Nucleic acids encoding improved lipase proteins

Abstract

The present invention relates to proteins having improved lipase activity, nucleic acid molecules encoding the corresponding proteins having improved lipase activity, and methods for the stereoselective synthesis of chiral amines or the increase of chiral amine isomers in enantiomer mixtures.

Description

[0001] The present invention relates to proteins with improved lipase activity, nucleic acid molecules encoding the corresponding proteins with improved lipase activity, and methods for stereoselective synthesis of chiral amines or for increasing chiral amine isomers in enantiomeric mixtures.

[0002] Biocatalysis can be based on enzymes available in nature. More often, the need to produce a specific product creates a demand for a specific enzyme that has been modified to enable large-scale, economically viable production of the desired product. Enzyme engineering is one of the options for optimizing enzymes to achieve economic production of a given product.

[0003] Amines are ubiquitous in nature and are of great importance not only as parts of nucleic acids, but also as building blocks for tissue hormones (e.g. histamine and serotonin), neurotransmitters (e.g. dopamine and norepinephrine), and active pharmaceutical ingredients or agricultural products. The absolute configuration of the stereocenter of a chiral amine is crucial for the synthesis of herbicidal active agents. Producing the correct chirality is often a challenge in the production of the desired target molecule.

[0004] Enzymes, such as lipases, are extremely important as biocatalysts for the production of chiral compounds. Kirchner et al. reported two lipases that are highly stereoselective practical catalysts in almost anhydrous organic solvents. Under such "unnatural" conditions, enzymes can asymmetrically catalyze esterification and transesterification reactions, which are not feasible in aqueous solutions due to the dominant role of hydrolysis. Therefore, many 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. recorded the development of an economically relevant method for the lipase-catalyzed synthesis of amides. They produced oleamide by directly using Candida antarctica lipase B to catalyze the amidation reaction of oleic acid and ammonia in 2-methyl-2-butanol (Slotema WF, Sandoval G, Guieysse D, Straath of 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. recorded the enzymatic resolution of two chiral amines, 2-heptylamine and 2-phenylethylamine by Candida antarctica lipase B, in which different acyl donor reagents with NH, O and S parts 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). Reetz and Schimossek reported a lipase-catalyzed dynamic kinetic resolution of chiral amines using palladium as a racemization catalyst. They used a biocatalyst (Candida antarctica lipase) and a transition metal catalyst (palladium) to make the dynamic kinetic resolution of racemic phenylethylamine possible.It is reported that the conversion rate to the enantiomerically pure N-acylated form is 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. recorded a lipase-catalyzed selective amidation reaction of phenylglycinol. They recorded that the enzymatic synthesis using Antarctic Candida lipase B showed high regioselectivity and conversion rate, and provided 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] WO9728271A1 describes a process for producing optically active amines which are useful intermediates for the manufacture 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, and in a second step, the mixture obtained is separated.

[0006] EP 0716712 B1 describes the lipase-catalyzed acylation of alcohols with diketene, in particular for the production of enantioselective acylated alcohols from racemic alcohols.

[0007] US 5,512,454 discloses a process for preparing β-lactam antibiotic intermediates which involves the enzymatic acylation of 3-hydroxymethylcephalosporin.

[0008] US 5,902,738 relates to a process for producing a starting compound for preparing the corresponding acylated vitamin A. The process comprises the following steps: firstly, reacting a compound with an acylating agent in a mixture comprising an organic solvent and a lipase in suspension to form the compound, and secondly, recovering the compound.

[0009] EP 2283144 B1 describes a process for preparing sphingolipids by N-acetylation of lysophingolipids using lipase.

[0010] WO2012146935A1 discloses modified lipase variants, polynucleotides encoding lipase variant polypeptides and recombinant expression vectors, and methods for producing such lipase variants in selected bacterial and fungal host cells. Specific lipase variants have improved enzyme specificity or enhanced trans selectivity. Further described are methods for reducing or eliminating trans fatty acids from substrates.

[0011] Although some improvements in lipases have been achieved so far, limitations arise in the asymmetric synthesis of amines or the resolution of racemic amines, such as unfavorable equilibrium, substrate and product inhibition, poor thermal stability, insufficient substrate specificity and sometimes low enantioselectivity of lipases, which still need to be overcome in order to efficiently produce a wide range of amines on an industrial scale.

[0012] Therefore, there is a need for further improved lipases, in particular with respect to the production of desired acylated or carboxylated, enantiomerically enriched or pure products, preferably under specific and / or economically viable production methods.

[0013] The present invention provides lipase variants comprising modifications in their amino acid sequences, which lipase variants have improved reaction kinetics, improved substrate acceptance, such as improved selectivity and / or improved specific activity compared to the corresponding wild-type lipase. Therefore, the lipase variants of the present invention can be used in the production method of new acylated or carboxylated products or corresponding product precursors to develop economical and efficient production methods for acylated or carboxylated products, which cannot be achieved by using the corresponding wild-type lipase.

[0014] The variant lipases described herein have advantages over known wild-type and other known lipases. In particular, the advantages of the modified or variant lipases described herein are that they are better than the corresponding wild-type lipases in producing enantiomerically enriched or enantiomerically nearly pure or pure compounds.

[0015] SEQ ID No. 1 represents the amino acid sequence of a wild-type lipolytic protein. The wild-type lipase is from an uncultured bacterium, the environmental sample of which is obtained from GenPept (PDB) with accession number QRD81023 (version ORD81023.1). If there is any ambiguity between the amino acid sequence shown in SEQ ID No. 1 and the sequence shown in the above database entry, SEQ ID No. 1 shall prevail.

[0016] Described herein are proteins having lipolytic or lipase activity, respectively, wherein the amino acid sequences of these proteins represent variants of known proteins having lipolytic or lipase activity, respectively. In particular, the amino acid sequences of the proteins having lipase activity as described herein represent variants of the amino acid sequence represented by the amino acids shown in SEQ ID No. 1, wherein in the amino acid sequence shown in SEQ ID No. 1, at least the amino acid at position 186, the amino acid at position 280, the amino acid at position 312, the amino acid at position 3, the amino acid at position 29, the amino acid at position 17, the amino acid at position 4, the amino acid at position 18, the amino acid at position 202, the amino acid at position 301, the amino acid at position 309, the amino acid at position 31, the amino acid at position 111, the amino acid at position 85, the amino acid at position 8, the amino acid at position 79 or the amino acid at position 40 are different from the amino acids given at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1.

[0017] The term "variant" as used herein refers to a subject that is different from a subject known in the prior art. With respect to nucleic acid molecules and proteins, variants are understood to include a nucleic acid sequence or an amino acid sequence that deviates from the corresponding known sequence, but still encodes a protein having the same function or catalyzing the same reaction, such as the function of encoding a protein having lipase activity. The "deviation" of nucleic acid molecule sequences and amino acid sequences from known nucleic acid sequences and protein sequences means that these sequences, compared with the corresponding known nucleic acid sequence or amino acid sequence, respectively include substitutions (alternatives) and / or deletions and / or insertions of nucleotides or amino acids.

[0018] A first embodiment of the present invention relates to a protein having lipase activity, wherein the protein is encoded by an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, further more preferably 95%, even more preferably 96%, even further more preferably 97%, particularly preferably 98%, and most preferably 99% identity with the amino acid sequence shown in SEQ ID No. 1, in addition to the amino acid sequence having at least 80%, preferably 85%, more preferably 90%, further more preferably 95%, even more preferably 96%, even further more preferably 97%, particularly preferably 98%, and most preferably 99% sequence identity with the amino acid sequence shown in SEQ ID No. 1, further comprising modifications selected from the following:

[0019] i. The amino acid at position 186 is different from L. Preferably, the amino acid at position 186 is F, W, Y, E, D, Q, T, H, P, C, K, S, N, I or V. More preferably, the amino acid at position 186 is F, W, Y, E, D or K. Particularly preferably, the amino acid at position 186 is W or Y. Most preferably, the amino acid at position 186 is Y.

