Nucleic acids encoding improved lipase proteins

Abstract

The present invention relates to proteins with improved lipase activity, nucleic acid molecules encoding the respective proteins with improved lipase activity, and methods for the stereoselective synthesis of chiral amines or for enriching the chiral amine isomer in an enantiomeric mixture.

Description

[Technical Field]

[0001] The present invention relates to proteins with improved lipase activity, nucleic acid molecules encoding the respective proteins with improved lipase activity, and methods for the stereoselective synthesis of chiral amines or for enriching the chiral amine isomer in an enantiomeric mixture. [Background technology]

[0002] Biocatalysis can be based on naturally available enzymes.More often, the desire to produce a specific product creates a demand for a specific enzyme, which is adapted to enable the economically feasible production of the desired product on a large scale.Enzyme engineering is one option for optimizing enzymes for the economical production of a given product.

[0003] Amines are widely present in nature, not only as part of nucleic acids, but also as tissue hormones (e.g., histamine and serotonin), neurotransmitters (e.g., dopamine and noradrenaline), active pharmaceutical ingredients, or constituents of agricultural products. The absolute configuration of the stereocenters of chiral amines is important for the synthesis of herbicidal active agents. In the production of desired target molecules, generating the correct chirality is often a challenge.

[0004] The use of enzymes, such as lipases, as biocatalysts is of great importance for the production of chiral compounds. Kirchner et al. reported two lipases that act as highly stereoselective and practical catalysts in nearly anhydrous organic solvents. Under these "non-native" conditions, enzymes can asymmetrically catalyze esterification and transesterification reactions that are not feasible in aqueous solutions due to the predominance of hydrolysis. As a result, numerous optically active alcohols, carboxylic acids, and esters have been prepared on a gram scale (Gerald Kirchner, Mark P. Scollar, and Alexander M. Klibanov, J. Am. Chem. Soc. (1985), 107, 7072-7076). Slotema et al. described the development of an economically viable method for the lipase-catalyzed synthesis of amides. They produced oleamide by the direct-catalyzed amidation of oleic acid and ammonia in 2-methyl-2-butanol using Candida antarctica lipase B (Slotema WF, Sandoval G, Guieysse D, Straathof AJ, Marty A. Economically pertinent continuous amide formation by direct lipase-catalyzed amidation with ammonia. Biotechnol Bioeng. (2003) 82(6):664-9. doi:10.1002 / bit.10613. PMID:12673765).Ismail et al. described the enzymatic resolution of two chiral amines, 2-heptylamine and 2-phenylethylamine, by Candida antarctica lipase B. Different acyl donor reagents bearing NH, O, and S moieties were tested (Hilda Ismail, Rute Madeira Lau, Fred van Rantwijk, Roger A. Sheldon; Fully Enzymatic Resolution of Chiral Amines: Acylation and Deacylation in the Presence of Candida antarctica Lipase B; Advanced Synthesis & Catalysis (2008) 350(10):1511-1516). Lipase-catalyzed kinetic resolution of chiral amines using palladium as a racemization catalyst was reported by Reetz and Schimossek. They used a biocatalyst (Candida antarctica lipase) and a transition metal catalyst (palladium) to enable the dynamic kinetic resolution of racemic phenylethylamine. Conversion to enantiomerically pure N-acylated forms has been reported to be 75–77% (ee=99%) (Manfred T. Reetz and Klaus Schimossek; Lipase-Catalyzed Dynamic Kinetic Resolution of Chiral Amines: Use of Palladium as the Racemization Catalyst; Chimia 50 (1996) 668–669). Sun et al. described a lipase-catalyzed reaction for the selective amidation of phenylglycinol. They noted that enzymatic synthesis using Candida antarctica lipase B exhibited high regioselectivity and conversion, providing a promising alternative strategy for the synthesis of aromatic alkanolamides (Sun M., Nie K., Wang F., and Deng L.; Optimization of the Lipase-Catalyzed Selective Amidation of Phenylglycinol. (2020) Front. Bioeng. Biotechnol. 7:486).

[0005] WO 9728271 describes a method for preparing optically active amines, which are useful intermediates for the preparation of pharmaceuticals or plant protection products. In a first step, a suitable racemic amine is reacted with an ester in the presence of a lipase from Candida antarctica and, optionally, in the presence of a diluent. In a second step, the resulting mixture is separated.

[0006] EP 0716712 describes the lipase-catalyzed acylation of diketene with alcohols, particularly to produce enantiomerically selective acylated alcohols from racemic alcohols.

[0007] US Patent No. 5,512,454 discloses a process for the preparation of β-lactam antibiotic intermediates involving the enzymatic acylation of 3-hydroxymethyl cephalosporins.

[0008] U.S. Patent No. 5,902,738 relates to a method for preparing a starting material compound used to prepare the corresponding vitamin A acylate, which comprises the steps of first reacting the compound with an acylating agent in a mixture containing an organic solvent and a lipase present in suspension to form the compound, and second recovering the compound.

[0009] EP 2283144 describes a method for producing sphingolipids by N-acetylation of lysofingolipids using lipase.

[0010] WO 2012146935 discloses modified lipase variants, as well as polynucleotides and recombinant expression vectors encoding the lipase variant polypeptides, and methods for producing such lipase variants in selected bacterial and fungal host cells. Certain lipase variants have increased enzyme specificity or enhanced trans-selectivity. Furthermore, methods for their use to reduce or remove trans-fatty acids from substrates are described.

[0011] Although several improvements in lipases have been achieved so far, limitations arising during the asymmetric synthesis of amines or the resolution of racemic amines, such as unfavorable equilibria, substrate and product inhibition, poor thermostability, poor substrate specificity, and sometimes low enantioselectivity of lipases, still have to be overcome for the efficient production of a wide range of amines on an industrial scale. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] International Publication No. 9728271 Brochure [Patent Document 2] European Patent No. 0716712 [Patent Document 3] U.S. Patent No. 5,512,454 [Patent Document 4] U.S. Patent No. 5,902,738 [Patent Document 5] European Patent No. 2283144 [Patent Document 6] International Publication No. 2012146935 Brochure [Non-patent literature]

[0013] [Non-Patent Document 1] Gerald Kirchner, Mark P. Scollar, and Alexander M. Klibanov, J. Am. Chem. Soc. (1985), 107, 7072-7076 [Non-patent document 2] Slotema WF, Sandoval G, Guieysse D, Straathof AJ, Marty A.Biotechnol Bioeng.(2003)82(6):664-9.doi:10.1002 / bit.10613.PMID:12673765 [Non-patent document 3] Hilda Ismail, Rute Madeira Lau, Fred van Rantwijk, Roger A.Sheldon;Advanced Synthesis & Catalysis (2008)350(10):1511-1516 [Non-patent document 4] Manfred T. Reetz and Klaus Schimossek; Chimia 50 (1996) 668-669 [Non-Patent Document 5] Sun M., Nie K., Wang F. and Deng L.; (2020) Front.Bioeng.Biotechnol.7:486 Summary of the Invention [Problem to be solved by the invention]

[0014] Thus, there is a need for further improvements in lipases, particularly with respect to producing desired acylated or carboxylated, enantiomerically enriched or pure products, preferably under specific and / or economically viable manufacturing processes, and there is a need for further improved lipases. [Means for solving the problem]

[0015] The present invention provides lipase variants containing modifications in their amino acid sequences, which have improved reaction rate, improved substrate acceptance, e.g., improved selectivity, and / or improved specific activity compared to the respective wild-type lipases. Thus, the lipase variants of the present invention enable the development of economically efficient methods for producing new acylated or carboxylated products or precursors of the respective products that cannot be achieved using the respective wild-type lipases.

[0016] The variant lipases described herein have advantages over known wild-type and other previously known lipases. In particular, the modified or variant lipases described herein have the advantage that they can produce enantiomerically enriched, or enantiomerically nearly pure, or pure compounds better than the respective wild-type lipases.

[0017] SEQ ID NO: 1 represents the amino acid sequence of a wild-type lipolytic protein. The wild-type lipase is derived from an uncultivated bacterium derived from an environmental sample from GenPept (PDB) under accession number QRD81023 (version ORD81023.1). In case of ambiguity regarding the amino acid sequence shown in SEQ ID NO: 1 and the sequence shown in the above-mentioned database entry, SEQ ID NO: 1 shall prevail.

