Lipases with improved stereoselectivity and lipase-based chiral resolution methods.

Abstract

The present invention relates to proteins with improved lipase activity, nucleic acid molecules encoding the respective proteins with improved lipase activity, and methods for the chiral resolution of secondary alcohols.

Description

[Technical Field]

[0001] The present invention relates to proteins with improved lipase activity, nucleic acid molecules encoding the proteins, and enzymatic methods for the chiral resolution of secondary alcohols. [Background technology]

[0002] Enantiomerically enriched or pure alcohols are important compounds for the production of pesticides or pharmaceutical compounds. Therefore, the absolute configuration of the stereocenter of a chiral alcohol is important for the synthesis of the corresponding active agent. In the production of desired target molecules, the generation of the correct chirality is often a challenge.

[0003] Various methods for preparing enantiomerically enriched alcohols are known. While chiral transition metal catalysts can be used, they are characterized by their high cost (G.R. Cook, Transition Metal-Mediated Kinetic Resolution, Current Organic Chemistry, 2000, 4, 869-885; Hirama et al., J. Org. Chem. 1988, 53, 708). In addition to metal catalysts, organocatalytic acyltransferase kinetic resolution for bulky ester substrates has been described (Deng et al., J. Org. Chem. 2015, 80, 6, 3159). The use of enzymes, such as lipases, as biocatalysts for the production of chiral compounds is also known. Kirchner et al. (J. Am. Chem. Soc. (1985), 107, 7072-7076) reported two lipases that act as highly stereoselective and practical catalysts in nearly anhydrous organic solvents. Under these "unnatural" conditions, enzymes can asymmetrically catalyze esterification and transesterification reactions that are impossible in aqueous solutions because hydrolysis predominates. As a result, many optically active alcohols, carboxylic acids, and esters have been prepared on a gram scale. Faber and Riva (1992; Synthesis 1992(10), 895-910) and Nascimento et al. (2003, Tetrahedron Asymmetry 14, 311-311) described the enzymatic resolution of secondary alcohols by transesterification. Furthermore, Patil et al. (J. Org. Chem. 2008, 73, 4476-4483) and Reddy et al. (Synth. Communications, 2003, 33, 3717-3726) demonstrated the use of esterases for enantioselective ester hydrolysis in buffered aqueous systems, although the yields were only moderate in the presence of DMSO.

[0004] US Pat. No. 4,732,853A describes a method for producing chiral epoxy alcohols by enantioselective hydrolysis using lipases.

[0005] EP 0 716 712 B1 describes the lipase-catalyzed acylation of alcohols with diketene, in particular the preparation of enantioselectively acylated alcohols from racemic alcohols.

[0006] WO2012146935A1 discloses modified lipase variants, as well as polynucleotides and recombinant expression vectors encoding the lipase variant polypeptides, and methods for producing such lipase variants in selected bacterial and fungal host cells. Certain lipase variants have increased enzyme specificity or enhanced trans-selectivity. Furthermore, methods for their use to reduce or remove trans-fatty acids from substrates are described. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent Application Publication No. 4,732,853 [Patent Document 2] European Patent No. 0716712 [Patent Document 3] International Publication No. 2012146935 Brochure [Non-patent literature]

[0008] [Non-Patent Document 1] GRCook,Current Organic Chemistry,2000,4,869-885 [Non-patent document 2] Hirama et al., J. Org. Chem. 1988, 53, 708 [Non-patent document 3] Deng et al., J.Org.Chem.2015,80,6,3159 [Non-patent document 4] Kirchner et al., J. Am. Chem. Soc. (1985), 107, 7072-7076 [Non-patent document 5] Faber and Riva, 1992; Synthesis 1992(10), 895-910 [Non-patent document 6] Nascimento et al., 2003, Tetrahedron Asymmetry 14, 311-311 [Non-Patent Document 7] Patil et al., J. Org. Chem. 2008, 73, 4476-4483 [Non-patent document 8] Reddy et al., Synth. Communications, 2003, 33, 3717-3726 Summary of the Invention [Problem to be solved by the invention]

[0009] Although several improvements in lipases have been achieved so far, several limitations that arise during the asymmetric synthesis of secondary alcohols or the resolution of racemic alcohols still need to be overcome, such as unfavorable equilibria, substrate and product inhibition, poor thermostability, poor substrate specificity, and especially low enantioselectivity of lipases. In addition, in large-scale applications, downstream processing and isolation of the chiral alcohol of interest are challenging, while atom economy becomes significantly more important at industrial scale.

[0010] Therefore, there is a need for further improvements in lipases and further process improvements, particularly with respect to the production of enantiomerically enriched or pure products with increased atom economy. [Means for solving the problem]

[0011] The present invention at least partially solves the above problems by providing novel lipases with excellent enantioselectivity, as well as novel methods for the chiral resolution of industrially relevant building blocks.

[0012] The lipases described herein have certain advantages over known wild-type and other known lipases, in particular, the modified or variant lipases described herein have the advantage that they are better able to produce enantiomerically enriched or enantiomerically nearly pure or pure compounds than their respective wild-type lipases.

[0013] A first aspect of the present invention relates to a protein having lipase activity, the protein being encoded by an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 92%, and even more preferably 95% identity to the amino acid sequence set forth in SEQ ID NO. 1; The amino acid sequence of the variant is characterized in that it differs from the amino acid sequence of SEQ ID NO: 1 in at least one of the following positions: i. the amino acid at position 44 is different from L, preferably the amino acid at position 44 is M, W or Y; ii. the amino acid at position 51 is different from F, preferably the amino acid at position 51 is N or M; iii. the amino acid at position 52 is different from V, preferably the amino acid at position 52 is L; iv. the amino acid at position 53 is different from T, preferably the amino acid at position 53 is S, P, I, E, or A; v. The amino acid at position 54 is different from D, preferably the amino acid at position 54 is Q, M, F, G, E, L, T or P; vi. The amino acid at position 55 is different from A, preferably the amino acid at position 55 is R, M, D, Y, S, or I; vii. the amino acid at position 109 is different from G, preferably the amino acid at position 109 is H or F; viii. the amino acid at position 110 is different from M, preferably the amino acid at position 110 is T or V; ix. the amino acid at position 111 is different from A, preferably the amino acid at position 111 is T or S; x. the amino acid at position 117 is different from Y, preferably the amino acid at position 117 is F or S; xi. The amino acid at position 121 is different from Y, preferably, the amino acid at position 121 is V; xii. the amino acid at position 122 is different from K, preferably the amino acid at position 122 is Q, A, Y, R, or V; xiii. the amino acid at position 153 is different from H, preferably the amino acid at position 153 is N, Y, D, E, or C; xiv. The amino acid at position 160 is different from T, preferably the amino acid at position 160 is E, C, D, P, I, Q, K, M, S, F, A, or N; xv. the amino acid at position 179 is different from D, preferably, the amino acid at position 179 is C; xvi. The amino acid at position 181 is different from A, preferably, the amino acid at position 181 is Q; xvii. the amino acid at position 184 is different from A, preferably the amino acid at position 184 is G or T; xviii. The amino acid at position 211 is different from Y, preferably, the amino acid at position 211 is E; xix. The amino acid at position 212 is different from A, preferably, the amino acid at position 212 is S or P; xx. the amino acid at position 216 is different from Y, preferably the amino acid at position 216 is K or A; xxi. the amino acid at position 234 is different from S, preferably the amino acid at position 234 is K, T or G; xxii. The amino acid at position 235 is different from S, preferably the amino acid at position 235 is V or M; xxiii. the amino acid at position 236 is different from K, preferably the amino acid at position 236 is T; xxiv. The amino acid at position 238 is different from R, preferably the amino acid at position 238 is A, K, D, E, or Q; xxv. the amino acid at position 240 is different from Y, preferably the amino acid at position 240 is F; xxvi. The amino acid at position 289 is different from D, preferably, the amino acid at position 289 is S or G; xxvii. The amino acid at position 291 is different from G, preferably the amino acid at position 291 is E or W; xxviii. the amino acid at position 317 is different from N, preferably the amino acid at position 317 is T; xxix. The amino acid at position 320 is different from N, preferably, the amino acid at position 320 is E or G; xxx. The amino acid at position 321 is different from L, preferably the amino acid at position 321 is F.

[0014] SEQ ID NO: 1 shows the reference protein sequence having lipase activity.

[0015] The meanings of the amino acid abbreviations A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y can be derived from Table 2 below in the paragraph subheaded "Description of Sequences" herein.

[0016] As used herein, an "amino acid corresponding to position x" in a first amino acid sequence (e.g., position 44 in SEQ ID NO: 1) means that when the amino acid numbering of the second amino acid sequence differs from the amino acid numbering of the first amino acid sequence, the amino acid of the second amino acid sequence, as compared to the first amino acid sequence, appears at position x in the first amino acid sequence in a pairwise sequence alignment of the first and second amino acid sequences.

[0017] In the context of the present invention, the term "identity" in relation to sequence identity or sequence identity should be understood to mean the number of identical amino acids or nucleotides, respectively, that a first nucleic acid or amino acid sequence shares with another (second) nucleic acid or amino acid sequence over the entire sequence length, expressed as a percentage.

