Lipase with improved stereoselectivity and lipase-based chiral resolution method

Abstract

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

Description

TECHNICAL FIELD

[0001] The present invention relates to a protein having improved lipase activity, nucleic acid molecules encoding said protein and a method for the enzymatic resolution of secondary alcohols. BACKGROUND

[0002] Enantiomerically enriched or pure alcohols are important compounds for the production of agrochemicals or pharmaceutical compounds. Thus, the absolute configuration of the stereogenic center of a chiral alcohol is of crucial importance for the synthesis of the respective active agent. The production of the correct chirality is often a challenge in the production of desired target molecules.

[0003] Various methods for the preparation of enantiomerically enriched alcohols are known in the art. On the one hand, chiral transition metal catalysts can be used, however, which are characterized by high costs (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 catalysis, organocatalytic acyl transfer kinetic resolutions suitable for bulkier ester substrates have also 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) report on two lipases, which act as highly stereoselective practical catalysts in almost anhydrous organic solvents. Under such "unnatural" conditions, the enzymes can catalyze esterification and transesterification reactions asymmetrically, which is not feasible in aqueous solutions, since hydrolysis dominates. Thus, a number of 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) describe the enzymatic resolution reaction 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) show the use of esterases for enantioselective ester hydrolysis in buffered aqueous systems, however, in the presence of DMSO and only with moderate yields.

[0004] US 4,732,853 A describes a process for the preparation of chiral epoxy alcohols by enantioselective hydrolysis with a lipase.

[0005] EP 0716712 B1 describes lipase-catalyzed acylation of alcohols with diketenes, in particular for the production of enantioselectively acylated alcohols from racemic alcohols.

[0006] WO2012146935A1 discloses modified lipase variants, as well as polynucleotides and recombinant expression vectors encoding lipase variant polypeptides, and methods of producing such lipase variants in selected bacterial and fungal host cells. The particular lipase variants have increased enzyme specificity or enhanced trans-selectivity. Methods for their use to reduce or eliminate trans fatty acids from substrates are also described.

[0007] Despite the several improvements of lipases achieved so far, several limitations occurring in the asymmetric synthesis of secondary alcohols or the resolution of racemic alcohols still have to be overcome, such as unfavourable equilibria, substrate and product inhibition, poor thermostability, insufficient substrate specificity and in particular low enantioselectivity of the lipases. Furthermore, the downstream processing and separation of the target chiral alcohol is challenging in large scale applications, while the importance of atom economy increases significantly on an industrial scale.

[0008] Therefore, further improvements of lipases are needed, in particular with regard to the production of enantiomerically enriched or pure products with increased atom economy, and further process improvements. SUMMARY

[0009] The present invention solves at least part of the above problems by providing novel lipases with superior enantiomeric selectivity and by providing novel methods for the chiral resolution of industrially relevant building blocks.

[0010] The lipases described herein have several advantages over known wild type and other known lipases. In particular, the modified or variant lipases described herein have the advantage that they can produce enantiomerically enriched or enantiomerically nearly pure or pure compounds better than the corresponding wild type lipases.

[0011] A first aspect of the present invention relates to a protein having lipase activity, wherein the protein is encoded by an amino acid sequence which is at least 80 %, preferably 85 %, more preferably 90 %, further preferably 92 %, even more preferably 95 % identical to the amino acid sequence shown in SEQ ID No. 1,

[0012] characterized in that the amino acid sequence of the variant differs from the amino acid sequence of SEQ ID No. 1 in at least one of the following positions:

[0013] i. the amino acid at position 44 is not L, preferably the amino acid at position 44 is M, W or Y;

[0014] ii. the amino acid at position 51 is not L, preferably the amino acid at position 51 is N or M;

[0015] iii. the amino acid at position 52 is not V, preferably the amino acid at position 52 is L;

[0016] iv. the amino acid at position 53 is not T, preferably the amino acid at position 53 is S, P, I, E or A;

[0017] v. the amino acid at position 54 is not D, preferably the amino acid at position 54 is Q, M, F, G, E, L, T or P;

[0018] vi. the amino acid at position 55 is not A, preferably the amino acid at position 55 is R, M, D, Y, S or I;

[0019] vii. the amino acid at position 109 is not G, preferably the amino acid at position 109 is H or F;

[0020] viii. the amino acid at position 110 is not M, preferably the amino acid at position 110 is T or V;

[0021] ix. the amino acid at position 111 is not A, preferably the amino acid at position 111 is T or S; x. the amino acid at position 117 is not Y, preferably the amino acid at position 117 is F or S;

[0022] xi. the amino acid at position 121 is not Y, preferably the amino acid at position 121 is V; xii. the amino acid at position 122 is not K, preferably the amino acid at position 122 is Q, A, Y, R or V;

[0023] xiii. the amino acid at position 153 is not H, preferably the amino acid at position 153 is N, Y, D, E or C;