[0020] 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;

[0021] 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;

[0022] 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;

[0023] v. the amino acid at position 29 is different from N, preferably, the amino acid at position 29 is H, W or Y, more preferably, the amino acid at position 29 is H or W, most preferably, the amino acid at position 29 is H;

[0024] 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;

[0025] 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;

[0026] 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;

[0027] 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;

[0028] x. The amino acid at position 301 is different from D, preferably, the amino acid at position 301 is A;

[0029] xi. the amino acid at position 309 is different from P, preferably, the amino acid at position 309 is C;

[0030] xii. The amino acid at position 31 is different from Q, preferably, the amino acid at position 31 is W;

[0031] xiii. The amino acid at position 111 is different from Q, preferably, the amino acid at position 111 is E;

[0032] xiv. The amino acid at position 85 is different from W, preferably, the amino acid at position 85 is H;

[0033] xv. The amino acid at position 8 is different from K, preferably, the amino acid at position 8 is E;

[0034] 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;

[0035] xvii. The amino acid at position 40 is different from K. Preferably, the amino acid at position 40 is M.

[0036] The meanings of the amino acid abbreviations A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y can be derived from Table 2 below, under the subheading "Description of the sequences".

[0037] Another embodiment of the present invention relates to a protein having lipase activity, wherein the protein is selected from the group consisting of:

[0038] a) a protein comprising the amino acid sequence as shown in SEQ ID No. 1, except that the amino acid at position 186 is different from L;

[0039] b) a protein having an amino acid sequence that is at least 80%, preferably 85%, more preferably 90%, further more preferably 95%, even more preferably 96%, even further more preferably 97%, particularly preferably 98%, and most preferably 99% identical to the amino acid sequence shown in a), with the proviso that the amino acid at position 186 is different from L.

[0040] 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.

[0041] The “amino acid corresponding to position x” in a first amino acid sequence (e.g., position 3 in SEQ ID No. 1) means herein that, when the second amino acid sequence is aligned pairwise with the first amino acid sequence, if the amino acid numbering of the second amino acid sequence is different from the amino acid numbering of the first amino acid sequence, then an amino acid in the second amino acid sequence occurs at position x of the first amino acid sequence (when compared to the first amino acid sequence).

[0042] In the context of the present invention, the term "identity" with respect to sequence identity or sequence homology is to be understood as referring to the number of identical amino acids or nucleotides shared by a first nucleic acid or amino acid sequence, respectively, with another (second) nucleic acid or amino acid sequence over the entire sequence length, expressed as a percentage.

[0043] "Sequence identity" can be determined by comparing two amino acids or two nucleotide sequences using a global or local alignment algorithm, which is included in known software such as GAP or BESTFIT, or in the Emboss program "Needle". These software use the Needleman and Wunsch global alignment algorithms to compare two sequences over their entire length, maximizing the number of matches and minimizing the number of gaps. Typically, default parameters are used, wherein gap creation penalty=10, gap extension penalty=0.5 (both applicable to nucleotide and protein comparisons). For nucleotides, the default scoring matrix used is DNAFULL, and for proteins, the default scoring matrix used is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 10915-10919). Sequence alignment and sequence identity percentage scores can be determined, for example, using software (such as EMBOSS), which can be accessed by the World Wide Web site of EBI (ebi.ac.uk / Tools / emboss / ). Alternatively, sequence similarity or identity can be determined by searching against databases (e.g., EMBL, GenBank) using generally known algorithms and output formats, such as FASTA, BLAST, etc., but preferably the hits are retrieved and aligned pairwise to ultimately determine sequence identity.

[0044] If the sequences to be compared are of different lengths, identity is determined by determining the percentage of the number of amino acids or nucleotides shared by the shorter sequence and the longer sequence, respectively. Preferably, identity is determined using the known and publicly available computer program ClustalW (Thompson et al., Nucleic Acids Research 22 (1994), 4673-4680). ClustalW is publicly available from Julie Thompson (Thompson@EMBL-Heidelberg.DE) and Toby Gibson (Gibson@EMBL-Heidelberg.DE) European Molecular Biology Laboratory, Meyerhofstrasse 1, D69117 Heidelberg, Germany. ClustalW can also be downloaded from various Internet pages, in particular from IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire, BP163, 67404 Illkirch Cedex, France; ftp: / / ftp-igbmc.u-strasbg.fr / pub / ) and EBI (ftp: / / ftp.ebi.ac.uk / pub / software / ) and all mirror Internet pages of EBI (European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB101SD, UK).

[0045] Preferably, the identity between the proteins described in the context of the present invention and other proteins is determined using the ClustalW computer program version 1.8. Here, the parameter settings should be as follows: KTUPLE=1, TOPDIAG=5, WINDOW=5, PAIRGAP=3, GAPOPEN=10, GAPEXTEND=0.05, GAPDIST=8, MAXDIV=40, MATRIX=GONNET, ENDGAPS(OFF), NOPGAP, NOHGAP.

[0046] Preferably, the ClustalW computer program of version 1.8 is used to determine the identity between the nucleotide sequence of the nucleic acid molecule recorded in the context of the present invention and the nucleotide sequence of other nucleic acid molecules. Here, the parameter settings should be as follows:

[0047] KTUPLE=2, TOPDIAGS=4, PAIRGAP=5, DNAMATRIX:IUB, GAPOPEN=10, GAPEXT=5, MAXDIV=40, TRANSITIONS:unweighted.

[0048] "Identity" further refers to the existence of functional and / or structural equivalence between the nucleic acid molecules in question or the proteins encoded by them. Functional equivalence refers to a protein with lipase activity encoded by a nucleic acid molecule sequence or an amino acid sequence. Nucleic acid molecules that are homologous to the above-mentioned molecules and represent derivatives of these molecules are generally variants of these molecules, and these variants represent modifications with the same biological function or catalyze the same reaction, i.e., encode a protein with lipase activity. They can be naturally occurring variants, such as sequences from other species, or mutations, wherein these mutations can occur in a natural manner or be introduced by targeted mutations. In addition, these variants can also be synthetically produced sequences. Allelic variants can be naturally occurring variants or synthetically produced variants or variants produced by recombinant DNA technology. However, for the present invention, it is decisive that these variants encode proteins with lipase activity and contain amino acid substitutions (substitutions), deletions or insertions related to proteins of the present invention as described herein.

[0049] A special type of derivative is a nucleic acid molecule which differs from the nucleic acid molecules described herein above and below, for example, due to the degeneracy of the genetic code.

[0050] According to the NC-IUBMB (Nomenclature Committee of the International Union of Biochemistry and Molecular Biology), lipases belong to the class of hydrolases (EC 3). Hydrolases are a class of enzymes that usually act as biochemical catalysts, using water to break chemical bonds, which usually results in breaking down larger molecules into smaller molecules. The class of hydrolases includes enzymes that act on ester bonds (EC 3.1), including carboxylic acid ester hydrolases (EC 3.1.1) and the subgroup lipases (EC 3.1.1.3). Lipases have been identified from plants, mammals and microorganisms, including, for example, Pseudomonas, Vibrio, Acinetobacter, Burkholderia, Chromobacterium, cutinase from Fusarium solani (FSC), Candida antarctica A (CalA), Rhizopus oryzae (ROL), Thermomyces lanuginosus (TLL), Rhizomucormiehei (RML), Aspergillus Niger, Fusarium heterosporum, Fusarium oxysporum or Fusarium culmorum.

[0051] If a protein has lipase activity, this can be detected by methods known and documented in the art.

[0052] The method used to detect whether the protein of the present invention has lipase activity is not critical. Preferably, with respect to the present invention, the method is described in the "Examples" section.

[0053] Compared to the amino acid sequence described herein above with respect to the amino acid sequence shown in SEQ ID No. 1, the lipase variant protein of the present invention may exhibit other amino acid modifications (amino acid substitutions, deletions or insertions).