[0018] "Sequence identity" can be determined by aligning two amino acid or two nucleotide sequences using known software such as GAP or BESTFIT or the global or local alignment algorithms included in the Emboss program "Needle." This software aligns two sequences over their entire length using the Needleman and Wunsch global alignment algorithm, maximizing the number of matches and minimizing the number of gaps. Generally, default parameters are used: a gap creation penalty of 10 and a gap extension penalty of 0.5 (for both nucleotide and protein alignments). For nucleotides, the default scoring matrix is ​​DNAFULL, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 10915-10919). Sequence alignment and percentage sequence identity scores can be determined using software such as EMBOSS, accessible at the EBI World Wide Web site (ebi.ac.uk / Tools / emboss / ). Alternatively, sequence similarity or identity can be determined by searching against databases (e.g., EMBL, GenBank) using commonly known algorithms and output formats such as FASTA, BLAST, etc., but preferably hits should be retrieved and ultimately aligned pairwise to determine sequence identity.

[0019] When the sequences being compared are of different lengths, identity is determined by determining the percentage of amino acids or nucleotides that the shorter sequence shares with the longer sequence. Preferably, identity is determined using the publicly available computer program ClustalW (Thompson et al., Nucleic Acids Research 22 (1994), 4673-4680). ClustalW is published by Julie Thompson (Thompson@EMBL-Heidelberg.DE) and Toby Gibson (Gibson@EMBL-Heidelberg.DE), European Molecular Biology Laboratory, Meyerhofstrasse 1, D69117, Heidelberg, Germany. ClustalW can also be downloaded from various internet sites. In particular, they can be downloaded from the IGBMC (Institut de Genetique et de Biologie Moleculaire et Cellulaire, BP163, 67404 Illkirch Cedex, France; ftp: / / ftp-igbmc.u-strasbg.fr / pub / ) and the European Bioinformatics Institute (EBI) (ftp: / / ftp.ebi.ac.uk / pub / software / ), as well as from all mirrored internet pages of the European Bioinformatics Institute (European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK).

[0020] Preferably, to determine the identity of the proteins described in this invention to other proteins, use the ClustalW computer program version 1.8 or Clustal2, where the following parameters should be set: KTUPLE=1, TOPDIAG=5, WINDOW=5, PAIRGAP=3, GAPOPEN=10, GAPEXTEND=0.05, GAPDIST=8, MAXDIV=40, MATRIX=GONNET, ENDGAPS(OFF), NOPGAP, NOHGAP.

[0021] Preferably, the ClustalW computer program version 1.8 or Clustal2 is used to determine identity between, for example, the nucleotide sequences of the nucleic acid molecules described in connection with the present invention and the nucleotide sequences of other nucleic acid molecules, where the parameters should be set as follows: KTUPLE=2,TOPDIAGS=4,PAIRGAP=5,DNAMATRIX:IUB,GAPOPEN=10,GAPEXT=5,MAXDIV=40,TRANSITIONS:unweighted.

[0022] "Identity" also means functional and / or structural equivalence between the nucleic acid molecules in question or the proteins encoded thereby. Functional equivalence means that the nucleic acid molecule sequence or amino acid sequence encodes a protein with lipase activity. Nucleic acid molecules that are homologous to the above-mentioned molecules and represent derivatives of these molecules are generally variants of these molecules that exhibit the same biological function or catalyze the same reaction, i.e., encode proteins with lipase activity. They can be either naturally occurring variants, e.g., sequences from other species, or mutants, and these mutations can occur naturally or be introduced by targeted mutagenesis. Furthermore, variants can be synthetically produced sequences. Allelic variants can be naturally occurring variants, synthetically produced variants, or variants produced by recombinant DNA technology. However, for the present invention, it is crucial that these variants encode proteins with lipase activity and contain the amino acid substitutions, deletions, or insertions described herein for the proteins according to the invention.

[0023] A particular type of derivative is a nucleic acid molecule that differs from the nucleic acid molecules described in connection with the present invention, for example, as a result of the degeneracy of the genetic code.

[0024] According to the NC-IUBMB (Commission on Nomenclature of the International Union of Biochemistry and Molecular Biology), lipases belong to the class of hydrolases (EC 3). Hydrolases are a class of enzymes that function as biochemical catalysts, using water to break chemical bonds, usually resulting in the splitting of larger molecules into smaller ones. Under non-native anhydrous conditions, these enzymes also catalyze esterification reactions, such as acetylation and transesterification. The hydrolase group includes enzymes that act on ester bonds (EC 3.1), including carboxylic ester hydrolases (EC 3.1.1) and the subgroup lipases (EC 3.1.1.3). Lipases include cutinases from plants, mammals, and bacteria such as Pseudomonas, Vibrio, Acinetobacter, Burkholderia, Chromobacterium, Fusarium solani (FSC), Candida antarctica A (CalA), Rhizopus oryzae (ROL), Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML), Aspergillus Niger, and Fusarium heterosporum. It has been identified from microorganisms such as Fusarium heterosporum, Fusarium oxysporum, or Fusarium culmorum.

[0025] If the protein has lipase activity, this can be detected by methods known and described in the art.

[0026] It is not critical which method is used to detect whether a protein according to the invention has lipase activity. Preferably, in the context of the present invention, the methods are those described in the "Examples" section.

[0027] In a further embodiment of the invention, the amino acid sequence of the variant differs from the amino acid sequence of SEQ ID NO: 1 by at least one of the following modifications: i. the amino acid at position 44 is W or Y; ii. the amino acid at position 54 is F; iii. the amino acid at position 55 is R; iv. The amino acid at position 109 is H; v. the amino acid at position 110 is T or V; vi. The amino acid at position 117 is F; vii. the amino acid at position 122 is Q or R; viii. The amino acid at position 160 is E; ix. The amino acid at position 216 is K; the amino acid at position x.236 is T; xi. The amino acid at position 238 is K or E; xii. The amino acid at position 240 is F.

[0028] Preferably, the amino acid sequence of the variant differs from the amino acid sequence of SEQ ID NO: 1 by at least the mutation G109H, ie the amino acid at position 109 is H.

[0029] The lipase variant proteins according to the invention may exhibit further amino acid modifications (amino acid substitutions, deletions or insertions) compared to the amino acid sequence described herein with respect to the amino acid sequence shown in SEQ ID NO:1.

[0030] In a further embodiment of the invention, the amino acid sequence of the variant differs from the amino acid sequence of SEQ ID NO: 1 by at least two, more preferably at least three, even more preferably at least four, and particularly preferably at least five, modifications selected from the following: i. the amino acid at position 57 is different from N, preferably the amino acid is P; ii. the amino acid at position 109 is different from G, preferably the amino acid is H; iii. the amino acid at position 122 is different from K, preferably the amino acid is R; iv. The amino acid at position 212 is different from A, preferably the amino acid is P; v. The amino acid at position 234 is different from S, preferably the amino acid is K; vi. The amino acid at position 289 is different from D, preferably the amino acid is G.

[0031] Preferably, the amino acid sequence of the variant differs from the amino acid sequence of SEQ ID NO: 1 by the following modifications: i. the amino acid at position 57 is P; ii. the amino acid at position 109 is H; iii. the amino acid at position 212 is P; iv. The amino acid at position 234 is K; and The amino acid at position v289 is G.

[0032] More preferably, the amino acid sequence of the variant differs from the amino acid sequence of SEQ ID NO: 1 by the following modifications: i. the amino acid at position 57 is P; ii. the amino acid at position 109 is H; iii. the amino acid at position 122 is R; iv. the amino acid at position 212 is P; v. The amino acid at position 234 is K; and vi. The amino acid at position 289 is G.

[0033] In a further embodiment of the invention, the amino acid sequence of the variant differs from the amino acid sequence of SEQ ID NO: 1 by the following modifications: - the amino acid at position 57 is P; the amino acid at position 109 is H; the amino acid at position 122 is R or K or Q, preferably R; the amino acid at position 212 is P; the amino acid at position 234 is K; the amino acid at position 289 is G; - further comprising at least one of the following modifications: the amino acid at position 44 is W or Y; the amino acid at position 54 is F, the amino acid at position 55 is R, the amino acid at position 110 is V or T; the amino acid at position 111 is T; the amino acid at position 117 is F; the amino acid at position 160 is E; the amino acid at position 216 is K, the amino acid at position 236 is T; the amino acid at position 238 is K or E; and the amino acid at position 240 is F.

[0034] Preferred embodiments of the present invention are SEQ ID NOs: 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, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 2 41, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239.

[0035] A further embodiment of the present invention relates to a nucleic acid molecule encoding a protein according to the invention.

[0036] The nucleic acid molecule 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 acid can be a ribonucleic acid molecule (e.g., RNA, mRNA) or a deoxyribonucleic acid molecule (DNA, including genomic DNA, which may or may not contain introns and coding DNA).

[0037] Of particular interest in the present invention are SEQ ID NOs: 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, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182 42, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, and 240.

[0038] Therefore, the present invention also relates to a nucleic acid molecule encoding a protein having the activity of a lipase selected from the group consisting of: a) SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138 , 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240; b) A nucleic acid molecule having at least 60%, preferably 70%, more preferably 80%, even more preferably 90%, even more preferably 95%, even more preferably 96%, particularly preferably 97%, most preferably 98%, or especially preferably 99% identity to the nucleic acid sequence shown in a).