[0024] xiv. the amino acid at position 160 is not T, preferably the amino acid at position 160 is E, C, D, P, I, Q, K, M, S, F, A or N;

[0025] xv. the amino acid at position 179 is not D, preferably the amino acid at position 179 is C; xvi. the amino acid at position 181 is not A, preferably the amino acid at position 181 is Q; xvii. the amino acid at position 184 is not A, preferably the amino acid at position 184 is G or T;

[0026] xviii. the amino acid at position 211 is not Y, preferably the amino acid at position 211 is E;

[0027] xix. the amino acid at position 212 is not A, preferably the amino acid at position 212 is S or P;

[0028] xx. the amino acid at position 216 is not Y, preferably the amino acid at position 216 is K or A;

[0029] xxi. the amino acid at position 234 is not S, preferably the amino acid at position 234 is K, T or G;

[0030] xxii. the amino acid at position 235 is not S, preferably the amino acid at position 235 is V or M;

[0031] xxiii. the amino acid at position 236 is not K, preferably the amino acid at position 236 is T;

[0032] xxiv. the amino acid at position 238 is not R, preferably the amino acid at position 238 is A, K, D, E or Q;

[0034] xxv. the amino acid at position 240 is not Y, preferably the amino acid at position 240 is F;

[0035] xxvi. the amino acid at position 289 is not D, preferably the amino acid at position 289 is S or G; xxvii. the amino acid at position 291 is not G, preferably the amino acid at position 291 is E or W;

[0037] xxviii. the amino acid at position 317 is not N, preferably the amino acid at position 317 is T;

[0038] xxix. the amino acid at position 320 is not N, preferably the amino acid at position 320 is E or G;

[0040] xxx. the amino acid at position 321 is not L, preferably the amino acid at position 321 is F.

[0041] SEQ ID No. 1 refers to a reference protein sequence having lipase activity.

[0042] The meaning 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 found in Table 2 below under the subheading "Sequence Description" in the paragraph below.

[0043] By "amino acid corresponding to position x" in a first amino acid sequence (e.g. position 44 in SEQ ID No. 1) is meant herein, in case the amino acid numbering of a second amino acid sequence differs from the amino acid numbering of the first amino acid sequence, the amino acid of the second amino acid sequence which, when compared to the first amino acid sequence, occurs in the pairwise sequence alignment of the first amino acid sequence with the second amino acid sequence at position x of the first amino acid sequence.

[0044] In the context of the present application, the term "identity" with respect to sequence identity or sequence similarity is to be understood as meaning the number of identical amino acids or nucleotides, respectively, which are shared by a first nucleic acid or amino acid sequence with another (second) nucleic acid or amino acid sequence, expressed in percentage over the entire sequence length.

[0045] "Sequence identity" can be determined by aligning two amino acid sequences or two nucleotide sequences using a global or local alignment algorithm, for example included in known software like GAP or BESTFIT or the program "Needle" of Emboss. This software uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizing the number of gaps. Typically, (for both nucleotide and protein alignments) the default parameters are used, gap creation penalty = 10, gap extension penalty = 0.5. For nucleotides, the default scoring matrix used is DNAFULL, and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 10915-10919). Sequence alignment and percentage score of sequence identity can be determined, for example, using software like EMBOSS, which is accessible on the web site of the EBI (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 like FASTA, BLAST etc., but preferably the hit sequences should be retrieved and pairwise aligned to finally determine sequence identity.

[0046] If the sequences to be compared with each other have different lengths, the identity is determined by separately determining the percentage of the number of amino acids or nucleotides which the shorter sequence has in common with the longer sequence. Preferably, 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 publicly available from Julie Thompson (Thompson@EMBL-Heidelberg.DE) and Toby Gibson (Gibson@EMBL-Heidelberg.DE), European Molecular Biology Laboratory, Meyerhofstrasse 1, D 69117 Heidelberg, Germany. ClustalW can also be downloaded from various internet pages, in particular from IGBMC (Institut de Genetique et de Biologie Moleculaire et Cellulaire, B.P. 163, 67404 Illkirch Cedex, France; ftp: / / ftp-igbmc.u-strasbg.fr / pub / ) and EBI (ftp: / / ftp.ebi.ac.uk / pub / software / ) and all mirror internet pages of EBI (European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK).

[0047] Preferably, the identity between the nucleotide sequences of the nucleic acid molecules described, for example, in the context of the application and the nucleotide sequences of other nucleic acid molecules is determined using the ClustalW computer program version 1.8 or Clustal 2. In this case, the parameters must be set as follows:

[0048] Preferably, the identity between the nucleotide sequences of the nucleic acid molecules described, for example, in the context of the application and the nucleotide sequences of other nucleic acid molecules is determined using the ClustalW computer program version 1.8 or Clustal 2. In this case, the parameters must be set as follows:

[0049] KTUPLE = 2, TOPDIAGS = 4, PAIRGAP = 5, DNAMATRIX: IUB, GAPOPEN = 10, GAPEXT = 5, MAXDIV = 40, TRANSITIONS: unweighted.

