or W, and most preferably the amino acid at position 29 is H;

[0058] 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;

[0059] 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;

[0060] 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;

[0061] 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;

[0062] x. The amino acid at position 301 is different from D, preferably the amino acid at position 301 is A;

[0063] xi. the amino acid at position 309 is different from P, preferably the amino acid at position 309 is C;

[0064] xii. The amino acid at position 31 is different from Q, preferably the amino acid at position 31 is W;

[0065] xiii. The amino acid at position 111 is different from Q, preferably the amino acid at position 111 is E;

[0066] xiv. The amino acid at position 85 is different from W, preferably the amino acid at position 85 is H;

[0067] xv. The amino acid at position 8 is different from K, preferably the amino acid at position 8 is E;

[0068] 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;

[0069] xvii. The amino acid at position 40 is different from K. Preferably, the amino acid at position 40 is M.

[0070] The meanings of the amino acid abbreviations A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y are shown in Table 2 in the "Sequence Description" section below.

[0071] Another embodiment according to the present invention relates to a protein having lipase activity, wherein the protein is selected from the group consisting of:

[0072] a) a protein comprising the amino acid sequence shown in SEQ ID No. 1, except that the amino acid at position 186 is different from L;

[0073] b) a protein having an amino acid sequence that is at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and most preferably 99% identical to the amino acid sequence shown in a), with the proviso that the amino acid at position 186 is different from L.

[0074] Preferably, in the protein according to a) or b), the amino acid at position 186 is preferably 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.

[0075] The “amino acid corresponding to position X” in the first amino acid sequence (e.g., position 3 in SEQ ID No. 1) means herein that if the amino acid numbering of the second amino acid sequence deviates from the amino acid numbering of the first amino acid sequence, then in a pairwise sequence alignment of the first amino acid sequence and the second amino acid sequence, the amino acid of the second amino acid sequence appears at position x in the first amino acid sequence compared to the first amino acid sequence.

[0076] For the purposes of the present invention, the term "identity" in relation to sequence identity or identical sequences is to be understood as referring to the number of identical amino acids or nucleotides shared by a first nucleic acid or amino acid sequence with another (second) nucleic acid or amino acid sequence over the entire length of the sequence, expressed as a percentage.

[0077] "Sequence identity" can be determined by comparing two amino acids or two nucleotide sequences using a global or local alignment algorithm (such as the algorithm included in known software (such as GAP or BESTFIT) or Emboss program "Needle". The software uses the global alignment algorithm of Needleman and Wunsch to compare over the entire length of the two sequences to maximize the number of matches and minimize the number of gaps. In general, using default parameters, gap generation penalty = 10, gap extension penalty = 0.5 (for nucleotide alignment 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). For example, the software (such as EMBOSS) available on the EBI website (ebi.ac.uk / Tools / emboss / ) can be used to determine the score of sequence alignment and percentage sequence identity. Alternatively, sequence similarity or identity can be determined by searching databases (e.g., EMBL, GenBank) using common algorithms and output formats (e.g., FASTA, BLAST, etc.), but for a definitive determination of sequence identity, preferably the hits should be retrieved and aligned pairwise.

[0078] If the sequences to be compared are of different lengths, the identity should be determined by determining the percentage of the number of amino acids or nucleotides shared by the shorter sequence and the longer sequence. Preferably, the identity can be determined by means of the known and public 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, Meyerhofstraβe 1, D 69117 Heidelberg, Germany. ClustalW can also be downloaded from multiple websites, such as IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire, BP163, 67404 Illkirch Cedex, France; ftp: / / ftp-igbmc.u-strasbg.fr / pub / ) and EBI (ftp: / / ftp.ebi.ac.uk / pub / software / ) and mirror sites of EBI (European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK).

[0079] Preferably, the identity between the proteins described in the present invention and other proteins is determined using version 1.8 of the computer program ClustalW. Here, 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.

[0080] Preferably, the 1.8 version of the computer program ClustalW is used to determine the identity between the nucleotide sequence of a nucleic acid molecule, for example, associated with the present invention and the nucleotide sequence of other nucleic acid molecules. Here, the parameters must be set as follows:

[0081] KTUPLE=2, TOPDIAGS=4, PAIRGAP=5, DNAMATRIX:IUB, GAPOPEN=10, GAPEXT=5, MAXDIV=40, TRANSITIONS: unweighted.

[0082] "Identity" also means that there is functional and / or structural equivalence between the nucleic acid molecules involved or the proteins encoded therein. Functional equivalence means that the nucleic acid molecule sequence or amino acid sequence encodes a protein with lipase activity. The nucleic acid molecules homologous to the above-mentioned molecules and being derivatives of these molecules are generally variants of these molecules, and these variants are modifications with the same biological function or catalysis of the same reaction, i.e., encoding proteins with lipase activity. They can be naturally occurring variants, such as sequences from other species, or mutants, wherein the mutation can be abiogenesis or can be introduced by directed mutagenesis. In addition, variants can be synthetically produced sequences. Allelic variants can be naturally occurring variants, synthetically produced variants, or variants produced by recombinant DNA technology. However, for the present invention, it is critical that these variants encode proteins with lipase activity, and include amino acid substitutions (replacements), deletions or insertions of proteins according to the present invention described now.

[0083] A specific type of derivative is a nucleic acid molecule which differs from the nucleic acid molecules described in the present invention, for example due to the degeneracy of the genetic code.

[0084] 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). Hydrolases are a class of enzymes that usually act as biochemical catalysts and use water to break chemical bonds, which usually results in the decomposition of larger molecules into smaller molecules. The class of hydrolases includes enzymes that act on ester bonds (EC 3.1), such as carboxylic ester hydrolases (EC 3.1.1), and lipases (EC 3.1.1.3) as a subgroup thereof. Lipases have been identified from plants, mammals and microorganisms, such as 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.

[0085] If a protein has lipase activity, it can be detected using methods known and described in the prior art. The method used to detect whether a protein according to the present invention has lipase activity is not critical. Preferably, the method is described in the "Examples" section in conjunction with the present invention.

[0086] The lipase variant protein according to the present invention may have further amino acid modifications (amino acid substitutions, deletions or insertions) compared to the amino acid sequence related to the amino acid sequence shown in SEQ ID No. 1 as described herein above.

[0087] The lipase variants described above in point a) related to the amino acid sequence shown in SEQ ID No. 1 may have at least one, two, three, four, five, six or seven further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. In other words, the protein having lipase activity according to the present invention is selected from the group consisting of: a) comprising SEQ ID 1, except that the amino acid at position 186 is different from L and 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) a protein having an amino acid sequence that is at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, most preferably 99% identical to the amino acid sequence directly indicated in point a) above, with the proviso that the amino acid at position 186 is different from L and has at least one further amino acid substitution selected from 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.

[0088] In addition, in addition to further amino acid modifications, the lipase variant protein according to the present invention may have additional amino acid substitutions compared to the amino acid sequence related to the amino acid sequence shown in SEQ ID No.1 described above. These additional amino acid substitutions involve different amino acid sequence positions 79, 202, 280, 301, 3, 11, 17, 40 or 111 related to further amino acid modifications. The lipase variant related to the amino acid sequence shown in SEQ ID No.1 described in point a) above 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.

[0089] Therefore, another embodiment according to the present invention relates to a protein according to the present invention with further amino acid modifications; preferably, these embodiments are proteins with lipase activity, wherein the protein is selected from the group consisting of:

[0090] A protein having the amino acid sequence shown in SEQ ID No. 1, except for the following:

[0091] - the amino acid at position 186 is different from L, and the amino acid at position 79 is different from E, wherein the amino acid at position 186 is preferably W or Y, more preferably Y, and the amino acid at position 79 is preferably S, W or I, more preferably S;

[0092] - the amino acid at position 186 is different from L, and the amino acid at position 202 is different from A, wherein the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N;

[0093] - the amino acid at position 186 is different from L, and the amino acid at position 280 is different from L, wherein the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A;

[0094] - 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;

[0095] - 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;

[0096] - 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;

[0097] - the amino acid at position 186 is different from L, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, and the amino acid at position 17 is preferably P;

[0098] - 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;

[0099] - 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;

[0100] - a protein having an amino acid sequence that is at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, most preferably 99% identical to the amino acid sequence shown in a), with the proviso that the amino acid at position 186 is different from L, wherein preferably the amino acid at position 186 is preferably W or Y, and wherein the protein has at least one further amino acid substitution selected from the group indicated directly below the symbols listed above.

