WO2024041972A1 - Acides nucléiques codant pour des protéines lipases améliorées - Google Patents

Acides nucléiques codant pour des protéines lipases améliorées Download PDF

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WO2024041972A1
WO2024041972A1 PCT/EP2023/072709 EP2023072709W WO2024041972A1 WO 2024041972 A1 WO2024041972 A1 WO 2024041972A1 EP 2023072709 W EP2023072709 W EP 2023072709W WO 2024041972 A1 WO2024041972 A1 WO 2024041972A1
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amino acid
different
protein
preferred
nucleic acid
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PCT/EP2023/072709
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English (en)
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Christian Pitzler
Oliver Kensch
Florian Richter
Marcus Ludwig
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Bayer Aktiengesellschaft
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Publication of WO2024041972A1 publication Critical patent/WO2024041972A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase

Definitions

  • the present invention concerns proteins having improved lipase activity, nucleic acid molecules encoding respective proteins having improved lipase activity and methods for stereo-selective synthesis of chiral amines or increasing of chiral amines isomers in enantiomer mixtures.
  • Biocatalysis can be based on enzymes available in nature. More often a desire to produce a specific product creates demand for a specific enzyme, which is adapted to allow for economically feasible production of the desired product in large scale. Enzyme engineering is one option for optimizing enzymes towards the economical production of a given product.
  • Amines are ubiquitous in nature not only as parts of nucleic acids but have e.g. also great importance as tissue hormones (e.g. histamine and serotonin), neurotransmitters (e.g. dopamine and noradrenaline), as building blocks for active pharmaceutical ingredients or agricultural products.
  • tissue hormones e.g. histamine and serotonin
  • neurotransmitters e.g. dopamine and noradrenaline
  • the absolute configuration of the stereocenters of chiral amines is crucial for the synthesis of the herbicidal active agents. In the production of a desired target molecule, generation of correct chirality is often a challenge.
  • Ismail et al. describe enzymatic resolution of two chiral amines, 2-heptylamine and 2-phenylethylamine by Candida antarctica Lipase B, where different acyl donor reagents with NH, O and S moieties were tested (Hilda Ismail, Rute Madeira Lan, 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).
  • WO9728271A1 describes a method for the production of optically active amines that are useful intermediates for manufacturing pharmaceuticals or plant protection products.
  • suitable racemic amines are allowed to react in the presence of a lipase from Candida antarctica and optionally in the presence of a diluting agent with an ester and in a second step, the mixture obtained is separated
  • EP 0716712 Bl describes the lipase -catalyzed acylation of alcohols with diketenes, especially for the production of enantioselective acylated alcohols from racemic alcohols.
  • US 5,512,454 discloses a process for preparing beta lactam antibiotic intermediates involving enzymatic acylation of 3 -hydroxymethyl cephalosporins.
  • US 5,902,738 is concerned with a process for making starting material compounds used for producing the corresponding vitamin A acylates.
  • the process comprises the steps of firstly, reacting a compound with an acylating agent which is in a mixture comprising an organic solvent and a lipase which is present in a suspension, so as to form the compound and secondly, recovering the compound.
  • EP 2283144 Bl describes a process for producing sphingolipids by N-acetylation of lysophingolipids employing a lipase.
  • WO2012146935A1 discloses modified lipase variants, as well as polynucleotides and recombinant expression vectors encoding the lipase variant polypeptides, as well as methods for producing such lipase variants in selected bacterial and fungal host cells.
  • the specified lipase variants have increased enzyme specificity or enhanced trans-selectivity. Further described are methods of their use for reducing or eliminating trans-fatty acids from substrates.
  • lipases Although several improvements of lipases have been achieved so far, limitations arising during the asymmetric synthesis of amines or resolution of racemic amines, such as unfavorable equilibrium, substrate and product inhibition, poor thermostability, insufficient substrate specificity and sometimes low enantioselectivity of the lipase, still have to be overcome for an efficient production of a wide range of amines on industrial scale.
  • lipases in particular in respect to production of desired acylated or carboxylated, enantiomerically enriched or pure products, preferably under specific and/or economically viable production processes improved further lipases are needed.
  • the present invention provides lipase variants comprising modifications in their amino acid sequence, these lipase variants having improved reaction kinetics, improved substrate acceptance e.g. improved selectivity and/or improved specific activity in comparison to the respective wild-type lipase.
  • the lipase variants of the invention therefore enable the development of economically efficient production processes for acylated or carboxylated products in production methods of new acylated or carboxylated products or precursors of respective products not achievable by the use of the respective wild-type lipase.
  • the variant lipases described herein have advantages over known wild-type and other already known lipases.
  • the modified or variant lipases described herein have the advantage that they can produce enantiomerically enriched or enantiomerically nearly pure or pure compounds better than the respective wild-type lipase.
  • SEQ ID No. 1 represents the amino acid sequence of a wild-type lipolytic protein.
  • Said wild-type lipase is derived from an uncultured bacterium from an environmental sample derivable from GenPept (PDB) under accession No. QRD81023 (version ORD81023.1).
  • PDB GenPept
  • QRD81023 version ORD81023.1
  • proteins having the activity of a lipolytic enzyme or lipase respectively wherein the amino acid sequences of these proteins represent variants of a known protein having the activity of a lipolytic enzyme or lipase respectively.
  • the amino acid sequence of proteins having the activity of a lipase described herein represent variants of the amino acid sequence represented by the amino acid shown in SEQ ID No. 1, wherein in the amino acid sequence shown under SEQ ID No.
  • 1 at least the amino acid at position 186, the amino acid at position 280, the amino acid at position 312, the amino acid at position 3, the amino acid at position 29, the amino acid at position 17, the amino acid at position 4, the amino acid at position 18, the amino acid at position 202, the amino acid at position 301, the amino acid at position 309, the amino acid at position 31, the amino acid at position 111, the amino acid at position 85, the amino acid at position 8, the amino acid at position 79 or the amino acid at position 40 is different from the amino acid given at the respective amino acid position in the sequence shown under SEQ ID No. 1.
  • variants as used herein means subject-matter which is different from subject-matter known in the art.
  • variants are understood to comprise a nucleic acid sequence or an amino acid sequence, respectively, which deviates from accordingly known sequences but encode a protein having the same function or catalyzing the same reaction e.g. the function of encoding a protein having the activity of an lipase.
  • “Deviation” of nucleic acid molecule sequences and amino acid sequences from known nucleic acid sequences and protein sequences means that the sequences comprise substitutions (replacements) and/or deletions and/or insertions of nucleotides or amino acids, respectively, in comparison to the correspondingly known nucleic acid sequences or amino acid sequences.