[0050] Furthermore, "identity" means that there is a functional and / or structural equivalence between the nucleic acid molecules or the proteins encoded by them. Functional equivalence refers to the nucleic acid molecule sequence or the amino acid sequence encoding a protein having lipase activity. Nucleic acid molecules which are homologous to the above-described molecules and represent derivatives of these molecules are typically variants of these molecules which represent modifications having the same biological function or catalyzing the same reaction, i.e. encoding a protein having lipase activity. They can be naturally occurring variants, e.g. sequences from other species, or mutations, wherein these mutations can occur in a natural way or be introduced by targeted mutagenesis. Furthermore, variants can be synthetically produced sequences. Allelic variants can be naturally occurring variants or synthetically produced variants or variants produced by recombinant DNA technology. However, with regard to the present application, it is decisive that those variants encode a protein having lipase activity and comprise the amino acid substitutions (replacements), deletions or insertions described herein with regard to the proteins according to the present application.

[0051] A special type of derivative is a nucleic acid molecule which differs from the nucleic acid molecules described in the context of the present application, e.g. due to the degeneracy of the genetic code.

[0052] According to the NC-IUBMB (Nomenclature Committee of the International Union of Biochemistry and Molecular Biology), lipases belong to the class of hydrolases (EC 3). Hydrolases are a class of enzymes that are commonly used as biochemical catalysts that use water to break chemical bonds, which often results in the division of larger molecules into smaller molecules. Under non-native, anhydrous conditions, these enzymes can also catalyze esterification reactions, such as acetylation and transesterification reactions. The hydrolase group includes enzymes acting on ester bonds (EC 3.1), which includes carboxylic ester hydrolases (EC 3.1.1) and as a subfamily lipases (EC 3.1.1.3). Lipases have been identified from plants, mammals, and microorganisms, including Pseudomonas, Vibrio, Acinetobacter, Burkholderia, Chromobacterium, cutinase from Fusarium solani (FSC), Candida antarctica A (Cal A), Rhizopus oryzae (ROL), Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML), Aspergillus Niger, Fusarium heterosporum, Fusarium oxysporum, or Fusarium culmorum.

[0053] If the protein has the activity of a lipase, this can be detected by methods known and described in the art.

[0054] The method used to detect whether the protein according to the application has the activity of a lipase is not decisive. Preferably, in connection with the present application, the method is described in the “Examples” section.

[0055] In a further embodiment of the application, 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:

[0056] i. the amino acid at position 44 is W or Y;

[0057] ii. the amino acid at position 54 is F;

[0058] iii. the amino acid at position 55 is R;

[0059] iv. the amino acid at position 109 is H;

[0060] v. the amino acid at position 110 is T or V;

[0061] vi. the amino acid at position 117 is F;

[0062] vii. the amino acid at position 122 is Q or R;

[0063] viii. the amino acid at position 160 is E;

[0064] ix. the amino acid at position 216 is K;

[0065] x. the amino acid at position 236 is T;

[0066] xi. the amino acid at position 238 is K or E;

[0067] xii. the amino acid at position 240 is F.

[0068] Preferably, the amino acid sequence of said variant differs from the amino acid sequence of SEQ ID NO. 1 at least by having the mutation G109H, i.e. the amino acid at position 109 is H.

[0069] In addition to the amino acid sequence corresponding to the amino acid sequence shown in SEQ ID No. 1 as described above, the lipase variant protein according to the present application can exhibit other amino acid modifications (amino acid substitutions, deletions or insertions).

[0070] In a further embodiment of the present application, the amino acid sequence of said variant differs from the amino acid sequence of SEQ ID NO. 1 by at least two, further preferably at least three, even further preferably at least four, particularly preferably at least five modifications selected from:

[0071] i. the amino acid at position 57 is not N, preferably the amino acid is P;

[0072] ii. the amino acid at position 109 is not G, preferably the amino acid is H;

[0073] iii. the amino acid at position 122 is not K, preferably the amino acid is R;

[0074] iv. the amino acid at position 212 is not A, preferably the amino acid is P;

[0075] v. the amino acid at position 234 is not S, preferably the amino acid is K;

[0076] vi. the amino acid at position 289 is not D, preferably the amino acid is G.

[0077] Preferably, the amino acid sequence of said variant differs from the amino acid sequence of SEQ ID NO. 1 by the following modifications:

[0078] i. the amino acid at position 57 is P;

[0079] ii. the amino acid at position 109 is H;

[0080] iii. the amino acid at position 212 is P;

[0081] iv. the amino acid at position 234 is K; and

[0082] v. the amino acid at position 289 is G.