[0101] Preferably, the lipase variants described above in point a) related to the amino acid sequence shown in SEQ ID No. 1 may have at least two further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. Preferably, the amino acid at position 79 is S, W or I, more preferably S; preferably, the amino acid at position 202 is N; preferably, the amino acid at position 280 is A; preferably, the amino acid at position 301 is A; preferably, the amino acid at position 3 is Q; preferably, the amino acid at position 11 is A; preferably, the amino acid at position 17 is P; preferably, the amino acid at position 40 is M; preferably, the amino acid at position 111 is E.

[0102] Therefore, another embodiment according to the present invention relates to a protein according to the present invention having further amino acid modifications; preferably, these embodiments are proteins having lipase activity, wherein the protein is selected from the group consisting of proteins having the amino acid sequence shown in SEQ ID No. 1, with the following exceptions:

[0103] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, and the amino acid at position 202 is different from A, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 202 is preferably N;

[0104] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, and the amino acid at position 280 is different from L, wherein the amino acid at position 186 is preferably W or Y,

[0105] The amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 280 is preferably A;

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

[0107] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 3 is preferably Q;

[0108] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 11 is preferably A;

[0109] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 17 is preferably P;

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

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

[0112] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, and the amino acid at position 280 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, and the amino acid at position 280 is preferably A;

[0113] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, and the amino acid at position 301 is preferably A;

[0114] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, and the amino acid at position 3 is preferably Q;

[0115] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, and the amino acid at position 11 is preferably A;

[0116] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, and the amino acid at position 17 is preferably P;

[0117] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, and the amino acid at position 40 is preferably M;

[0118] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, and the amino acid at position 111 is preferably E;

[0119] - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, and the amino acid at position 301 is preferably A;

[0120] - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, and the amino acid at position 3 is preferably Q;

[0121] - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, and the amino acid at position 11 is preferably A;

[0122] - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, and the amino acid at position 17 is preferably P;

[0123] - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, and the amino acid at position 40 is preferably M;

[0124] - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, and the amino acid at position 111 is preferably E;

[0125] - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, and the amino acid at position 3 is preferably Q;

[0126] - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, and the amino acid at position 11 is preferably A;

[0127] - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, and the amino acid at position 17 is preferably P;

[0128] - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, and the amino acid at position 40 is preferably M;

[0129] - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, and the amino acid at position 111 is preferably E;

[0130] - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, and the amino acid at position 11 is preferably A;

[0131] - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, and the amino acid at position 17 is preferably P;

[0132] - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, and the amino acid at position 40 is preferably M;

[0133] - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, and the amino acid at position 111 is preferably E;

[0134] - the amino acid at position 186 is different from L, the amino acid at position 11 is different from C, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 11 is preferably A, and the amino acid at position 17 is preferably P;

[0135] - the amino acid at position 186 is different from L, the amino acid at position 11 is different from C, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 11 is preferably A, and the amino acid at position 40 is preferably M;

[0136] - the amino acid at position 186 is different from L, the amino acid at position 11 is different from C, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 11 is preferably A, and the amino acid at position 111 is preferably E;

[0137] - the amino acid at position 186 is different from L, the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 17 is preferably P, and the amino acid at position 40 is preferably M;

[0138] - the amino acid at position 186 is different from L, the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 17 is preferably P, and the amino acid at position 111 is preferably E;

[0139] - the amino acid at position 186 is different from L, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E;

[0140] - a protein having an amino acid sequence that is at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, most preferably 99% identical to the amino acid sequence shown in a), with the proviso that the amino acid at position 186 is different from L, wherein the amino acid at position 186 is preferably W or Y, and wherein the protein has at least two further amino acid substitutions selected from the group indicated directly below the symbols listed above.

[0141] Preferably, the lipase variants described above in point a) in relation to the amino acid sequence shown in 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. 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.

[0142] Therefore, another embodiment according to the present invention relates to a protein according to the present invention having further amino acid modifications; preferably, these embodiments are proteins having lipase activity, wherein the protein is selected from the group consisting of proteins having the amino acid sequence shown in SEQ ID No. 1, with the following exceptions:

[0143] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 202 is different from A, and the amino acid at position 280 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 202 is preferably N, and the amino acid at position 280 is preferably A;

[0144] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 202 is different from A, and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 202 is preferably N, and the amino acid at position 301 is preferably A;

[0145] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 202 is different from A, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 202 is preferably N, and the amino acid at position 3 is preferably Q;

[0146] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 202 is different from A, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 202 is preferably N, and the amino acid at position 11 is preferably A;

[0147] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 202 is different from A, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 202 is preferably N, and the amino acid at position 17 is preferably P;

[0148] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 202 is different from A, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 202 is preferably N, and the amino acid at position 40 is preferably M;

[0149] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 202 is different from A, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 202 is preferably N, and the amino acid at position 111 is preferably E;

[0150] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 280 is different from L, and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 280 is preferably A, and the amino acid at position 301 is preferably A;

[0151] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 280 is different from L, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 280 is preferably A, and the amino acid at position 3 is preferably Q;

[0152] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 280 is different from L, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 280 is preferably A, and the amino acid at position 11 is preferably A;

[0153] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 280 is different from L, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 280 is preferably A, and the amino acid at position 17 is preferably P;

[0154] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 280 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 280 is preferably A, and the amino acid at position 40 is preferably M;

[0155] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 280 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 280 is preferably A, and the amino acid at position 111 is preferably E;

[0156] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 301 is different from D, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 301 is preferably A, and the amino acid at position 3 is preferably Q;

[0157] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 301 is different from D, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 301 is preferably A, and the amino acid at position 11 is preferably A;

[0158] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 301 is different from D, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 301 is preferably A, and the amino acid at position 17 is preferably P;

[0159] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 301 is different from D, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, more preferably Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 301 is preferably A, and the amino acid at position 40 is preferably M, wherein it is particularly preferred that the amino acid at position 186 is Y, the amino acid at position 79 is S, the amino acid at position 301 is A, and the amino acid at position 40 is M;

[0160] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 301 is different from D, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 301 is preferably A, and the amino acid at position 111 is preferably E;

[0161] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 3 is different from M, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 3 is preferably Q, and the amino acid at position 11 is preferably A;

[0162] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 3 is different from M, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 3 is preferably Q, and the amino acid at position 17 is preferably P;

[0163] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 3 is different from M, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 3 is preferably Q, and the amino acid at position 40 is preferably M;

[0164] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 3 is different from M, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 3 is preferably Q, and the amino acid at position 111 is preferably E;

[0165] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 11 is different from C, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 11 is preferably A, and the amino acid at position 17 is preferably P;

[0166] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 11 is different from C, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 11 is preferably A, and the amino acid at position 40 is preferably M;

[0167] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 11 is different from C, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 11 is preferably A, and the amino acid at position 111 is preferably E;

[0168] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 17 is preferably P, and the amino acid at position 40 is preferably M;

[0169] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 17 is preferably P, and the amino acid at position 111 is preferably E;

[0170] - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E;

[0171] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 280 is different from L, and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 280 is preferably A, and the amino acid at position 301 is preferably A;

[0172] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 280 is different from L, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 280 is preferably A, and the amino acid at position 3 is preferably Q;

[0173] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 280 is different from L, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 280 is preferably A, and the amino acid at position 11 is preferably A;

[0174] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 280 is different from L, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 280 is preferably A, and the amino acid at position 17 is preferably P;

[0175] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 280 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 280 is preferably A, and the amino acid at position 40 is preferably M;

[0176] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 280 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 280 is preferably A, and the amino acid at position 111 is preferably E;

[0177] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 301 is different from D, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 301 is preferably A, and the amino acid at position 3 is preferably Q;

[0178] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 301 is different from D, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 301 is preferably A, and the amino acid at position 11 is preferably A;