[0039] In the context of the present invention, the term "hybridize" means hybridization under conventional hybridization conditions, preferably under stringent conditions, as described, for example, by Sambrook et al. (Molecular Cloning, A Laboratory Manual, 3rd Edition (2001), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. ISBN: 0879695773) or Ausubel et al. (Short Protocols in Molecular Biology, John Wiley & Sons; 5th Edition (2002), ISBN: 0471250929). Particularly preferably, "hybridization" means hybridization under the following conditions: Hybridization Buffer: 2xSSC; 10x Denhardt's solution (Fikoll400+PEG+BSA; ratio 1:1:1); 0.1% SDS; 5mM EDTA; 50mM Na2HPO4; 250μg / ml herring sperm DNA; 50μg / ml tRNA; or 25M sodium phosphate buffer pH 7.2; 1mM EDTA; 7% SDS Hybridization temperature: T=65~68℃ Wash buffer: 0.1xSSC; 0.1% SDS Wash temperature: T=65~68℃.

[0040] The nucleic acid molecule that hybridizes with the nucleic acid molecule encoding a protein having the activity of a lipase may be derived from any organism; it may therefore be derived from a bacterium, a fungus, an animal, a human, a plant or a virus.

[0041] The nucleic acid molecule that hybridizes with the nucleic acid molecule encoding a protein having lipase activity is preferably derived from a microorganism, more preferably from a fungus or bacterium, and most preferably from a bacterium.

[0042] Nucleic acid molecules that hybridize with the above-mentioned molecules can be isolated, for example, from genomic or cDNA libraries. Such nucleic acid molecules can be identified and isolated using the nucleic acid molecules described herein, or can be identified and isolated using portions of these molecules or their reverse complements, for example, by hybridization or PCR amplification according to standard methods (e.g., 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).

[0043] The fragments used as hybridization samples may be synthetic fragments or oligonucleotides prepared using conventional synthesis techniques, and their sequences are essentially identical to the nucleic acid molecules described in connection with the present invention. Once a gene that hybridizes with the nucleic acid sequence described in connection with the present invention has been identified and isolated, its sequence must be determined, and the properties of the protein encoded by this sequence must be analyzed to determine whether it is a protein with lipase activity. Methods for determining whether a protein has the activity of a protein with lipase activity are known to those skilled in the art.

[0044] Molecules that hybridize with the nucleic acid molecules described in connection with the present invention include, in particular, specific fragments, derivatives and allelic variants of the described nucleic acid molecules. In the context of the present invention, the term "derivative" means that the sequences of these molecules differ from the sequences of the above-mentioned nucleic acid molecules at one or more positions and are highly identical to these sequences. The differences from the above-mentioned nucleic acid molecules can be due, for example, to deletions, additions, substitutions, insertions or recombinations.

[0045] The meanings of the nucleotide abbreviations a, c, g, t and the degenerate nucleotide abbreviations r, y, s, w, k, m, b, d, h, v, n are derived from Table 1 in the section subheaded "Sequence Description" herein. Which amino acids are encoded by codons containing degenerate nucleotides are derived from Table 3 in the section subheaded "Sequence Description" herein.

[0046] Furthermore, the present invention relates to recombinant nucleic acid molecules comprising the nucleic acid molecules according to the invention.

[0047] In the context of the present invention, the term "recombinant nucleic acid molecule" is understood to mean a nucleic acid molecule that contains additional sequences in addition to the nucleic acid molecule according to the invention and that does not occur in nature in the combination present in the recombinant nucleic acid according to the invention. Here, said additional sequences may be any sequences, preferably they are functional or regulatory sequences (promoters, termination signals, enhancers, ribosome binding sites (rbs), leader sequences that enhance transcription, translation or RNA stability, intracellular targeting sequences, etc.), particularly preferably they are functional or regulatory sequences that are active in microorganisms, and especially preferably they are regulatory sequences that are active in fungi, in particular yeasts or bacteria. Methods for producing recombinant nucleic acid molecules according to the invention are known to those skilled in the art and include genetic methods such as combining nucleic acid molecules by ligation, genetic recombination or de novo synthesis of nucleic acid molecules. These methods are described, for example, in Sambrok et al. (Molecular Cloning, A Laboratory Manual, 3rd Edition (2001) Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY. ISBN: 0879695773) or Ausubel et al. (Short Protocols in Molecular Biology, John Wiley & Sons; 5th Edition (2002) ISBN: 0471250929).

[0048] In a further embodiment, a recombinant nucleic acid molecule according to the invention comprises a nucleic acid molecule according to the invention linked to a regulatory sequence that initiates transcription in a prokaryotic or eukaryotic cell.

[0049] A "regulatory sequence that initiates transcription" in a cell is also known as a promoter.

[0050] Information regarding regulatory sequences and plasmids is well known to those skilled in the art and is described, for example, by the Registry of Standard Biological Parts, sponsored by The International Genetically Engineered Machine (iGEM) Foundation (One Kendall Square, Suite B6104, Cambridge, MA 02139, USA) on the World Wide Web (http: / / parts.igem.org / Catalog).

[0051] Regulatory sequences for initiating transcription in prokaryotes, such as E. coli, and eukaryotes are well documented, particularly for expression in yeast, such as Saccharomyces cerevisiae. Overviews of various systems for protein expression in various host organisms can be found, for example, in Methods in Enzymology 153 (1987), 383-516 and Bitter et al. (Methods in Enzymology 153 (1987), 516-544) or Gomes et al. (2016, Advances in Animal and Veterinary Sciences, 4 (4), 346) and Baghban et al. (2018, Current Pharmaceutical Biotechnology, 19 (6)). Common yeast promoters include pAOX1, pHIS4, pGAL, and pScADH2 (Baghban et al., 2018, see above). Common bacterial promoters are T5, T7, rhamnose-inducible, arabinose-inducible, PhoA, and the artificial trc (trp-lac) promoter described by Marschall et al. (2017, Appl Microbiol Biotechnol 101, 501-512) and Tegel et al. (2011, FEBS Journal 278, 729-739).

[0052] A further embodiment of a recombinant nucleic acid molecule of the present invention is a vector or plasmid comprising a nucleic acid molecule according to the present invention.

[0053] "Vector" is commonly understood in the field of molecular biology and, as used herein, refers to a nucleic acid sequence or a vehicle containing a nucleic acid sequence used to introduce genetic material (DNA or RNA) into a target cell. A vector can be a plasmid, e.g., a T-DNA or binary vector for generating transgenic plants, an expression vector for expression of a nucleic acid sequence in a host cell, a shuttle vector competent for propagation in a different host, or the vector can be a viral particle or bacteriophage modified to deliver foreign genetic material into a host.

[0054] "Plasmid" is commonly understood in the field of molecular biology and, as used herein, refers to an autonomously self-replicating, often circular, DNA molecule when present in a host cell separated from chromosomal DNA.

[0055] A nucleic acid molecule according to the invention, a recombinant nucleic acid molecule according to the invention, a vector or a plasmid according to the invention can be used for the production of a protein according to the invention, e.g. by expressing a nucleic acid molecule according to the invention in a host cell.

[0056] Another embodiment of the present invention relates to a host or host cell which comprises or expresses a nucleic acid molecule according to the present invention, which comprises a protein according to the present invention, which comprises a recombinant nucleic acid molecule according to the present invention, which comprises a vector according to the present invention, or which comprises a plasmid according to the present invention.

[0057] The nucleic acid molecules according to the invention encoding proteins with lipase activity can be expressed in host cells, for example for their propagation or for the production of proteins according to the invention. For expression in host cells, the nucleic acid molecules according to the invention can be included on a vector or plasmid, or they can be stably integrated into the genome of the respective host cell. The nucleic acid molecules according to the invention can also be included in a vector that supports their introduction into the host cell.

[0058] Further embodiments of the present invention relate to a host or host cell according to the invention which comprises a nucleic acid molecule according to the invention, or which comprises a recombinant nucleic acid molecule according to the invention, or which comprises a vector according to the invention, or which comprises a plasmid according to the invention, and in each case comprises a protein according to the invention.

[0059] Another embodiment of the present invention relates to a host or host cell according to the present invention, which comprises a nucleic acid molecule according to the present invention, which comprises a recombinant nucleic acid molecule according to the present invention, which comprises a vector according to the present invention, or which comprises a plasmid according to the present invention, in each case expressing a protein according to the present invention.

[0060] Another embodiment of the present invention relates to a host or host cell according to the present invention, which comprises a nucleic acid molecule according to the present invention, which comprises a recombinant nucleic acid molecule according to the present invention, which comprises a vector according to the present invention, or which comprises a plasmid according to the present invention, and in each case expresses a protein, which protein has the activity of a lipase.

[0061] "Expressing a nucleic acid molecule" is understood to mean that, if the nucleic acid molecule is RNA or mRNA, the nucleic acid molecule is translated into a protein, preferably translated into a protein having the activity of a lipase, or, if the nucleic acid molecule is DNA or cDNA, the nucleic acid molecule is transcribed into mRNA (and processed in the case of genomic DNA containing introns), preferably transcribed into mRNA encoding a protein having the activity of a lipase, and subsequently translated into a protein having the activity of a lipase.

[0062] Transcription of a given nucleic acid molecule in the host can be demonstrated by methods known to those skilled in the art, for example, by detection of specific transcripts (mRNA) of the foreign nucleic acid molecule by Northern blot analysis or RT-PCR.

[0063] Whether a host or host cell contains a given protein or contains a protein derived from the expression of a nucleic acid molecule can be determined by methods known to those skilled in the art, for example, immunological methods such as Western blot analysis, ELISA (enzyme-linked immunosorbent assay), or RIA (radioimmunoassay). Those skilled in the art are familiar with methods for preparing antibodies that specifically react with, i.e., bind to, a particular protein (see, for example, Lottspeich and Zorbas (eds.), 1998, Bioanalytik, Spektrum akad, Verlag, Heidelberg, Berlin, ISBN 3-8274-0041-4). Several companies (Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA USA 0245; GenScript, 60 Centennial Ave., Piscataway, NJ 08854, USA) offer custom antibody preparation services.