[0054] In addition to the lipase variant described in item a) above with respect to the amino acid sequence shown in SEQ ID No. 1, it may also have at least one, two, three, four, five, six or seven other amino acid substitutions at positions 79, 202, 280, 301, 3, 11, 17, 40 or 111. In other words, the protein of the present invention having lipase activity is selected from the following: a) comprising SEQ ID a protein having an amino acid sequence as shown in No. 1, which, in addition to the amino acid at position 186 being different from L, has at least one, two, three, four, five, six, seven or more other amino acid substitutions selected from the following: (i) the amino acid at position 79 is different from E; (ii) the amino acid at position 202 is different from A; (iii) the amino acid at position 280 is different from L; (iv) the amino acid at position 301 is different from D; (v) the amino acid at position 3 is different from M; (vi) the amino acid at position 11 is different from C; (vii) the amino acid at position 17 is different from L; (viii) the amino acid at position 40 is different from K; (ix) the amino acid at position 111 is different from Q; and b) a protein having an amino acid sequence that is at least 80%, preferably 85%, more preferably 90%, further more preferably 95%, even more preferably 96%, even further more preferably 97%, particularly preferably 98%, and most preferably 99% identical to the amino acid sequence given directly in item a) above, provided that the amino acid at position 186 is different from L and has at least one other amino acid substitution selected from the group consisting of (i) to (ix) mentioned directly above. Preferably, the amino acid at position 79 is S, W or I, more preferably S; preferably, the amino acid at position 202 is N; preferably, the amino acid at position 280 is A; preferably, the amino acid at position 301 is A; preferably, the amino acid at position 3 is Q; preferably, the amino acid at position 11 is A; preferably, the amino acid at position 17 is P; preferably, the amino acid at position 40 is M; preferably, the amino acid at position 111 is E.

[0055] In addition, compared to the above amino acid sequence of the amino acid sequence shown in SEQ ID No.1, the lipase variant protein of the present invention may also show additional amino acid substitutions in addition to other amino acid modifications. These additional amino acid substitutions involve amino acid sequence positions different from positions 79, 202, 280, 301, 3, 11, 17, 40 or 111 involving other amino acid modifications. The lipase variant in item a) above herein, with respect to the amino acid sequence shown in SEQ ID No.1, may have at least one, two, three, four, five, six, seven or more additional amino acid substitutions at positions 4, 8, 18, 29, 31, 42, 84, 85, 192, 217, 309 or 312. The amino acid at position 4 is different from S, preferably, the amino acid at this position is P. The amino acid at position 8 is different from K, preferably, the amino acid at this position is E. The amino acid at position 18 is different from V, preferably, the amino acid at this position is C. The amino acid at position 29 is different from N, preferably, the amino acid at this position is W or H. The amino acid at position 31 is different from Q, preferably the amino acid at this position is W. The amino acid at position 42 is different from L, preferably the amino acid at this position is D. The amino acid at position 84 is different from N, preferably the amino acid at this position is T. The amino acid at position 85 is different from W, preferably the amino acid at this position is H. The amino acid at position 192 is different from F, preferably the amino acid at this position is A or V. The amino acid at position 217 is different from Q, preferably the amino acid at this position is M. The amino acid at position 309 is different from P, preferably the amino acid at this position is C. The amino acid at position 312 is different from P, preferably the amino acid at this position is N.

[0056] Therefore, another embodiment of the present invention relates to a protein of the present invention comprising other amino acid modifications, preferably, these embodiments are proteins with lipase activity, wherein the protein is selected from the following:

[0057] A protein comprising the amino acid sequence shown in SEQ ID No. 1, in addition,

[0058] - the amino acid at position 186 is different from L, and the amino acid at position 79 is different from E, wherein preferably, 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;

[0059] - the amino acid at position 186 is different from L, and the amino acid at position 202 is different from A, wherein preferably the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N;

[0060] - the amino acid at position 186 is different from L, and the amino acid at position 280 is different from L, wherein preferably the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A;

[0061] - the amino acid at position 186 is different from L, and the amino acid at position 301 is different from D, wherein preferably the amino acid at position 186 is preferably W or Y, and the amino acid at position 301 is preferably A;

[0062] - the amino acid at position 186 is different from L, and the amino acid at position 3 is different from M, wherein preferably, the amino acid at position 186 is preferably W or Y, and the amino acid at position 3 is preferably Q;

[0063] - the amino acid at position 186 is different from L, and the amino acid at position 11 is different from C, wherein preferably the amino acid at position 186 is preferably W or Y, and the amino acid at position 11 is preferably A;

[0064] - the amino acid at position 186 is different from L, and the amino acid at position 17 is different from L, wherein preferably the amino acid at position 186 is preferably W or Y, and the amino acid at position 17 is preferably P;

[0065] - the amino acid at position 186 is different from L, and the amino acid at position 40 is different from K, wherein preferably, the amino acid at position 186 is preferably W or Y, and the amino acid at position 40 is preferably M;

[0066] - the amino acid at position 186 is different from L, and the amino acid at position 111 is different from Q, wherein preferably the amino acid at position 186 is preferably W or Y, and the amino acid at position 111 is preferably E;

[0067] - A protein having an amino acid sequence that is at least 80%, preferably 85%, more preferably 90%, further more preferably 95%, even more preferably 96%, even further more preferably 97%, particularly preferably 98%, and most preferably 99% identical to the amino acid sequence shown in a), with the proviso that the amino acid at position 186 is different from L, wherein preferably, the amino acid at position 186 is preferably W or Y, and the protein has at least one other amino acid substitution selected from the group indicated by the punctuation symbols listed above.

[0068] Preferably, the lipase variant in item a) above, with respect to the amino acid sequence shown in SEQ ID No. 1, may have at least two other 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.

[0069] Therefore, another embodiment of the present invention relates to a protein of the present invention comprising other amino acid modifications, preferably these embodiments are proteins having lipase activity, wherein the protein is selected from the protein comprising the amino acid sequence shown in SEQ ID NO.1, in addition

[0070] - the amino acid at position 186 is different from L, and the amino acid at position 79 is different from E, and the amino acid at position 202 is different from A, wherein preferably the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 202 is preferably N;

[0071] - the amino acid at position 186 is different from L, and the amino acid at position 79 is different from E, and the amino acid at position 280 is different from L, wherein preferably 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;

[0072] - the amino acid at position 186 is different from L, and the amino acid at position 79 is different from E, and the amino acid at position 301 is different from D, wherein preferably 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;

[0073] - the amino acid at position 186 is different from L, and the amino acid at position 79 is different from E, and the amino acid at position 3 is different from M, wherein preferably, 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;

[0074] - the amino acid at position 186 is different from L, and the amino acid at position 79 is different from E, and the amino acid at position 11 is different from C, wherein preferably the amino acid at position 186 is preferably W or Y, the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 11 is preferably A;

[0075] - the amino acid at position 186 is different from L, and the amino acid at position 79 is different from E, and the amino acid at position 17 is different from L, wherein preferably 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;

[0076] - the amino acid at position 186 is different from L, and the amino acid at position 79 is different from E, and the amino acid at position 40 is different from K, wherein preferably, 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;

[0077] - the amino acid at position 186 is different from L, and the amino acid at position 79 is different from E, and the amino acid at position 111 is different from Q, wherein preferably 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;

[0078] - the amino acid at position 186 is different from L, and the amino acid at position 202 is different from A, and the amino acid at position 280 is different from L, wherein preferably 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;

[0079] - the amino acid at position 186 is different from L, and the amino acid at position 202 is different from A, and the amino acid at position 301 is different from D, wherein preferably 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;

[0080] - the amino acid at position 186 is different from L, and the amino acid at position 202 is different from A, and the amino acid at position 3 is different from M, wherein preferably, 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;

[0081] - the amino acid at position 186 is different from L, and the amino acid at position 202 is different from A, and the amino acid at position 11 is different from C, wherein preferably 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;