[0179] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 301 is different from D, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 301 is preferably A, and the amino acid at position 17 is preferably P;

[0180] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 301 is different from D, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 301 is preferably A, and the amino acid at position 40 is preferably M;

[0181] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 301 is different from D, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 301 is preferably A, and the amino acid at position 111 is preferably E;

[0182] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 3 is different from M, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 3 is preferably Q, and the amino acid at position 11 is preferably A;

[0183] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 3 is different from M, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 3 is preferably Q, and the amino acid at position 17 is preferably P;

[0184] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 3 is different from M, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 3 is preferably Q, and the amino acid at position 40 is preferably M;

[0185] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 3 is different from M, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 3 is preferably Q, and the amino acid at position 111 is preferably E;

[0186] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 11 is different from C, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 11 is preferably A, and the amino acid at position 17 is preferably P;

[0187] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 11 is different from C, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 11 is preferably A, and the amino acid at position 40 is preferably M;

[0188] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 11 is different from C, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 11 is preferably A, and the amino acid at position 111 is preferably E;

[0189] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 17 is preferably P, and the amino acid at position 40 is preferably M;

[0190] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 17 is preferably P, and the amino acid at position 111 is preferably E;

[0191] - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E;

[0192] - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 301 is preferably A, and the amino acid at position 3 is preferably Q;

[0193] - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 301 is preferably A, and the amino acid at position 11 is preferably A;

[0194] - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 301 is preferably A, and the amino acid at position 17 is preferably P;

[0195] - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 301 is preferably A, and the amino acid at position 40 is preferably M;

[0196] - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 301 is preferably A, and the amino acid at position 111 is preferably E;

[0197] - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 11 is preferably A;

[0198] - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 17 is preferably P;

[0199] - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 40 is preferably M;

[0200] - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 111 is preferably E;

[0201] - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 11 is different from C, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 11 is preferably A, and the amino acid at position 17 is preferably P;

[0202] - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 11 is different from C, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 11 is preferably A, and the amino acid at position 40 is preferably M;

[0203] - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 11 is different from C, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 11 is preferably A, and the amino acid at position 111 is preferably E;

[0204] - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 17 is preferably P, and the amino acid at position 40 is preferably M;

[0205] - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 17 is preferably P, and the amino acid at position 111 is preferably E;

[0206] - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E;

[0207] - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 3 is different from M, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 11 is preferably A;

[0208] - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 3 is different from M, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 17 is preferably P;

[0209] - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 3 is different from M, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 40 is preferably M;

[0210] - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 3 is different from M, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 111 is preferably E;

[0211] - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 11 is different from C, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 11 is preferably A, and the amino acid at position 17 is preferably P;

[0212] - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 11 is different from C, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 11 is preferably A, and the amino acid at position 40 is preferably M;

[0213] - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 11 is different from C, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 11 is preferably A, and the amino acid at position 111 is preferably E;

[0214] - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 17 is preferably P, and the amino acid at position 40 is preferably M;

[0215] - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 17 is preferably P, and the amino acid at position 111 is preferably E;

[0216] - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E;

[0217] - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, the amino acid at position 11 is different from C, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, the amino acid at position 11 is preferably A, and the amino acid at position 17 is preferably P;

[0218] - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, the amino acid at position 11 is different from C, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, the amino acid at position 11 is preferably A, and the amino acid at position 40 is preferably M;

[0219] - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, the amino acid at position 11 is different from C, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, the amino acid at position 11 is preferably A, and the amino acid at position 111 is preferably E;

[0220] - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, the amino acid at position 17 is preferably P, and the amino acid at position 40 is preferably M;

[0221] - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, the amino acid at position 17 is preferably P, and the amino acid at position 111 is preferably E;

[0222] - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E;

[0223] - the amino acid at position 186 is different from L, the amino acid at position 11 is different from C, the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 11 is preferably A, the amino acid at position 17 is preferably P, and the amino acid at position 40 is preferably M;

[0224] - the amino acid at position 186 is different from L, the amino acid at position 11 is different from C, the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 11 is preferably A, the amino acid at position 17 is preferably P, and the amino acid at position 111 is preferably E;

[0225] - the amino acid at position 186 is different from L, the amino acid at position 11 is different from C, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 11 is preferably A, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E;

[0226] - the amino acid at position 186 is different from L, the amino acid at position 17 is different from L, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 17 is preferably P, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E;

[0227] - a protein having an amino acid sequence that is at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, most preferably 99% identical to the amino acid sequence shown in a), with the proviso that the amino acid at position 186 is different from L, wherein the amino acid at position 186 is preferably W or Y, more preferably Y, and wherein the protein has at least three further amino acid substitutions selected from the group indicated directly below the symbols listed above.

[0228] The lipase variants described above in point a) in relation to the amino acid sequence shown in SEQ ID No. 1 may have at least four further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. Preferably, the amino acid at position 79 is S, W or I, more preferably S; preferably, the amino acid at position 202 is N; preferably, the amino acid at position 280 is A; preferably, the amino acid at position 301 is A; preferably, the amino acid at position 3 is Q; preferably, the amino acid at position 11 is A; preferably, the amino acid at position 17 is P; preferably, the amino acid at position 40 is M; preferably, the amino acid at position 111 is E.

[0229] The lipase variants described above in point a) in relation to the amino acid sequence shown in 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.

[0230] The lipase variants described above in point a) in relation to the amino acid sequence shown in 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.

[0231] The lipase variants described above in point a) in relation to the amino acid sequence shown in SEQ ID No. 1 may have at least four further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. Preferably, the amino acid at position 79 is S, W or I, more preferably S; preferably, the amino acid at position 202 is N; preferably, the amino acid at position 280 is A; preferably, the amino acid at position 301 is A; preferably, the amino acid at position 3 is Q; preferably, the amino acid at position 11 is A; preferably, the amino acid at position 17 is P; preferably, the amino acid at position 40 is M; preferably, the amino acid at position 111 is E.

[0232] The lipase variants described above in point a) in relation to the amino acid sequence shown in SEQ ID No. 1 may have at least seven further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. Preferably, the amino acid at position 79 is S, W or I, more preferably S; preferably, the amino acid at position 202 is N; preferably, the amino acid at position 280 is A; preferably, the amino acid at position 301 is A; preferably, the amino acid at position 3 is Q; preferably, the amino acid at position 11 is A; preferably, the amino acid at position 17 is P; preferably, the amino acid at position 40 is M; preferably, the amino acid at position 111 is E.

[0233] According to a preferred embodiment of the present invention is a protein encoding a lipase having SEQ ID No. 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, 120 1, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 31 1, 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, and the amino acid sequence shown in 401.

[0234] Particularly preferred embodiments according to the present invention are proteins encoding lipases having the amino acid sequences shown in EQ ID Nos. 233 and 399.

[0235] Other proteins with lipolytic or lipase activity were tested. The amino acid sequences of these other proteins are variants of the amino acid sequence described by the amino acids in SEQ ID No. 1, wherein:

[0236] - with respect to the amino acid sequence shown in SEQ ID No. 1, the two amino acids at positions 40 and 79 are different from the amino acids shown at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific tested variant related to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 40 is M and the amino acid at position 79 is S;

[0237] - with respect to the amino acid sequence shown in SEQ ID No. 1, the two amino acids at positions 40 and 186 are different from the amino acids shown at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific tested variant related to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 40 is M and the amino acid at position 186 is Y;

[0238] - with respect to the amino acid sequence shown in SEQ ID No. 1, the two amino acids at positions 40 and 301 are different from the amino acids shown at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific tested variant related to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 40 is M and the amino acid at position 301 is A;

[0239] - With respect to the amino acid sequence shown in SEQ ID No. 1, the two amino acids at positions 79 and 186 are different from the amino acids shown at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific tested variant related to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 79 is S and the amino acid at position 186 is Y;

[0240] - with respect to the amino acid sequence shown in SEQ ID No. 1, the two amino acids at positions 79 and 301 are different from the amino acids shown at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific tested variant related to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 79 is S and the amino acid at position 301 is A;

[0241] - With respect to the amino acid sequence shown in SEQ ID No. 1, the two amino acids at positions 186 and 301 are different from the amino acids shown at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific tested variant related to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 186 is Y and the amino acid at position 301 is A.