[0064] Furthermore, a person skilled in the art can test whether a host or host cell contains a protein according to the invention by detecting the (additional) activity of a protein having lipase activity in the respective host cell. Preferably, the activity of a protein having lipase activity in the respective host cell is detected by comparing the lipase activity of the host cell according to the invention with the respective activity of a host cell not containing the protein according to the invention.

[0065] Testing whether a protein has lipase activity can be carried out by methods known in the art.

[0066] A host or host cell according to the invention can be prepared by one skilled in the art by known methods for genetically modifying or transforming organisms.

[0067] A further subject of the present invention is therefore a host or host cell according to the invention, in particular a prokaryotic or eukaryotic host or host cell which is genetically modified (or transformed) by a nucleic acid molecule according to the invention or a recombinant nucleic acid molecule according to the invention or a vector according to the invention or a plasmid according to the invention. Preferably, the genetically modified (transformed) host or host cell according to the invention expresses a protein having lipase activity, more preferably the genetically modified (transformed) host or host cell according to the invention expresses a protein according to the invention.

[0068] As used herein, "recombined with a nucleic acid molecule" or "transformed with a nucleic acid molecule" is understood to mean that the nucleic acid molecule is or has been introduced into a host or host cell by technical and / or non-naturally occurring means, preferably by technical methods in the fields of molecular biology, biotechnology or genetic recombination.

[0069] Descendants, offspring or progeny of a host or host cell 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, comprise a recombinant nucleic acid molecule according to the invention, comprise a vector according to the invention, 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, comprise a recombinant nucleic acid molecule according to the invention, comprise a vector according to the invention or comprise a plasmid according to the invention, in each case expressing a protein which has the activity of a lipase, and even more preferably these descendants, offspring or progeny comprise a nucleic acid molecule according to the invention, comprise a recombinant nucleic acid molecule according to the invention, comprise a vector according to the invention or comprise a plasmid according to the invention, in each case expressing a protein which has the activity of a lipase according to the invention.

[0070] A host or host cell according to the present invention can be of any prokaryotic or eukaryotic origin. The host or host cell may be a bacterium or bacterial cell (e.g., E. coli, bacteria of the genus Bacillus, in particular Bacillus subtilis, Agrobacterium, in particular Agrobacterium tumefaciens or Agrobacterium rhizogenes, Pseudomonas, in particular Pseudomonas fluorescens, Streptomyces spp., Rhodococcus spp., in particular Rhodococcus rhodochrous, Vibrio natriegens, or any of the following: natrigens, Corynebacterium, in particular Corynebacterium glutamicum), fungi or fungal cells (e.g., Agaricus, in particular Agaricus bisporus, Aspergillus, Trichoderma or yeasts, in particular Pichia ssp. such as S. cerevisiae, P. pastoris), as well as plants or plant cells, or they may be animals or animal cells.

[0071] Preferred host cells according to the invention are cells of microorganisms, which within the framework of the present patent application are understood to include all bacteria and all protists (e.g. fungi, in particular yeasts and algae), as defined, for example, in Schlegel's "General Microbiology" (Georg Thieme Publishing House (1985), 1-2).

[0072] With respect to microorganisms, the host or host cell according to the invention is preferably a bacterium / bacterial cell or a yeast / yeast cell, most preferably a bacterium / bacterial cell. With respect to bacteria / bacterial cells, the host or host cell according to the invention is preferably a Bacillus spp. / Bacillus spp. cell or an Escherichia coli / E. coli cell, most preferably an E. coli / E. coli cell.

[0073] Alternatively, Pseudomonas, in particular Pseudomonas fluorescens, Streptomyces spp., Rhodococcus spp., in particular Rhodococcus rhodochrous, Vibrio spp., in particular Vibrio natrigens, Corynebacterium spp., in particular Corynebacterium glutamicum or others may be hosts or host cells according to the invention.

[0074] A preferred embodiment of the present invention relates to a host or host cell according to the invention comprising a nucleic acid molecule according to the invention, wherein the codons of said nucleic acid molecule are altered to match the codon usage of the host or host cell, respectively.

[0075] The host cells according to the invention can be used for the production of the proteins according to the invention. The proteins according to the invention can be used in a process for the production of enantiomerically enriched or nearly enantiomerically pure secondary alcohols.

[0076] A second aspect of the present invention relates to a method for the enantiomerically selective hydrolysis of a racemic substrate of formula (II) to isolate an enantiomerically enriched or pure compound of formula (I), which method comprises contacting the substrate with a mutein having the activity of a lipase according to the first aspect of the invention or a lipase according to SEQ ID NO: 1. [ka] In the formula, R 1 and R 2 are each independently selected from substituted or unsubstituted (n)-alkyl, isoalkyl, unsubstituted aryl, alkyl-substituted or aryl-substituted aryl.

[0077] Preferably, the substrate is contacted with a mutein having the activity of a lipase according to the first aspect of the invention.

[0078] R 1 and R 2 are preferably, independently of one another, linear or branched C 1-10 is selected as the residue.

[0079] More preferably, R 1 is selected from methyl, isobutyl, tert-butyl, and isopropyl; and / or R 2 is methyl.

[0080] Even more preferably, R 1 is selected from methyl, isobutyl, tert-butyl, and isopropyl; R 2 is methyl.

[0081] In a further embodiment of the invention, the method comprises the enantiomerically selective hydrolysis of a racemic substrate of formula (II-1) to isolate an enantiomerically enriched or pure compound of formula (I-1). [ka] The method preferably comprises isolating compound (I) or (I-1), and / or (III) or (III-1) after hydrolysis. Isolation of compound (I-1) can be carried out by any means known to those skilled in the art, preferably by extraction and / or direct distillation.

[0082] The lipase variants or protein variants according to the invention exhibit improved selectivity and / or improved specific activity for the stereoselective hydrolysis of methyl-3-hydroxy-2-methylene-butanoate (formula II-1) and are more suitable for isolating enantiomerically enriched or nearly pure substrate methyl(3S)-3-hydroxy-2-methylene-butanoate (formula I-1) compared to wild-type lipases.

[0083] Formula (I): [ka] Compounds of formula (I-1): are important building blocks in the synthesis of complex pesticide compounds. [ka] Methyl (3S)-3-hydroxy-2-methylene-butanoate is the S-enantiomer of racemic methyl 3-hydroxy-2-methylene-butanoate and is a key intermediate in the synthesis of each of the agrochemical compounds described in WO 2018 / 228985. The lipase variant according to the present invention selectively hydrolyzes the R-enantiomer of racemic methyl 3-hydroxy-2-methylene-butanoate, allowing for the separation of the S-enantiomer of the racemic methyl 3-hydroxy-2-methylene-butanoate.

[0084] In a further embodiment of the invention, the method is carried out in an aqueous solution.

[0085] Preferably, the process is carried out in a mixture or two-phase liquid system of water and an organic solvent, such as methyl tert-butyl ether, toluene, 2-methyltetrahydrofuran, methyl isobutyl ketone, cyclohexane, cyclopentyl methyl ether, chlorobenzene, tert-amyl methyl ether, ethyl acetate, or isopropyl acetate. More preferably, the organic solvent is methyl tert-butyl ether (MTBE).

[0086] The ratio of organic solvent to water is more preferably in the range of 1:1 to 6:1, and even more preferably in the range of 2:1 to 4:1.

[0087] Surprisingly, it was found that drastically reducing the aqueous phase and partially replacing it with organic solvents not only increased the concentration of the final product and improved productivity, but also reduced the enzyme / lipase load.

[0088] Additionally, the presence of an organic solvent such as MTBE facilitates efficient downstream processing of the reaction product, i.e., isolation of the compound of formula (I) or (I-1).

[0089] In a further embodiment of the invention, the process is carried out at a temperature between 20° C. and 60° C., preferably between 30° C. and 55° C. More preferably, the process is carried out at a temperature between 35° C. and 50° C.

[0090] In a further embodiment of the invention, the method is carried out for at least 1 hour, preferably at least 2 hours, more preferably at least 3 hours.

[0091] In a further embodiment of the invention, the method is carried out for a period of at most 40 hours, preferably at most 30 hours, more preferably at most 20 hours.

[0092] In a further embodiment of the invention, the process is carried out at a pH between 7 and 8.5, preferably between 7.2 and 8.0, more preferably between 7.4 and 7.6. The pH can be adjusted by the addition of an organic or inorganic base, preferably by the addition of an inorganic base, such as bicarbonate or carbonate, hydrogen phosphate and hydroxide.

[0093] At pH above 8.5, the enzyme is inactivated; furthermore, the ester of formula (I) is partially hydrolyzed. At pH below 7, the reaction rate slows down and eventually stops. At pH below 4.5, the enzyme is further inactivated.

[0094] The pH can be controlled by the addition of a base such as NaOH, KOH, K2CO3, KHCO3, Na2CO3, or NaHCO3. Preferred bases are carbonates or bicarbonates. The base can be added as a solid or aqueous solution, diluted or saturated. The base can be added to the reaction before the substrate is added, or it can be added during the reaction in parallel with the substrate. The substrate and / or base can be added all at once or in addition.

[0095] The lipase of SEQ ID NO: 1 or protein variant according to the first aspect of the invention may be provided as a purified enzyme, or in the form of spray-dried or freeze-dried biomass, or as a broth. Preferably, the protein variant is provided as a broth, i.e., as a cell culture, or the broth is centrifuged and the cell pellet freeze-dried to obtain freeze-dried cells or biomass.