[0083] Further preferably, the amino acid sequence of said variant differs from the amino acid sequence of SEQ ID NO. 1 by the following modifications:

[0084] i. the amino acid at position 57 is P;

[0085] ii. the amino acid at position 109 is H;

[0086] iii. the amino acid at position 122 is R;

[0087] iv. the amino acid at position 212 is P;

[0088] v. the amino acid at position 234 is K; and

[0089] vi. the amino acid at position 289 is G.

[0090] In a further embodiment of the application, the amino acid sequence of said variant differs from the amino acid sequence of SEQ ID NO. 1 by the following modifications:

[0091] - 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;

[0092] - and additionally 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, the amino acid at position 240 is F.

[0093] A preferred embodiment of the present application is a protein according to the present application encoding a lipase having the amino acid sequence shown as SEQ ID No. 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.

[0094] A further embodiment of the present application relates to a nucleic acid molecule encoding a protein according to the present application.

[0095] The nucleic acid molecule according to the present application can be any kind of nucleic acid as long as it encodes a protein according to the present application. The nucleic acid can be a ribonucleic acid molecule (such as RNA, mRNA) or a deoxyribonucleic acid molecule (DNA, including genomic DNA, which can or can not comprise introns and coding DNA).

[0096] Of particular interest in the present application are nucleic acid molecules encoding a protein having lipase activity comprising the amino acid sequence as shown in SEQ ID No. 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.

[0097] Accordingly, the present application also relates to a nucleic acid molecule encoding a protein having lipase activity selected from the group consisting of

[0098] a) a nucleic acid molecule comprising the nucleic acid sequence as shown in SEQ ID No. 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;

[0099] b) a nucleic acid molecule having at least 60%, preferably 70%, more preferably 80%, further preferably 90%, even more preferably 95%, even further preferably 96%, particularly preferably 97%, most preferably 98% or especially preferably 99% identity to the nucleic acid sequence as indicated in a).

[0100] In the context of the present application, the term "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). Particularly preferably, "hybridization" means hybridization under the following conditions:

[0101] Hybridization buffer:

[0102] 2xSSC; 10xDenhardt's solution (Fikoll 400 + PEG + BSA; ratio 1 : 1 : 1); 0.1% SDS; 5mM EDTA; 50mM Na2HP04; 250μg / ml herring sperm DNA; 50μg / mL tRNA;

[0103] or

[0104] 25M sodium phosphate buffer, pH 7.2; ImM EDTA; 7% SDS

[0105] Hybridization temperature: T = 65-68°C

[0106] Washing buffer: 0.1 x SSC; 0.1% SDS

[0107] Washing temperature: T = 65-68°C.

[0108] The nucleic acid molecules hybridizing with nucleic acid molecules encoding a protein having lipase activity can be derived from any organism; thus, they can be derived from bacteria, fungi, animals, humans, plants or viruses.

[0109] The nucleic acid molecules hybridizing to nucleic acid molecules encoding a protein having lipase activity are preferably derived from microorganisms, more preferably from fungi or bacteria, most preferably from bacteria.

[0110] The nucleic acid molecules hybridizing to said molecules can be isolated from, for example, a genomic library or an eDNA library. These 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 using PCR amplification.

[0111] The fragments used as hybridization samples can also be synthetic fragments or oligonucleotides prepared using conventional synthesis techniques, which are essentially identical in sequence to the nucleic acid molecules described in the context of the present application. When genes hybridizing to the nucleic acid sequences described in the context of the present application are identified and isolated, the sequences should be identified and the properties of the proteins encoded by the sequences should be analyzed in order to identify whether they are proteins having lipase activity. Methods of how to identify whether a protein has lipase activity are known to the person skilled in the art.

[0112] The molecules hybridizing to the nucleic acid molecules described in the context of the present application include in particular fragments, derivatives and allelic variants of said nucleic acid molecules. In the context of the present application, the term "derivative" means that the sequences of these molecules differ from the sequences of the above-mentioned nucleic acid molecules in one or more positions and have a high identity with these sequences. The differences from the above-mentioned nucleic acid molecules can be, for example, due to deletions, additions, substitutions, insertions or recombination.

[0113] The meaning of the abbreviations of the nucleotides a, c, g, t and the abbreviations of the degenerate nucleotides r, y, s, w, k, m, b, d, h, v, n can be found in Table 1 below under the subheading "Sequence Description" under the paragraph below. Which amino acid is encoded by a codon comprising a degenerate nucleotide can be found in Table 3 below under the subheading "Sequence Description" under the paragraph below.

[0114] Furthermore, the present application relates to recombinant nucleic acid molecules comprising the nucleic acid molecules according to the present application.