  • a first embodiment of the invention concerns proteins having the activity of a lipase, wherein the proteins are 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%, particular preferably 98%, most preferably 99% identity with the amino acid sequence shown under SEQ ID No.
  • amino acid sequence shown under SEQ ID No. 1 comprises a modification selected from the group consisting of i.
  • the amino acid at position 186 is different from L, preferably, the amino acid at position 186 is F, W, Y, E, D, Q, T, H, P, C, K, S, N, I, or V, more preferably, the amino acid at position 186 is F, W, Y, E, D or K, particular preferably, the amino acid at position 186 is W or Y, most preferably the amino acid at position 186 is Y; ii. the amino acid at position 280 is different from L, preferably, the amino acid at position 280 is E, S, K, D or A, more preferably, the amino acid at position 280 is A; iii.
  • the amino acid at position 312 is different from P, preferably, the amino acid at position 312 is N, F, D, Q or K, more preferably, the amino acid at position 312 is N; iv. the amino acid at position 3 is different from M, preferably, the amino acid at position 3 is L, Q or C, more preferably, the amino acid at position 3 is Q; v. the amino acid at position 29 is different from N, preferably, the amino acid at position 29 is H, W or Y, more preferably, the amino acid at position 29 is H or W, most preferably, the amino acid at position 29 is H; vi. the amino acid at position 17 is different from L, preferably, the amino acid at position
  • the amino acid at position 17 is P or T, more preferably, the amino acid at position 17 is P; vii. the amino acid at position 4 is different from S, preferably, the amino acid at position 4 is P or L, more preferably, the amino acid at position 4 is P; viii. the amino acid at position 18 is different from V, preferably, the amino acid at position
  • the amino acid at position 18 is A, T, C or S, more preferably, the amino acid at position 18 is A or C; ix. the amino acid at position 202 is different from A, preferably, the amino acid at position 202 is Q or N. More preferably, the amino acid at position 202 is N; x. the amino acid at position 301 is different from D, preferably, the amino acid at position 301 is A; xi. the amino acid at position 309 is different from P, preferably, the amino acid at position 309 is C; xii. the amino acid at position 31 is different from Q, preferably, the amino acid at position 31 is W; xiii. the amino acid at position 111 is different from Q preferably, the amino acid at position 111 is E; xiv.
  • the amino acid at position 85 is different from W, preferably, the amino acid at position 85 is H; xv. the amino acid at position 8 is different from K, preferably, the amino acid at position 8 is E; xvi. the amino acid at position 79 is different from E, preferably, the amino acid at position 79 is S, I or W, more preferably, the amino acid at position 79 is S; xvii. the amino acid at position 40 is different from K, preferably, the amino acid at position 40 is M.
  • a further embodiment of the invention concerns proteins having the activity of a lipase, wherein the proteins are selected from the group consisting of a) proteins comprising the amino acid sequence as shown in SEQ ID No. 1 apart from that the amino acid at position 186 is different from L; b) proteins having 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%, particular preferably 98%, most preferably 99% identity with the amino acid sequence shown under a) given that the amino acid at position 186 is different from L.
  • 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. Particular preferably, the amino acid at position 186 is W or Y. Most preferably, the amino acid at position 186 is Y.
  • amino acid corresponding to position x in a first amino acid sequence means herein that an amino acid of a second amino acid sequence, when compared with the first amino acid sequence, appears at position x of the first amino acid sequence in a pairwise sequence alignment of the first amino acid sequence with the second amino acid sequence in case the numbering of the amino acids of the second amino acid sequence differs from the amino acid numbering of the first amino acid sequence.
  • identity in respect to sequence identity or sequences being identical to is to be understood as meaning the number of identical amino acids or nucleotides shared over the entire sequence length by a first nucleic or amino acid sequence with another (second) nucleic or amino acid sequence, respectively, expressed in percent.
  • Sequence alignments and scores for percentage sequence identity may for example be determined using software, such as EMBOSS, accessible at world wide web site of the EBI (ebi.ac.uk/Tools/emboss/).
  • sequence similarity or identity may be determined by searching against databases (e.g. EMBL, GenBank) by using commonly known algorithms and output formats such as FASTA, BLAST, etc., but preferably hits should be retrieved and aligned pairwise to finally determine sequence identity.
  • the identity is to be determined by determining the identity in percent of the number of amino acids or nucleotides, respectively, which the shorter sequence shares with the longer sequence.
  • the identity is determined using the known and publicly available computer program ClustalW (Thompson et al., Nucleic Acids Research 22 (1994), 4673-4680).
  • ClustalW is made publicly available by Julie Thompson (Thompson@EMBL- Heidelberg.DE) and Toby Gibson (Gibson@EMBL-Heidelberg.DE), European Molecular Biology Laboratory, Meyerhofstrasse 1, D 69117 Heidelberg, Germany.
  • ClustalW can also be downloaded from various Internet pages, inter aha from IGBMC (Institut de Genetique et de Biologic Moleisme et Cellulaire, B.P.163, 67404 Illkirch Cedex, France; ftp://ftp-igbmc.u-strasbg.fr/pub/) and from EBI (ftp://ftp.ebi.ac.uk/pub/software/) and all mirrored Internet pages of the EBI (European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK).
  • the ClustalW computer program of version 1.8 determines the identity between proteins described in the context of the present invention and other proteins.
  • the ClustalW computer program of version 1.8 determines the identity for example between the nucleotide sequence of the nucleic acid molecules described in the context of the present invention and the nucleotide sequence of other nucleic acid molecules.
  • the parameters have to be set as follows:
  • “Identity” furthermore means that there is a functional and/or structural equivalence between the nucleic acid molecules in question or the proteins encoded by them.
  • Functional equivalence means that the nucleic acid molecule sequences or the amino acid sequences encode a protein having the activity of a lipase.
  • the nucleic acid molecules which are homologous to the molecules described above and represent derivatives of these molecules are generally variants of these molecules which represent modifications having the same biological function or catalyzing the same reaction, i.e. coding for a protein having the activity of a lipase. They may be either naturally occurring variants, for example sequences from other species, or mutations, where these mutations may have occurred in a natural manner or were introduced by targeted mutagenesis.
  • variants may be synthetically produced sequences.
  • allelic variants may be either naturally occurring variants or synthetically produced variants or variants generated by recombinant DNA techniques.
  • those variants encode proteins having lipase -activity and comprise the amino acid substitutions (replacements), deletions or insertions described herein concerning the proteins according to the invention.