[0096] While freeze-dried biomass is easier to handle with regard to timing independence for carrying out the claimed method, it is cost and time efficient to use the broth / cell culture directly to carry out the claimed method.

[0097] For downstream processing, the reaction is preferably carried out in a mixture of water and a water-immiscible solvent. If the reaction is carried out in a two-phase mixture of water and an immiscible organic solvent, the phases are separated and the aqueous phase is finally back-extracted. The biomass can be separated by known technical means (e.g., centrifuge, filtration or decantation) before distillation, or the distillation can be carried out without separating the biomass.

[0098] It is to be understood that in the context of the present invention, all embodiments relating to the process according to the second aspect can be combined with all different lipase variants, particularly preferred lipase variants, according to the first aspect of the invention, i.e., the process for the enantiomerically selective hydrolysis of a racemic substrate of formula (II) and isolation of an enantiomerically enriched or pure compound of formula (I) can be carried out using a protein with lipase activity, which protein is encoded by an amino acid sequence having at least 80%, preferably 85%, more preferably 90%, even more preferably 92%, even more preferably 95% identity with the amino acid sequence shown in SEQ ID NO: 1, The amino acid sequence of the variant is characterized in that it differs from the amino acid sequence of SEQ ID NO: 1 in at least one of the following positions: i. the amino acid at position 44 is different from L, preferably the amino acid at position 44 is M, W or Y; ii. the amino acid at position 51 is different from F, preferably the amino acid at position 51 is N or M; iii. the amino acid at position 52 is different from V, preferably the amino acid at position 52 is L; iv. the amino acid at position 53 is different from T, preferably the amino acid at position 53 is S, P, I, E, or A; v. The amino acid at position 54 is different from D, preferably the amino acid at position 54 is Q, M, F, G, E, L, T or P; vi. The amino acid at position 55 is different from A, preferably the amino acid at position 55 is R, M, D, Y, S, or I; vii. the amino acid at position 109 is different from G, preferably the amino acid at position 109 is H or F; viii. the amino acid at position 110 is different from M, preferably the amino acid at position 110 is T or V; ix. the amino acid at position 111 is different from A, preferably the amino acid at position 111 is T or S; x. the amino acid at position 117 is different from Y, preferably the amino acid at position 117 is F or S; xi. The amino acid at position 121 is different from Y, preferably, the amino acid at position 121 is V; xii. the amino acid at position 122 is different from K, preferably the amino acid at position 122 is Q, A, Y, R, or V; xiii. the amino acid at position 153 is different from H, preferably the amino acid at position 153 is N, Y, D, E, or C; xiv. The amino acid at position 160 is different from T, preferably the amino acid at position 160 is E, C, D, P, I, Q, K, M, S, F, A, or N; xv. the amino acid at position 179 is different from D, preferably, the amino acid at position 179 is C; xvi. The amino acid at position 181 is different from A, preferably, the amino acid at position 181 is Q; xvii. the amino acid at position 184 is different from A, preferably the amino acid at position 184 is G or T; xviii. The amino acid at position 211 is different from Y, preferably, the amino acid at position 211 is E; xix. The amino acid at position 212 is different from A, preferably, the amino acid at position 212 is S or P; xx. the amino acid at position 216 is different from Y, preferably the amino acid at position 216 is K or A; xxi. the amino acid at position 234 is different from S, preferably the amino acid at position 234 is K, T or G; xxii. The amino acid at position 235 is different from S, preferably the amino acid at position 235 is V or M; xxiii. the amino acid at position 236 is different from K, preferably the amino acid at position 236 is T; xxiv. The amino acid at position 238 is different from R, preferably the amino acid at position 238 is A, K, D, E, or Q; xxv. the amino acid at position 240 is different from Y, preferably the amino acid at position 240 is F; xxvi. The amino acid at position 289 is different from D, preferably, the amino acid at position 289 is S or G; xxvii. The amino acid at position 291 is different from G, preferably the amino acid at position 291 is E or W; xxviii. the amino acid at position 317 is different from N, preferably the amino acid at position 317 is T; xxix. The amino acid at position 320 is different from N, preferably, the amino acid at position 320 is E or G; xxx. The amino acid at position 321 is different from L, preferably the amino acid at position 321 is F.

[0099] The amino acid sequence of a mutein lipase used in the method for enantiomerically selectively hydrolyzing a racemic substrate of formula (II) to isolate an enantiomerically enriched or pure compound of formula (I) may differ from the amino acid sequence of SEQ ID NO: 1 by at least one of the following modifications: i. the amino acid at position 44 is W or Y; ii. the amino acid at position 54 is F; iii. the amino acid at position 55 is R; iv. The amino acid at position 109 is H; v. the amino acid at position 110 is T or V; vi. The amino acid at position 117 is F; vii. the amino acid at position 122 is Q or R; viii. The amino acid at position 160 is E; ix. The amino acid at position 216 is K; the amino acid at position x.236 is T; xi. The amino acid at position 238 is K or E; xii. The amino acid at position 240 is F.

[0100] Preferably, the amino acid sequence of the mutein lipase used in the method for enantiomerically selectively hydrolyzing a racemic substrate of formula (II) to isolate an enantiomerically enriched or pure compound of formula (I) differs from the amino acid sequence of SEQ ID NO: 1 by at least the mutation G109H, i.e., the amino acid at position 109 is H.

[0101] The amino acid sequence of a mutein lipase used in a method for enantiomerically selectively hydrolyzing a racemic substrate of formula (II) to isolate an enantiomerically enriched or pure compound of formula (I) can differ from the amino acid sequence of SEQ ID NO: 1 by at least two, more preferably at least three, even more preferably at least four, and particularly preferably at least five of the following modifications selected from: i. the amino acid at position 57 is different from N, preferably the amino acid is P; ii. the amino acid at position 109 is different from G, preferably the amino acid is H; iii. the amino acid at position 122 is different from K, preferably the amino acid is R; iv. The amino acid at position 212 is different from A, preferably the amino acid is P; v. The amino acid at position 234 is different from S, preferably the amino acid is K; vi. The amino acid at position 289 is different from D, preferably the amino acid is G.

[0102] Preferably, the amino acid sequence of the variant differs from the amino acid sequence of SEQ ID NO: 1 by the following modifications: i. the amino acid at position 57 is P; ii. the amino acid at position 109 is H; iii. the amino acid at position 212 is P; iv. The amino acid at position 234 is K; and The amino acid at position v289 is G.

[0103] More preferably, the amino acid sequence of the variant differs from the amino acid sequence of SEQ ID NO: 1 by the following modifications: i. the amino acid at position 57 is P; ii. the amino acid at position 109 is H; iii. the amino acid at position 122 is R; iv. the amino acid at position 212 is P; v. The amino acid at position 234 is K; and vi. The amino acid at position 289 is G.

[0104] The amino acid sequence of a mutein lipase used in the method for enantiomerically selectively hydrolyzing a racemic substrate of formula (II) to isolate an enantiomerically enriched or pure compound of formula (I) may differ from the amino acid sequence of SEQ ID NO: 1 by the following modifications: - the amino acid at position 57 is P; the amino acid at position 109 is H; the amino acid at position 122 is R or K or Q, preferably R; the amino acid at position 212 is P; the amino acid at position 234 is K; the amino acid at position 289 is G; - further comprising at least one of the following modifications: the amino acid at position 44 is W or Y; the amino acid at position 54 is F, the amino acid at position 55 is R, the amino acid at position 110 is V or T; the amino acid at position 111 is T; the amino acid at position 117 is F; the amino acid at position 160 is E; the amino acid at position 216 is K, the amino acid at position 236 is T; the amino acid at position 238 is K or E, and the amino acid at position 240 is F.

[0105] Preferred proteins of the invention encoding lipases that can be used in a method for enantiomerically selectively hydrolyzing a racemic substrate of formula (II) to isolate an enantiomerically enriched or pure compound of formula (I) are shown in SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 1 77, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239.

[0106] A further embodiment of the invention is the use of a protein according to the first aspect of the invention for the stereoselective hydrolysis of racemic methyl-3-hydroxy-2-methylene-butanoate to isolate methyl (3S)-3-hydroxy-2-methylene-butanoate.

[0107] By "enantiomerically enriched" herein is meant that one of the two enantiomers is present in a greater amount than the other enantiomer, preferably at least 60% of one enantiomer is present in the composition, more preferably at least 65% of one enantiomer is present in the composition, even more preferably at least 70% of one enantiomer is present in the composition, even more preferably at least 75% of one enantiomer is present in the composition, even more preferably at least 80% of one enantiomer is present in the composition, particularly preferably at least 85% of one enantiomer is present in the composition, most preferably at least 90% of one enantiomer is present in the composition, or especially preferably at least 94% of one enantiomer is present in the composition.

[0108] By "substantially enantiomerically pure" herein is meant that one of the two enantiomers is present in the composition in an amount of at least 95.0%, preferably one of the two enantiomers is present in the composition in an amount of at least 95.5%, more preferably one of the two enantiomers is present in the composition in an amount of at least 96.0%, even more preferably one of the two enantiomers is present in the composition in an amount of at least 96.5%, even more preferably one of the two enantiomers is present in the composition in an amount of at least 97.0%, even more preferably one of the two enantiomers is present in the composition in an amount of at least 98.0%, particularly preferably one of the two enantiomers is present in the composition in an amount of at least 98.5%, most preferably one of the two enantiomers is present in the composition in an amount of at least 99.0%, and especially preferably one of the two enantiomers is present in the composition in an amount of at least 99.5%.