[0115] In connection with the present application, the term "recombinant nucleic acid molecule" is to be understood as meaning a nucleic acid molecule which, in addition to the nucleic acid molecule according to the application, also contains additional sequences which do not naturally occur in the combination in which they occur in the recombinant nucleic acid according to the application. In the present context, the above-mentioned additional sequences can be any sequences, preferably they are functional or regulatory sequences (promoters, termination signals, enhancers, ribosome binding sites (rbs), leader sequences which enhance transcription, translation or RNA stability, subcellular targeting sequences and the like), particularly preferably they are functional or regulatory sequences which are active in microorganisms, and especially particularly preferably they are regulatory sequences which are active in fungi, in particular yeasts, or in bacteria. Methods for producing recombinant nucleic acid molecules according to the application are known to the person skilled in the art and include genetic methods, for example the joining, genetic recombination or new 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).

[0116] In a further embodiment, the recombinant nucleic acid molecule according to the application comprises the nucleic acid molecule according to the application linked to a regulatory sequence which initiates transcription in a prokaryotic or eukaryotic cell.

[0117] A "regulatory sequence which initiates transcription" in a cell is also referred to as a promoter.

[0118] Information on regulatory sequences and plasmids is well known to the person skilled in the art and is described, for example, in the Registry of Standard Biological Parts (One Kendall Square, Suite B6104, Cambridge, MA 02139, USA) supported by the International Genetically Engineered Machine (iGEM) Foundation on the World Wide Web (http: / / parts.igem.org / Catalog).

[0119] Regulatory sequences that direct transcription of the coding sequence in prokaryotic organisms such as E. coli and in eukaryotic organisms are well described in the literature, in particular those for expression in yeast such as Saccharomyces cerevisiae. An overview of various systems for expressing proteins 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 are pAOX1, pHIS4, pGAL, pScADH2 (Baghban et al., 2018, see above). Common bacterial promoters are T5, T7, rhamnose-inducible, arabinose-inducible, PhoA, artificial trc (trp-lac) promoters, as described by Marschall et al. (2017, Appl Microbiol Biotechnol 101, 501 -512) and Tegel et al. (201 1, FEBS Journal 278, 729-739).

[0120] A further embodiment of the recombinant nucleic acid molecule of the present application is a vector or plasmid comprising the nucleic acid molecule according to the present application.

[0121] “Vectors” are generally understood in the field of molecular biology to represent a nucleic acid sequence or a vehicle comprising a nucleic acid sequence for the transfer of genetic material (DNA or RNA) into target cells. Vectors can be plasmids, such as T-DNA or binary vectors for the production of transgenic plants, expression vectors for the expression of nucleic acid sequences in host cells, shuttle vectors suitable for propagation in different hosts, or viruses or bacteriophages that have been modified to deliver foreign genetic material into a host.

[0122] “Plasmids” are generally understood in the field of molecular biology and herein to represent a self-replicating, usually circular DNA molecule that is separate from chromosomal DNA when present in a host cell.

[0123] The nucleic acid molecule according to the application, the recombinant nucleic acid molecule according to the application, the vector or plasmid according to the application can be used for the production of a protein according to the application, for example by expressing the nucleic acid molecule according to the application in a host cell.

[0124] A further embodiment of the present application relates to a host or host cell comprising or expressing a nucleic acid molecule according to the application, or comprising a protein according to the application, or comprising a recombinant nucleic acid molecule according to the application, or comprising a vector according to the application, or comprising a plasmid according to the application.

[0125] The nucleic acid molecule according to the application encoding a protein having lipase activity can be expressed in a host cell, for example for their propagation or for the production of a protein according to the application. For expression in a host cell, the nucleic acid molecule according to the application can be comprised on a vector or plasmid, or they can be stably integrated into the genome of the respective host cell. The nucleic acid molecule according to the application can also be comprised in a vector supporting its introduction into a host cell.

[0126] A further embodiment of the present application relates to a host or host cell according to the application comprising a nucleic acid molecule according to the application or comprising a recombinant nucleic acid molecule according to the application or comprising a vector according to the application or comprising a plasmid according to the application, and in each case comprising a protein according to the application.

[0127] A further embodiment of the present application relates to a host or host cell according to the application comprising a nucleic acid molecule according to the application or comprising a recombinant nucleic acid molecule according to the application or comprising a vector according to the application or comprising a plasmid according to the application, and in each case expressing a protein according to the application.

[0128] A further embodiment of the present application relates to a host or host cell according to the application comprising a nucleic acid molecule according to the application or comprising a recombinant nucleic acid molecule according to the application or comprising a vector according to the application or comprising a plasmid according to the application, and in each case expressing a protein, wherein the protein has the activity of a lipase.

[0129] "Expressing a nucleic acid molecule" is to be understood herein to mean that, in the case of a nucleic acid molecule which is RNA or mRNA, the nucleic acid molecule is translated into a protein, preferably into a protein having the activity of a lipase, or, in the case of a nucleic acid molecule which is DNA or cDNA, it is transcribed (and, in the case of genomic DNA containing introns, processed) into mRNA, preferably into mRNA encoding a protein having the activity of a lipase, and subsequently translated into a protein, preferably into a protein having the activity of a lipase.