  • a special type of derivatives are, for example, nucleic acid molecules which differ from the nucleic acid molecules described in the context of the present invention as a result of the degeneracy of the genetic code.
  • hydrolase is a class of enzymes that commonly perform as biochemical catalysts that use water to break a chemical bond, which typically results in dividing a larger molecule into smaller molecules.
  • the group of hydrolases comprises enzymes acting on ester bonds (EC 3.1) encompassing carboxylic ester hydrolases (EC 3.1.1) and as a subgroup lipases (EC 3. 1.1.3).
  • Lipases have been identified from plants, mammals and microorganisms including e.g.
  • Pseudomonas Vibrio, Acinetobacter, Burkholderia, Chromobacterium, cutinase from Fusarium solani (FSC), Candida antarctica A (CalA), Rhizopus oryzae (ROL), Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML), Aspergillus Niger, Fusarium heterosporum, Fusarium oxysporum or Fusarium culmorum.
  • a protein has the activity of a lipase, this can be detected with methods known and described in the art.
  • Lipase variant proteins according to the invention may exhibit further amino acid modifications (amino acid substitutions, deletions or insertions) compared to the amino acid sequences described herein above in respect to the amino acid sequence shown under SEQ ID No. 1.
  • the inventive protein having the activity of a lipase is selected from the group consisting of a) proteins comprising the amino acid sequence shown in SEQ ID No.
  • amino acid at position 186 1 apart from that the amino acid at position 186 is different from L and have at least one, two, three, four, five, six, seven or more further 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; and b) proteins having 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%, particular preferably 98%, most preferably 99% identity with the amino acid sequence shown directly
  • 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.
  • the lipase variant proteins according to the invention may exhibit, apart from the further amino acid modifications, additional amino acid substitutions compared to the amino acid sequences described herein above in respect to the amino acid sequence shown under SEQ ID No. 1. These additional amino acid substitutions relate to positions of the amino acid sequence that are different than the position/s 79, 202, 280, 301, 3, 11, 17, 40 or 111 which relate to the further amino acid modifications.
  • the lipase variants described herein above under item a) with reference to the amino acid sequence shown under SEQ ID No. 1, can have at least one, two, three, four, five, six, seven or more additional amino acid substitution/s at position/s 4, 8, 18, 29, 31, 42, 84, 85, 192, 217, 309 or 312.
  • the amino acid at position 4 is different from S, preferably the amino acid at that position is P.
  • the amino acid at position 8 is different from K, preferably the amino acid at that position is E.
  • the amino acid at position 18 is different from V, preferably the amino acid at that position is C.
  • the amino acid at position 29 is different from N, preferably the amino acid at that position is W or H.
  • the amino acid at position 31 is different from Q, preferably the amino acid at that position is W.
  • the amino acid at position 42 is different from L, preferably the amino acid at that position is D.
  • the amino acid at position 84 is different from N, preferably the amino acid at that position is T.
  • the amino acid at position 85 is different from W, preferably the amino acid at that position is H.
  • the amino acid at position 192 is different from F, preferably the amino acid at that position is A or V.
  • the amino acid at position 217 is different from Q, preferably the amino acid at that position is M.
  • the amino acid at position 309 is different from P, preferably the amino acid at that position is C.
  • the amino acid at position 312 is different from P, preferably the amino acid at that position is N.
  • a further embodiment of the invention therefore concerns proteins according to the invention comprising further amino acid modifications, preferably those embodiments are proteins having the activity of a lipase, wherein the proteins are selected from the group consisting of proteins comprising the amino acid sequence shown in SEQ ID No. 1 apart from that
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E, wherein it is preferred that the amino acid at position 186 is preferably W or Y, more preferably Y, and the amino acid at position 79 is preferably S, W or I, more preferably S;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N;
  • the amino acid at position 186 is different from L and the amino acid at position 280 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 301 is different from D, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 301 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 3 is different from M, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 3 is preferably Q;
  • the amino acid at position 186 is different from L and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 11 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 17 is preferably P;
  • the amino acid at position 186 is different from L and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 111 is preferably E;
  • amino acid sequence being at least 80%, preferably 85%, more preferably 90%, further more preferably 95%, even more preferably 96%, even further more preferably 97%, particular preferably 98%, most preferably 99% identity with the amino acid sequence shown under a) given that the amino acid at position 186 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the proteins have at least one further amino acid substitution selected from the group shown under the listed punctuation marks directly here above.
  • the lipase variants described herein above under item a) with reference to the amino acid sequence shown under SEQ ID No. 1, can have at least two further amino acid substitution/s at position/s 79, 202, 280, 301, 3, 11, 17, 40 or 111.
  • 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.
  • a further embodiment of the invention therefore concerns proteins according to the invention comprising further amino acid modifications, preferably those embodiments are proteins having the activity of a lipase, wherein the proteins are selected from the group consisting of proteins comprising the amino acid sequence shown in SEQ ID NO. 1 apart from that
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 202 is preferably N;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 280 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 280 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 301 is different from D, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 301 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 3 is different from M, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 3 is preferably Q;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 17 is preferably P;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 79 is preferably S, W or I, more preferably S, and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 280 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 301 is different from D, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 301 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 3 is different from M, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 3 is preferably Q;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 11 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 17 is preferably P;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A and the amino acid at position 301 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 3 is different from M, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A and the amino acid at position 3 is preferably Q;
  • the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A and the amino acid at position 11 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A and the amino acid at position 17 is preferably P;
  • the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 301 is preferably A and the amino acid at position 3 is preferably Q;
  • the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 301 is preferably A and the amino acid at position 11 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 301 is preferably A and the amino acid at position 17 is preferably P;
  • the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 301 is preferably A and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 301 is preferably A and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 3 is preferably Q and the amino acid at position 11 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 3 is preferably Q and the amino acid at position 17 is preferably P;
  • the amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 3 is preferably Q and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 3 is preferably Q and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 11 is preferably A and the amino acid at position 17 is preferably P;
  • the amino acid at position 186 is different from L and the amino acid at position 11 is different from C and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 11 is preferably A and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L and the amino acid at position 11 is different from C and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 11 is preferably A and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 17 is preferably P and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 17 is different from L and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 17 is preferably P and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 40 is preferably M and the amino acid at position 111 is preferably E;
  • amino acid sequence being at least 80%, preferably 85%, more preferably 90%, further more preferably 95%, even more preferably 96%, even further more preferably 97%, particular preferably 98%, most preferably 99% identity with the amino acid sequence shown under a) given that the amino acid at position 186 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the proteins have at least two further amino acid substitutions selected from the group shown under the listed punctuation marks directly here above.