[0018] Described herein are proteins having lipolytic enzyme or lipase activity, respectively, wherein the amino acid sequences of these proteins represent variants of known proteins having lipolytic enzyme or lipase activity, respectively. In particular, the amino acid sequences of the proteins having lipase activity described herein represent variants of the amino acid sequence set forth in SEQ ID NO: 1, wherein at least the amino acid at positions 186, 280, 312, 3, 29, 17, 4, 18, 202, 301, 309, 31, 111, 85, 8, 79, or 40 in the amino acid sequence set forth in SEQ ID NO: 1 differs from the amino acid at each amino acid position in the sequence set forth in SEQ ID NO: 1.

[0019] The term "variant" as used herein refers to a subject matter that differs from that known in the art. With respect to nucleic acid molecules and proteins, variants are understood to include nucleic acid or amino acid sequences, respectively, that deviate from the known sequence but encode a protein having the same function or catalyzing the same reaction, e.g., a protein having lipase activity. The "deviation" of nucleic acid molecule sequences and amino acid sequences from known nucleic acid and protein sequences means that the sequences contain nucleotide or amino acid substitutions (replacements) and / or deletions and / or insertions, respectively, compared to the corresponding known nucleic acid or amino acid sequences.

[0020] A first embodiment of the present invention is a protein having the activity of a lipase, wherein said protein is encoded by an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and most preferably 99% identity with the amino acid sequence shown under SEQ ID NO: 1, wherein said amino acid sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and most preferably 99% identity with the amino acid sequence shown under SEQ ID NO: 1 is i. the amino acid at position 186 is different from L, preferably the amino acid at position 186 is F, W, Y, E, D, Q, T, H, P, C, K, S, N, I or V, more preferably the amino acid at position 186 is F, W, Y, E, D or K, particularly preferably the amino acid at position 186 is W or Y, and most preferably the amino acid at position 186 is Y; ii. the amino acid at position 280 is different from L, preferably the amino acid at position 280 is E, S, K, D or A, more preferably the amino acid at position 280 is A; iii. the amino acid at position 312 is different from P, preferably the amino acid at position 312 is N, F, D, Q or K, more preferably the amino acid at position 312 is N; iv. the amino acid at position 3 is different from M, preferably the amino acid at position 3 is L, Q or C, more preferably the amino acid at position 3 is Q; v. the amino acid at position 29 is different from N, preferably the amino acid at position 29 is H, W or Y, more preferably the amino acid at position 29 is H or W, and most preferably the amino acid at position 29 is H; vi. the amino acid at position 17 is different from L, preferably the amino acid at position 17 is P or T, more preferably the amino acid at position 17 is P; vii. the amino acid at position 4 is different from S, preferably the amino acid at position 4 is P or L, more preferably the amino acid at position 4 is P; viii. the amino acid at position 18 is different from V, preferably the amino acid at position 18 is A, T, C, or S, more preferably the amino acid at position 18 is A or C; ix. the amino acid at position 202 is different from A, preferably the amino acid at position 202 is Q or N, more preferably the amino acid at position 202 is N; x. the amino acid at position 301 is different from D, preferably the amino acid at position 301 is A; xi. the amino acid at position 309 is different from P, preferably the amino acid at position 309 is C; xii. the amino acid at position 31 is different from Q, preferably the amino acid at position 31 is W; xiii. the amino acid at position 111 is different from Q, preferably the amino acid at position 111 is E; xiv. the amino acid at position 85 is different from W, preferably the amino acid at position 85 is H; xv. the amino acid at position 8 is different from K, preferably the amino acid at position 8 is E; xvi. the amino acid at position 79 is different from E, preferably the amino acid at position 79 is S, I, or W, more preferably the amino acid at position 79 is S; xvii. the amino acid at position 40 is different from K, preferably the amino acid at position 40 is M; The present invention relates to a method for producing a medicament for the treatment of a pulmonary arthritis, the method comprising the steps of:

[0021] The meanings of the amino acid abbreviations A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y are derived from Table 2 herein under the paragraph subtitled "Sequence Description."

[0022] A further embodiment of the present invention relates to a protein having the activity of a lipase, wherein said protein a) a protein comprising the amino acid sequence shown in SEQ ID NO: 1, except that the amino acid at position 186 differs from L; b) a protein having an amino acid sequence that is at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and most preferably 99% identical to the amino acid sequence shown under a), provided that the amino acid at position 186 is different from L. is selected from the group consisting of:

[0023] Preferably, the amino acid at position 186 is F, W, Y, E, D, Q, T, H, P, C, K, S, N, I, or V. More preferably, the amino acid at position 186 is F, W, Y, E, D, or K. Particularly preferably, the amino acid at position 186 is W or Y. Most preferably, the amino acid at position 186 is Y.

[0024] As used herein, an "amino acid corresponding to position x" in a first amino acid sequence (e.g., position 3 in SEQ ID NO: 1) means that, when the amino acid numbering of a second amino acid sequence differs from the amino acid numbering of the first amino acid sequence, the amino acid of the second amino acid sequence, when compared to the first amino acid sequence, appears at position x in the first amino acid sequence in a pairwise sequence alignment of the first and second amino acid sequences.

[0025] In the context of the present invention, the term "identity" with respect to sequence identity or identical sequences should be understood to mean the number of identical amino acids or nucleotides, respectively, that a first nucleic acid or amino acid sequence shares with another (second) nucleic acid or amino acid sequence over the entire sequence length, expressed as a percentage.

[0026] "Sequence identity" can be determined by aligning two amino acid or two nucleotide sequences, for example, by using a global or local alignment algorithm included in known software such as GAP or BESTFIT or the Emboss program "Needle." This software uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizing the number of gaps. Generally, the default parameters are used: gap creation penalty = 10 and gap extension penalty = 0.5 (for both nucleotide and protein alignment). For nucleotides, the default scoring matrix used is DNAFULL, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 10915-10919). Sequence alignments and percent sequence identity scores can be determined using software such as EMBOSS, accessible at the EBI worldwide website (ebi.ac.uk / Tools / emboss / ). Alternatively, sequence similarity or identity can be determined by searching against databases (e.g., EMBL, GenBank) using commonly known algorithms and output formats such as FASTA, BLAST, etc., although hits should preferably be searched and aligned pairwise to ultimately determine sequence identity.

[0027] When the lengths of the sequences to be compared are different, identity should be determined by determining the percentage of the number of amino acids or nucleotides that the shorter sequence shares with the longer sequence.Preferably, identity is determined using the publicly available computer program ClustalW (Thompson et al., Nucleic Acids Research 22 (1994), 4673-4680).ClustalW is publicly available from Julie Thompson (Thompson@EMBL-Heidelberg.DE) and Toby Gibson (Gibson@EMBL-Heidelberg.DE), European Molecular Biology Laboratory, Meyerhofstrasse 1, D 69117 Heidelberg, Germany. ClustalW can also be downloaded from various internet pages, namely IGBMC (Institut de Genetique et de Biologie Moleculaire et Cellulaire, BP163, 67404 Illkirch Cedex, France; ftp: / / ftp-igbmc.u-strasbg.fr / pub / ) and EBI (ftp: / / ftp.ebi.ac.uk / pub / software / ) and all mirrored internet pages of EBI (European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK).

[0028] Preferably, the ClustalW computer program version 1.8 is used to determine identity between the proteins described in connection with the present invention and other proteins, with the following parameters set: KTUPLE=1, TOPDIAG=5, WINDOW=5, PAIRGAP=3, GAPOPEN=10, GAPEXTEND=0.05, GAPDIST=8, MAXDIV=40, MATRIX=GONNET, ENDGAPS(OFF), NOPGAP, NOHGAP.

[0029] Preferably, the ClustalW computer program, version 1.8, is used to determine identity between, for example, the nucleotide sequences of the nucleic acid molecules described in accordance with the present invention and the nucleotide sequences of other nucleic acid molecules, where the parameters should be set as follows: KTUPLE=2, TOPDIAGS=4, PAIRGAP=5, DNAMATRIX:IUB, GAPOPEN=10, GAPEXT=5, MAXDIV=40, TRANSITIONS: Unweighted.