[0130] Transcription of a given nucleic acid molecule in the host can be confirmed by methods known to those skilled in the art, such as detecting specific transcripts (mRNA) of exogenous nucleic acid molecules by Northern blot analysis or RT-PCR.

[0131] Whether a host or host cell contains a given protein or a protein derived from an expressed nucleic acid molecule can be determined by methods known to those skilled in the art, such as immunological methods, including Western blot analysis, ELISA (Enzyme-Linked Immunosorbent Assay), or RIA (Radio Immunoassay). Those skilled in the art are familiar with methods for preparing antibodies that specifically react with a particular protein, i.e., bind specifically to a particular 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, MAUSA 0245; GenScript, 60 Centennial Ave., Piscataway, NJ 08854, USA) offer the preparation of such antibodies as an order service.

[0132] Furthermore, those skilled in the art can test whether a host or host cell contains the protein according to the invention by detecting the (additional) activity of a protein with lipase activity in the corresponding host cell. Preferably, the activity of the protein with additional lipase activity in the corresponding host cell is detected by comparing the lipase activity of a host cell according to the invention with the corresponding activity of a host cell that does not contain the protein according to the invention.

[0133] The presence of lipase activity in a protein can be tested using methods known in the art.

[0134] The host or host cell according to the invention can be produced by those skilled in the art using known methods for genetic modification or transformation of organisms.

[0135] A further subject-matter of the present application is therefore a host or host cell according to the application, in particular a prokaryotic or eukaryotic host or host cell, which is genetically modified (or transformed) using a nucleic acid molecule according to the application or using a recombinant nucleic acid molecule according to the application or a vector according to the application or a plasmid according to the application. Preferably, the genetically modified (transformed) host or host cell according to the application expresses a protein having lipase activity, more preferably the genetically modified (transformed) host or host cell according to the application expresses a protein according to the application.

[0136] "Genetically modified with a nucleic acid molecule" or "transformed with a nucleic acid molecule" is to be understood herein to mean that a nucleic acid molecule has been introduced into or has been 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.

[0137] Descendants, offspring or progeny of a host or host cell according to the application are also an embodiment of the present application, preferably these descendants, offspring or progeny comprise a nucleic acid molecule according to the application or comprise a recombinant nucleic acid molecule according to the application or comprise a vector according to the application or comprise a plasmid according to the application or comprise a protein according to the application, more preferably these descendants, offspring or progeny comprise a nucleic acid molecule according to the application or comprise a recombinant nucleic acid molecule according to the application or comprise a vector according to the application or comprise a plasmid according to the application 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 application or comprise a recombinant nucleic acid molecule according to the application or comprise a vector according to the application or comprise a plasmid according to the application and in each case express a protein, wherein the protein has the activity of a lipase according to the application.

[0138] The host or host cell according to the present application can be a host or host cell derived from any prokaryotic or eukaryotic organism. The host or host cell can be a bacterium or bacterial cell (such as E. coli; a bacterium of the genus Bacillus, in particular Bacillus subtilis; a bacterium of the genus Agrobacterium, in particular Agrobacterium tumefaciens or Agrobacterium rhizogenes; a bacterium of the genus Pseudomonas, in particular Pseudomonas fluorescens; a bacterium of the genus Streptomyces spp; a bacterium of the genus Rhodococcus spp, in particular Rhodococcus rhodochrous, Vibrio natrigens; a bacterium of the genus Corynebacterium, in particular Corynebacterium glutamicum), or a fungus or fungal cell (such as a mushroom of the genus Agaricus, in particular Agaricus bisporus; a fungus of the genus Aspergillus; a fungus of the genus Trichoderma; or a yeast, in particular S. cerevisiae; a yeast of the genus Pichia ssp., such as P. pastoris), as well as a plant or plant cell, or they can be an animal or animal cell.

[0139] A preferred host cell according to the present application is a cell of a microorganism. Within the scope of the present patent application, this is to be understood as including all bacteria and all protists (e.g. fungi, in particular yeasts and algae) as defined in Schlegel "General Microbiology" (Georg Thieme Publishing House (1985), 1-2).

[0140] With regard to microorganisms, the host or host cell according to the present application is preferably a bacterium / bacterial cell or a yeast / yeast cell, most preferably it is a bacterium / bacterial cell. With regard to a bacterium / bacterial cell, the host or host cell according to the present application is preferably a bacterium of the genus Bacillus / Bacillus cell or a bacterium of E. coli / E. coli cell, most preferably an E. coli / E. coli cell.

[0141] Alternatively, Pseudomonas, in particular Pseudomonas fluorescens; Streptomyces; Rhodococcus, in particular Rhodococcus globerulus; Vibrio, in particular Vibrio natriqum; Corynebacterium, in particular Corynebacterium glutamicum and the like can be the host or host cell according to the present application.