[0082] - the amino acid at position 186 is different from L, and the amino acid at position 202 is different from A, and the amino acid at position 17 is different from L, wherein preferably 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;

[0083] - the amino acid at position 186 is different from L, and the amino acid at position 202 is different from A, and the amino acid at position 40 is different from K, wherein preferably 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;

[0084] - the amino acid at position 186 is different from L, and the amino acid at position 202 is different from A, and the amino acid at position 111 is different from Q, wherein preferably 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;

[0085] - the amino acid at position 186 is different from L, and the amino acid at position 280 is different from L, and the amino acid at position 301 is different from D, wherein preferably 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;

[0086] - the amino acid at position 186 is different from L, and the amino acid at position 280 is different from L, and the amino acid at position 3 is different from M, wherein preferably 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;

[0087] - the amino acid at position 186 is different from L, and the amino acid at position 280 is different from L, and the amino acid at position 11 is different from C, wherein preferably 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;

[0088] - the amino acid at position 186 is different from L, and the amino acid at position 280 is different from L, and the amino acid at position 17 is different from L, wherein preferably 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;

[0089] - the amino acid at position 186 is different from L, and the amino acid at position 280 is different from L, and the amino acid at position 40 is different from K, wherein preferably 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;

[0090] - the amino acid at position 186 is different from L, and the amino acid at position 280 is different from L, and the amino acid at position 111 is different from Q, wherein preferably 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;

[0091] - the amino acid at position 186 is different from L, and the amino acid at position 301 is different from D, and the amino acid at position 3 is different from M, wherein preferably 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;

[0092] - the amino acid at position 186 is different from L, and the amino acid at position 301 is different from D, and the amino acid at position 11 is different from C, wherein preferably 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;

[0093] - the amino acid at position 186 is different from L, and the amino acid at position 301 is different from D, and the amino acid at position 17 is different from L, wherein preferably 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;

[0094] - the amino acid at position 186 is different from L, and the amino acid at position 301 is different from D, and the amino acid at position 40 is different from K, wherein preferably 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;

[0095] - the amino acid at position 186 is different from L, and the amino acid at position 301 is different from D, and the amino acid at position 111 is different from Q, wherein preferably 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;

[0096] - the amino acid at position 186 is different from L, and the amino acid at position 3 is different from M, and the amino acid at position 11 is different from C, wherein preferably 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;

[0097] - the amino acid at position 186 is different from L, and the amino acid at position 3 is different from M, and the amino acid at position 17 is different from L, wherein preferably, 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;

[0098] - the amino acid at position 186 is different from L, and the amino acid at position 3 is different from M, and the amino acid at position 40 is different from K, wherein preferably, 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;

[0099] - the amino acid at position 186 is different from L, and the amino acid at position 3 is different from M, and the amino acid at position 111 is different from Q, wherein preferably 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;

[0100] - the amino acid at position 186 is different from L, and the amino acid at position 11 is different from C, and the amino acid at position 17 is different from L, wherein preferably 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;

[0101] - the amino acid at position 186 is different from L, and the amino acid at position 11 is different from C, and the amino acid at position 40 is different from K, wherein preferably 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;

[0102] - the amino acid at position 186 is different from L, and the amino acid at position 11 is different from C, and the amino acid at position 111 is different from Q, wherein preferably 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;

[0103] - the amino acid at position 186 is different from L, and the amino acid at position 17 is different from L, and the amino acid at position 40 is different from K, wherein preferably, 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;

[0104] - the amino acid at position 186 is different from L, and the amino acid at position 17 is different from L, and the amino acid at position 111 is different from Q, wherein preferably 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;

[0105] - the amino acid at position 186 is different from L, and the amino acid at position 40 is different from K, and the amino acid at position 111 is different from Q, wherein preferably 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;

[0106] - A protein having an amino acid sequence that is at least 80%, preferably 85%, more preferably 90%, further more preferably 95%, even more preferably 96%, even further more preferably 97%, particularly preferably 98%, and most preferably 99% identical to the amino acid sequence shown in a), taking into account that the amino acid at position 186 is different from L, wherein preferably, the amino acid at position 186 is preferably W or Y, and the protein has at least two further amino acid substitutions selected from the group indicated by the punctuation symbols listed above.

[0107] Preferably, the lipase variant in item a) above, with respect to the amino acid sequence shown in SEQ ID No. 1, may have at least three other 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.

[0108] Therefore, another embodiment of the present invention relates to a protein of the present invention comprising other amino acid modifications, preferably these embodiments are proteins having lipase activity, wherein the protein is selected from the protein comprising the amino acid sequence shown in SEQ ID NO.1, in addition

[0109] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0110] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0111] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0112] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0113] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0114] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0115] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0116] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0117] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0118] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0119] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0120] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0121] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0122] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0123] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0124] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0125] - the amino acid at position 186 is different from L, and 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 preferably, the amino acid at position 186 is preferably W or Y, more preferably Y, the amino acid at position 79 is preferably S, W or I, more preferably S, the amino acid at position 301 is preferably A, and the amino acid at position 40 is preferably M; wherein particularly 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;

[0126] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0127] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0128] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0129] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0130] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0131] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0132] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0133] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0134] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0135] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0136] - 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 preferably, 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;

[0137] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0138] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0139] - the amino acid at position 186 is different from L, and 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 preferably 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;

[0140] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0141] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0142] - the amino acid at position 186 is different from L, and 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 preferably 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;

[0143] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0144] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0145] - the amino acid at position 186 is different from L, and 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 preferably, the amino acid at position 186 is preferably W or Y, the amino acid at position 202 is preferably N, the amino acid at position 301 is preferably A, and the amino acid at position 17 is preferably P;

[0146] - 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 preferably, 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;

[0147] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0148] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0149] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0150] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0151] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0152] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0153] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0154] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0155] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0156] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0157] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0158] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0159] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0160] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0161] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0162] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0163] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0164] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0165] - the amino acid at position 186 is different from L, and 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 preferably, the amino acid at position 186 is preferably W or Y, the amino acid at position 280 is preferably A, the amino acid at position 3 is preferably Q, and the amino acid at position 40 is preferably M;

[0166] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0167] - the amino acid at position 186 is different from L, and 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 preferably 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;

[0168] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0169] - the amino acid at position 186 is different from L, and 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 preferably 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;

[0170] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0171] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0172] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0173] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0174] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0175] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0176] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0177] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0178] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0179] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0180] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0181] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0182] - 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 preferably, 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;

[0183] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0184] - 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 preferably, 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;

[0185] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0186] - 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 preferably, 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;

[0187] - 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 preferably, 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;

[0188] - 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 preferably, 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;

[0189] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0190] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0191] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0192] - the amino acid at position 186 is different from L, and 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 preferably, 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;

[0193] - A protein having an amino acid sequence that is at least 80%, preferably 85%, more preferably 90%, further more preferably 95%, even more preferably 96%, even further more preferably 97%, particularly preferably 98%, and most preferably 99% identical to the amino acid sequence shown in a), with the proviso that the amino acid at position 186 is different from L, wherein preferably, the amino acid at position 186 is preferably W or Y, more preferably Y, and the protein has at least three other amino acid substitutions selected from the group indicated by the punctuation symbols listed above.

[0194] The lipase variant in item a) above, with respect to the amino acid sequence shown in SEQ ID No. 1, may have at least four other 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.

[0195] The lipase variant in item a) above, with respect to the amino acid sequence shown in SEQ ID No. 1, may have at least five other 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.

[0196] The lipase variant in item a) above, with respect to the amino acid sequence shown in SEQ ID No. 1, may have at least six other 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.

[0197] The lipase variant in item a) above, with respect to the amino acid sequence shown in SEQ ID No. 1, may have at least seven other 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.

[0198] A preferred embodiment of the present invention is a protein of the present invention, which encodes a protein having 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, 21 7, 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, 3 13, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, and 401.