[0030] "Identity" also refers to functional and / or structural equivalence between the nucleic acid molecules in question or the proteins encoded thereby. Functional equivalence means that the nucleic acid molecule sequence or amino acid sequence encodes a protein with lipase activity. Nucleic acid molecules that are homologous to the above-mentioned molecules and represent derivatives of these molecules are usually variants of these molecules, representing variants that have the same biological function or catalyze the same reaction, i.e., encode proteins with lipase activity. They can be naturally occurring variants, e.g., sequences from other species, or mutations, where these mutations can occur naturally or be introduced by targeted mutagenesis. Furthermore, variants can be synthetically produced sequences. Allelic variants can be naturally occurring or synthetically produced, or variants produced by recombinant DNA technology. However, for the purposes of the present invention, it is crucial that these variants encode proteins with lipase activity and contain the amino acid substitutions, deletions, or insertions described herein for the proteins of the present invention.

[0031] A particular type of derivative is a nucleic acid molecule that differs from the nucleic acid molecules described in accordance with the present invention, for example, as a result of the degeneracy of the genetic code.

[0032] According to the NC-IUBMB (Commission on Nomenclature of the International Union of Biochemistry and Molecular Biology), lipases belong to the class of hydrolases (EC 3). Hydrolases are a class of enzymes that typically act as biochemical catalysts, using water to break chemical bonds, typically resulting in the splitting of larger molecules into smaller ones. The group of hydrolases includes enzymes that act on ester bonds (EC 3.1), including, for example, carboxylic ester hydrolases (EC 3.1.1) and the subgroup lipases (EC 3.1.1.3). Lipases have been identified from plants, mammals, and microorganisms, such as cutinases from Pseudomonas, Vibrio, Acinetobacter, Burkholderia, Chromobacterium, Fusarium solani (FSC), Candida antarctica A (CalA), Rhizopus oryzae (ROL), Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML), Aspergillus Niger, and Fusarium heterosporum. heterosporum, Fusarium oxysporum or Fusarium culmorum.

[0033] If the protein has lipase activity, this can be detected by methods known and described in the art.

[0034] It is not critical which method is used to detect whether a protein according to the invention has lipase activity, but preferably in the context of the present invention the method is described in the "Examples" section.

[0035] 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 described herein above with respect to the amino acid sequence shown under SEQ ID NO: 1.

[0036] In addition to the lipase variants described herein under item a) above with reference to the amino acid sequence shown under SEQ ID NO: 1, they may have at least 1, 2, 3, 4, 5, 6 or 7 further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. In other words, a protein according to the present invention having lipase activity is a protein comprising the amino acid sequence set forth in SEQ ID NO: 1, apart from the fact that the amino acid at position 186 is different from L and that it has at least one, two, three, four, five, six, seven or more further amino acid substitutions selected from the group consisting of: (i) the amino acid at position 79 is different from E; (ii) the amino acid at position 202 is different from A; (iii) the amino acid at position 280 is different from L; (iv) the amino acid at position 301 is different from D; (v) the amino acid at position 3 is different from M; (vi) the amino acid at position 11 is different from C; (vii) the amino acid at position 17 is different from L; (viii) the amino acid at position 40 is different from K; (ix) the amino acid at position 111 is different from Q; and b) The amino acid sequence is selected from the group consisting of proteins having an amino acid sequence that is at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and most preferably 99% identical to the amino acid sequence shown directly under a), provided that the amino acid at position 186 is different from L and has at least one additional amino acid substitution selected from 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; and preferably, the amino acid at position 111 is E.

[0037] Furthermore, the lipase variant proteins according to the present invention can have, apart from the further amino acid modifications, further amino acid substitutions compared to the amino acid sequence described herein above with respect to the amino acid sequence shown under SEQ ID NO: 1. These further amino acid substitutions relate to positions in the amino acid sequence that are different from positions 79, 202, 280, 301, 3, 11, 17, 40, or 111 associated with the further amino acid modifications. The lipase variants described herein above under item a) with reference to the amino acid sequence shown under SEQ ID NO: 1 can have at least one, two, three, four, five, six, seven, or more further amino acid substitutions at positions 4, 8, 18, 29, 31, 42, 84, 85, 192, 217, 309, or 312. The amino acid at position 4 is different from S, preferably the amino acid at that position is P. The amino acid at position 8 is different from K, preferably the amino acid at that position is E. The amino acid at position 18 is different from V, preferably the amino acid at that position is C. The amino acid at position 29 is different from N, preferably the amino acid at that position is W or H. The amino acid at position 31 is different from Q, preferably the amino acid at that position is W. The amino acid at position 42 is different from L, preferably the amino acid at that position is D. The amino acid at position 84 is different from N, preferably the amino acid at that position is T. The amino acid at position 85 is different from W, preferably the amino acid at that position is H. The amino acid at position 192 is different from F, preferably the amino acid at that position is A or V. The amino acid at position 217 is different from Q, preferably the amino acid at that position is M. The amino acid at position 309 is different from P, preferably the amino acid at that position is C. The amino acid at position 312 is different from P, preferably the amino acid at that position is N.

[0038] Therefore, a further embodiment of the invention relates to proteins according to the invention comprising further amino acid modifications; preferably these embodiments are proteins with the activity of a lipase, wherein said protein comprises: - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E, wherein the amino acid at position 186 is preferably W or Y, more preferably Y, and the amino acid at position 79 is preferably S, W or I, more preferably S; - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A, whereby the amino acid at position 186 is preferably W or Y and the amino acid at position 202 is preferably N; - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L, preferably wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 280 is preferably A; - the amino acid at position 186 is different from L and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 301 is preferably A; - the amino acid at position 186 is different from L and the amino acid at position 3 is different from M, whereby the amino acid at position 186 is preferably W or Y and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L and the amino acid at position 11 is different from C, whereby it is preferred that the amino acid at position 186 is preferably W or Y and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L and the amino acid at position 17 is different from L, preferably wherein the amino acid at position 186 is preferably W or Y and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L and the amino acid at position 40 is different from K, whereby the amino acid at position 186 is preferably W or Y and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L and the amino acid at position 111 is different from Q, whereby the amino acid at position 186 is preferably W or Y and the amino acid at position 111 is preferably E; Apart from that, a protein comprising the amino acid sequence set forth in SEQ ID NO: 1, - a protein having an amino acid sequence which is at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and most preferably 99% identical to the amino acid sequence shown under a), with the proviso that the amino acid at position 186 is different from L, wherein the amino acid at position 186 is preferably W or Y, and wherein said protein has at least one further amino acid substitution selected from the group shown under the punctuation listed immediately above. is selected from the group consisting of:

[0039] Preferably, the lipase variants described herein above under item a) referring to the amino acid sequence shown under 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.

[0040] Therefore, a further embodiment of the present invention relates to proteins according to the invention comprising further amino acid modifications, preferably these embodiments are proteins with the activity of a lipase, wherein said protein - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, 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, preferably S, and the amino acid at position 202 is preferably N; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, and the amino acid at position 280 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 280 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 301 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 11 is more preferably A; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, and the amino acid at position 280 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, and the amino acid at position 280 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, and the amino acid at position 301 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A and the amino acid at position 301 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 11 is different from C, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 11 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 11 is different from C, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 11 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 11 is different from C, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 11 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 17 is preferably P, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 17 is preferably P, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E; Apart from that, a protein having the amino acid sequence set forth in SEQ ID NO: 1, - a protein having an amino acid sequence that is at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and most preferably 99% identical to the amino acid sequence shown under a), with the proviso that the amino acid at position 186 is different from L, wherein the amino acid at position 186 is preferably W or Y, and wherein said protein has at least two further amino acid substitutions selected from the group shown under the punctuation listed immediately above here. is selected from the group consisting of:

[0041] Preferably, the lipase variants described herein under item a) referring to the amino acid sequence shown under SEQ ID NO: 1 may have at least three further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. Preferably, the amino acid at position 79 is S, W or I, more preferably S; preferably, the amino acid at position 202 is N; preferably, the amino acid at position 280 is A; preferably, the amino acid at position 301 is A; preferably, the amino acid at position 3 is Q; preferably, the amino acid at position 11 is A; preferably, the amino acid at position 17 is P; preferably, the amino acid at position 40 is M; and preferably, the amino acid at position 111 is E.