[0142] A preferred embodiment of the present application relates to a host or host cell according to the present application comprising a nucleic acid molecule according to the present application, wherein the nucleic acid molecule according to the present application is characterized in that the codons of said nucleic acid molecule are altered such that they are adapted to the frequency of usage of codons of the host or host cell, respectively.

[0143] The host cell according to the present application can be used for the production of a protein according to the present application. The protein according to the present application can be used in a process for the production of an enantiomerically enriched or almost enantiomerically pure secondary alcohol.

[0144] A second aspect of the present application relates to a process for the resolution of an enantiomerically enriched or pure compound of formula (I) by hydrolyzing a racemic substrate of formula (II) in an enantioselective manner, which process comprises contacting the substrate with a variant protein having lipase activity according to the first aspect of the present application or with a lipase according to SEQ ID N o .1,

[0145]

[0146] wherein R 1 and R 2 are independently from each other selected from substituted or unsubstituted (n)-alkyl, isoalkyl, unsubstituted aryl, alkyl-substituted or aryl-substituted aryl.

[0147] Preferably, the substrate is contacted with a variant protein having lipase activity according to the first aspect of the present application.

[0148] R 1 and R 2 are preferably selected independently from each other as linear or branched C 1-10 residues.

[0149] Further preferably, R 1 is selected from methyl, isobutyl, tert-butyl and isopropyl; and / or R 2 is methyl.

[0150] Even further preferably, R 1 is selected from methyl, isobutyl, tert-butyl and isopropyl; and R 2 is methyl.

[0151] In another embodiment of the present application, the process comprises the resolution of an enantiomerically enriched or pure compound of formula (I-1) by hydrolyzing a racemic substrate of formula (II-1) in an enantioselective manner,

[0152]

[0153] The method preferably comprises isolating compound (I) or (I-1) and / or (III) or (III-1) after hydrolysis. The isolation of compound (I-1) can be performed by any method known to the person skilled in the art and is preferably performed by extraction and / or direct distillation.

[0154] The lipase variant or protein variant according to the present application shows an improved selectivity and / or an improved specific activity in the stereoselective hydrolysis of 3-hydroxy-2-methylene-butyric acid methyl ester (formula II-1) and is more suitable for isolating the enantiomerically enriched or almost pure substrate (3S)-3-hydroxy-2-methylene-butyric acid methyl ester (formula I-1) compared to the wild-type lipase.

[0155] Compound of formula (I)

[0156]

[0157] represents an important building block in the synthesis of complex agrochemical compounds. In particular, (3S)-3-hydroxy-2-methylene-butyric acid methyl ester of formula (I-1)

[0158]

[0159] which is the S-enantiomer of racemic 3-hydroxy-2-methylene-butyric acid methyl ester, is an important intermediate in the corresponding synthesis of agrochemical compounds described in WO 2018 / 228985. The selective hydrolysis of the R-enantiomer of racemic 3-hydroxy-2-methylene-butyric acid methyl ester by the lipase variant according to the present application allows the isolation of the S-enantiomer of said racemic 3-hydroxy-2-methylene-butyric acid methyl ester.

[0160] In a further embodiment of the present application, the method is performed in aqueous solution.

[0161] Preferably, the method is performed in water and an organic solvent such as a mixture of methyl tert-butyl ether, toluene, 2-methyltetrahydrofuran, methyl isobutyl ketone, cyclohexane, cyclopentyl methyl ether, chlorobenzene, tert-amyl methyl ether, ethyl acetate, isopropyl acetate or a biphasic liquid system. Even further preferably, the organic solvent is methyl tert-butyl ether (MTBE).

[0162] The ratio of organic solvent to water is further preferably from 1 : 1 to 6: 1, even more preferably from 2: 1 to 4: 1.

[0163] Surprisingly, it was found that a significant reduction of the aqueous phase, which is partially replaced by an organic solvent, not only leads to a higher concentration of the final product, thus increasing the productivity, but also to a reduced enzyme / lipase load.

[0164] Furthermore, the presence of an organic solvent, such as MTBE, facilitates the efficient downstream processing of the reaction product, i.e. the isolation of the compound of formula (I) or (I-1).

[0165] In a further embodiment of the present application, the method is carried out at a temperature between 20 °C and 60 °C, preferably between 30 °C and 55 °C. Further preferred, the method is carried out at a temperature between 35 °C and 50 °C.

[0166] In a further embodiment of the present application, the method is carried out for at least 1 h, preferably for at least 2 h, further preferred for at least 3 h.

[0167] In a further embodiment of the present application, the method is carried out for at most 40 h, preferably for at most 30 h, further preferred for at most 20 h.