[0242] Other proteins with lipolytic or lipase activity were also tested. The amino acid sequences of these other proteins are variants of the amino acid sequence described by the amino acids in SEQ ID No. 1, wherein:

[0243] - with respect to the amino acid sequence shown in SEQ ID No. 1, the three amino acids at positions 40, 79 and 186 are different from the amino acids shown at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific tested variant related to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 40 is M, the amino acid at position 79 is S, and the amino acid at position 186 is Y;

[0244] - with respect to the amino acid sequence shown in SEQ ID No. 1, the three amino acids at positions 40, 79 and 301 are different from the amino acids shown at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific tested variant related to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 40 is M, the amino acid at position 79 is S, and the amino acid at position 301 is A;

[0245] - With respect to the amino acid sequence shown in SEQ ID No. 1, the three amino acids at positions 40, 186 and 301 are different from the amino acids shown at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific tested variant related to the amino acid sequence shown in 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.

[0246] The lipase and lipase variant according to the present invention have high selectivity and / or high specificity for stereoselective acylation or carboxylation of 2,6-dimethyl-1-aminoindan (DMAI) and are capable of producing enantiomerically enriched or substantially pure [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamic acid methyl ester. [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamic acid methyl ester is an important intermediate for the synthesis of the herbicidal compound indoxazolidinone.

[0247] As used herein, "enantiomerically enriched" means that the content of one of the two enantiomers in the composition is greater than the other enantiomer, preferably the content of one enantiomer in the composition is at least 60%, more preferably the content of one enantiomer in the composition is at least 65%, even more preferably the content of one enantiomer in the composition is at least 70%, even more preferably the content of one enantiomer in the composition is at least 75%, even more preferably the content of one enantiomer in the composition is at least 80%, particularly preferably the content of one enantiomer in the composition is at least 85%, most preferably the content of one enantiomer in the composition is at least 90%, or very particularly preferably the content of one enantiomer in the composition is at least 94%.

[0248] As used herein, "enantiomerically substantially pure" means that one of the two enantiomers is present in an amount of at least 95.0%, preferably one of the two enantiomers is present in an amount of at least 95.5%, more preferably one of the two enantiomers is present in an amount of at least 96.0%, even more preferably one of the two enantiomers is present in an amount of at least 96.5%, even more preferably one of the two enantiomers is present in an amount of at least 97.0%, even more preferably one of the two enantiomers is present in an amount of at least 98.0%, particularly preferably one of the two enantiomers is present in an amount of at least 98.5%, most preferably one of the two enantiomers is present in an amount of at least 99.0%, or very particularly preferably one of the two enantiomers is present in an amount of at least 99.5%.

[0249] Another embodiment according to the invention relates to a nucleic acid molecule encoding a protein according to the invention.

[0250] The nucleic acid molecule according to the present invention can be any kind of nucleic acid, as long as the nucleic acid encodes the protein according to the present invention. The nucleic acid can be a ribonucleic acid molecule (e.g., RNA, mRNA) or a deoxyribonucleic acid molecule (DNA, including genomic DNA, which may or may not include introns and coding DNA).

[0251] Of particular interest to the present invention are nucleic acid molecules encoding proteins having lipase activity comprising SEQ ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215 , 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 310 11, 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, and the amino acid sequence shown in 401.

[0252] The present invention further relates to a nucleic acid molecule encoding a protein having lipase activity selected from the group consisting of:

[0253] a) comprising SEQ ID No. 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, 1 22, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 2 18, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 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;

[0254] b) a nucleic acid molecule which is at least 60%, preferably 70%, more preferably 80%, even more preferably 90%, even more preferably 95%, even more preferably 96%, particularly preferably 97%, most preferably 98% or very particularly preferably 99% identical to the nucleic acid sequence shown in a).

[0255] For the purposes of the present invention, "hybridization to" refers to hybridization under conventional hybridization conditions, preferably hybridization under stringent conditions, such as the conditions described in Sambrook et al. (Molecular Cloning, A Laboratory Manual, 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ISBN: 0879695773) or Ausubel et al. (Short Protocols in Molecular Biology, John Wiley & Sons; 5th edition (2002), ISBN: 0471250929). Particularly preferably, "hybridization" refers to hybridization under the following conditions:

[0256] Hybridization Buffer:

[0257] 2x SSC; 10x Denhardt's solution (Fikoll 400+PEG+BSA; 1:1:1 ratio); 0.1% SDS; 5mM EDTA; 50mM Na2HPO4; 250μg / ml herring sperm DNA; 50μg / ml tRNA;

[0258] or

[0259] 25M sodium phosphate buffer, pH 7.2; 1mM EDTA; 7% SDS

[0260] Hybridization temperature: T = 65°C to 68°C

[0261] Wash buffer: 0.1x SSC; 0.1% SDS

[0262] Washing temperature: T = 65°C to 68°C.

[0263] The nucleic acid molecules hybridizing to the nucleic acid molecule encoding the protein having lipase activity may originate from any organism; thus, they may be of bacterial, fungal, animal, human, plant or viral origin.

[0264] The nucleic acid molecule that hybridizes with the nucleic acid molecule encoding the protein having lipase activity is preferably derived from a microorganism, more preferably from a fungus or a bacterium, and most preferably from a bacterium.

[0265] Nucleic acid molecules hybridized to the aforementioned molecules can be separated, for example, from genomic DNA or cDNA libraries. These nucleic acid molecules can be identified and separated using nucleic acid molecules as described herein, or can be identified and separated using a portion of these molecules or the reverse complement of these molecules, for example, hybridized according to standard methods (see, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ISBN: 0879695773; Ausubel et al., Short Protocols in Molecular Biology, John Wiley & Sons; 5th edition (2002), ISBN: 0471250929) or amplified by PCR.

[0266] The fragment used as hybridization probe can also be a synthetic fragment, or an oligonucleotide produced using conventional synthesis techniques, whose sequence is substantially identical to the nucleic acid molecules described in the context of the present invention. When identifying and separating genes that hybridize with nucleotide sequences of the present invention, the sequence should be determined and the properties of the protein encoded by the sequence should be analyzed to determine whether it is a protein with lipase activity. Methods for determining whether a protein has the activity of a protein with lipase activity are known to those of ordinary skill in the art.

[0267] Molecules hybridizing with the nucleic acid molecules described in the context of the present invention particularly include fragments, derivatives and allelic variants of the aforementioned nucleic acid molecules. For the purposes of the present invention, the term "derivative" refers to sequences of these molecules that differ from the sequences of the aforementioned nucleic acid molecules at one or more positions and are identical to these sequences to a great extent. The differences associated with the aforementioned nucleic acid molecules may be attributed to, for example, deletions, additions, substitutions, insertions or reorganizations.

[0268] Preferred nucleic acid molecules according to the present invention are SEQ ID No. 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, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216 6, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, The nucleic acid molecules shown in 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.

[0269] The meanings of the nucleotide abbreviations a, c, g, t and the meanings of the degenerate nucleotide abbreviations r, y, s, w, k, m, b, d, h, v, n are shown in Table 1 in the section entitled "Sequence Description" below. The amino acids encoded by the degenerate nucleotides can be found in Table 3 in the section entitled "Sequence Description" below.

[0270] Also disclosed are recombinant nucleic acid molecules comprising a nucleic acid molecule suitable for use in a method according to the invention.

[0271] The term "recombinant nucleic acid molecule" is to be understood as a nucleic acid molecule that not only comprises a nucleic acid molecule suitable for use in the method according to the invention, but also comprises further sequences that are not naturally present in combination, which are present in the combination in the recombinant nucleic acid used in the method according to the invention. The above-mentioned additional sequences can be any sequence; preferably, they are 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.), particularly preferably functional or regulatory sequences that are active in microorganisms, very particularly preferably regulatory sequences that are active in fungi (especially yeast) or bacteria. Methods for producing recombinant nucleic acid molecules suitable for use in the method according to the invention are well known to those of ordinary skill in the art. These methods include genetic methods, such as combining nucleic acid molecules by connection, genetic recombination or de novo synthesis of nucleic acid molecules. These methods are 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 Ausubel et al. (Short Protocols in Molecular Biology, John Wiley & Sons; 5th edition (2002), ISBN: 0471250929).