[0109] A third aspect of the present invention is a compound of formula (I): [ka] A method for enantiomerically enriching a compound of formula (II) by reacting a compound of formula (II) with a compound of formula (IV) in the presence of a lipase under anhydrous conditions. [ka] and in particular, [ka] [In the formula, R 1 and R 2 is defined as above, and R 3 is a substituted or unsubstituted (n)-alkyl or isoalkyl. The method relates to the above method, comprising:

[0110] "Anhydrous conditions" refers to a reaction system containing a maximum of 3% water in the liquid phase.

[0111] The reaction is preferably carried out in an organic solvent such as hexane or n-heptane, or without any additional solvent.

[0112] R 3 is preferably a linear C 1-16 Residues, especially methyl or unsubstituted and saturated C8-C 12 Compound (IV) may in particular be a residue of vinyl acetate or vinyl laurate.

[0113] The lipase is preferably immobilized on a solid support during operation, which results in increased enzyme stability in non-aqueous solutions.

[0114] The reaction is preferably carried out at a temperature between 20° C. and 40° C., preferably between 25° C. and 35° C. Such a temperature range allows for optimal enzyme reactivity.

[0115] The reaction is preferably carried out for at least 1 hour, more preferably at least 3 hours, and even more preferably at least 5 hours.

[0116] The lipase is preferably recycled after the reaction is carried out, which allows the process to be designed in a cost-effective manner. Thus, the lipase can be used multiple times for the reaction according to the third aspect or for further reactions.

[0117] The compound of formula (I) is preferably isolated by distillation either directly from the reaction mixture or from the supernatant obtained after decanting the reaction mixture, with the lipase preferably remaining in the remaining reaction mixture and being able to be reused in another reaction.

[0118] The lipase is preferably CALB lipase. CALB is a nonspecific lipase derived from Candida antarctica B and was first described in 1994 (Uppenberg J, Patkar S, Bergfors T, Jones TA (1994) J Mol Biol 235(2):790-792).

[0119] The lipase is more preferably immobilized on a hydrophobic support such as an acrylic resin. A commercially available version of CALB is, for example, Novozym® 435, which can be used to carry out the reaction.

[0120] Detailed Description The polypeptide (enzyme, i.e. lipase) and the method according to the invention are of formula (I) [ka] This allows for efficient enantiomeric enrichment of compounds of the present invention.

[0121] The introduction of specific amino acid modifications into the protein variants according to the present invention has been found to improve the activity of the lipase, particularly with regard to its substrate specificity, which means that these further modified lipase variants are more suitable for producing enantiomerically enriched or nearly pure products compared to known lipase variants. This particularly refers to the substrate (I-1): enantiomerically pure or at least enriched methyl (3S)-3-hydroxy-2-methylene-butanoate: [ka] is the S-enantiomer of racemic methyl 3-hydroxy-2-methylene-butanoate and is a key intermediate in the synthesis of each of the compounds described in WO 2018 / 228985. The lipase variants of the present invention enantioselectively catalyze the hydrolysis of racemic methyl 3-hydroxy-2-methylene-butanoate to (3R)-3-hydroxy-2-methylene-butanoic acid, leaving methyl (3S)-3-hydroxy-2-methylene-butanoate unhydrolyzed.

[0122] The terms used herein are known to those skilled in the art. Otherwise, the following definitions apply.

[0123] For the purposes of the present invention, the term "alkyl" includes saturated hydrocarbon residues which may be branched or straight-chain and unsubstituted or at least monosubstituted. Examples of suitable alkyl groups, which may be unsubstituted, monosubstituted or polysubstituted, are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, 2-butyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, isopentyl, neopentyl, n-hexyl, 2-hexyl, 3-hexyl, n-heptyl, n-octyl, -C(H)(C2H5)2, -C(H)(n-C3H7)2 and -CH2-CH2-C(H)(CH3)-(CH2)3-CH3.

[0124] Unless otherwise defined elsewhere, the term "aryl", alone or in combination with other terms, is to be understood as a monocyclic or polycyclic, preferably monocyclic or bicyclic, aromatic hydrocarbon radical, preferably having 6, 10 or 14 carbon atoms. Aryl radicals can be unsubstituted or mono- or polysubstituted with the same or different substituents.

[0125] Array Description Throughout the application, nucleotide and amino acid abbreviations are used according to the following IUPAC code:

[0126] Table 1 [Table 1] To distinguish between amino acids and nucleotides, the capitalized abbreviations for nucleotides given in the table above are written in lower case herein.

[0127] Table 2 [Table 2] Codon usage herein follows the so-called "universal genetic code", according to the table below, where in ribonucleic acid (RNA) sequences "t" is replaced by "u".

[0128] Table 3 [Table 3] TIFF2026508596000013.tif106125 The sequence listing associated with this application has been submitted in electronic format and is hereby incorporated by reference in its entirety. "PRT" stands for "protein" and "NUC" stands for "nucleic acid."

[0129] Table 4a: Lipase variants according to the first aspect of the invention (SEQ ID NOs: 7-169) and further lipases (SEQ ID NOs: 1-6) [Table 4a] TIFF2026508596000015.tif192134TIFF2026508596000016.tif191133TIFF2026508596000017.tif161134Table 4b: Further lipase variants according to the invention (protein SEQ ID NOs: 171-239). The variants described comprise the backbone mutations N57P, K122R, A212P, S234K, D289G and G109H (SEQ ID NOs: 171, 172), with the proviso that (i) where the mutation "R122K" is additionally shown, i.e., the K122R mutation has undergone a back mutation, with position 122 now being K again, or (ii) where the mutation "R122Q" is additionally shown, i.e., the K122R mutation has undergone a further mutation, with position 122 now being Q. [Table 4b] TIFF2026508596000019.tif190127TIFF2026508596000020.tif193128TIFF2026508596000021.tif186127TIFF2026508596000022.tif22130 [Example]

[0130] The following examples illustrate the invention without limiting it.

[0131] Starting Materials and Protocols Analytical grade reagents and ready-to-use kits were sourced and used from common suppliers such as Sigma Aldrich, Acros Organics, Fisher Scientific, Qiagen or Stratagene. Novozym® 435 was purchased from Sigma Aldrich.

[0132] Example 1 Cloning of lipase variants: The nucleic acid sequence of SEQ ID NO: 1 or variants thereof was cloned into a pKA81-based expression vector. Genetic elements were incorporated into the vector by means known in the art. For expression of the various lipase variants, the vector was introduced into electrocompetent E. coli W3110 cells.

[0133] Mutant library: To allow for amino acid substitution, nucleotides were substituted in the wild-type sequence or a sequence derived therefrom. This substitution can be achieved by various molecular biology methods. One method for substituting nucleic acids is site-directed mutagenesis, which can introduce mutations at one or more sites in the amino acid sequence. The method of site-directed mutagenesis is state-of-the-art and has been described in the literature (e.g., Directed Mutagenesis: A Practical Approach, 1991, Edited by MJ McPHERSON, IRL PRESS) and can be purchased as a ready-made kit (e.g., QUIKCHANGE™ lightening mutagenesis kit from Qiagen or Stratagene). After inserting the mutation into the gene sequence and culturing it in a suitable E. coli cloning strain, the resulting plasmid was transformed into E. coli W3110.

[0134] Transformants were tested in appropriate biotransformation reactions to determine product yield and selectivity. Suitable biotransformation reactions are described below and see Example 2. Sequence verification was performed as known in the art.

[0135] Glycerol stocks of E. coli cultures transformed with each expression plasmid were prepared by adding 1 volume of 40% glycerol solution to 1 volume of E. coli culture.

[0136] To isolate single bacterial colonies, appropriate dilutions of E. coli cultures were plated onto LB-agar plates containing the appropriate concentration of kanamycin and incubated at 37°C until single colonies were obtained.

[0137] Example 2 culture: To prepare the precultures, sterile 2 mL 96-well deep-well plates (Eppendorf, Hamburg, Germany) were used to mix 590 μL of TB medium (kanamycin 50 μg / mL) and 10 μL of the glycerol stock filled with each E. coli strain. Alternatively, 590 μL of TB medium (kanamycin 50 μg / mL) was inoculated with cell material from a colony on an agar plate. The precultures were incubated at 37°C and 250 rpm for 17 hours.

[0138] Sterile 2 mL 96-well deep well plates with 510 μL of TB medium (kanamycin 50 mg / L) were used to prepare the main culture. The expression culture was inoculated with 30 μL of preculture and incubated at 37°C and 250 rpm. After 4 hours of incubation, enzyme expression was induced by adding 60 μL of IPTG (10 mM IPTG supplemented with 50 mg / L kanamycin and diluted in expression medium). The expression culture plate was then incubated at 28°C and 250 rpm for 20 hours.

[0139] The cells were collected by centrifugation at 4°C and 2500 x g for 15 minutes. The culture supernatant was discarded, and the cell pellet was suspended in 200 µL of PBS. The cells were then lyophilized for 24 hours and stored at 4°C until use.