[0042] Therefore, a further embodiment of the present invention relates to proteins according to the invention comprising further amino acid modifications, preferably these embodiments are proteins with the activity of a lipase, wherein said protein - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 202 is different from A, and the amino acid at position 280 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 202 is preferably N, and the amino acid at position 280 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 202 is different from A, and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 202 is preferably N, and the amino acid at position 301 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 202 is different from A, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 202 is preferably N, and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 202 is different from A, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 202 is preferably N, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 202 is different from A, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 202 is preferably N, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 202 is different from A, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 202 is preferably N, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 202 is different from A, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 202 is preferably N, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 280 is different from L, and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 280 is preferably A, and the amino acid at position 301 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 280 is different from L, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 280 is preferably A, and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 280 is different from L, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 280 is preferably A, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 280 is different from L, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 280 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 280 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 280 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 280 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 280 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 301 is different from D, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 301 is preferably A, and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 301 is different from D, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 301 is preferably A, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 301 is different from D, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 301 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 301 is different from D, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, more preferably Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 301 is preferably A, and the amino acid at position 40 is preferably M, and 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; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 301 is different from D, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 301 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 3 is different from M, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 3 is preferably Q, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 3 is different from M, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 3 is preferably Q, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 3 is different from M, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 3 is preferably Q, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 3 is different from M, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 3 is preferably Q, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 11 is different from C, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 11 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 11 is different from C, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 11 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 11 is different from C, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 11 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 17 is preferably P, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 17 is preferably P, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 79 is different from E, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 280 is different from L, and the amino acid at position 301 is different from D, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 280 is preferably A, and the amino acid at position 301 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 280 is different from L, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 280 is preferably A, and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 280 is different from L, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 280 is preferably A, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 280 is different from L, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 280 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 280 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 280 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 280 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 280 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 301 is different from D, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 301 is preferably A, and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 301 is different from D, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 301 is preferably A, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 301 is different from D, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is N, the amino acid at position 301 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 301 is different from D, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 301 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 301 is different from D, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 301 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 3 is different from M, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 3 is preferably Q, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 3 is different from M and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 3 is preferably Q and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 3 is different from M, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 3 is preferably Q, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 3 is different from M, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 3 is preferably Q, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 11 is different from C, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 11 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 11 is different from C, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 11 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 11 is different from C, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 11 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 17 is preferably P, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 17 is preferably P, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 202 is different from A, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 3 is different from M, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 301 is preferably A, and the amino acid at position 3 is preferably Q; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 301 is preferably A, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 301 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 301 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 301 is different from D, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 301 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is A, the amino acid at position 3 is preferably Q, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 3 is different from M, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 11 is different from C, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 11 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 11 is different from C, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 11 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 11 is different from C, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 11 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 17 is preferably P, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 17 is preferably P, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 280 is different from L, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 3 is different from M, and the amino acid at position 11 is different from C, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 11 is preferably A; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 3 is different from M, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 3 is different from M, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 3 is different from M, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 11 is different from C, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 11 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 11 is different from C, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 11 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 11 is different from C, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 11 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 17 is preferably P, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 17 is preferably P, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 301 is different from D, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 301 is preferably A, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, the amino acid at position 11 is different from C, and the amino acid at position 17 is different from L, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, the amino acid at position 11 is preferably A, and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, the amino acid at position 11 is different from C, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, the amino acid at position 11 is preferably A, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, the amino acid at position 11 is different from C, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, the amino acid at position 11 is preferably A, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, the amino acid at position 17 is preferably P, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, the amino acid at position 17 is preferably P, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 3 is different from M, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 3 is preferably Q, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 11 is different from C, the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 11 is preferably A, the amino acid at position 17 is preferably P, and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L, the amino acid at position 11 is different from C, the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 11 is preferably A, the amino acid at position 17 is preferably P, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 11 is different from C, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 11 is preferably A, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L, the amino acid at position 17 is different from L, the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein the amino acid at position 186 is preferably W or Y, the amino acid at position 17 is preferably P, the amino acid at position 40 is preferably M, and the amino acid at position 111 is preferably E; Apart from that, a protein comprising the amino acid sequence set forth in SEQ ID NO: 1, - a protein having an amino acid sequence that is at least 80%, preferably 85%, more preferably 90%, even more preferably 95%, even more preferably 96%, even more preferably 97%, particularly preferably 98%, and most preferably 99% identical to the amino acid sequence shown under a), with the proviso that the amino acid at position 186 is different from L, wherein the amino acid at position 186 is preferably W or Y, more preferably Y, and wherein said protein has at least three further amino acid substitutions selected from the group shown under the punctuation listed immediately above here. is selected from the group consisting of:

[0043] The lipase variants described herein above under item a) with reference to the amino acid sequence shown under SEQ ID NO: 1 may have at least four further amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. Preferably, the amino acid at position 79 is S, W or I, more preferably S; preferably, the amino acid at position 202 is N; preferably, the amino acid at position 280 is A; preferably, the amino acid at position 301 is A; preferably, the amino acid at position 3 is Q; preferably, the amino acid at position 11 is A; preferably, the amino acid at position 17 is P; preferably, the amino acid at position 40 is M; and preferably, the amino acid at position 111 is E.

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

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

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

[0047] A preferred embodiment according to the present invention is a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 19, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 31 9, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401.

[0048] Additional proteins with lipolytic enzyme or lipase activity have been tested, the amino acid sequences of which are variants of the amino acid sequence represented by the amino acid sequence of SEQ ID NO: 1, wherein: - in the amino acid sequence shown under SEQ ID NO: 1, the two amino acids at positions 40 and 79 are different from the amino acids shown at the corresponding amino acid positions in the sequence shown under SEQ ID NO: 1. With reference to the amino acid sequence shown under SEQ ID NO: 1, in the specific variant being tested, the amino acid at position 40 is M and the amino acid at position 79 is S; - in the amino acid sequence shown under SEQ ID NO: 1, two amino acids at positions 40 and 186 differ from the amino acids shown at the corresponding amino acid positions in the sequence shown under SEQ ID NO: 1. With reference to the amino acid sequence shown under SEQ ID NO: 1, in the specific variant being tested, the amino acid at position 40 is M and the amino acid at position 186 is Y; - in the amino acid sequence shown under SEQ ID NO: 1, the two amino acids at positions 40 and 301 are different from the amino acids shown at the corresponding amino acid positions in the sequence shown under SEQ ID NO: 1. With reference to the amino acid sequence shown under SEQ ID NO: 1, in the specific variant being tested, the amino acid at position 40 is M and the amino acid at position 301 is A; - in the amino acid sequence shown under SEQ ID NO: 1, the two amino acids at positions 79 and 186 differ from the amino acids shown at the corresponding amino acid positions in the sequence shown under SEQ ID NO: 1. With reference to the amino acid sequence shown under SEQ ID NO: 1, in the specific variant being tested, the amino acid at position 79 is S and the amino acid at position 186 is Y; - in the amino acid sequence shown under SEQ ID NO: 1, the two amino acids at positions 79 and 301 are different from the amino acids shown at the corresponding amino acid positions in the sequence shown under SEQ ID NO: 1. With reference to the amino acid sequence shown under SEQ ID NO: 1, in the specific variant being tested, the amino acid at position 79 is S and the amino acid at position 301 is A; - In the amino acid sequence shown under SEQ ID NO: 1, two amino acids at positions 186 and 301 differ from the amino acids shown at the corresponding amino acid positions in the sequence shown under SEQ ID NO: 1. With reference to the amino acid sequence shown under SEQ ID NO: 1, in the particular variant being tested, the amino acid at position 186 is Y and the amino acid at position 301 is A.

[0049] Additional proteins with lipolytic enzyme or lipase activity have been tested, the amino acid sequences of which are variants of the amino acid sequence represented by the amino acid sequence of SEQ ID NO: 1, wherein: - in the amino acid sequence shown under SEQ ID NO: 1, three amino acids at positions 40, 79 and 186 differ from the amino acids shown at the corresponding amino acid positions in the sequence shown under SEQ ID NO: 1. With reference to the amino acid sequence shown under SEQ ID NO: 1, in the particular variant being tested, the amino acid at position 40 is M, the amino acid at position 79 is S, and the amino acid at position 186 is Y; - in the amino acid sequence shown under SEQ ID NO: 1, the three amino acids at positions 40, 79 and 301 are different from the amino acids shown at the corresponding amino acid positions in the sequence shown under SEQ ID NO: 1. With reference to the amino acid sequence shown in SEQ ID NO: 1, in the particular variant being tested, the amino acid at position 40 is M, the amino acid at position 79 is S, and the amino acid at position 301 is A; - in the amino acid sequence shown under SEQ ID NO: 1, three amino acids at positions 40, 186 and 301 differ from the amino acids shown at the corresponding amino acid positions in the sequence shown under SEQ ID NO: 1. With reference to the amino acid sequence shown under SEQ ID NO: 1, in the specific variant being tested, the amino acid at position 40 is M, the amino acid at position 186 is Y, and the amino acid at position 301 is A; - In the amino acid sequence shown under SEQ ID NO: 1, three amino acids at positions 79, 186 and 301 differ from the amino acids shown at the corresponding amino acid positions in the sequence shown under SEQ ID NO: 1. With reference to the amino acid sequence shown under SEQ ID NO: 1, in the particular variant being tested, the amino acid at position 79 is S, the amino acid at position 186 is Y, and the amino acid at position 301 is A.