[0272] Recombinant nucleic acid molecules which can be used in the method according to the invention comprise suitable nucleic acid molecules linked to regulatory sequences which promote transcription in prokaryotic or eukaryotic cells.

[0273] Regulatory sequences that initiate transcription in a cell are also referred to as promoters. Information about 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).

[0274] Regulatory sequences that initiate transcription in prokaryotes (such as Escherichia coli) and eukaryotes are widely described in the literature; those related to expression in yeast (such as Saccharomyces cerevisiae) are particularly described. An overview of different systems for expressing proteins in different host organisms can be found in, for example, Methods in Enzymology 153 (1987), 383–516 and Bitter et al. (Methods in Enzymology 153 (1987), 516–544) or Gomes et al. (2016, Advances in Animal and Veterinary Sciences, 4 (4), 346) and Baghban et al. (2018, Current Pharmaceutical Biotechnology, 19 (6)). Commonly used yeast promoters are pAOX1, pHIS4, pGAL, pScADH2 (Baghban et al., 2018, see above). Commonly used bacterial promoters are T5 promoter, T7 promoter, rhamnose inducible promoter, arabinose inducible promoter, PhoA promoter, artificial trc (trp-lac) promoter, as described in Marschall et al. (2017, Appl Microbiol Biotechnol 101, 501-512) and Tegel et al. (2011, FEBS Journal 278, 729-739).

[0275] Another embodiment of a recombinant nucleic acid molecule which can be used in the method according to the invention is a plasmid vector comprising a suitable nucleic acid molecule.

[0276] "Vector" is well known in the field of molecular biology and is herein a nucleic acid sequence or a vector comprising a nucleic acid sequence used to transfer genetic material (DNA or RNA) to a target cell. A vector may be a plasmid, such as a T-DNA or binary vector for producing transgenic plants, an expression vector for expressing a nucleic acid sequence in a host cell, a shuttle vector capable of propagation in different hosts, or a vector may be a viral particle or bacteriophage modified to deliver foreign genetic material to a host.

[0277] A "plasmid" is well known in the art of molecular biology, and as used herein is an autonomous self-replicating, usually circular DNA molecule that, when present in a host cell, is separate from the chromosomal DNA.

[0278] Suitable nucleic acid molecules, recombinant nucleic acid molecules, vectors or plasmids can be used to produce proteins for use in the methods according to the invention, for example by expressing suitable nucleic acid molecules in host cells.

[0279] Also disclosed are hosts or host cells comprising or expressing a nucleic acid molecule suitable for the method according to the invention, or comprising a suitable protein with lipase activity, 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.

[0280] Suitable nucleic acid molecules encoding proteins with lipase activity can be expressed in host cells, for example, for their propagation or for producing proteins with lipase activity. For expression in host cells, suitable nucleic acid molecules can be present on vectors or plasmids, or stably integrated into the genome of a particular host cell. Suitable nucleic acid molecules can also be present in vectors to facilitate their introduction into host cells.

[0281] 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, each of which comprises 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, each expressing a protein suitable for the method according to the invention, wherein the protein preferably has lipase activity.

[0282] "Expression of a nucleic acid molecule" is 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 with lipase activity, or, if the nucleic acid molecule is DNA or cDNA, it is translated into mRNA (and processed in the case of genomic DNA containing introns), preferably into mRNA encoding a protein with lipase activity, and subsequently translated into a protein, preferably into a protein with lipase activity.

[0283] The transcription of a specific nucleic acid molecule in a host can be detected by methods known to those of ordinary skill in the art, such as detecting specific transcripts (mRNA) of foreign nucleic acid molecules by Northern blot analysis or RT-PCR.

[0284] Whether the host or host cell comprises a specific protein or comprises 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 (radioimmunoassay). Those of ordinary skill in the art are familiar with methods for producing antibodies that specifically react with a specific protein, that is, antibodies that specifically bind to a specific protein (see, for example, Lottspeich and Zorbas (eds.), 1998, Bioanalytik, Spektrum akad, Verlag, Heidelberg, Berlin, ISBN 3-8274-0041-4). Some companies (Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA USA 0245; GenScript, 60 Centennial Ave., Piscataway, NJ 08854, USA) provide custom services for the production of the antibodies.

[0285] Furthermore, a person skilled in the art can check whether a host or a host cell comprises a protein suitable for the method according to the invention by detecting the (additional) activity of a protein having lipase activity in the corresponding host cell. Preferably, the activity of the protein having lipase additional activity in the corresponding host cell is detected by comparing the lipase activity in the host cell used in the method according to the invention with the corresponding activity of a host cell not comprising the protein suitable for the method according to the invention. Methods known from the prior art can be used to check whether a protein has lipase activity.

[0286] The host or host cell used in the method of the present invention can be produced by a person skilled in the art using known biological genetic modification or transformation methods.

[0287] Therefore, a host or host cell suitable for the method of the present invention is also disclosed, in particular a prokaryotic or eukaryotic host or host cell that has been genetically modified (or transformed) using a suitable nucleic acid molecule or using a suitable recombinant nucleic acid molecule or using a suitable vector or using a suitable plasmid. Preferably, the genetically modified (transformed) host or host cell used in the method of the present invention expresses a protein having lipase activity; more preferably, the genetically modified (transformed) host or host cell expresses a protein suitable for the method of the present invention. "Genetic modification using a nucleic acid molecule" or "transformation using a nucleic acid molecule" is understood herein as a nucleic acid molecule being introduced or has been introduced into a host or host cell by a technical and / or non-naturally occurring method, preferably by a technical method in the field of molecular biology, biotechnology or gene technology.

[0288] Also disclosed are progeny or descendants of a host or host cell used in the method of the invention; preferably, the progeny or descendants comprise a nucleic acid molecule suitable for the method of 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, the progeny or descendants 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 they in any case express a protein with lipase activity, even more preferably, the progeny or descendants 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 they in any case express a protein with lipase activity that can be used in the method of the invention.

[0289] The host or host cell used in the method of the invention may be a host or host cell from a prokaryotic or eukaryotic organism. The host or host cell may be a bacterium or bacterial cell (e.g., Escherichia coli, a bacterium of the genus Bacillus, in particular Bacillus subtilis, Agrobacterium, in particular Agrobacterium tumefaciens or Agrobacterium rhizogenes, Pseudomonas, in particular Pseudomonas fluorescens, Streptomyces, Rhodococcus, in particular Rhodococcus rhodochrous, Vibrio natrigens, Corynebacterium, in particular Corynebacterium glutamicum), or a fungus or fungal cell (e.g., Agaricus, in particular Agaricus bisporus). bisporus), Aspergillus, Trichoderma or yeast, in particular S. cerevisiae, Pichia ssp., for example Pichia pastoris), can also be a plant or plant cell, or an animal or animal cell. Preferred host cells are microbial cells. In the context of the present patent application, it is assumed that all bacteria and protists (such as fungi, in particular yeasts and algae), as defined in Schlegel "General Microbiology" (Georg Thieme Verlag (1985), 1-2), are included. Regarding microorganisms, the host or host cell used in the method of the present invention is preferably a bacterium / bacterial cell or a yeast / yeast cell, more preferably a bacterium / bacterial cell, even more preferably a Bacillus / Bacillus cell or an Escherichia coli / E. coli cell, most preferably an E. coli / E. coli cell. Alternatively, Pseudomonas, in particular Pseudomonas fluorescens, Streptomyces, Rhodococcus, in particular Rhodococcus rhodochrous, Vibrio, in particular Vibrio natrigens, Corynebacterium, in particular Corynebacterium glutamicum, or other hosts or host cells may also be used in the method of the invention.

[0290] Preferred hosts or host cells for use in the methods of the invention comprise a suitable nucleic acid molecule, characterized in that the codons of the nucleic acid molecule have been altered so as to adapt them to the frequency of codon usage in the host or host cell.