  • the lipase variants described herein above under item a) with reference to the amino acid sequence shown under SEQ ID No. 1, can have at least three further amino acid substitution/s at position/s 79, 202, 280, 301, 3, 11, 17, 40 or 111.
  • 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.
  • a further embodiment of the invention therefore concerns proteins according to the invention comprising further amino acid modifications, preferably those embodiments are proteins having the activity of a lipase, wherein the proteins are selected from the group consisting of proteins comprising the amino acid sequence shown in SEQ ID No. 1 apart from that - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 280 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A and the amino acid at position 301 is different from D, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 301 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A and the amino acid at position 3 is different from M, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 3 is preferably Q;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 11 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 17 is preferably P;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 202 is different from A and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 301 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 280 is different from L and the amino acid at position 3 is different from M, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 3 is preferably Q;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 280 is different from L and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 11 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 280 is different from L and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 17 is preferably P;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 280 is different from L and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 280 is different from L and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 3 is preferably Q;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 301 is different from D and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 11 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 301 is different from D and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 17 is preferably P;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 301 is different from D and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, more preferably Y, and 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 and the amino acid at position 40 is preferably M, wherein it is especially preferred that the amino acid at position 186 is Y, and the amino acid at position 79 is S, and the amino acid at position 301 is A and the amino acid at position 40 is M;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 301 is different from D and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 11 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 3 is different from M and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 17 is preferably P;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 3 is different from M and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 3 is different from M and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 17 is preferably P;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 11 is different from C and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 11 is different from C and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 17 is different from L and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 79 is different from E and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and 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 and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 280 is preferably A and the amino acid at position 301 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L and the amino acid at position 3 is different from M, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 280 is preferably A and the amino acid at position 3 is preferably Q;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 280 is preferably A and the amino acid at position 11 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 280 is preferably A and the amino acid at position 17 is preferably P;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 280 is preferably A and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 280 is different from L and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 280 is preferably A and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 301 is preferably A and the amino acid at position 3 is preferably Q;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 301 is different from D and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 301 is preferably A and the amino acid at position 11 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 301 is different from D and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 301 is preferably A and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 301 is different from D and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 301 is preferably A and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 301 is different from D and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 301 is preferably A and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 3 is preferably Q and the amino acid at position 11 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 3 is different from M and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 3 is preferably Q and the amino acid at position 17 is preferably P;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 3 is different from M and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 3 is preferably Q and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 3 is different from M and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 3 is preferably Q and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 11 is preferably A and the amino acid at position 17 is preferably P;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 11 is different from C and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 11 is preferably A and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 11 is different from C and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 11 is preferably A and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 17 is preferably P and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 17 is different from L and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 17 is preferably P and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 202 is different from A and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 202 is preferably N and the amino acid at position 40 is preferably M and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A and the amino acid at position 301 is preferably A and the amino acid at position 3 is preferably Q;
  • the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A and the amino acid at position 301 is preferably A and the amino acid at position 11 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A and the amino acid at position 301 is preferably A and the amino acid at position 17 is preferably P; - the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A and the amino acid at position 301 is preferably A and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 301 is different from D and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A and the amino acid at position 301 is preferably A and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A and the amino acid at position 3 is preferably Q and the amino acid at position 11 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 3 is different from M and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A and the amino acid at position 3 is preferably Q and the amino acid at position 17 is preferably P;
  • the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 3 is different from M and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A and the amino acid at position 3 is preferably Q and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 3 is different from M and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A and the amino acid at position 3 is preferably Q and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A and the amino acid at position 11 is preferably A and the amino acid at position 17 is preferably P;
  • the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 11 is different from C and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A and the amino acid at position 11 is preferably A and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 11 is different from C and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A and the amino acid at position 11 is preferably A and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A and the amino acid at position 17 is preferably P and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 17 is different from L and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A and the amino acid at position 17 is preferably P and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 280 is different from L and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 280 is preferably A and the amino acid at position 40 is preferably M and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 301 is preferably A and the amino acid at position 3 is preferably Q and the amino acid at position 11 is preferably A;
  • the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 301 is preferably A and the amino acid at position 3 is preferably Q and the amino acid at position 17 is preferably P;
  • the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 301 is preferably A and the amino acid at position 3 is preferably Q and the amino acid at position 40 is preferably M; - the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 3 is different from M and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 301 is preferably A and the amino acid at position 3 is preferably Q and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 301 is preferably A and the amino acid at position 11 is preferably A and the amino acid at position 17 is preferably P;
  • the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 11 is different from C and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 301 is preferably A and the amino acid at position 11 is preferably A and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 11 is different from C and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 301 is preferably A and the amino acid at position 11 is preferably A and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 301 is preferably A and the amino acid at position 17 is preferably P and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 17 is different from L and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 301 is preferably A and the amino acid at position 17 is preferably P and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 301 is different from D and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 301 is preferably A and the amino acid at position 40 is preferably M and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 3 is preferably Q and the amino acid at position 11 is preferably A and the amino acid at position 17 is preferably P;
  • the amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 3 is preferably Q and the amino acid at position 11 is preferably A and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 11 is different from C and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 3 is preferably Q and the amino acid at position 11 is preferably A and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 3 is preferably Q and the amino acid at position 17 is preferably P and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 17 is different from L and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 3 is preferably Q and the amino acid at position 17 is preferably P and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 3 is different from M and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 3 is preferably Q and the amino acid at position 40 is preferably M and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 11 is preferably A and the amino acid at position 17 is preferably P and the amino acid at position 40 is preferably M;
  • the amino acid at position 186 is different from L and the amino acid at position 11 is different from C and the amino acid at position 17 is different from L and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 11 is preferably A and the amino acid at position 17 is preferably P and the amino acid at position 111 is preferably E; - the amino acid at position 186 is different from L and the amino acid at position 11 is different from C and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 11 is preferably A and the amino acid at position 40 is preferably M and the amino acid at position 111 is preferably E;
  • the amino acid at position 186 is different from L and the amino acid at position 17 is different from L and the amino acid at position 40 is different from K and the amino acid at position 111 is different from Q, wherein it is preferred that the amino acid at position 186 is preferably W or Y, and the amino acid at position 17 is preferably P and the amino acid at position 40 is preferably M and the amino acid at position 111 is preferably E;
  • amino acid sequence being at least 80%, preferably 85%, more preferably 90%, further more preferably 95%, even more preferably 96%, even further more preferably 97%, particular preferably 98%, most preferably 99% identity with the amino acid sequence shown under a) given that the amino acid at position 186 is different from L wherein it is preferred that the amino acid at position 186 is preferably W or Y, more preferably Y, and the proteins have at least three further amino acid substitutions selected from the group shown under the listed punctuation marks directly here above.