[0199] Other proteins with lipolytic enzyme or lipase activity were tested separately. The amino acid sequences of these other proteins represent variants of the amino acid sequence represented by the amino acids in SEQ ID No. 1, wherein:

[0200] - in the amino acid sequence shown in SEQ ID No. 1, the two amino acids at positions 40 and 79 are different from the amino acids given at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific experimental variant, with respect to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 40 is M and the amino acid at position 79 is S;

[0201] - in the amino acid sequence shown in SEQ ID No. 1, the two amino acids at positions 40 and 186 are different from the amino acids given at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific experimental variant, with respect to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 40 is M and the amino acid at position 186 is Y;

[0202] - in the amino acid sequence shown in SEQ ID No. 1, the two amino acids at positions 40 and 301 are different from the amino acids given at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific test variant, with respect to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 40 is M and the amino acid at position 301 is A;

[0203] - in the amino acid sequence shown in SEQ ID No. 1, the two amino acids at positions 79 and 186 are different from the amino acids given at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific test variant, with respect to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 79 is S and the amino acid at position 186 is Y;

[0204] - in the amino acid sequence shown in SEQ ID No. 1, the two amino acids at positions 79 and 301 are different from the amino acids given at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific test variant, with respect to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 79 is S and the amino acid at position 301 is A;

[0205] - In the amino acid sequence shown in SEQ ID No. 1, the two amino acids at positions 186 and 301 are different from the amino acids given at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific experimental variant, with respect to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 186 is Y and the amino acid at position 301 is A.

[0206] More other proteins with lipolytic enzyme or lipase activity were tested respectively. The amino acid sequences of these other proteins represent variants of the amino acid sequence represented by the amino acids in SEQ ID No. 1, wherein:

[0207] - in the amino acid sequence shown in SEQ ID No. 1, the three amino acids at positions 40, 79 and 186 are different from the amino acids given at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific test variant, with respect to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 40 is M, the amino acid at position 79 is S, and the amino acid at position 186 is Y;

[0208] - in the amino acid sequence shown in SEQ ID No. 1, the three amino acids at positions 40, 79 and 301 are different from the amino acids given at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific test variant, with respect to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 40 is M, the amino acid at position 79 is S, and the amino acid at position 301 is A;

[0209] - in the amino acid sequence shown in SEQ ID No. 1, the three amino acids at positions 40, 186 and 301 are different from the amino acids given at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific test variant, with respect to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 40 is M, the amino acid at position 186 is Y, and the amino acid at position 301 is A;

[0210] - In the amino acid sequence shown in SEQ ID No. 1, the three amino acids at positions 79, 186 and 301 are different from the amino acids given at the corresponding amino acid positions in the sequence shown in SEQ ID No. 1. In a specific experimental variant, with respect to the amino acid sequence shown in SEQ ID No. 1, the amino acid at position 79 is S, the amino acid at position 186 is Y, and the amino acid at position 301 is A.

[0211] The lipase variant or protein variant of the present invention exhibits improved selectivity and / or improved specific activity in the stereoselective acylation or carboxylation of 2,6-dimethyl-1-indanamine (DMAI), and is more suitable for producing enantiomerically enriched or almost pure methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate than wild-type lipase. Methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate is an important intermediate in the synthesis of the herbicidally active compound indaziflam.

[0212] "Enantiomerically enriched" herein means that one of the two enantiomers is present in the composition in a higher 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, further 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 further 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 especially preferably at least 94% of one enantiomer is present in the composition.

[0213] "Enantiomerically almost pure" means in the present context 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%, further 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 further 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 especially preferably one of the two enantiomers is present in the composition in an amount of at least 99.5%.

[0214] Another embodiment of the present invention relates to a nucleic acid molecule encoding a protein of the present invention.

[0215] The nucleic acid molecule of the present invention can be any type of nucleic acid, as long as the nucleic acid encodes the protein of 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 and coding DNA that may or may not contain introns).

[0216] Of particular interest herein are nucleic acid molecules encoding proteins having lipase activity comprising 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, 120 1, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 31 1, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, and the amino acid sequence shown in 401.

[0217] Therefore, the present invention also relates to a nucleic acid molecule encoding a protein having lipase activity, wherein the lipase is selected from the group consisting of:

[0218] a) comprising 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, 121 2, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 2 18, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402;

[0219] b) a nucleic acid molecule having at least 60%, preferably 70%, more preferably 80%, further more preferably 90%, even more preferably 95%, even further more preferably 96%, particularly preferably 97%, most preferably 98%, or especially preferably 99% identity with the nucleic acid sequence shown in a).

[0220] In the context of the present invention, the term "hybridizes with" refers to hybridization under conventional hybridization conditions, preferably hybridization under stringent conditions, for example, as described in Sambrook et al. (Molecular Cloning, A Laboratory Manual, 3rd edition (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ISBN: 0879695773) or Ausubel et al. (Short Protocols in Molecular Biology, John Wiley & Sons; 5th edition (2002), ISBN: 0471250929). Particularly preferably, "hybridization" refers to hybridization under the following conditions:

[0221] Hybridization Buffer:

[0222] 2xSSC; 10xDenhardt solution (Fikoll 400+PEG+BSA; ratio 1:1:1); 0.1% SDS; 5mMEDTA; 50mM Na2HPO4; 250μg / ml herring sperm DNA; 50μg / ml tRNA;

[0223] or

[0224] 25 mM sodium phosphate buffer pH 7.2; 1 mM EDTA; 7% SDS

[0225] Hybridization temperature: T = 65 to 68°C

[0226] Wash buffer: 0.1xSSC; 0.1% SDS

[0227] Washing temperature: T = 65 to 68°C.

[0228] The nucleic acid molecules hybridizing to the nucleic acid molecule encoding the protein having lipase activity may originate from any organism; thus, they may originate from bacteria, fungi, animals, humans, plants or viruses.

[0229] The nucleic acid molecule hybridizing with the nucleic acid molecule encoding a protein having lipase activity is preferably derived from a microorganism, more preferably from a fungus or a bacterium, most preferably from a bacterium.

[0230] Nucleic acid molecules that hybridize to the above molecules can be isolated from, for example, a genome or cDNA library. These nucleic acid molecules can be identified and isolated using the nucleic acid molecules described herein, or can be identified and isolated using a portion of these molecules or the reverse complement of these molecules, for example by hybridization according to standard methods (see, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd edition (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ISBN: 0879695773; Ausubel et al., Short Protocols in Molecular Biology, John Wiley & Sons; 5th edition (2002), ISBN: 0471250929) or by PCR amplification.

[0231] The fragment used as hybridization sample can also be a synthetic fragment or oligonucleotide prepared using conventional synthesis techniques, whose sequence is substantially the same as the nucleic acid molecules recorded in the context of the present invention. Once the gene hybridizing with the nucleic acid sequence recorded in the context of the present invention is identified and isolated, its sequence should be determined, and the characteristics of the protein encoded by the sequence should be analyzed to determine whether they are proteins with lipase activity. How to determine whether a protein has the activity of a protein (the protein has lipase activity) is known to those skilled in the art.

[0232] Molecules hybridizing with the nucleic acid molecules described in the context of the present invention particularly include fragments, derivatives and allelic variants of the above-mentioned nucleic acid molecules. In the context of the present invention, the term "derivative" refers to molecules whose sequences differ from the sequences of the above-mentioned nucleic acid molecules at one or more positions and are highly identical to these sequences. The differences from the above-mentioned nucleic acid molecules may be due to, for example, deletion, addition, displacement, insertion or recombination.

[0233] Preferred nucleic acid molecules according to the present invention are 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, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 136, 137, 138, 139, 140, 141 22, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216 , 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, 3 The nucleic acid molecules shown in 12, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402.