[0291] Sequence Description

[0292] Throughout this application, abbreviations for nucleotides and amino acids are used according to the following IUPAC codes:

[0293] Table 1

[0294]

[0295]

[0296] To distinguish amino acids from nucleotides, the abbreviated nucleotide codes capitalized in the table above are not capitalized here.

[0297] Table 2

[0298]

[0299]

[0300] The codons used herein follow the so-called "universal genetic code" in the table below, replacing "t" with "u" in ribonucleic acid (RNA) sequences.

[0301] Table 3

[0302]

[0303]

[0304]

[0305] Table 4

[0306] The sequence listing associated with this application is filed in electronic format and is hereby incorporated by reference in its entirety into this patent document."PRT" refers to "protein" and "NUC" refers to "nucleic acid."

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327] The protein with lipase activity is preferably used in an amount of 0.1-50% by weight, based on mixture (I). A preferred amount is 0.5-10% by weight. A particularly preferred amount is 1-5% by weight.

[0328] Step 1 of the process according to the invention can be carried out in the absence of a solvent or in the solvent. Preferred solvents are selected from the group consisting of methyl tert-butyl ether, heptane, toluene, xylene, mesitylene, anisole, chlorobenzene, n-butanol, isopropanol, n-propanol and ethanol and mixtures thereof. Particularly preferred are solvents selected from the group consisting of toluene, xylene, mesitylene, n-butanol and ethanol and mixtures thereof. It is also particularly preferred to carry out the reaction in the absence of a solvent.

[0329] Based on the molar amount of the mixture (I) used, the acylating or carboxylating agent RC(=O)R 1 The amount of is preferably 0.4-25 equivalents. Particularly preferably, the amount of is 0.6-10 equivalents. Particularly preferably, the amount of is 1-5 equivalents.

[0330] The reaction according to step 1 is usually carried out at a temperature 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.

[0331] The separation of component (II) defined in step 2 of the process according to the invention is carried out by crystallization known to those skilled in the art.

[0332] The base defined in step 3 of the method of the present invention is generally selected 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. It is particularly preferred to use a base selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, lithium ethoxide, sodium ethoxide and potassium ethoxide.

[0333] Very particularly preferably, a base selected from the group consisting of lithium hydroxide, sodium hydroxide and potassium hydroxide is used.

[0334] Typically, the base is used in a stoichiometric amount of 1.00-3.00 equivalents, based on the molar 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.

[0335] Preferably, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, tert-butanol and 1-methoxypropan-2-ol, as well as toluene, xylene and veratrole are used as solvents. Particularly preferably, ethanol, n-butanol, isopropanol, 1-methoxypropan-2-ol, toluene, xylene and veratrole are used. Particularly preferably, ethanol, n-butanol and xylene are used.

[0336] The reaction according to step 3 is usually carried out at a temperature 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.

[0337] Another advantage of the method of the present invention is that the mixture of 2,6-dimethyl-1-aminoindan (III) remaining in step 1 and step 2 can be isomerized again under metal catalysis to form a mixture of four stereoisomers of 2,6-dimethyl-1-aminoindan (I) in a resource-saving manner.

[0338] The isomerization can be carried out as follows:

[0339] Preferably, palladium on carbon (Pd / C), palladium on calcium carbonate (Pd / CaCO3) or palladium on aluminum oxide (Pd / Al2O3) with a palladium loading of 1-20% by weight is used as a metal catalyst. It is also preferred to use the Shvo catalyst, whose IUPAC name is 1-hydroxytetraphenylcyclopentadienyl (tetraphenyl-2,4-cyclopentadiene-1-one)-μ-hydrogen tetracarbonyl diruthenium (II), with a stoichiometric amount of 1-10mol%. It is particularly preferred to use a palladium on carbon (Pd / C) or palladium on aluminum oxide (Pd / Al2O3) catalyst with a palladium loading of 1-20% by weight. It is particularly preferred to use a palladium on aluminum oxide (Pd / Al2O3) catalyst with a palladium loading of 1-20% by weight. The amount of the supported catalyst is 0.1-10% by weight based on the compound of formula (III); preferably 0.5-5% by weight is used. The amount of the catalyst used is calculated based on the dry mass of the catalyst.

[0340] Preferably, pyridine, N-methylmorpholine, morpholine, cyclohexylamine, di-n-butylamine, tri-n-butylamine, triethylamine, diisopropylethylamine, piperidine, potassium carbonate, sodium carbonate, lithium carbonate, potassium ethoxide, sodium ethoxide and lithium ethoxide are used as base, or no base is used. Particularly preferably, piperidine, morpholine, diisopropylethylamine, tri-n-butylamine, potassium carbonate and sodium ethoxide are used, or no base is used. Particularly preferably, piperidine and sodium ethoxide are used, or no base is used.

[0341] Preferably, toluene, xylene, mesitylene, anisole, chlorobenzene, n-butanol, isopropanol, n-propanol and ethanol are used as solvents, or the reaction is carried out without solvent. Particularly preferably, toluene, xylene, mesitylene, n-butanol and ethanol are used as solvents, or the reaction is carried out without solvent. Particularly preferably, the reaction is carried out in the absence of a solvent.

[0342] The reaction is preferably carried out at a pressure of 0-20 bar gauge. Particularly preferably, the reaction is carried out at a pressure of 0-10 bar gauge. Particularly preferably, the reaction is carried out at a pressure of 1-6 bar gauge.

[0343] The reaction pressure is substantially achieved by injecting a gas / gas mixture. Preferably, hydrogen, nitrogen or argon or a mixture of hydrogen and nitrogen or hydrogen and argon is injected. Particularly preferably, hydrogen or a mixture of nitrogen and hydrogen is injected. Particularly preferably, pure hydrogen is injected.

[0344] The reaction is preferably operated at a temperature of 80-150° C. Particularly preferably, the reaction is operated at 100-130° C. Particularly preferably, the reaction is operated at 110-125° C.

[0345] Therefore, the present invention further provides a method for producing a mixture of all four stereoisomers of 2,6-dimethyl-1-aminoindan by metal-catalyzed isomerization of a mixture of three stereoisomers of (1R,2R)-, (1S,2R)- and (1S,2S)-2,6-dimethyl-1-aminoindan:

[0346]

[0347] The following examples illustrate the present invention in more detail:

[0348] Terrific Broth (TB) medium was prepared using 47.6 g / l of granular medium and 4 ml / l of glycerol in demineralized water and sterilized at 121°C for 20 minutes.

[0349] Cloning of lipase

[0350] The nucleotide sequences encoding the lipase and lipase variants as described herein can be synthesized according to the methods known in the prior art, for example, provided by relevant service providers, such as Eurofins Genomics GmbH (EurofinsGenomics GmbH, Anzinger Str.7a, 85560 Ebersberg, Germany). In short, as described herein, the nucleic acid sequence of the wild-type lipase (SEQ ID No.2) or the relevant variant is cloned into an expression vector based on the vector pKA81a. The genetic elements are introduced into the modified pKA81a vector by well-known methods. The wild-type lipase and lipase variants are expressed by introducing the expression vector into the resistive Escherichia coli MG1655 cells.

[0351] Generation of enzyme variants

[0352] Nucleotide substitutions (replacements) are introduced into the nucleic acid parent sequence, for example, in order to replace one amino acid with another amino acid. These substitutions can be achieved using a variety of molecular biological methods. According to the present invention, a useful method for generating mutant nucleic acids and corresponding mutant proteins is to perform site-directed mutagenesis on codons encoding one or more amino acids, which codons are pre-selected. Methods for achieving these site-directed mutagenesis are well known to those of ordinary skill in the art and are fully described in the literature (particularly: Directed Mutagenesis: A Practical Approach, 1991, edited by MJ McPHERSON, IRL PRESS), or can be performed using commercial kits (e.g., QUIKCHANGE from Qiagen or Stratagene). TMAfter site-directed mutagenesis, the nucleic acid was transformed into E. coli MG1655 cells.

[0353] The transformed cells are tested in suitable bioconversion reactions to determine product yield and product selectivity. Suitable bioconversion reactions are described below. Sequence verification is performed as known in the prior art.