[0050] The lipases and lipase variants according to the present invention have high selectivity and / or high specific activity for the stereoselective acylation or carboxylation of 2,6-dimethyl-1-indanamine (DMAI) compared to wild-type lipases and are suitable for producing enantiomerically enriched or substantially pure methyl[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate, which is a key intermediate for the synthesis of the herbicidal compound indaziflam.

[0051] As used herein, "enantiomerically enriched" means that one of the two enantiomers is present in the composition in greater amount than the other enantiomer, preferably at least 60% of one enantiomer is present in the composition, more preferably at least 65% of one enantiomer is present in the composition, even more preferably at least 70% of one enantiomer is present in the composition, even more preferably at least 75% of one enantiomer is present in the composition, even more preferably at least 80% of one enantiomer is present in the composition, particularly preferably at least 85% of one enantiomer is present in the composition, most preferably at least 90% of one enantiomer is present in the composition, or very particularly preferably at least 94% of one enantiomer is present in the composition.

[0052] As used herein, "enantiomerically substantially pure" means that one of the two enantiomers is present in the composition in an amount of at least 95.0%, preferably one of the two enantiomers is present in the composition in an amount of at least 95.5%, more preferably one of the two enantiomers is present in the composition in an amount of at least 96.0%, even more preferably one of the two enantiomers is present in the composition in an amount of at least 96.5%, even more preferably one of the two enantiomers is present in the composition in an amount of at least 97.0%, even more preferably one of the two enantiomers is present in the composition in an amount of at least 98.0%, particularly preferably one of the two enantiomers is present in the composition in an amount of at least 98.5%, most preferably one of the two enantiomers is present in the composition in an amount of at least 99.0%, or very particularly preferably one of the two enantiomers is present in the composition in an amount of at least 99.5%.

[0053] A further embodiment according to the invention relates to a nucleic acid molecule encoding a protein according to the invention.

[0054] The nucleic acid molecule according to the present invention can be any type of nucleic acid, provided that it encodes a protein according to the present invention. The nucleic acid can be a ribonucleic acid molecule (e.g., RNA, mRNA) or a deoxyribonucleic acid molecule (DNA, including genomic DNA, which may or may not contain introns and coding DNA).

[0055] Of particular interest for the present invention are SEQ ID NOs: 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, 311, 313, 315, 317, 319, 321, 322 3, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401.

[0056] The present invention further comprises: a) SEQ ID NOs: 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, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, a nucleic acid molecule comprising a nucleic acid sequence set forth under 16, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402; b) a nucleic acid molecule having at least 60%, preferably 70%, more preferably 80%, even more preferably 90%, even more preferably 95%, even more preferably 96%, particularly preferably 97%, most preferably 98% or especially preferably 99% identity with the nucleic acid sequence shown under a). The present invention relates to a nucleic acid molecule encoding a protein having lipase activity selected from the group consisting of:

[0057] In the context of the present invention, the term "hybridizes to" means hybridization under conventional hybridization conditions, preferably under stringent conditions, such as those described, for example, by Sambrook et al. (Molecular Cloning, A Laboratory Manual, 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ISBN: 0879695773) or Ausubel et al. (Short Protocols in Molecular Biology, John Wiley & Sons, 5th Edition (2002), ISBN: 0471250929). Particularly preferably, "hybridization" means hybridization under the following conditions: Hybridization buffer: 2xSSC; 10x Denhardt's solution (Fikoll 400 + PEG + BSA; ratio 1:1:1); 0.1% SDS; 5mM EDTA; 50mM Na2HPO4; 250μg / ml herring sperm DNA; 50μg / ml tRNA; or 25M sodium phosphate buffer, pH 7.2; 1mM EDTA; 7% SDS Hybridization temperature: T=65~68℃ Wash buffer: 0.1xSSC; 0.1% SDS Washing temperature: T=65~68℃.

[0058] The nucleic acid molecules which hybridize with the nucleic acid molecule encoding a protein having the activity of a lipase may be derived from any organism; therefore, they may be derived from bacteria, fungi, animals, humans, plants or viruses.

[0059] The nucleic acid molecule that hybridizes with the nucleic acid molecule encoding a protein having lipase activity is preferably derived from a microorganism, more preferably from a fungus or bacterium, and most preferably from a bacterium.

[0060] The nucleic acid molecule that hybridizes with the aforementioned molecule can be isolated from, for example, genome or cDNA library.Such nucleic acid molecule can be identified and isolated by using the nucleic acid molecule described herein, or can be identified and isolated by using the part of these molecules or the reverse complement of these molecules, for example, by standard hybridization (for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ISBN: 0879695773; Ausubel et al., Short Protocols in Molecular Biology, John Wiley & Sons; 5th Edition (2002), ISBN: 0471250929) or PCR amplification.

[0061] The fragments used as hybridization samples may also be synthetic fragments or oligonucleotides prepared using conventional synthesis techniques, whose sequences are essentially identical to the nucleic acid molecules described in connection with the present invention. Once genes that hybridize with the nucleic acid sequences described in connection with the present invention are identified and isolated, their sequences should be determined to determine whether they are proteins with lipase activity, and the properties of the proteins encoded by these sequences should be analyzed. Methods for determining whether a protein has the activity of a protein with lipase activity are known to those skilled in the art.

[0062] Molecules that hybridize with the nucleic acid molecules described in the present invention include, in particular, fragments, derivatives, and allelic variants of the aforementioned nucleic acid molecules. In the context of the present invention, the term "derivative" means that the sequences of these molecules differ from the sequences of the above-mentioned nucleic acid molecules at one or more positions and are highly identical to these sequences. Differences from the above-mentioned nucleic acid molecules can be due to, for example, deletions, additions, substitutions, insertions, or recombinations.

[0063] Preferred nucleic acid molecules according to the present invention are those set forth in SEQ ID NOs: 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, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 362, 364, 366, 368, 270, 14, 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.

[0064] The meanings of the nucleotide abbreviations a, c, g, t and their abbreviations r, y, s, w, k, m, b, d, h, v, n for degenerate nucleotides can be derived herein from Table 1 in the section subtitled "Sequence Description" that follows. Which amino acids are encoded by codes containing degenerate nucleotides can be derived herein from Table 3 in the section subtitled "Sequence Description" that follows.

[0065] Furthermore, the present invention relates to recombinant nucleic acid molecules comprising the nucleic acid molecules according to the invention.

[0066] In the context of the present invention, the term "recombinant nucleic acid molecule" is understood to mean a nucleic acid molecule that, in addition to the nucleic acid molecule according to the invention, contains additional sequences that do not naturally occur in the combination that occurs in the recombinant nucleic acid according to the invention. While these additional sequences may be any sequences, they are preferably functional or regulatory sequences (promoters, termination signals, enhancers, ribosome binding sites (rbs), leader sequences that increase transcription, translation, or RNA stability, intracellular targeting sequences, etc.), and are particularly preferably functional or regulatory sequences that are active in microorganisms, and very particularly preferably regulatory sequences that are active in fungi, in particular yeasts, or bacteria. Methods for creating recombinant nucleic acid molecules according to the invention are known to those skilled in the art and include genetic methods such as linking nucleic acid molecules by ligation, genetic recombination, or de novo synthesis of nucleic acid molecules. These methods are described, for example, in Sambrok et al. (Molecular Cloning, A Laboratory Manual, 3rd Edition (2001) Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY. ISBN: 0879695773) or Ausubel et al. (Short Protocols in Molecular Biology, John Wiley & Sons; 5th Edition (2002), ISBN: 0471250929).

[0067] In a further embodiment, a recombinant nucleic acid molecule according to the invention comprises a nucleic acid molecule according to the invention linked to a regulatory sequence that initiates transcription in a prokaryotic or eukaryotic cell.

[0068] The regulatory sequence that initiates transcription in a cell is also known as a promoter.