[0234] The meanings of the nucleotide abbreviations a, c, g, t, and the meanings of the degenerate nucleotide abbreviations r, y, s, w, k, m, b, d, h, v, n can be derived from Table 1 below under the subheading "Description of the sequence". The amino acids encoded by the codons containing degenerate nucleotides can be derived from Table 3 below under the subheading "Description of the sequence".

[0235] Furthermore, the present invention relates to a recombinant nucleic acid molecule comprising a nucleic acid molecule according to the invention.

[0236] In conjunction with the present invention, the term "recombinant nucleic acid molecule" should be understood as a nucleic acid molecule that, in addition to comprising the nucleic acid molecule of the present invention, also comprises additional sequences that do not naturally occur in the combination in which they occur in the recombinant nucleic acid of the present invention. In this article, the above-mentioned additional sequence can be any sequence, preferably a functional or regulatory sequence (promoter, termination signal, enhancer, ribosome binding site (rbs), a leader sequence that enhances transcription, translation or RNA stability, a subcellular targeting sequence, etc.), particularly preferably a functional or regulatory sequence that is active in a microorganism, especially particularly preferably a regulatory sequence that is active in a fungus, particularly a yeast or a bacterium. The method for creating the recombinant nucleic acid molecule of the present invention known to those skilled in the art includes genetic methods such as combining nucleic acid molecules by connection, genetic recombination or new synthesis of nucleic acid molecules. These methods are described in, for example, 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).

[0237] In another embodiment, a recombinant nucleic acid molecule of the invention comprises a nucleic acid molecule of the invention linked to regulatory sequences that promote transcription in a prokaryotic or eukaryotic cell.

[0238] Regulatory sequences that initiate transcription in cells are also called promoters.

[0239] Information concerning regulatory sequences and plasmids is known to those skilled in the art and is described, for example, in the Registry of Standard Biological Parts supported by The International Genetically Engineered Machine (iGEM) Foundation (One Kendall Square, Suite B6104, Cambridge, MA 02139, USA) on the World Wide Web (http: / / parts.igem.org / Catalog).

[0240] Regulatory sequences that initiate transcription in prokaryotes (such as E. coli) and eukaryotes are well documented in the literature, particularly regulatory sequences expressed in yeast (such as Saccharomyces cerevisiae). For example, overviews of various systems for protein expression in various host organisms can be found 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, pScADH2 (Baghban et al., 2018, see above). Common bacterial promoters include T5, T7, rhamnose-inducible, arabinose-inducible, PhoA, and the artificial trc (trp-lac) promoter described by Marschall et al. (2017, Appl Microbiol Biotechnol 101, 501-512) and Tegel et al. (2011, FEBS Journal 278, 729-739).

[0241] Another embodiment of the recombinant nucleic acid molecule of the present invention is a vector or plasmid, which comprises the nucleic acid molecule of the present invention.

[0242] "Vector" is generally understood in the field of molecular biology and herein to represent a nucleic acid sequence or a tool containing a nucleic acid sequence for transferring genetic material (DNA or RNA) into a target cell. A vector may be a plasmid, such as a T-DNA or binary vector for generating transgenic plants, an expression vector for expressing a nucleic acid sequence in a host cell, a shuttle vector capable of propagation in different hosts, or a vector may also be a virus particle or a bacteriophage modified to deliver foreign genetic material to a host.

[0243] "Plasmid" is generally understood in the art of molecular biology and herein to mean an autonomously replicating, usually circular DNA molecule that, when present in a host cell, is separate from the chromosomal DNA.

[0244] The nucleic acid molecules, recombinant nucleic acid molecules, vectors or plasmids of the present invention can be used to produce the protein of the present invention, for example, by expressing the nucleic acid molecules of the present invention in a host cell.

[0245] Another embodiment of the present invention relates to a host or host cell comprising or expressing a nucleic acid molecule of the present invention, or comprising a protein of the present invention, or comprising a recombinant nucleic acid molecule of the present invention, or comprising a vector of the present invention, or comprising a plasmid of the present invention.

[0246] The protein nucleic acid molecules of coding of the present invention with lipase activity can be expressed in host cells, for example, for their amplification or for producing protein of the present invention. In order to express in host cells, nucleic acid molecules of the present invention can be contained in vectors or plasmids, or they can be stably integrated in the genome of the corresponding host cells. Nucleic acid molecules of the present invention can also be contained in the vector that supports its introduction into host cells.

[0247] Another embodiment of the invention relates to a host or host cell of the invention, comprising a nucleic acid molecule of the invention, or comprising a recombinant nucleic acid molecule of the invention, or comprising a vector of the invention, or comprising a plasmid of the invention, and in each case comprising a protein of the invention.

[0248] Another embodiment of the invention relates to a host or host cell of the invention, comprising a nucleic acid molecule of the invention, or comprising a recombinant nucleic acid molecule of the invention, or comprising a vector of the invention, or comprising a plasmid of the invention, and in each case expressing a protein of the invention.

[0249] Another embodiment of the invention relates to a host or host cell according to the invention, comprising a nucleic acid molecule according to the invention, or comprising a recombinant nucleic acid molecule according to the invention, or comprising a vector according to the invention, or comprising a plasmid according to the invention, and in each case expressing a protein, wherein the protein has lipase activity.

[0250] "Expressed 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 into a protein with lipase activity, or if the nucleic acid molecule is DNA or cDNA, it is transcribed (or processed if it is genomic DNA containing introns) into mRNA, preferably into mRNA encoding a protein with lipase activity, and subsequently translated into a protein, preferably into a protein with lipase activity.

[0251] Transcription of a given nucleic acid molecule in a host can be demonstrated by methods known to those skilled in the art, such as detection of specific transcripts (mRNA) of the exogenous nucleic acid molecule by Northern blot analysis or RT-PCR.

[0252] Whether a host or host cell contains a given protein or contains a protein derived from an expressed 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 preparing antibodies that specifically react with certain proteins, i.e. antibodies that are able to specifically bind to certain proteins (see, e.g., Lottspeich and Zorbas (eds.), 1998, Bioanalytik, Spektrum akad, Verlag, Heidelberg, Berlin, ISBN 3-8274-0041-4). Some companies (Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA USA 0245; GenScript, 60 Centennial Ave., Piscataway, NJ 08854, USA) provide order services for the preparation of such antibodies.

[0253] In addition, a person skilled in the art can test whether a host or a host cell comprises a protein of the invention by detecting the (additional) activity of a protein having lipase activity in a corresponding host cell. Preferably, the activity of a protein having additional lipase activity in a corresponding host cell is detected by comparing the lipase activity of a host cell of the invention with that of a host cell not comprising the protein of the invention.

[0254] Testing whether a protein has lipase activity can be performed by methods known in the art.

[0255] The host or host cell of the present invention can be prepared by those skilled in the art through known methods of genetic modification or transformation of organisms.

[0256] Therefore, another subject of the present invention is a host or host cell of the present invention, in particular a prokaryotic or eukaryotic host or host cell, which is genetically modified (or transformed) with a nucleic acid molecule of the present invention, or a recombinant nucleic acid molecule of the present invention, or a vector of the present invention, or a plasmid of the present invention. Preferably, the genetically modified (transformed) host or host cell of the present invention expresses a protein having lipase activity, more preferably, the genetically modified (transformed) host or host cell of the present invention expresses a protein of the present invention.

[0257] "Genetic modification with a nucleic acid molecule" or "transformation with a nucleic acid molecule" is understood herein to mean the introduction of a nucleic acid molecule into a host or a host cell by technical and / or non-naturally occurring methods, preferably by technical methods in the fields of molecular biology, biotechnology or genetic modification.