[0354] Glycerol stocks of E. coli cultures transformed with the corresponding expression plasmids were prepared by adding one volume of 40% glycerol solution to one volume of E. coli culture.

[0355] To isolate single bacterial colonies, appropriately diluted E. coli cultures were plated on LB agar plates containing appropriate concentrations of kanamycin and cultured at 37 °C until single colonies were obtained.

[0356] Synthesis of Ethyl [(1R,2S)-2,6-Dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate

[0357] Embodiment 1:

[0358] In a comparative experiment, the suitability of three different proteins with lipase activity was tested: 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-1-amine (racemic mixture of 82% trans- and 18% cis-2,6-dimethylindan-1-amine) were initially introduced into a 25 mL three-necked flask with a magnetic stirrer under argon and diluted with 13.6 g of diethyl carbonate in each case. The solution was heated to an internal temperature of 110° C. and then 250 mg of the protein with lipase activity was added once. The progress of the reaction was monitored by HPLC measurement. After complete conversion of the (1R,2S)-2,6-dimethylindan-1-amine isomer, the reaction was terminated by adding 5 mL of diethyl carbonate, cooled to 100° C. and the reaction mixture was filtered through glass sand (porosity 3). The filter cake was washed with a further 5 mL of diethyl carbonate. The combined orange filtrate was then distilled at 50° C. and a vacuum of 35-10 mbar. 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 was formed which was filtered through glass sand (porosity 3). The filter cake was washed with 5 mL of n-heptane and then dried under reduced pressure. Then, the mixture was quantitatively concentrated to 100% by volume. 1 The chemical purity of the obtained white solid was determined by H-NMR measurement, and the purity of the stereoisomers was determined by chiral HPLC measurement. The results are shown in Table 5 below.

[0359] Table 5

[0360]

[0361] The yields here are based on the content of pure ethyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate.

[0362] Embodiment 2:

[0363] In a 500 mL four-necked 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-1-amine (a racemic mixture of 82% trans- and 18% cis-2,6-dimethylindan-1-amine) was dissolved in 130.0 g of diethyl carbonate under argon. The solution was heated to an internal temperature of 110° C. with stirring. Thereafter, 1.2 g of the E. coli culture transformed with SEQ ID No. 234 was added in one portion, and the reaction was continued at 110° C. with stirring and argon. After 7 hours of reaction, the conversion of (1R, 2S)-2,6-dimethylindan-1-amine was determined to be substantially complete by HPLC measurement, and the reaction was terminated by cooling to 80° C. and then filtering through glass sand (porosity 3). The filter cake was then washed with 2×40 mL of diethyl carbonate and the combined filtrates were distilled at 50° C. and a vacuum of 35-10 mbar. This removed a total of 157.4 g of distillate from the initial 264.2 g of filtrate. 120 mL of methylcyclohexane were then added to the distillation residue and the resulting mixture was cooled to 2° C. to give a suspension. The solid was removed by filtration through glass sand (porosity 3) and washed with a small amount of methylcyclohexane. After vacuum drying, 37.0 g of a white solid were obtained. Quantitative 1 H-NMR measurements confirmed that the chemical purity of ethyl [(1R, 2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate was 85.4%. The purity of the stereoisomers was determined to be 98.5% by chiral HPLC. This corresponds to a yield of 27% pure ethyl [(1R, 2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate. 1 H-NMR (400MHz; CDCl3) δ=7.08-7.01(m,3H),4.81-4.71(m,2H),4.19(q,J=8.0Hz,2H),3.00(dd,J= 8.0,14.0Hz,1H),2.51-2.45(m,1H),2.33(s,3H),2.16(dt,J=8.0,14.0Hz,1H),1.31-1.26(m,6H).

[0364] Prepared similarly using dimethyl carbonate, and the derived ethyl [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate was obtained as a white solid. 1 H-NMR (400MHz; CDCl3) δ = 7.08-7.01 (m, 3H), 4.79-4.78 (m, 2H), 3.74 (s, 3H), 3.00 (dd, J = 8 .0,16.0Hz,1H),2.51-2.45(m,1H),2.32(s,3H),2.20-2.10(m,1H),1.27(d,J=8.0Hz,3H).

[0365] Embodiment 3:

[0366] In a 25 mL three-necked 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. Thereafter, 10.00 g of 2,6-dimethylindan-1-amine (a racemic mixture of 82% trans and 18% cis-2,6-dimethylindan-1-amine) and 0.25 g of the E. coli culture transformed with SEQ ID No. 234 were added in one portion, and the reaction was stirred continuously at 110° C. for 10 hours under argon until HPLC measurement showed that (1R, 2S)-2,6-dimethylindan-1-amine was substantially completely converted. The reaction mixture was subsequently diluted with another 5 mL of diethyl carbonate and then filtered through glass sand (porosity 3) at 100° C. The filter cake was then washed with 5 mL of diethyl carbonate and the temperature was adjusted to 80° C. The combined filtrate was then distilled at 50°C and a vacuum of 35-10 mbar. Thereafter, 50 mL of n-heptane was added to the distillation residue to form a thick suspension. The suspension was heated to 40°C and stirred at this temperature for 20 minutes. Thereafter, the suspension was cooled to 21°C within 30 minutes. Subsequently, the suspension was further cooled to 2°C and filtered through glass sand (porosity 3). The filter cake was washed with 20 mL of n-heptane, cooled to 5°C, and then dried at 40°C and 10 mbar vacuum. 5.1 g of a white solid was obtained. Quantitative 1 H-NMR measurements confirmed that the chemical purity of ethyl [(1R, 2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate was 99%. The purity of the stereoisomers was determined to be 98.5% by chiral HPLC measurements. This corresponds to a yield of 34% pure ethyl [(1R, 2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate.

[0367] Embodiment 4:

[0368] In a 600 mL autoclave, 100.0 g of 2,6-dimethylindan-1-amine (a racemic mixture of 82% trans- and 18% cis-2,6-dimethylindan-1-amine), 139.2 g of diethyl carbonate and 2 g of biomass were added. Stir at 110°C under argon for 7 hours. After cooling to 80°C, filter through glass frit and wash the filter cake with diethyl carbonate. Concentrate the combined filtrate under reduced pressure at 50°C and crystallize the distillation residue in an ice bath by adding 150 mL of methylcyclohexane. Remove the solid by filtration through glass frit and wash with a small amount of methylcyclohexane. After vacuum drying, 47.6 g of a white solid are obtained. Quantitative 1 H-NMR measurements confirmed that the chemical purity of [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl] ethyl carbamate was 91.9%. The purity of the stereoisomers was determined to be 96.1% by chiral HPLC. This corresponds to a yield of 31.9% pure [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl] ethyl carbamate. The filtrate was concentrated under reduced pressure at 50°C, and the content of 2,6-dimethylindan-1-amine isomers was determined by chiral GC, and the isomer ratio was R,R:S,S:S,R:R,S=6.0:16.2:77.2:0.

[0369] Synthesis of (1R,2S)-2,6-dimethylinden-1-amine from [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]methyl carbamate

[0370] Embodiment 5:

[0371] In a 250 mL jacketed reactor inertized with argon at 21 ° C, 25 g of [(1R, 2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl] methyl carbamate (98% stereoisomer purity, 98% chemical purity) was suspended in 80 mL of n-butanol. The suspension was heated to an internal temperature of 65 ° C to obtain a clear solution. Thereafter, 15 g of potassium hydroxide (85% chemical purity) was added in one go, and the solution was further heated to an internal temperature of 100 ° C. The solution was further stirred at this temperature for 1 hour, while insoluble solids precipitated. HPLC measurements showed complete conversion. Subsequently, a reduced pressure of 360 mbar was applied and 55 mL of solvent was distilled off. Thereafter, 100 mL of p-xylene was added and the reaction mixture was cooled to 20 ° C. The mixture was washed with 1×50 mL of deionized water. The organic phase was then distilled at 40° C. to a reduced pressure of 5 mbar to obtain 17.8 g of (1R,2S)-2,6-dimethylindan-1-amine as a pale yellow oil. 1Its chemical purity was determined to be 95% by H-NMR measurement, corresponding to a yield of 95%. The purity of the stereoisomers was determined to be 98% by chiral GC measurement. 1 H NMR (500MHz, CDCl3): δ7.12(s,1H),7.05(d,J=7.5Hz,1H),6.99(d,J=7.5Hz,1H),3.74(d,J=8.5Hz,1H),2.98(dd, J=7.5,15.5Hz,1H),2.42(dd,J=9.5,15.5Hz,1H),2.34(s,3H),1.98(m,1H),1.72(bs,2H),1.24(d,J=6.5Hz,3H).