  • the lipase variants described herein above under item a) with reference to the amino acid sequence shown under SEQ ID No. 1, can have at least four further amino acid substitution/s at position/s 79, 202, 280, 301, 3, 11, 17, 40 or 111.
  • 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.
  • the lipase variants described herein above under item a) with reference to the amino acid sequence shown under SEQ ID No. 1, can have at least five further amino acid substitution/s at position/s 79, 202, 280, 301, 3, 11, 17, 40 or 111.
  • 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.
  • the lipase variants described herein above under item a) with reference to the amino acid sequence shown under SEQ ID No. 1, can have at least six further amino acid substitution/s at position/s 79, 202, 280, 301, 3, 11, 17, 40 or 111.
  • 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.
  • the lipase variants described herein above under item a) with reference to the amino acid sequence shown under SEQ ID No. 1, can have at least seven further amino acid substitution/s at position/s 79, 202, 280, 301, 3, 11, 17, 40 or 111.
  • the amino acid at position 79 is S, W or I, more preferably S; preferably the amino acid at position 202 is N; preferably the amino acid at position 280 is A; preferably the amino acid at position 301 is A; preferably the amino acid at position 3 is Q; preferably the amino acid at position 11 is A; preferably the amino acid at position 17 is P; preferably the amino acid at position 40 is M; preferably the amino acid at position 111 is E.
  • Preferred embodiments of the invention are proteins according to the invention encoding lipases having the amino acid sequences shown under 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,
  • amino acid sequences of these further proteins represent variants of the amino acid sequence represented by the amino acid in SEQ ID No. 1, wherein:
  • the two amino acids at positions 40 and 79 are different from the amino acids given at the respective amino acid positions in the sequence shown under SEQ ID No. 1.
  • the amino acid at position 40 is M and the amino acid at position 79 is S;
  • the two amino acids at positions 40 and 186 are different from the amino acids given at the respective amino acid positions in the sequence shown under SEQ ID No. 1.
  • the amino acid at position 40 is M and the amino acid at position 186 is Y;
  • the amino acid at positions 40 and 301 are different from the amino acids given at the respective amino acid positions in the sequence shown under SEQ ID No. 1.
  • the amino acid at position 40 is M and the amino acid at position 301 is A;
  • the two amino acids at positions 79 and 186 are different from the amino acids given at the respective amino acid positions in the sequence shown under SEQ ID No. 1.
  • the amino acid at position 79 is S and the amino acid at position 186 is Y;
  • the two amino acids at positions 79 and 301 are different from the amino acids given at the respective amino acid positions in the sequence shown under SEQ ID No. 1.
  • the amino acid at position 79 is S and the amino acid at position 301 is A;
  • the two amino acids at positions 186 and 301 are different from the amino acids given at the respective amino acid positions in the sequence shown under SEQ ID No. 1.
  • the amino acid at position 186 is Y and the amino acid at position 301 is A.
  • amino acid sequences of these further proteins represent variants of the amino acid sequence represented by the amino acid in SEQ ID No. 1, wherein:
  • the three amino acids at positions 40, 79 and 186 are different from the amino acids given at the respective amino acid positions in the sequence shown under SEQ ID No. 1.
  • the amino acid at position 40 is M and the amino acid at position 79 is S and the amino acid at position 186 is Y;
  • the three amino acids at positions 40, 79 and 301 are different from the amino acids given at the respective amino acid positions in the sequence shown under SEQ ID No. 1.
  • the amino acid at position 40 is M and the amino acid at position 79 is S and the amino acid at position 301 is A;
  • the three amino acids at positions 40, 186 and 301 are different from the amino acids given at the respective amino acid positions in the sequence shown under SEQ ID No. 1.
  • the amino acid at position 40 is M and the amino acid at position 186 is Y and the amino acid at position 301 is A;
  • the three amino acids at positions 79, 186 and 301 are different from the amino acids given at the respective amino acid positions in the sequence shown under SEQ ID No. 1.
  • the amino acid at position 79 is S and the amino acid at position 186 is Y and the amino acid at position 301 is A;
  • the lipase variants or protein variants according to the invention show improved selectivity and/or improved specific activity regarding the stereoselective acylation or carboxylation of 2,6-Dimethyl-l- indanamine (DMAI) and are better adapted to produce enantiomerically enriched or nearly pure methyl- [(lR,2S)-2,6-dimethyl-2,3-dihydro-lH-inden-l-yl] carbamate compared to the wild-type lipase.
  • Methyl- [(lR,2S)-2,6-dimethyl-2,3-dihydro-lH-inden-l-yl] carbamate is an important intermediate for the synthesis of the herbicidal active compound Indaziflam.
  • Enantiomerically enriched means herein that one of two enantiomers is present in a composition in higher amounts 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, particular 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.
  • Enantiomerically nearly pure means herein that one of two enantiomers is present in a composition in amounts of at least 95.0%, preferably one of two enantiomers is present in a composition in amounts of at least 95.5%, more preferably one of two enantiomers is present in a composition in amounts of at least 96.0%, further more preferably one of two enantiomers is present in a composition in amounts of at least 96.5%, even more preferably one of two enantiomers is present in a composition in amounts of at least 97.0%, even further more preferably one of two enantiomers is present in a composition in amounts of at least 98.0%, particular preferably one of two enantiomers is present in a composition in amounts of at least 98.5%, most preferably one of two enantiomers is present in a composition in amounts of at least 99.0%, or especially preferably one of two enantiomers is present in a composition in amounts of at least 99.5%.
  • One further embodiment of the invention concerns nucleic acid molecules encoding a protein according to the invention.
  • Nucleic acid molecules according to the invention can be any kind of nucleic acid, as long as the nucleic acid encodes a protein according to the invention.
  • the nucleic acids can be ribonucleic nucleic acid molecules (e.g. RNA, mRNA) or deoxyribonucleic nucleic acid molecules (DNA, including genomic DNA which may or may not comprise introns and coding DNA).
  • nucleic acid molecules encoding proteins having the activity of a lipase comprising the amino acid sequences shown under 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,
  • the invention therefore also concerns nucleic acid molecules encoding a protein having the activity of a lipase selected from the group consisting of a) nucleic acid molecules comprising the nucleic acid sequences shown under SEQ ID Nos. 4, 6, 8,
  • nucleic acid molecules having at least 60%, preferably 70%, more preferably 80%, further more preferably 90%, even more preferably 95%, even further more preferably 96%, particular preferably 97%, most preferably 98% or especially preferably 99% identity with the nucleic acid sequences shown under a).