[0258] Descendants, offspring or progeny of the host or host cell of the present invention are also an embodiment of the present invention, preferably these offspring, offspring or progeny comprise the nucleic acid molecule of the present invention, or comprise the recombinant nucleic acid molecule of the present invention, or comprise the vector of the present invention, or comprise the plasmid of the present invention, or comprise the protein of the present invention, more preferably these offspring, offspring or progeny comprise the nucleic acid molecule of the present invention, or comprise the recombinant nucleic acid molecule of the present invention, or comprise the vector of the present invention, or comprise the plasmid of the present invention, and in each case express a protein, wherein the protein has lipase activity, even more preferably these offspring, offspring or progeny comprise the nucleic acid molecule of the present invention, or comprise the recombinant nucleic acid molecule of the present invention, or comprise the vector of the present invention, or comprise the plasmid of the present invention, and in each case express a protein, wherein the protein has lipase activity of the present invention.

[0259] The host or host cell of the present invention can be a host or host cell from any prokaryotic or eukaryotic organism. The host or host cell can be a bacterium or bacterial cell (e.g., E. coli; a bacterium of the genus Bacillus, in particular Bacillus subtilis; Agrobacterium, in particular Agrobacterium tumefaciens or Agrobacterium rhizogenes; Pseudomonas, in particular Pseudomonas fluorescens; Streptomyces spp; Rhodococcus spp, in particular Rhodococcus rhodochrous; Vibrio natrigens; Corynebacterium, in particular Corynebacterium glutamicum; glutamicum); or fungi or fungal cells (for example Agaricus, in particular Agaricus bisporus; Aspergillus; Trichoderma or yeast, in particular Saccharomyces cerevisiae; Pichia ssp. such as Pichia pastoris), as well as plants or plant cells, or they may be animals or animal cells.

[0260] Preferred host cells of the present invention are microbial cells. Within the framework of the present patent application, this is understood to include all bacteria and all protists (e.g. fungi, particularly yeast and algae) as they are defined in, for example, Schlegel's "General Microbiology" (Georg Thieme Publishing House (1985), 1-2).

[0261] With regard to microorganisms, the host or host cell of the present invention is preferably a bacterium / bacterial cell or a yeast / yeast cell, most preferably a bacterium / bacterial cell. With regard to bacteria / bacterial cells, the host or host cell of the present invention is preferably a Bacillus / Bacillus cell or an Escherichia coli / E. coli cell, most preferably an E. coli / E. coli cell.

[0262] Alternatively, Pseudomonas, especially Pseudomonas fluorescens; Streptomyces species; Rhodococcus species, especially Rhodococcus rhodochrous; Vibrio species (Vibrio spp), especially Vibrio nautilus; Corynebacterium, especially Corynebacterium glutamicum; or other bacterial species can also be used as the host or host cell of the present invention.

[0263] A preferred embodiment of the present invention relates to a host or a host cell according to the present invention comprising a nucleic acid molecule according to the present invention, wherein said nucleic acid molecule according to the present invention is characterized in that the codons of said nucleic acid molecule are altered to adapt to the codon usage frequency of the host or host cell, respectively.

[0264] The host cells of the present invention can be used to produce the proteins of the present invention. The proteins of the present invention can be used in a method for producing an enantiomerically enriched or nearly enantiomerically pure acylated or carboxylated product from an acyl or carboxyl acceptor in the presence of an acyl or carboxyl donor.

[0265] Therefore, another embodiment of the present invention relates to a method for producing an acylated or carboxylated product, comprising the steps of:

[0266] a) providing an acyl or carboxyl acceptor molecule;

[0267] b) providing an acyl or carboxyl donor molecule;

[0268] c) contacting the acyl or carboxyl acceptor molecule provided in step a) and the acyl or carboxyl donor molecule provided in step b) with the protein of the present invention;

[0269] d) Optionally, obtaining an acylated or carboxylated product.

[0270] Another embodiment of the present invention is the use of a protein according to the present invention for the production of amines, preferably for the production of (1R,2S)-amines.

[0271] Another embodiment of the invention is the use of a protein according to the invention for stereoselective acylation or carboxylation of racemic 2,6-dimethyl-1-indanylamine.

[0272] Another embodiment of the present invention is the use of a protein of the present invention for stereoselective acylation or carboxylation of racemic 2,6-dimethyl-1-indanamine, wherein the substrate is racemic 2,6-dimethyl-1-indanamine.

[0273] Description of the sequence

[0274] In this application, nucleotide and amino acid abbreviations are used according to the following IUPAC code:

[0275] Table 1

[0276] IUPAC nucleotide code Base A Adenine C Cytosine G Guanine T(or U) Thymine (or uracil) R A or G Y C or T S G or C W A or T K G or T M A or C B C or G or T D A or G or T H A or C or T V A or C or G N Any base - Gap

[0277] To distinguish amino acids from nucleotides, the uppercase nucleotide code abbreviations given in the above table are written herein in lowercase letters.

[0278] Table 2

[0279]

[0280]

[0281] Codon usage herein follows the so-called "universal genetic code" according to the following table, in which "t" in ribonucleic acid (RNA) sequences is replaced by "u".

[0282] Table 3

[0283]

[0284]

[0285]

[0286]

[0287] Table 4

[0288] The sequence listing associated with this application is submitted in electronic format and is hereby incorporated by reference in its entirety into this specification. "PRT" stands for "protein" and "NUC" stands for "nucleic acid".

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314] Example

[0315] Terrific broth (TB) was prepared using 47.6 g / l of granular medium and 4 ml / L of glycerol in demineralized water and sterilized at 121°C for 20 minutes.

[0316] Example 1

[0317] Cloning of lipase variants

[0318] The nucleotide sequences encoding the lipase variants as described herein can be synthesized according to methods known in the art, for example, provided by corresponding service providers such as Eurofins Genomics GmbH (Eurofins Genomics GmbH, Anzinger Str. 7a, 85560 Ebersberg, Germany). In short, the nucleic acid sequence of the wild-type lipase (SEQ ID NO. 2) or the related variants as described herein is cloned into an expression vector based on the vector pKA81a. Genetic elements are introduced into the modified vector pKA81a by generally known methods. In order to express the wild-type lipase and the lipase variants, respectively, the expression vectors are introduced into electrocompetent Escherichia coli MG1655 cells.

[0319] Generation of enzyme variants

[0320] Nucleotide substitutions (alternatives) are introduced into the nucleic acid parent sequence, for example to obtain an amino acid exchange for another amino acid. A variety of molecular biological methods can be used to achieve these substitutions. A useful method of preparing mutant nucleic acids and corresponding mutant proteins of the present invention is to perform site-directed mutagenesis on the codons encoding one or more pre-selected amino acids. Methods for obtaining these site-directed mutagenesis are well known to those skilled in the art and are widely documented in the literature (especially: Directed Mutagenesis: A Practical Approach, 1991, edited by MJ McPHERSON, IRL PRESS), or those methods that can use commercial kits (e.g., QUIKCHANGE from Qiagen or Stratagene). TM After site-directed mutagenesis, the nucleic acid was transformed into Escherichia coli MG1655 cells.

[0321] To determine product yield and selectivity, the transformed cells are tested in appropriate biotransformation reactions. Suitable biotransformation reactions are described below, see for example Example 2. Sequence verification is performed as known in the art.

[0322] Glycerol stocks of E. coli cultures transformed with the corresponding expression plasmids were prepared by adding one volume of 40% glycerol solution to one volume of E. coli culture.

[0323] To isolate a single bacterial colony, appropriately diluted E. coli culture was spread on LB agar plates containing appropriate concentrations of kanamycin and cultured at 37°C until a single colony was obtained.

[0324] Example 2

[0325] Conversion of racemic DMAI to DMAI carbamate using wild-type lipase or its variants

[0326] nourish

[0327] For screening purposes, Escherichia coli MG1655 was used as the host of expression plasmid. For pre-culture, TB culture medium (TB, Fisher Bioreagents, 22711022) supplemented with 50 μg / ml kanamycin (kanamycin solution, K0254, Sigma-Aldrich, St.Louis, MO, USA) of 590 μL was filled with sterilized 2mL 96-hole deep-well plates (0030501306, Eppendorf, Hamburg, Germany), and the glycerol storage solution of 10 μL corresponding variants was inoculated, or the TB culture medium of 590 μL was inoculated with the cell material from agar plate bacterium colony. Pre-culture was cultivated 17 hours at 37°C and in the climo shaker ISF1-X (Kühner AG, Birsfelden, Switzerland) at 250rpm.