[0372] Embodiment 6:

[0373] Synthesis of (1R,2S)-2,6-dimethylinden-1-amine hydrochloride from [(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamic acid methyl ester

[0374] In a 250 mL jacketed reactor inertized with argon at 21 ° C, 50 g of [(1R, 2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl] methyl carbamate (98% stereoisomer purity, 98% chemical purity) was suspended in 160 mL of n-butanol. The suspension was heated to an internal temperature of 65 ° C to obtain a clear solution. Thereafter, 31.1 g of potassium hydroxide (85% chemical purity) was added in one go, and the solution was further heated to an internal temperature of 100 ° C. The solution was further stirred at this temperature for 1 hour, while insoluble solids precipitated. HPLC measurement showed complete conversion. Subsequently, a reduced pressure of 360 mbar was applied and 110 mL of solvent was distilled off. Thereafter, 200 mL of p-xylene was added and the reaction mixture was cooled to 20 ° C. The mixture was washed with 1×100 mL of deionized water. The organic phase was then cooled to 10° C. and admixed with 22.8 g of concentrated hydrochloric acid (37% by weight in water) so that the temperature did not exceed 25° C. 100 mL of solvent were then distilled off from the resulting suspension at 50° C. and the solids were removed by filtration through glass frit (porosity 3) at 20° C. The filter cake was washed with 1×100 mL of toluene and then dried at 40° C. and under reduced pressure of 10 mbar to obtain 40 g of (1R,2S)-2,6-dimethylindan-1-amine hydrochloride in the form of a white solid, which was quantitatively determined by 1 Its chemical purity was determined to be 99% by H-NMR measurements, corresponding to a yield of 95%. The purity of the stereoisomers was determined to be 98% by chiral GC measurements. 1H-NMR (400MHz; DMSO-d6) δ = 8.66 (bs, 3H), 7.45 (s, 1H), 7.14 (dd, J = 8.0, 16.0Hz, 2H), 4.20 ( d,J=8.0Hz,1H),3.18-3.12(m,1H),2.50-2.40(m,1H),2.30(s,3H),1.22(d,J=8.0Hz,3H).

[0375] The 2,6-dimethylindan-1-amine mixture (III) isomerized to form the 2,6-dimethylindan-1-amine mixture (I)

[0376] Embodiment 7:

[0377] In a 25 ml autoclave, 10 g of the obtained 2,6-dimethylindan-1-amine mixture (III) (concentrated filtrate of Example 4) was mixed with 396 mg of a palladium catalyst (5% Pd / Al2O3). The autoclave was purged three times with 5 bar of argon, followed by injection of 3 bar of hydrogen and stirred at 120°C and 3 bar of hydrogen for 10 hours. After cooling to room temperature and venting the autoclave, the content of 2,6-dimethylindan-1-amine isomers in the obtained mixture was determined by chiral GC, and the isomer ratio was R,R:S,S:S,R:R,S=9.2:8.9:43.0:39.0.

[0378] (1R,2S)-2,6-dimethylindan-1-amine isomerizes to 2,6-dimethylindan-1-amine (racemic mixture of 82% trans and 18% cis isomers)

[0379] Embodiment 8-27:

[0380] 1.0 g of (1R,2S)-2,6-dimethylindan-1-amine, 39.6 mg of palladium catalyst (5% Pd / Al2O3), 1.47 g of diethyl carbonate and 10-20 mol% of base were charged into a 6 mL Wheaton screw cap vial. Thereafter, the vial was closed with a screw cap and shaken at 400 rpm for 6 hours at 90-110°C. It was then cooled to 21°C and analyzed by chiral GC analysis and achiral HPLC measurement. The results are listed in the table. The activity defines the isomerization process from an isomer ratio of 0:0:0:100 up to an equilibrium ratio of 9:9:41:41, mainly measured by the degradation process of the 1R,2S isomer from 100% to 41%. Therefore, the activity is determined based on the measured ratio not exceeding 59% degradation. Chemical selectivity describes the extent of the desired components (all DMAI reactant isomers and all DMAI carbamate product isomers) relative to the sum of all components (including secondary components) formed in the isomerization reaction. The relevant data are shown in Table 6 below:

[0381] Table 6

[0382]

[0383]

Claims

1. A process for preparing substantially enantiopure (1R,2S)-2,6-dimethyl-1-aminoindan, characterized in that 1. In the first step, a mixture of four stereoisomers of 2,6-dimethyl-1-aminoindan (I) is reacted with an acylating or carboxylating agent RC(=O)R in the presence of a protein having lipase activity. 1 Reaction to form the corresponding amide or carbamate (II) and a mixture of stereoisomers of unreacted 2,6-dimethyl-1-aminoindan (III): wherein the protein is encoded by an amino acid sequence selected from the group consisting of: I. A protein having at least 80% identity with the amino acid sequence shown in SEQ ID No. 1, II. A protein having at least 80% identity to the amino acid sequence shown in 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. In a second step, the amide or carbamate (II) is separated from the remaining 2,6-dimethyl-1-aminoindan (III) and secondary components by crystallization, and 3. In the third step, the amide or carbamate (II) is converted into substantially enantiomerically pure (1R,2S)-2,6-dimethyl-1-aminoindan (IV) using an acid or a base, wherein R represents a group selected from the group consisting of CH2OCH3, CH2OCH2CH3, CH3, OCH3, OCH2CH3, OCH(CH3)2, OCH2CH2CH2CH3, OCH2CHCH2 and OCH2(C6H5), and Where R 1 It represents a compound selected from OCH3, OCH2CH3, OCH(CH3)2, A group consisting of OCH2CH2CH2CH3, OCH2CHCH2 and OCH2(C6H5).

2. The method according to claim 1, wherein R represents a group selected from the group consisting of OCH3 and OCH2CH3, and R 1 represents a group selected from the group consisting of 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) a protein comprising the amino acid sequence shown in SEQ ID No. 1, except that the amino acid at position 186 is different from L; b) a protein having an amino acid sequence that is at least 80% identical to the amino acid sequence shown in a), with the proviso that the amino acid at position 186 is different from L, wherein according to a) or b) In the protein, the amino acid 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 amino acid substitutions at positions 186, 79, 301 and 40, wherein preferably the amino acid at position 186 is Y, the amino acid at position 79 is S, the amino acid at position 301 is A, and the amino acid at position 40 is M.

6. The method according to any 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. The method according to any 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. The method according to any one of claims 1 to 7, characterized in that The protein is used in an amount of 1-5% by weight based on the mixture (I).

9. The method according to any one of claims 1 to 8, characterized in that Step 1 is performed without a solvent or in the presence of a solvent selected from the group consisting of toluene, xylene, mesitylene and n-butanol.

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

11. The method according to any one of claims 1 to 10, characterized in that In step 1, based on the molar amount of the mixture (I) used, the acylating or carboxylating agent RC(=O)R 1 The usage amount is preferably 1.0-5.0 equivalents.

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

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

14. The method 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 method according to any one of claims 1 to 14, characterized in that In step 3, the base is used in a stoichiometric amount of 1.00 to 3.00 equivalents based on the molar amount of component (II).

16. A process for producing a mixture of all four stereoisomers of 2,6-dimethyl-1-aminoindan by metal-catalyzed isomerization of a mixture of three stereoisomers of (1R,2R)-, (1S,2R)- and (1S,2S)-2,6-dimethyl-1-aminoindan:

17. The method according to claim 16, characterized in that A palladium-supported carbon (Pd / C) or palladium-supported alumina (Pd / Al2O3) catalyst with a palladium loading of 0.5-10 wt % was used.

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

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