  • hybridizing with means hybridization under conventional hybridization conditions, preferably under stringent conditions, as described, for example, in Sambrook et al. (Molecular Cloning, A Laboratory Manual, 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ISBN: 0879695773) or Ausubel et al. (Short Protocols in Molecular Biology, John Wiley & Sons; 5th edition (2002), ISBN: 0471250929).
  • “hybridization” means a hybridization under the following conditions: hybridization buffer:
  • 2xSSC 2xSSC; lOxDenhardt solution (Fikoll 400+PEG+BSA; ratio 1: 1: 1); 0.1% SDS; 5 mM EDTA; 50 mM Na2HPO4; 250 pg/ml of herring sperm DNA; 50 pg/ml of tRNA; or
  • Nucleic acid molecules which hybridize with nucleic acid molecules coding for a protein having the activity of a lipase may originate from any organism; accordingly, they may originate from bacteria, fungi, animals, humans, plants or viruses.
  • Nucleic acid molecules which hybridize with nucleic acid molecules coding for a protein having the activity of a lipase preferably originate from microorganisms, more preferably from fungi or bacteria, most preferably from bacteria.
  • Nucleic acid molecules which hybridize with the molecules mentioned may be isolated, for example, from genomic or from cDNA libraries. Such nucleic acid molecules can be identified and isolated using the nucleic acid molecules described herein or they can be identified and isolated using parts of these molecules or the reverse complements 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 amplification using PCR.
  • the fragments used as hybridization samples may also be synthetic fragments or oligonucleotides prepared using the customary synthesis techniques, whose sequence is essentially identical to the nucleic acid molecule described in the context of the present invention.
  • the molecules hybridizing with the nucleic acid molecules described in the context of the present invention comprise in particular fragments, derivatives and allelic variants of the nucleic acid molecules mentioned.
  • the term “derivative” means that the sequences of these molecules differ in one or more positions from the sequences of the nucleic acid molecules described above and are highly identical to these sequences.
  • the differences to the nucleic acid molecules described above may, for example, be due to deletion, addition, substitution, insertion or recombination.
  • Preferred nucleic acid molecules according to the invention are those nucleic acid molecules shown under SEQ ID Nos. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,
  • nucleotide abbreviations a, c, g, t, and those of abbreviations for degenerate nucleotides r, y, s, w, k, m, b, d, h, v, n is derivable herein below from Table 1 under the paragraph sub-titled “Description of the Sequences”. Which amino acids are encoded by codons comprising degenerate nucleotides is derivable herein below from Table 3 under the paragraph sub-titled “Description of the Sequences”.
  • the invention relates to recombinant nucleic acid molecules comprising a nucleic acid molecule according to the invention.
  • the term “heldrecombinant nucleic acid molecule“ is to be understood to mean a nucleic acid molecule, which contains additional sequences in addition to nucleic acid molecules according to the invention, which do not naturally occur in the combination in which they occur in recombinant nucleic acids according to the invention.
  • the abovementioned additional sequences can be any sequences, preferably they are functional or regulatory sequences (promoters, termination signals, enhancers, ribosome binding sites (rbs), leader sequences enhancing transcription, translation or RNA stability, subcellular targeting sequences etc.), particularly preferably they are functional or regulatory sequences that are active in microorganisms, and especially particularly preferably they are regulatory sequences that are active in fungi, in particular yeasts or in bacteria.
  • Methods for the creation of recombinant nucleic acid molecules according to the invention are known to the person skilled in the art and include genetic methods such as bonding nucleic acid molecules by way of ligation, genetic recombination, or new synthesis of nucleic acid molecules.
  • the recombinant nucleic acid molecules according to the invention comprise a nucleic acid molecule according to the invention which is linked with regulatory sequences, which initiate transcription in prokaryotic or eukaryotic cells.
  • Regulatory sequences, which initiate transcription” in a cell are also known as promoters.
  • Regulatory sequences which initiate transcription in prokaryotic organisms e.g. E. coli, and in eukaryotic organisms are sufficiently described in literature, in particular such for expression in yeast are described, e.g. Saccharomyces cerevisiae.
  • An overview of various systems for expression for proteins in various host organisms can be found, for example, in Methods in Enzymology 153 (1987), 383-516 and in Bitter et al. (Methods in Enzymology 153 (1987), 516-544) or in Gomes et al. (2016, Advances in Animal and Veterinary Sciences, 4(4), 346) and Baghban et al. (2018, Current Pharmaceutical Biotechnology, 19(6)).
  • Common yeast promoters are pAOXl, pHIS4, pGAL, pScADH2 (Baghban et al., 2018, see above).
  • Common bacterial promoters are T5, T7, rhamnose-inducible, arabinose-inducible, PhoA, artificial trc (trp-lac) promoter as described by Marschall et al. (2017, Appl Microbiol Biotechnol 101, 501-512) and Tegel et al. (2011, FEBS Journal 278, 729-739).
  • a further embodiment of recombinant nucleic acid molecules of the present invention are vectors or plasmids, which comprise the nucleic acid molecules according to the invention.
  • Vectors are commonly understood in the field of molecular biology and herein to represent a nucleic acid sequence or a vehicle comprising a nucleic acid sequence used to transfer genetic material (DNA or RNA) into a target cell.
  • Vectors can be plasmids, e.g. T-DNA or binary vectors for generating transgenic plants, expression vectors for expression of nucleic acid sequences in a host cell, shuttle vectors which are eligible to propagate in different hosts, or vectors can be virus particles or bacteriophages having been modified to deliver foreign genetic material into a host.
  • “Plasmids” are commonly understood in the field of molecular biology and herein to represent an autonomously self-replicating, often circular DNA molecule which is when present in a host cell separated from the chromosomal DNA.
  • Nucleic acid molecules according to the invention, recombinant nucleic acid molecules according to the invention, vectors or plasmids according to the invention can be used for production of proteins according to the invention, e.g. by expressing the nucleic acid molecules according to the invention in host cells.
  • Another embodiment of the invention concerns hosts or host cells comprising or expressing a nucleic acid molecule according to invention or comprising proteins 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.
  • nucleic acid molecules according to the invention encoding a protein having the activity of a lipase can be expressed in host cells for e.g. their multiplication or for production of proteins according to the invention.
  • nucleic acid molecules according to the invention can be comprised on vectors or plasmids or they can be stably integrated into the genome of a respective host cell.