[0140] Biotransformation, Chiral Resolution and Analysis: Chiral resolution of methyl (3S)-3-hydroxy-2-methylenebutanoate was performed in a microtiter plate using the lyophilized supernatant. For chiral resolution, 100 μl of racemic methyl 3-hydroxy-2-methylenebutanoate, 105 μl of MTBE (methyl tert-butyl ether), and 45 μl of ultrapure water (containing 416.6 g / L KHCO3) were used in each well. Each plate was sealed and incubated at 45°C for 6 hours in a shaker at 300 rpm. The reaction was then stopped by adding 22.5 μl of 20% H2SO4 solution. For further extraction, 1 ml of MTBE was added per well in a deep-well plate. The plate was shaken at room temperature for 10 minutes and then centrifuged at 2500 × g for 10 minutes. Next, 10 μl of the organic phase was transferred to a 96-well PCR plate containing 100 μl of MTBE and analyzed by HPLC.

[0141] Analytical HPLC method The samples were analyzed using HPLC with the following settings:

[0142] Instrument: Agilent Technologies 1290 Infinity II; Column: Lux cellulose-2, 100 x 4.6 mm, 3 μm; Eluent A: mixed heptanes (+0.05% formic acid); Eluent B: ethanol; Flow: isocratic (90% eluent A / 10% eluent B), flow rate: 0.8 mL / min; Temperature: 25 °C. Sample injection volume: 1 μL; Detection: absorbance at 210 nm.

[0143] Racemic methyl 3-hydroxy-2-methylene-butanoate and racemic 3-hydroxy-2-methylene-butanoic acid (prepared as biotransformation samples) were used as reference materials and standards for quantification. Appropriate dilutions of the standards were used so that the substrates used and the resulting products could be quantified using standard lines. Samples were analyzed for substrate conversion and product formation. Evaluation of individual samples and comparison with each other was performed by determining the substrate and product ee [%].

[0144] Example 3: Enzymatic hydrolysis of racemic methyl 3-hydroxy-2-methylene-butanoate to (3R)-3-hydroxy-2-methylene-butanoic acid Cultivation, biotransformation, and HPLC analysis were performed as described in Example 2. Glycerol culture was used for inoculation during cultivation. Activity and selectivity results for enzyme variants with individual point mutations are shown in Tables 5 and 6 below. Activity and selectivity results for enzyme variants with compound mutations are shown in Tables 7 and 8. Activity and selectivity results for enzyme variants with additional compound mutations are shown in Tables 9 and 10.

[0145] The high selectivity and activity of each enzyme variant play a crucial role in efficiently converting racemic methyl 3-hydroxy-2-methylene-butanoate to (3R)-3-hydroxy-2-methylene-butanoic acid, producing a nearly enantiomerically pure product in high yield.

[0146] The enzyme selectivity ee [%] 3-hydroxy-2-methylene-butanoic acid is defined as the difference between the molar fractions of (3R)-3-hydroxy-2-methylene-butanoic acid and (3S)-3-hydroxy-2-methylene-butanoic acid divided by the sum of the molar fractions of (3R)-3-hydroxy-2-methylene-butanoic acid and (3S)-3-hydroxy-2-methylene-butanoic acid.

number

number

[0147] Table 5: Lipase variants showing relative improvement in enzymatic activity compared to lipase of SEQ ID NO: 1. The relative improvement in enzymatic activity is defined as the quotient, in percentage, of the enantiomeric excess ee [%] of each variant and the enantiomeric excess ee [%] of the reference lipase (SEQ ID NO: 1). The substrate ee [%] of lipase of SEQ ID NO: 1 is 9.8.

[0148] [Table 5] Table 6: Lipase variants showing relative improvement in enzyme selectivity compared to the reference lipase of SEQ ID NO: 1. The relative improvement in enzyme selectivity is defined as the quotient, expressed as a percentage, of the ee [%] of 3-hydroxy-2-methylene-butanoic acid for each variant and the ee [%] of 3-hydroxy-2-methylene-butanoic acid for the reference lipase of SEQ ID NO: 1. The product ee [%] for the reference lipase is 75.8.

[0149] [Table 6] TIFF2026508596000027.tif171129 Table 7: Lipase variants showing relative improvement in enzymatic activity compared to the reference lipase of SEQ ID NO: 1. The relative improvement in enzymatic activity is defined as the quotient, in percentage, of the enantiomeric excess ee [%] of each variant and the enantiomeric excess [%] of the reference lipase (SEQ ID NO: 1). [Table 7] Table 8: Lipase variants showing relative improvement in enzyme selectivity compared to the reference lipase of SEQ ID NO: 1. The relative improvement in enzyme selectivity is defined as the quotient, expressed as a percentage, of the ee [%] of 3-hydroxy-2-methylene-butanoic acid of each variant and the ee [%] of 3-hydroxy-2-methylene-butanoic acid of the reference lipase of SEQ ID NO: 1. [Table 8] Example 4 Mutants based on the backbone mutations N57P, K122R, A212P, S234K, D289G, and G109H were supplemented with additional mutations in the sequences described in the examples above and tested for enzymatic activity and selectivity as described in Example 3.

[0150] Table 9: Lipase variants showing relative improvement in enzymatic activity compared to the reference lipase of SEQ ID NO: 171. The relative improvement in enzymatic activity is defined as the quotient, in percentage, of the enantiomeric excess ee [%] of each variant and the enantiomeric excess ee [%] of the reference lipase (SEQ ID NO: 171). All lipase variants in the table further have the mutations N57P, K122R, A212P, S234K, D289G, G109H. [Table 9] Table 10: Lipase variants showing relative improvement in enzyme selectivity compared to the reference lipase of SEQ ID NO: 171. The relative improvement in enzyme selectivity is defined as the quotient, expressed as a percentage, of the ee [%] of 3-hydroxy-2-methylene-butanoic acid for each variant and the ee [%] of 3-hydroxy-2-methylene-butanoic acid for the reference lipase of SEQ ID NO: 171. All lipase variants in the table further have the mutations N57P, K122R, A212P, S234K, D289G, G109H. [Table 10] Example 5: Enzymatic hydrolysis of different esters The enzymatic hydrolysis of the methyl ester (a) of 3-hydroxy-2-methylene-butanoic acid, followed by the respective tert-butyl ester (b), iso-butyl ester (c) and iso-propyl ester (d) was also tested under appropriate conditions (1 mL scale, 5 g / L ester compound, 5 g / L lyophilisate of enzyme SEQ ID NO: 1, 100 mM KPi buffer pH 8): [ka] All esters (a) to (d) allow for efficient enantiomeric resolution, with the (R)-ester being preferably hydrolyzed. [Table 11] Example 6: Alternative Solvents Several different solvents were tested for the chiral resolution of methyl 3-hydroxy-2-methylene-butanoate. The reaction conditions were: total volume 100 mL, 300 g / L racemic methyl 3-hydroxy-2-methylene-butanoate, 20 g / L spray-dried lipase of SEQ ID NO: 1, 70% solvent, 0.54 equivalents of KHCO, pH 8.5 (titrated with 40% w / v KCO), 45 °C, 6 h. In particular, MTBE, CPME, MIBK, and toluene enabled high enantiomeric excess of the hydrolysis product (3R)-3-hydroxy-2-methylene-butanoic acid (ee). [Table 12] Example 7: Further example of the enzymatic hydrolysis of racemic methyl 3-hydroxy-2-methylene-butanoate to (3R)-3-hydroxy-2-methylene-butanoic acid Next to the lipase of SEQ ID NO: 1, three further related variants have also been tested: a variant with the mutations Q295V and K298A (SEQ ID NO: 3); a variant with the mutations V33I and I254S (SEQ ID NO: 5); and a variant with the mutations T188S, I254S, P302L and Q304E (SEQ ID NO: 241).

[0151] Cultivation, biotransformation, and HPLC analysis were performed as described in Example 2. As described in Example 3, the enzyme selectivity ee [%] for 3-hydroxy-2-methylene-butanoic acid is defined as the difference between (3R)-3-hydroxy-2-methylene-butanoic acid and (3S)-3-hydroxy-2-methylene-butanoic acid divided by the sum of (3R)-3-hydroxy-2-methylene-butanoic acid and (3S)-3-hydroxy-2-methylene-butanoic acid. The enzyme activity ee [%] for the conversion of the substrate methyl(3R)3-hydroxy-2-methylene-butanoate is expressed as the difference between methyl-(3S)3-hydroxy-2-methylene-butanoate and methyl-(3R)3-hydroxy-2-methylene-butanoate divided by the sum of methyl-(3S)3-hydroxy-2-methylene-butanoate and methyl-(3R)3-hydroxy-2-methylene-butanoate. The reference lipase of SEQ ID NO: 1 shows 100% selectivity and 100% activity.

[0152] Table 11: Comparative lipase variants showing similar enzymatic activity profiles compared to the reference lipase of SEQ ID NO: 1. The relative difference in enzymatic activity is defined as the quotient, in percentage, of the enantiomeric excess ee [%] of each variant and the enantiomeric excess ee [%] of the reference lipase (SEQ ID NO: 1). [Table 13] Table 12: Comparative lipase variants showing similar enzyme selectivity compared to the reference lipase of SEQ ID NO: 1. The relative difference in enzyme selectivity is defined as the quotient, expressed as a percentage, of the ee [%] of 3-hydroxy-2-methylene-butanoic acid of each variant and the ee [%] of 3-hydroxy-2-methylene-butanoic acid of the reference lipase of SEQ ID NO: 1. [Table 14] As shown in Tables 11 and 12, the comparative lipases worsen the activity of the lipase and do not improve the selectivity for the substrates discussed.