[0069] Information regarding regulatory sequences and plasmids is well known to those skilled in the art and can be found, for example, in the standard biological parts registry supported by The International Genetically Engineered Machine (iGEM) Foundation (One Kendall Square, Suite B6104, Cambridge, MA 02139, USA) on the World Wide Web (http: / / parts.igem.org / Catalog).

[0070] Regulatory sequences for initiating transcription in prokaryotes such as Escherichia coli and eukaryotes are well documented, particularly in yeasts such as Saccharomyces cerevisiae. Overviews of various systems for protein expression in various host organisms are found, for example, in Methods in Enzymology 153 (1987), 383-516 and Bitter et al. (Methods in Enzymology 153 (1987), 516-544) or Gomes et al. (2016, Advances in Animal and Veterinary Sciences, 4(4), 346) and Baghban et al. (2018, Current Pharmaceutical Biotechnology, 19(6)). Common yeast promoters include pAOX1, pHIS4, pGAL, and pScADH2 (Baghban et al., 2018, see above). Conventional bacterial promoters are the T5, T7, rhamnose-inducible, arabinose-inducible, PhoA, and artificial trc (trp-lac) promoters, as described by Marschall et al. (2017, Appl Microbiol Biotechnol 101, 501-512) and Tegel et al. (2011, FEBS Journal 278, 729-739).

[0071] A further embodiment of a recombinant nucleic acid molecule of the present invention is a vector or plasmid comprising a nucleic acid molecule of the present invention.

[0072] A "vector" is commonly understood in the field of molecular biology, and as used herein, refers to a nucleic acid sequence or a vehicle containing a nucleic acid sequence used to transfer genetic material (DNA or RNA) into a target cell. A vector can be a plasmid, e.g., a T-DNA or binary vector for producing a transgenic plant, an expression vector for expressing a nucleic acid sequence in a host cell, a shuttle vector capable of propagating in different hosts, or a vector can be a virus particle or bacteriophage modified to deliver foreign genetic material into a host.

[0073] "Plasmid" is commonly understood in the field of molecular biology and, as used herein, refers to an autonomously replicating, often circular, DNA molecule that is separate from chromosomal DNA when present in a host cell.

[0074] A nucleic acid molecule according to the invention, a recombinant nucleic acid molecule according to the invention, a vector or a plasmid according to the invention can be used for example to produce a protein according to the invention, by expressing the nucleic acid molecule according to the invention in a host cell.

[0075] Another embodiment of the present invention relates to a host or host cell which comprises or expresses a nucleic acid molecule according to the invention, or which comprises a protein according to the invention, or which comprises a recombinant nucleic acid molecule according to the invention, or which comprises a vector according to the invention, or which comprises a plasmid according to the invention.

[0076] The nucleic acid molecule of the present invention encoding a protein having lipase activity can be expressed in a host cell, for example, for its propagation or for the production of the protein of the present invention. For expression in a host cell, the nucleic acid molecule of the present invention may be contained on a vector or plasmid, or may be stably integrated into the genome of the respective host cell. The nucleic acid molecule of the present invention may be contained in a vector that supports its introduction into the host cell.

[0077] A further embodiment of the present invention relates to a host or host cell of the invention, which comprises a nucleic acid molecule of the invention, or which comprises a recombinant nucleic acid molecule of the invention, or which comprises a vector of the invention, or which comprises a plasmid of the invention, and in any case which comprises a protein of the invention.

[0078] Another embodiment of the present invention relates to a host or host cell of the present invention, which comprises a nucleic acid molecule of the present invention, or which comprises a recombinant nucleic acid molecule of the present invention, or which comprises a vector of the present invention, or which comprises a plasmid of the present invention, and in each case expresses a protein of the present invention.

[0079] Another embodiment of the present invention relates to a host or host cell of the present invention, which comprises a nucleic acid molecule of the present invention, or which comprises a recombinant nucleic acid molecule of the present invention, or which comprises a vector of the present invention, or which comprises a plasmid of the present invention, and in each case expresses a protein, wherein the protein has the activity of a lipase.

[0080] "Expressing a nucleic acid molecule" is understood herein to mean that, if the nucleic acid molecule is RNA or mRNA, the nucleic acid molecule is translated into a protein, preferably translated into a protein having lipase activity, or, if the nucleic acid molecule is DNA or cDNA, is transcribed into mRNA (and processed in the case of genomic DNA containing introns), preferably transcribed into mRNA encoding a protein having lipase activity, which is subsequently translated into a protein, preferably translated into a protein having lipase activity.

[0081] Transcription of a given nucleic acid molecule in the host can be demonstrated by methods known to those skilled in the art, for example, by detection of specific transcripts (mRNA) of the foreign nucleic acid molecule by Northern blot analysis or RT-PCR.

[0082] Whether a host or host cell contains a given protein or a protein derived from the expression of a nucleic acid molecule can be determined by methods known to those skilled in the art, for example, by immunological methods such as Western blot analysis, ELISA (enzyme-linked immunosorbent assay) or RIA (radioimmunoassay).Those skilled in the art are familiar with methods for producing antibodies that specifically react with, i.e., specifically bind to, a specific protein (see, for example, Lottspeich and Zorbas (eds.), 1998, Bioanalytik, Spektrum akad, Verlag, Heidelberg, Berlin, ISBN 3-8274-0041-4).Several companies (Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA USA 0245; GenScript, 60 Centennial Ave., Piscataway, NJ 08854, USA) offer the preparation of such antibodies as a custom service.

[0083] Furthermore, a person skilled in the art can test whether a host or host cell contains a protein according to the invention by detecting the (additional) activity of a protein having lipase activity in the respective host cell. Preferably, the activity of a protein having lipase activity in the respective host cell is detected by comparing the lipase activity of the host cell according to the invention with the respective activity of a host cell not containing the protein according to the invention.

[0084] Testing whether a protein has lipase activity can be performed by methods known in the art.

[0085] A host or host cell according to the invention can be produced by a person skilled in the art by known methods for genetically modifying or transforming organisms.

[0086] A further subject of the present invention is therefore a host or host cell according to the invention, in particular a prokaryotic or eukaryotic host or host cell, which has been genetically modified (or transformed) with a nucleic acid molecule according to the invention or a recombinant nucleic acid molecule according to the invention, or a vector according to the invention or a plasmid according to the invention. Preferably, the genetically modified (transformed) host or host cell according to the invention expresses a protein having lipase activity, more preferably the genetically modified (transformed) host or host cell according to the invention expresses a protein according to the invention.

[0087] "Genetically modified with a nucleic acid molecule" or "transformed with a nucleic acid molecule" should be understood herein to mean that a nucleic acid molecule is introduced or has been introduced into a host or host cell by technical and / or non-natural means, preferably by technical methods in the fields of molecular biology, biotechnology or genetic modification.

[0088] Progeny, offspring or descendants of a host or host cell according to the invention are also an embodiment of the invention, preferably these progeny, offspring or descendants comprise a nucleic acid molecule of the invention, comprise a recombinant nucleic acid molecule of the invention, comprise a vector of the invention, comprise a plasmid of the invention or comprise a protein of the invention, more preferably these progeny, offspring or descendants comprise a nucleic acid molecule of the invention, comprise a recombinant nucleic acid molecule of the invention, comprise a vector of the invention or comprise a plasmid of the invention, and in either case express a protein, which protein has the activity of a lipase, and even more preferably these progeny, offspring or descendants comprise a nucleic acid molecule of the invention, comprise a recombinant nucleic acid molecule of the invention, comprise a vector of the invention or comprise a plasmid of the invention, and in either case express a protein, which protein has the activity of a lipase according to the invention.

[0089] A host or host cell according to the present invention may be of any prokaryotic or eukaryotic origin. The host or host cell may be a bacterium or bacterial cell (e.g., Escherichia coli, in particular a bacterium of the genus Bacillus, in particular Bacillus subtilis, in particular Agrobacterium subtilis, in particular Agrobacterium tumefaciens or Agrobacterium rhizogenes, Pseudomonas, in particular Pseudomonas fluorescens, Streptomyces spp., Rhodococcus spp., in particular Rhodococcus rhodochrous, Vibrio natrigens, natrigens, Corynebacterium, in particular Corynebacterium glutamicum) or fungi or fungal cells (e.g. Agaricus, in particular Agaricus bisporus, Aspergillus, Trichoderma or yeasts, in particular S. cerevisiae, Pichia ssp., for example P. pastoris), as well as plants or plant cells, or they may be animals or animal cells.