[0328] For expression cultures, a sterilized 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); the expression culture was inoculated with 30 μL of pre-culture. The expression plates were cultured at 37°C and 250 rpm in a climo shaker. After 4 hours of culture, 60 μL of 10 mM IPTG diluted in expression medium (supplemented with 50 mg / L kanamycin) was added to each well to induce enzyme expression (yielding a final concentration of 1 mM IPTG). Subsequently, the expression plates were cultured at 28°C in a shaker incubator for 20 hours.

[0329] Harvest cells by centrifuging the expression culture at 4°C and 2500 x g for 15 min. Discard the culture supernatant and resuspend the remaining cell pellet in 200 μL PBS (Gibco PBS, Mg-free). 2+ , No Ca 2+ ). The resuspended culture was then lyophilized for 24 h. The deep well plates were stored at 4°C until use.

[0330] Biotransformation

[0331] The deep-well plate containing the cell supernatant was equilibrated to room temperature. 13.2 μL of racemic 2,6-dimethyl-1-indanamine (DMAI) and 186.8 μL of dimethyl carbonate were added to the lyophilized culture to start the bioconversion reaction, which was then cultured in a shaking incubator at 70°C or 80°C at 250 rpm for 8 to 22 hours. 600 μL of acetonitrile was added to terminate the reaction. Subsequently, the plate was centrifuged at 4°C and 2500×50 for 15 min. 100 μL of supernatant was transferred from each well to a 96-well plate. The plate was stored at -20°C or directly subjected to HPLC analysis.

[0332] Analytical HPLC method

[0333] Analytical HPLC was performed using the following settings:

[0334] Instrument: Agilent Technologies 1290 Infinity; Column: Poroshell, 100x4.6mm, 2.7μm; Eluent A: water (+0.001% formic acid); Eluent B: acetonitrile; Flow: isocratic (10% eluent A / 90% eluent B), flow rate: 1.4mL / min; Temperature: 25°C; Injection volume: 1μL; Detection: absorption at 210nm.

[0335] 1R,2SDMAI carbamate (dissolved in acetonitrile) was used as reference material and quantitative standard. Appropriate dilutions were prepared to cover the maximum concentration range used in the biotransformation. Samples were analyzed to detect acylated or carboxylated product isomers as well as non-acylated and non-carboxylated substrates. The concentration of the corresponding compound in the sample was calculated by comparing the obtained peak area with the peak area of ​​the corresponding compound standard.

[0336] Example 3

[0337] Yield of 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 variants.

[0338] Cultivation, bioconversion and HPLC analysis were performed as described in Example 2. Bioconversion was performed at 70°C for 22 h. The glycerol stock was used as inoculum for cultivation. The yield of methyl-[(1R, 2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate and the results of 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 yield of methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate is defined as the yield of methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate of the variant divided by the yield of methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate of the wild-type lipase. dr is defined as the yield of 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, expressed as a percentage.

[0339]

[0340]

[0341] surface

[0342] The key to an efficient production process is the high selectivity of the corresponding enzyme variants to produce nearly enantiomerically pure 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 and a dr of 61.7% for the methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate product.

[0343] Table 5: Relative 1R,2SDMAI carbamate yields of lipase variants with improved 1R,2SDMAI carbamate product yields.

[0344]

[0345]

[0346]

[0347] Table 6: Lipase variants with improved diastereoisomerization ratio (dr).

[0348]

[0349]

[0350] Example 4

[0351] The racemic 2,6-dimethyl-1-indenamine was converted to methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate using a lipase variant based on the wild-type lipase. Cultivation, bioconversion and HPLC analysis were performed as described in Example 2.

[0352] Biotransformation was carried out at 80°C for 8 h. The glycerol stock was used as inoculum for the culture. The results of the yield of methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate product and the diastereoisomer ratio (dr) of methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate are shown in Tables 7 and 8.

[0353] The wild type lipase (SEQ ID NO. 1) showed a product yield of 2.04 mg / ml and a dr of 64.6%.

[0354] Table 7: Relative methyl-[(1R,2S)-2,6-dimethyl-2,3-dihydro-1H-inden-1-yl]carbamate yields of lipase variants with improved 1R,2SDMAI carbamate product yield.

[0355]

[0356]

[0357]

[0358]

[0359]

[0360] Table 8: Selection of variants with improved dr.

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

Claims

1. A protein having lipase activity, the protein being encoded by an amino acid sequence having at least 80% identity to the amino acid sequence shown in SEQ ID No. 1, wherein the amino acid sequence has at least 80% sequence identity to the amino acid sequence shown in SEQ ID No. 1, and further comprising a modification selected from the following: i. The amino acid at position 186 is different from L; ii. the amino acid at position 280 is different from L; iii. the amino acid at position 312 is different from P; iv. The amino acid at position 3 is different from M; v. The amino acid at position 29 is different from N; vi. the amino acid at position 17 is different from L; vii. The amino acid at position 4 is different from S; viii. The amino acid at position 18 is different from V; ix. The amino acid at position 202 is different from A; x. The amino acid at position 301 is different from D; xi. the amino acid at position 309 is different from P; xii. The amino acid at position 31 is different from Q; xiii. The amino acid at position 111 is different from Q; xiv. The amino acid at position 85 is different from W; xv. The amino acid at position 8 is different from K; xvi. The amino acid at position 79 is different from E; xvii. The amino acid at position 40 is different from K.

2. The protein according to claim 1, wherein the protein is selected from the group consisting of: a) a protein comprising the amino acid sequence as shown in SEQ ID No. 1, except that the amino acid at position 186 is different from L; b) a protein having an amino acid sequence that is at least 80% identical to the amino acid sequence shown in a), with the proviso that the amino acid at position 186 is different from L.

3. The protein of 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 protein of claim 3, wherein the protein has at least one additional amino acid substitution 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.

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

6. The protein of claim 4, wherein the protein has at least three additional amino acid substitutions selected from the group consisting of: moieties (i), (ii), (iii), (iv), (v), (vi), (vii), (viii) and (ix) as defined in claim 4.

7. The protein according to any one of claims 4 to 6, wherein the other amino acid substitutions are: (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; (viii) the amino acid at position 40 is M; (ix) The amino acid at position 111 is E.

8. The protein according to any one of claims 2 to 7, wherein the protein has an amino acid substitution at position 186 and position 79, preferably, the amino acid at position 186 is Y and the amino acid at position 79 is S.

9. The protein according to any one of claims 2 to 8, wherein the protein has amino acid substitutions at position 186, position 79, position 301 and position 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 9, wherein the protein has at least one additional amino acid substitution.

11. A nucleic acid molecule encoding the protein according to any one of claims 1 to 9.

12. The nucleic acid molecule according to claim 11, encoding a protein having lipase activity, wherein the lipase is selected from the group consisting of: a) comprising 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, 121 2, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 2 18, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402; b) having a nucleic acid molecule that is at least 60%, preferably 70%, more preferably 80%, further more preferably 90%, even more preferably 95%, even further more preferably 96%, particularly preferably 97%, most preferably 98%, or especially preferably 99% identical to the nucleic acid sequence shown in a).

13. A recombinant nucleic acid molecule comprising the nucleic acid molecule according to claim 11 or 12. 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 the protein according to any one of claims 1 to 10 or the nucleic acid molecule according to claim 11 or 12 or the recombinant nucleic acid molecule according to claim 13 or 14.

16. Use of the protein according to any one of claims 1 to 10 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 racemic 2,6-dimethyl-1-indanamine.

18. A method for producing an acylated or carboxylated product, comprising 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 the protein of the present invention; d) Optionally, obtaining an acylated or carboxylated product.

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