[0153] Example 8: Enzymatic hydrolysis of racemic methyl 3-hydroxy-2-methylene-butanoate to (3R)-3-hydroxy-2-methylene-butanoic acid 8.0 g of spray-dried lipase SEQ ID NO: 1 was suspended in 50 g of water and diluted with 100 mL of MTBE. After warming to 45°C, a mixture of 120 g of racemic methyl 3-hydroxy-2-methylene-butanoate (99% purity) in 100 g of MTBE was dosed to the suspension over 3 hours. After complete dosing, the mixture was further stirred overnight at 45°C. During the dosing and additional stirring period, the pH was maintained at 7.5 by the parallel addition of 40% aqueous potassium carbonate. The biomass was then separated by centrifugation, and the phases were separated. After extracting the aqueous phase with 2 × 125 g of MTBE each, the combined organic extracts were concentrated under reduced pressure at 45°C to give methyl (3S)-3-hydroxy-2-methylene-butanoate with a purity of 91%, ee of 96%, and a corrected yield of 40%.

[0154] Example 9: Enzymatic hydrolysis of racemic methyl 3-hydroxy-2-methylene-butanoate to (3R)-3-hydroxy-2-methylene-butanoic acid 120 g of water was heated to 45°C, and 2.0 g of spray-dried lipase SEQ ID NO: 1 was added, followed by 280 mL of MTBE. At 45°C, 120 g of racemic methyl 3-hydroxy-2-methylenebutanoate (99% purity) was dosed to the suspension over 2 hours. After complete dosing, the mixture was stirred for an additional 8.5 hours at 45°C. During the dosing and additional stirring period, the pH was maintained at 7.5 by the parallel addition of 40% aqueous potassium carbonate. The lower aqueous phase was then separated and back-extracted once with 100 mL of MTBE. The extract was combined with the upper organic phase, and the mixture was distilled at 33-55°C, reducing the pressure to 10 mbar. After addition of 50 g of high-boiling Marlotherm, the residue was further distilled to 150 °C at 3-10 mbar to give 58.2 g (92% purity, 96% ee, 45% yield) of methyl (3S)-3-hydroxy-2-methylene-butanoate with a purity of 91%, 96% ee and a corrected yield of 40%.

[0155] Example 10: Enantioselective acylation of racemic methyl-3-hydroxy-2-methylene-butanoate A suspension of 1400 g of racemic methyl 3-hydroxy-2-methylene-butanoate [10.62 mol, 98.7% purity] and 105 g of Novozyme 435 is heated to an internal temperature of 25 °C. 1373 g of vinyl dodecanoate (5.95 mol, 98%) is added over 3 hours using a dosing pump under a vacuum of 50 mbar. The reaction mixture is then heated to an internal temperature of 35 °C and at 50 mbar for an additional 8 hours. Acetaldehyde is distilled under vacuum. The suspension is then allowed to proceed at 35 °C and 50 mbar for an additional 8 hours. The reaction mixture is heated under vacuum to a jacket temperature of 115 °C, and methyl (3S)-3-hydroxy-2-methylene-butanoate is distilled from the suspension. The vacuum is then gradually reduced to 3 mbar and the jacket temperature is increased to 135 °C. The product is analyzed using chiral HPLC standard methods. The isolated yield of methyl (3S)-3-hydroxy-2-methylene-butanoate is 41% with a chemical purity of >99% and an enantiomeric excess of >98% ee.

Claims

1. A protein variant having lipase activity, wherein the protein is encoded by an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO: 1; said protein variant, characterized in that the amino acid sequence of said protein variant differs from the amino acid sequence of SEQ ID NO: 1 at least in one of the following positions: i. the amino acid at position 44 is M, W, or Y; ii. the amino acid at position 51 is N or M; iii. the amino acid at position 52 is L; iv. the amino acid at position 53 is S, P, I, E, or A; v. the amino acid at position 54 is Q, M, F, G, E, L, T, or P; vi. the amino acid at position 55 is R, M, D, Y, S, or I; vii. the amino acid at position 109 is H or F; viii. the amino acid at position 110 is T or V; ix. the amino acid at position 111 is S; x. the amino acid at position 117 is F or S; xi. the amino acid at position 121 is V; xii. the amino acid at position 122 is Q, A, Y, R, or V; xiii. the amino acid at position 153 is N, Y, D, E, or C; xiv. the amino acid at position 160 is E, C, D, P, I, Q, K, M, S, F, A, or N; xv. the amino acid at position 179 is C; xvi. The amino acid at position 181 is Q; xvii. the amino acid at position 184 is G or T; xviii. the amino acid at position 211 is E; xix. The amino acid at position 212 is S; xx. the amino acid at position 216 is K or A; xxi. the amino acid at position 234 is K, T, or G; xxii. the amino acid at position 235 is V or M; xxiii. the amino acid at position 236 is T; xxiv. the amino acid at position 238 is A, K, D, E, or Q; xxv. the amino acid at position 240 is F; xxvi. the amino acid at position 289 is S or G; xxvii. the amino acid at position 291 is E or W; xxviii. the amino acid at position 317 is T; xxix. the amino acid at position 320 is E or G; xxx. The amino acid at position 321 is F.

2. 2. The protein variant according to claim 1, characterized in that the amino acid sequence of the protein variant differs from the amino acid sequence of SEQ ID NO: 1 by at least one of the following mutations: i. the amino acid at position 44 is W or Y; ii. the amino acid at position 54 is F; iii. the amino acid at position 55 is R; iv. the amino acid at position 109 is H; v. the amino acid at position 110 is T or V; vi. The amino acid at position 117 is F; vii. the amino acid at position 122 is Q or R; viii. the amino acid at position 160 is E; ix. the amino acid at position 216 is K; x. the amino acid at position 236 is T; xi. the amino acid at position 238 is K or E; xii. The amino acid at position 240 is F.

3. 3. The protein variant according to claim 1 or 2, characterized in that the amino acid sequence of the protein variant differs from the amino acid sequence of SEQ ID NO: 1 by at least the following mutation: the amino acid at position 109 is H.

4. 10. The protein variant according to any one of the preceding claims, wherein the protein variant comprises at least two, preferably at least three of the amino acid substitutions listed.

5. 10. The protein variant according to any one of the preceding claims, wherein the amino acid sequence of the protein variant differs from the amino acid sequence of SEQ ID NO: 1 by at least two, more preferably at least three, even more preferably at least four, particularly preferably at least five, and most preferably all of the following modifications selected from: i. the amino acid at position 57 is P; ii. the amino acid at position 109 is H; iii. the amino acid at position 122 is R; iv. the amino acid at position 212 is P; v. The amino acid at position 234 is K; vi. The amino acid at position 289 is G.

6. 10. The protein variant according to any one of the preceding claims, wherein the amino acid sequence of the protein variant differs from the amino acid sequence of SEQ ID NO: 1 by the following modification: i. the amino acid at position 57 is P; the amino acid at position 109 is H; the amino acid at position 122 is R, K, or Q, preferably R; the amino acid at position 212 is P; the amino acid at position 234 is K; and the amino acid at position 289 is G; ii. and further comprising at least one of the following modifications: the amino acid at position 44 is W or Y; the amino acid at position 54 is F; the amino acid at position 55 is R; the amino acid at position 110 is V or T; the amino acid at position 111 is T; the amino acid at position 117 is F; the amino acid at position 160 is E; the amino acid at position 216 is K, the amino acid at position 236 is T, the amino acid at position 238 is K or E; and the amino acid at position 240 is F.

7. 10. The protein variants are selected from the group consisting of SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239.

8. A nucleic acid molecule encoding a protein having the activity of a lipase according to any one of the preceding claims.

9. 9. The nucleic acid molecule of claim 8, which encodes a protein having lipase activity selected from the group consisting of: a) SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138 , 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240; b) A nucleic acid molecule having at least 60%, preferably 70%, more preferably 80%, even more preferably 90%, even more preferably 95%, even even more preferably 96%, particularly preferably 97%, most preferably 98%, or especially preferably 99% identity to the nucleic acid sequence shown in a).

10. A recombinant nucleic acid molecule comprising the nucleic acid molecule of claim 8 or 9.

11. The recombinant nucleic acid molecule of claim 10, wherein the recombinant nucleic acid molecule is a vector or a plasmid.

12. A host cell comprising a protein according to any one of claims 1 to 7, or a nucleic acid molecule according to claim 8 or 9, or a recombinant nucleic acid molecule according to claim 10 or 11.

13. Use of a protein according to any one of claims 1 to 7 for the stereoselective hydrolysis of racemic methyl 3-hydroxy-2-methylene-butanoate to (3R)-3-hydroxy-2-methylene-butanoic acid.

14. A method for enantiomerically hydrolyzing a substrate of formula (II) to a compound of formula (III), comprising contacting the substrate with a protein mutant according to any one of claims 1 to 7: 【Chemistry 1】 [In the formula, R １ and R ２ are independently selected from substituted or unsubstituted (n)-alkyl, isoalkyl, aryl, alkyl-substituted or aryl-substituted aryl.

15. The substrate of formula (II-1) is enantiomerically hydrolyzed to a compound of formula (III-1): 【Chemistry 2】 The method of claim 14, further comprising isolating the compound of formula (I-1).

16. 16. The process according to claim 14 or 15, wherein the process is carried out in a two-phase system of water and an organic solvent, preferably selected from methyl tert-butyl ether, toluene, 2-methyltetrahydrofuran, methyl isobutyl ketone, cyclohexane, cyclopentyl methyl ether, chlorobenzene, tert-amyl methyl ether, ethyl acetate, and isopropyl acetate.

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