  • the nucleic acid molecules according to the invention can also be comprised by vectors which support their introduction into host cells.
  • a further embodiment of the present invention concerns 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 comprising a protein according to the invention.
  • Another embodiment of the present invention concerns 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 according to the invention.
  • Another embodiment of the present invention concerns 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 the activity of a lipase.
  • “Expressing a nucleic acid molecule” shall be understood herein to mean that in case the nucleic acid molecule is RNA or mRNA the nucleic acid molecule is translated into a protein, preferably translated into a protein having the activity of a lipase or in case of the nucleic acid molecule is DNA or cDNA it is transcribed (and in case of genomic DNA containing introns is processed) into mRNA, preferably into a mRNA encoding a protein having the activity of a lipase and subsequently translated into a protein, preferably translated into a protein having the activity of a lipase.
  • Transcription of a given nucleic acid molecule in a host can be demonstrated by methods known to a person skilled in the art, for example, by detection of specific transcripts (mRNA) of foreign nucleic acid molecules by Northern blot analysis or RT-PCR.
  • mRNA specific transcripts
  • hosts or host cells comprise a given protein or comprise a protein which is derived from expressing a nucleic acid molecule can be determined by methods known to a person skilled in the art, for example, by immunological methods, such as Western blot analysis, ELISA (Enzyme Linked Immuno Sorbent Assay) or RIA (Radio Immune Assay).
  • immunological methods such as Western blot analysis, ELISA (Enzyme Linked Immuno Sorbent Assay) or RIA (Radio Immune Assay).
  • the person skilled in the art is familiar with methods for preparing antibodies which react specifically with a certain protein, i.e. which bind specifically to a certain protein (see, for example, Lottspeich and Zorbas (eds.), 1998, Bioanalytik, Spektrum akad, Verlag, Heidelberg, Berlin, ISBN 3-8274-0041-4).
  • Some companies (Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA USA 0245; GenScript, 60 Centennial Ave., Pi
  • a person skilled in the art can test if a host or host cell comprises a protein according to the invention by detecting (additional) activity of proteins having the activity of a lipase in a respective host cell.
  • activity of proteins having additional activity of a lipase in a respective host cell is detected by comparing the activities of lipases of a host cell according to the invention with the respective activity of host cell not comprising a protein according to the invention.
  • Testing if a protein has the activity of a lipase can be done by methods known in the art.
  • Host or host cells according to the invention can be produced by a person skilled in the art by known methods for genetically modifying or transforming organisms.
  • a further subject of the present invention therefore is a host or host cell according to the invention, particularly a prokaryotic or eukaryotic host or host cell, which is genetically modified (or transformed) with a nucleic acid molecule according to the invention or with a recombinant nucleic acid molecule according to the invention or with a vector according to the invention or a plasmid according to the invention.
  • the genetically modified (transformed) host or host cell according to the invention expresses a protein having the activity of a lipase, more preferably, the genetically modified (transformed) host or host cell according to the invention expresses a protein according to the invention.
  • Genetically modified with a nucleic acid molecule or “transformed with a nucleic acid molecule” shall be understood herein to mean that a nucleic acid molecule is or was introduced into a host or host cell by technical and/or non-naturally occurring means, preferably by technical methods in the field of molecular biology, biotechnology or genetic modification.
  • Descendants, offspring or progeny of hosts or host cells according to the invention are also an embodiment of the invention, preferably these descendants, offspring or progeny comprise a nucleic acid molecule according to the invention or comprise a recombinant nucleic acid molecule according to the invention or comprise a vector according to the invention or comprise a plasmid according to the invention or comprise a protein according to the invention, more preferably these descendants, offspring or progeny comprise a nucleic acid molecule according to the invention or comprise a recombinant nucleic acid molecule according to the invention or comprise a vector according to the invention or comprise a plasmid according to the invention and, in each case express a protein, wherein the protein has the activity of a lipase, even more preferably these descendants, offspring or progeny comprise a nucleic acid molecule according to the invention or comprise a recombinant nucleic acid molecule according to the invention or comprise a vector according to the invention or comprise a plasmid
  • the host or host cell according to the invention can be a host or host cell from any prokaryotic or eucaryotic organism.
  • the hosts or host cells can be bacteria or bacteria cells (e.g. E. coli, bacteria of the genus Bacillus, in particular Bacillus subtilis, Agrobacterium, particularly Agrobacterium tumefaciens or Agrobacterium rhizogenes, Pseudomonas, particularly Pseudomonas fluorescens, Streptomyces spp, Rhodococcus spp, in particular Rhodococcus rhodochrous, Vibrio natrigens, Corynebacterium, particularly Corynebacterium glutamicum) or fungi or fungal cells (e.g.
  • Agaricus in particular Agaricus bisporus, Aspergillus, Trichoderma or yeasts, particularly .S', cerevisiae, Pichia ssp. like P. pastoris), as well as plants or plant cells or they can be animals or animal cells.
  • Preferred host cells according to the invention are cells of microorganisms.
  • this is understood to include all bacteria and all protists (e.g. fungi, particularly yeasts and algae), as they are defined in Schlegel "General Microbiology” (Georg Thieme Publishing House (1985), 1-2), for example.
  • the hosts or host cells according to the invention are preferably bacteria/bacteria cells or yeast/yeast cells, most preferably they are bacteria/bacteria cells.
  • the hosts or host cells according to the invention are preferably Bacillus species/ Bacillus species cells or Escherichia coli I Escherichia coli cells cells most preferably Escherichia coli I Escherichia coli cells.
  • Pseudomonas particularly Pseudomonas fluorescens, Streptomyces spp, Rhodococcus spp, in particular Rhodococcus rhodochrous
  • Vibrio spp particularly Vibrio natrigens
  • Corynebacterium particularly Corynebacterium glutamicum or others
  • hosts or host cells according to the invention can be hosts or host cells according to the invention.
  • a preferred embodiment of the invention concerns hosts or host cells according to the invention comprising a nucleic acid molecule according to the invention, wherein the nucleic acid molecule according to the invention is characterized in that the codons of said nucleic acid molecule are changed such that they are adapted to the frequency of use of the codons of the host or a host cell, respectively.
  • Host cells according to the invention can be used for production of proteins according to the invention.
  • Proteins according to the invention can be used in methods for production of enantiomerically enriched or nearly enantiomerically pure acylated or carboxylated products from a acyl or carboxyl acceptor in the presence of an acyl or carboxyl donor.