[0090] Preferred host cells according to the present invention are cells of microorganisms, which in the context of this patent application are understood to include all bacteria and all protists (e.g., fungi, especially yeasts and algae), as defined, for example, in Schlegel "General Microbiology" (Georg Thieme Publishing House (1985) 1-2).

[0091] With regard to microorganisms, the hosts or host cells according to the invention are preferably bacteria / bacterial cells or yeast / yeast cells, most preferably they are bacteria / bacterial cells. With regard to bacteria / bacterial cells, the hosts or host cells according to the invention are preferably Bacillus sp. / Bacillus sp. cells or E. coli / E. coli cells, most preferably E. coli / E. coli cells.

[0092] Alternatively, Pseudomonas, particularly Pseudomonas fluorescens, Streptomyces, Rhodococcus, particularly Rhodococcus rhodochrous, Vibrio, particularly Vibrio natrigens, Corynebacterium, particularly Corynebacterium glutamicum, or others may be hosts or host cells of the present invention.

[0093] A preferred embodiment of the present invention relates to a host or host cell according to the invention comprising a nucleic acid molecule according to the invention, characterized in that the codons of said nucleic acid molecule are modified to match the codon usage of the host or host cell, respectively.

[0094] Host cells according to the invention can be used for the production of proteins according to the invention. The proteins according to the invention can be used in methods for producing enantiomerically enriched or nearly enantiomerically pure acylated or carboxylated products from acyl or carboxyl acceptors in the presence of acyl or carboxyl donors.

[0095] Therefore, another embodiment of the present invention comprises the steps of: a) providing an acyl or carboxyl acceptor molecule; b) providing an acyl or carboxyl donor molecule; c) contacting the acyl or carboxyl acceptor molecule provided in step a) and the acyl or carboxyl donor molecule provided in step b) with a protein according to the invention; d) optionally obtaining the acylated or carboxylated product. The present invention relates to a method for producing an acylated or carboxylated product, comprising:

[0096] A further embodiment of the present invention is the use of the preparation of a protein according to the invention for the preparation of an amine, preferably for the preparation of a (1R,2S)-amine.

[0097] A further embodiment of the present invention is the use of a protein according to the invention for the stereoselective acylation or carboxylation of racemic 2,6-dimethyl-1-indanylamine.

[0098] A further embodiment of the present invention is the use of a protein according to the invention for the stereoselective acylation or carboxylation of racemic 2,6-dimethyl-1-indanylamine, wherein the substrate is racemic 2,6-dimethyl-1-indanylamine.

[0099] Array Description Throughout this application, abbreviations for nucleotides and amino acids are used according to the following IUPAC code: Table 1 [Table 1] To distinguish between amino acids and nucleotides, the capitalized nucleotide code abbreviations shown in the table above are written in lower case herein.

[0100] Table 2 [Table 2] Codon usage herein is according to the so-called "universal genetic code" according to the table below, with "t" replaced by "u" in ribonucleic acid (RNA) sequences.

[0101] Table 3 [Table 3] TIFF2025527619000004.tif254153TIFF2025527619000005.tif222152Table 4 The Sequence Listing associated with this application has been filed in electronic format and is incorporated herein by reference in its entirety. "PRT" stands for "protein" and "NUC" stands for "nucleic acid." [Table 4] TIFF2025527619000007.tif255166TIFF2025527619000008.tif254165 TIFF2025527619000009.tif253165TIFF2025527619000010.tif255166TIFF202 5527619000011.tif254166TIFF2025527619000012.tif255167TIFF20255276190 00013.tif255166TIFF2025527619000014.tif255165TIFF2025527619000015.t if243166TIFF2025527619000016.tif253166TIFF2025527619000017.tif255166 TIFF2025527619000018.tif255166TIFF2025527619000019.tif255166TIFF2025527619000020.t if255163TIFF2025527619000021.tif249166TIFF2025527619000022.tif254167TIFF2025527619 000023.tif253166TIFF2025527619000024.tif253166TIFF2025527619000025.tif253166TIFF20 25527619000026.tif253167TIFF2025527619000027.tif254166TIFF2025527619000028.tif40166 [Example]

[0102] Terrific Broth Culture (TB) medium was prepared in demineralized water using 47.6 g / L granulation medium and 4 ml / L glycerol and sterilized at 121°C for 20 minutes.

[0103] Example 1 Cloning of lipase mutants Nucleotide sequences encoding the lipases and lipase variants described herein can be synthesized according to methods known in the art, for example, those provided by service providers such as Eurofins Genomics GmbH (Eurofins Genomics GmbH, Anzinger Str. 7a, 85560 Ebersberg, Germany). Briefly, the nucleic acid sequence of the wild-type lipase (SEQ ID NO: 2) or related variants described herein was cloned into an expression vector based on the vector pKA81a. Genetic elements were introduced into the modified pKA81a vector by commonly known methods. The expression vectors were then introduced into electrocompetent E. coli MG1655 cells to express the wild-type lipase and lipase variants, respectively.

[0104] Generation of enzyme mutants Nucleotide substitutions (replacements) were introduced into the parent nucleic acid sequence, for example, to obtain an amino acid exchange with another amino acid. Several molecular biology methods can be used to achieve these substitutions. One useful method for producing mutant nucleic acids and corresponding mutant proteins according to the present invention is site-directed mutagenesis in codons encoding one or more preselected amino acids. Methods for obtaining these site-directed mutations are well known to those skilled in the art and are widely described in the literature (in particular: Directed Mutagenesis: A Practical Approach, 1991, edited by MJ McPHERSON, IRL PRESS) or can be obtained using commercially available kits (e.g., QUIKCHANGE™ lightening Mutagenesis Kit from Qiagen or Stratagene). After site-directed mutagenesis, the nucleic acid was transformed into E. coli MG1655 cells.

[0105] The transformed cells were tested in appropriate biotransformation reactions to determine product yield and selectivity. Suitable biotransformation reactions are described below, see, e.g., Example 2. Sequence verification was performed as known in the art.

[0106] Glycerol stocks of E. coli cultures transformed with each expression plasmid were prepared by adding 1 volume of 40% glycerol solution to 1 volume of E. coli culture.

[0107] To isolate single bacterial colonies, suitable dilutions of E. coli cultures were plated onto LB agar plates containing an appropriate concentration of kanamycin and incubated at 37°C until single colonies were obtained.

[0108] Example 2 Conversion of racemic DMAI to DMAI carbamate using wild-type lipase or its mutants culture For screening purposes, E. coli MG1655 was used as a host for the expression plasmids. For precultures, sterile 2 mL 96-well deep-well plates (product code 0030501306, Eppendorf, Hamburg, Germany) were filled with 590 μL of TB medium (TB, Fisher Bioreagents, product code 22711022) supplemented with 50 μg / mL kanamycin (kanamycin solution, K0254, Sigma-Aldrich, St. Louis, MO, USA) and inoculated with 10 μL of a glycerol stock of each mutant. Alternatively, 590 μL of TB medium was inoculated with cell material from an agar plate colony. Precultures were incubated for 17 h at 37 °C and 250 rpm in a Climoshaker ISF1-X (Kühner AG, Birsfelden, Switzerland).

[0109] For the expression culture, a sterile 2 mL 96-well deep-well plate (0030501306, Eppendorf, Hamburg, Germany) was filled with 510 μL of TB expression medium supplemented with kanamycin (50 mg / L), and the expression culture was inoculated with 30 μL of preculture. The expression culture plate was incubated at 37°C and 250 rpm in a Climoshaker. After 4 hours of incubation, 60 μL of 10 mM IPTG diluted in expression medium (supplemented with 50 mg / L kanamycin) was added per well to induce enzyme expression (to a final concentration of 1 mM IPTG). The expression culture plate was then incubated at 28°C in a shaking incubator for 20 hours.

[0110] Cells were harvested by centrifugation of the expression culture at 4°C and 2500 x g for 15 minutes. The culture supernatant was discarded and the remaining cell pellet was resuspended in 200 μL of PBS (Gibco PBS, Mg 2+ No Ca 2+ The cultures were then resuspended in PBS (without PBS). The resuspended cultures were then freeze-dried for 24 hours. The deep-well plates were stored at 4°C until use.