  • Another embodiment of the invention therefore concerns a method for the production of 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 a protein according to the invention; d) optionally, obtaining the acylated or carboxylated product.
  • a further embodiment of the invention is the use of a protein according to the invention production of an amine, preferably for production of an ( l/?.2.S')-aminc.
  • a further embodiment of the invention is the use of a protein according to the invention for the stereoselective acylation or carboxylation of racemic 2,6-dimethyl-l-indanylamine.
  • a further embodiment of the invention is the use of a protein according to the invention for the stereoselective acylation or carboxylation of racemic 2,6-dimethyl-l-indanylamine, wherein the substrate is racemic 2,6-dimethyl-l-indanylamine.
  • Codon usage follows herein the so called “general genetic code” according to the following Table, wherein “t” is to be substituted by “u” in ribonucleic acid (RNA) sequences.
  • TB media were prepared in demineralized water with the use of 47.6 g/1 of granulated medium and 4ml/L glycerol and sterilized at 121 °C for 20 minutes.
  • Nucleotide sequences encoding lipase variants as described herein can be synthesized as known in the art, e.g. as offered by respective service providers such as Eurofins Genomics GmbH (Eurofins Genomics GmbH, Anzinger Str. 7a, 85560 Ebersberg, Germany).
  • nucleic acid sequences of wild-type lipase (SEQ ID NO. 2) or related variants as described herein were cloned into an expression vector based on the vector pKA81a. Genetic elements were introduced into the modified pKA81a vector by means of commonly known methods.
  • the expression vectors were introduced into electro-competent Escherichia coli MG 1655 cells.
  • Nucleotide substitutions were introduced into the nucleic acid parent sequences, e.g. to obtain an amino acid exchange into other amino acids.
  • Several Molecular Biological methods can be used to achieve these replacements.
  • a useful method for preparing a mutated nucleic acid and the corresponding mutated protein according to the invention is site-directed mutagenesis on codons encoding one or more amino acids, which are selected in advance. The methods for obtaining these site-directed mutations are well known to the skilled person and widely described in the literature (in particular: Directed Mutagenesis: A Practical Approach, 1991, Edited by M.J.
  • McPHERSON, IRL PRESS are methods for which it is possible to employ commercial kits (for example the QUIKCHANGETM lightening mutagenesis kit from Qiagen or Stratagene). After site-directed mutagenesis, nucleic acids were transformed into the Escherichia coli MG 1655 cells.
  • Transformed cells were tested in appropriate biotransformation reactions in order to determine product yield and selectivity. Appropriate biotransformation reactions are described below, see e.g. Example 2. Sequence verification was performed as known in the art.
  • Glycerol stocks of the E. coli cultures transformed with the respective expression plasmids were prepared by adding one volume of 40% glycerol solution to one volume of E. coli culture.
  • Escherichia coli MG1655 was used as host for expression plasmids.
  • TB TB
  • Fisher Bioreagents, 22711022 50pg/ml kanamycin
  • 10 pL of glycerol stocks of the respective variants or alternatively 590 pL TB-medium were inoculated with cell material from Agar plate colonies.
  • Pre-cultures were incubated for 17 hours at 37°C and 250 rpm in a climo shaker ISF1-X (Ktihner AG, Birsfelden, Switzerland).
  • Deep-well plates containing the cell suspensions were equilibrated to room temperature. Biotransformation reactions were started by addition of 13.2 pL of racemic 2,6-dimethyl-l-indanylamine (DMAI) and 186.8 pl of dimethylcarbonate to the lyophilized culture and incubation occurred for 8h-22 h at 70°C or 80°C in a shaking incubator at 250rpm. Reactions were stopped by adding 600 pl acetonitrile. Afterwards the plates were centrifuged for 15 min at 4°C and 2500x g. From each well 100 pL of the supernatant was transferred to a 96-well plate. The plates were stored at -20°C or directly submitted to HPLC analysis.
  • DMAI racemic 2,6-dimethyl-l-indanylamine
  • Analytical HPLC was performed with the following setup: Instrument: Agilent Technologies 1290 Infinity; Column: Poroshell, 100 x 4.6mm, 2.7 gm; Eluent A: Water (+0.001% formic acid); Eluent B: acetonitrile; Flow: isocratic (10% Eluent A/90% Eluent B), flow rate: 1.4 mL/min; Temperature: 25 °C; sample injection volume: 1 pL; detection: Absorption at 210 nm.
  • 1R,2S DMAI carbamate (dissolved in acetoniltrile) was used as reference substance and as a standard for quantification. Appropriate dilutions were prepared covering the range up to the maximum concentration used in the biotransformation. Samples were analyzed for acylated or caboxylated product isomers and non-acylated and non-carboxylated substrate. The concentration of the respective compounds in samples was calculated by comparing the obtained peak areas with those of the respective compound standards.
  • the relative methyl-[(lR,2S)-2,6-dimethyl-2,3-dihydro-IH- inden-l-yl] carbamate yield is defined as methyl-[(lR,2S)-2,6-dimethyl-2,3-dihydro-lH-inden-l-yl] carbamate yield of variant divided by methyl-[(lR,2S)-2,6-dimethyl-2,3-dihydro-lH-inden-l-yl] carbamate of the wild-type lipase, d.r.
  • Table Key for an efficient production process is a high selectivity of the respective enzyme variant in order to generate almost enantiopure methyl-[(lR,2S)-2,6-dimethyl-2,3-dihydro-lH-inden-l-yl] carbamate.
  • Wild-type lipase (SEQ ID NO. 1) showed a methyl-[(lR,2S)-2,6-dimethyl-2,3-dihydro-lH- inden-l-yl] carbamate product yield of 0.96 mg/ml and a d.r. of 61.7 %.
  • Wild type lipase (SEQ ID NO. 1) showed a product yield of 2.04 mg/ml and a d.r. of 64.6 %.
  • Table 7 Relative methyl-[(lR,2S)-2,6-dimethyl-2,3-dihydro-lH-inden-l-yl]carbamate yield for lipase variants with improved 1R,2S DMAI carbamate product yield.
  • Table 8 Selection of variants with an improvement in d.r..

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Abstract

La présente invention concerne des protéines ayant une activité lipase améliorée, des molécules d'acide nucléique codant pour des protéines respectives ayant une activité lipase améliorée et des procédés de synthèse stéréosélective d'amines chirales ou d'augmentation d'isomères d'amines chirales dans des mélanges d'énantiomères.
PCT/EP2023/072709 2022-08-23 2023-08-17 Acides nucléiques codant pour des protéines lipases améliorées WO2024041972A1 (fr)

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