[0111] Biotransformation The deep-well plate containing the cell suspension was equilibrated to room temperature. The biotransformation reaction was initiated by adding 13.2 μL of racemic 2,6-dimethyl-1-indanylamine (DMAI) and 186.8 μL of dimethyl carbonate to the lyophilized culture and incubated at 70°C or 80°C in a shaking incubator at 250 rpm for 8 to 22 hours. The reaction was stopped by adding 600 μL of acetonitrile. The plate was then centrifuged at 4°C and 2500 × g for 15 minutes. 100 μL of supernatant from each well was transferred to a 96-well plate. The plate was either stored at -20°C or directly subjected to HPLC analysis.

[0112] Analytical HPLC method Analytical HPLC was performed with the following settings: Instrument: Agilent Technologies 1290 Infinity; Column: Poroshell, 100 × 4.6 mm, 2.7 μm; Eluent A: water (+0.001% formic acid); Eluent B: acetonitrile; Flow rate: isocratic (10% eluent A / 90% eluent B); Flow rate: 1.4 mL / min; Temperature: 25 °C; Sample injection volume: 1 μL; Detection: absorbance at 210 nm.

[0113] 1R,2S-DMAI carbamate (dissolved in acetonitrile) was used as a reference substance and as a standard for quantification. Appropriate dilutions were prepared to cover the range up to the maximum concentration used for biotransformation. Samples were analyzed for acylated or carboxylated product isomers, as well as nonacylated and noncarboxylated substrates. The concentration of each compound in the sample was calculated by comparing the resulting peak area with that of the respective compound standard.

[0114] Example 3 Yields of the conversion of racemic 2,6-dimethyl-1-indanylamine (DMAI) to methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate using lipase mutants Cultivation, biotransformation, and HPLC analysis were performed as described in Example 2. Biotransformation was carried out at 70°C for 22 hours. Glycerol stock was used as inoculum for cultivation. The results of the yield of methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate product and the diastereomeric ratio (dr) of methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate are shown in Tables 5 and 6. The relative methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate yield is defined as the methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate yield of the mutant divided by the methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate yield of the wild-type lipase. dr is defined as methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate divided by the sum of methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate, methyl-[(1S,2R)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate, methyl-[(1R,2R)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate and methyl-[(1S,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate. [Table 5] The key to an efficient manufacturing process is the high selectivity of each enzyme variant to produce nearly enantiopure methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate. The wild-type lipase (SEQ ID NO: 1) showed a yield of 0.96 mg / ml of methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate product and a dr of 61.7%.

[0115] Table 5: Relative 1R,2S DMAI carbamate yields for lipase variants with improved 1R,2S DMAI carbamate product yields. [Table 6] TIFF2025527619000032.tif209119Table 6: Lipase variants with improved diastereomeric ratio (dr). [Table 7] TIFF2025527619000034.tif218116 Example 4 Conversion of racemic 2,6-dimethyl-1-indanamine to methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate using a lipase variant based on wild-type lipase. Cultivation, biotransformation, and HPLC analysis were performed as described in Example 2.

[0116] Biotransformation was carried out at 80°C for 8 hours. The glycerol stock was used as inoculum for the cultivation. The yield of the methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate product and the diastereomeric ratio (dr) of methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate are shown in Tables 7 and 8.

[0117] The wild-type lipase (SEQ ID NO: 1) gave a product yield of 2.04 mg / ml and a dr of 64.6%.

[0118] Table 7: Relative methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate yields for lipase variants with improved 1R,2S DMAI carbamate product yields. [Table 8] TIFF2025527619000036.tif255154TIFF2025527619000037.tif255154TIFF2025527619000038.tif255154TIFF2025527619000039.tif255154TIFF2025527619000040.tif71156Table 8: Selection of mutants with improved dr. [Table 9] TIFF2025527619000042.tif254145TIFF2025527619000043.tif255142TIFF2025527619000044.tif251145 TIFF2025527619000045.tif254145TIFF2025527619000046.tif255142TIFF2025527619000047.tif160153

Claims

1. A protein having lipase activity, wherein the protein is encoded by an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in Sequence ID No. 1, wherein the amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in Sequence ID No. 1 is as follows: 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; viiii. The amino acid at position 18 is different from that of V; The amino acid at position 202 is different from that of A; x. The amino acid at position 301 is different from that of D; xi. The amino acid at position 309 is different from P; xi. The amino acid at position 31 is different from Q; xiiii. The amino acid at position 111 is different from Q; xiv. The amino acid at position 85 is different from that of W; xv. The amino acid at position 8 is different from K; xvi. The amino acid at position 79 is different from that of E; xvii. The amino acid at position 40 is different from K. A protein containing modifications selected from the group consisting of the following.

2. The aforementioned protein, a) A protein having the amino acid sequence shown in Sequence ID No. 1, wherein 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), provided that the amino acid at position 186 is different from L. A protein according to claim 1, selected from the group consisting of the following.

3. The protein according to claim 2, wherein the amino acid at position 186 is F, W, Y, E, D, Q, T, H, P, C, K, S, N, I, or V.

4. The aforementioned protein, (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 that of 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; (viiii) The amino acid at position 40 is different from K; (ix) The amino acid at position 111 is different from Q. The protein according to claim 3, having at least one further amino acid substitution selected from the group consisting of the following.

5. The protein according to claim 4, wherein the protein has at least two further amino acid substitutions selected from the group consisting of sections (i), (ii), (iii), (iv), (v), (vi), (vii), (viiii), and (ix) as described in claim 4.

6. The protein according to claim 4, wherein the protein has at least three further amino acid substitutions selected from the group consisting of sections (i), (ii), (iii), (iv), (v), (vi), (vii), (viiii), and (ix) as described in claim 4.

7. Further amino acid substitutions, (i) The amino acid at position 79 is S, W, or I; (ii) The amino acid at position 202 is N; (iii) The amino acid at position 280 is A; (iv) The amino acid at position 301 is A; (v) The amino acid at position 3 is Q; (vi) The amino acid at position 11 is A; (vii) The amino acid at position 17 is P; (viiii) The amino acid at position 40 is M; (ix) The amino acid at position 111 is E. The protein according to any one of claims 4 to 6.

8. The protein according to any one of claims 2 to 6, wherein the protein has amino acid substitutions at the 186th and 79th positions, preferably the amino acid at the 186th position is Y and the amino acid at the 79th position is S.

9. The protein according to any one of claims 2 to 6, wherein the protein has amino acid substitutions at positions 186, 79, 301, and 40, 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.

10. The protein according to any one of claims 1 to 6, wherein the protein has at least one additional amino acid substitution.

11. A nucleic acid molecule encoding a protein according to any one of claims 1 to 6.

12. a) Sequence numbers 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 ,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,164,166,168,170,172,174,176,178,180,182,184,186,188,190,192,194,196,198,200,202,204,206,208,210,212,214,216,218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 3 Nucleic acid molecules containing the nucleic acid sequences shown below: 16, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402; b) The nucleic acid molecule according to claim 11, which encodes a protein having lipase activity selected from the group consisting of nucleic acid molecules having at least 60%, preferably 70%, more preferably 80%, even more preferably 90%, even more preferably 95%, even more preferably 96%, particularly preferably 97%, most preferably 98%, or especially preferably 99% identity with the nucleic acid sequence shown in a).

13. Recombinant nucleic acid molecules comprising the nucleic acid molecule described in claim 11.

14. The recombinant nucleic acid molecule according to claim 13, wherein the recombinant nucleic acid molecule is a vector or a plasmid.

15. A host cell comprising a protein according to any one of claims 1 to 6, a nucleic acid molecule encoding the protein according to any one of claims 1 to 6, or a recombinant nucleic acid molecule containing a nucleic acid molecule encoding the protein according to any one of claims 1 to 6.

16. Use of the protein according to any one of claims 1 to 6 for stereoselective acylation or carboxylation of 2,6-dimethyl-1-indanamine.

17. The use of the protein according to claim 16, wherein the substrate is a racemic 2,6-dimethyl-1-indanylamine.

18. a) A step of providing an acyl- or carboxy-receptor molecule; b) A step of providing an acyl- or carboxyl-donor molecule; c) A step of contacting the acyl- or carboxy-receptor molecule provided in step a) and the acyl- or carboxy-donor molecule provided in step b) with the protein according to the present invention; d) Optionally, a step to obtain an acylation or carboxylation product. A method for producing acylation or carboxylation products, including the product itself.