[0168] In a further embodiment of the present application, the method is carried out at a pH between 7 and 8.5, preferably between 7.2 and 8.0, further preferred 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 bicarbonates or carbonates, hydrogen phosphates and hydroxides.

[0169] At a pH above 8.5, the enzyme is inactivated; further, partial hydrolysis of the ester of formula (I) occurs. At a pH below 7, the reaction rate is reduced and the final reaction stops. Further, at a pH of 4.5 or below, the enzyme is inactivated.

[0170] The pH can be controlled by the addition of a base such as NaOH, KOH, K2CO3, KHCO3, Na2CO3, NaHCO3. Preferred bases are carbonates or bicarbonates. The base can be added to the reaction as a solid or as an aqueous solution (diluted or saturated). The base can be added to the reaction before the substrate is dosed or the base is dosed to the reaction in parallel to the substrate. The substrate and / or the base can be dosed or added in one portion.

[0171] The lipase or protein variant of SEQ ID No. 1 according to the first aspect of the present application can be provided as a purified enzyme or in the form of a 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 is freeze-dried to obtain a freeze-dried cell or biomass.

[0172] While lyophilized biomass is easy to handle in terms of independent timing of the claimed process, the direct use of broth / cell culture for the claimed process is cost- and time-saving.

[0173] For downstream processing, the reaction is preferably carried out in a mixture of water and a water-immiscible solvent. In case the reaction is carried out in a biphasic mixture comprising water and an immiscible organic solvent, the phases are separated and the aqueous phase is eventually back-extracted. The biomass can be separated by known means of separation (such as centrifugation, filtration or decantation) prior to distillation, or the distillation can be carried out without separation of the biomass.

[0174] In the context of the present application it is understood that all embodiments referring to the method according to the second aspect can be combined with all different lipase variants according to the first aspect of the present application and in particular with the preferred lipase variants. That is, the method of hydrolyzing a racemic substrate of formula (II) in an enantioselective manner to isolate an enantiomerically enriched or pure compound of formula (I) can be carried out by means of a protein having lipase activity, wherein the protein is encoded by an amino acid sequence which has at least 80 %, preferably 85 %, more preferably 90 %, further preferably 92 %, even more preferably 95 % of the amino acid sequence shown in SEQ ID No. 1,

[0175] characterized in that the amino acid sequence of the variant differs from the amino acid sequence of SEQ ID No. 1 in at least one of the following positions:

[0176] i. the amino acid at position 44 is not L, preferably the amino acid at position 44 is M, W or Y;

[0177] ii. the amino acid at position 51 is not L, preferably the amino acid at position 51 is N or M;

[0178] iii. the amino acid at position 52 is not V, preferably the amino acid at position 52 is L;

[0179] iv. the amino acid at position 53 is not T, preferably the amino acid at position 53 is S, P, I, E or A;

[0180] v. the amino acid at position 54 is not D, preferably the amino acid at position 54 is Q, M, F, G, E, L, T or P;

[0181] vi. the amino acid at position 55 is not A, preferably the amino acid at position 55 is R, M, D, Y, S or I;

[0182] vii. the amino acid at position 109 is not G, preferably the amino acid at position 109 is H or F;

[0183] viii. the amino acid at position 110 is not M, preferably the amino acid at position 110 is T or V;

[0184] ix. the amino acid at position 111 is not A, preferably the amino acid at position 111 is T or S;

[0185] x. the amino acid at position 117 is not Y, preferably the amino acid at position 117 is F or S;

[0186] xi. the amino acid at position 121 is not Y, preferably the amino acid at position 121 is V;

[0187] xii. the amino acid at position 122 is not K, preferably the amino acid at position 122 is Q, A, Y, R or V;

[0188] xiii. the amino acid at position 153 is not H, preferably the amino acid at position 153 is N, Y, D, E or C;

[0189] xiv. the amino acid at position 160 is not T, preferably the amino acid at position 160 is E, C, D, P, I, Q, K, M, S, F, A or N;

[0190] xv. the amino acid at position 179 is not D, preferably the amino acid at position 179 is C;

[0191] xvi. the amino acid at position 181 is not A, preferably the amino acid at position 181 is Q; xvii. the amino acid at position 184 is not A, preferably the amino acid at position 184 is G or T;

[0192] xviii. the amino acid at position 211 is not Y, preferably the amino acid at position 211 is E;

[0193] xix. the amino acid at position 212 is not A, preferably the amino acid at position 212 is S or P;

[0194] xx. the amino acid at position 216 is not Y, preferably the amino acid at position 216 is K or A;

[0195] xxi. the amino acid at position 234 is not S, preferably the amino acid at position 234 is K, T or G;

[0196] xxii. the amino acid at position 235 is not S, preferably the amino acid at position 235 is V or M;

[0197] xxiii. the amino acid at position 236 is not K, preferably the amino acid at position 236 is T; xxiv. the amino acid at position 238 is not R, preferably the amino acid at position 238 is A, K, D, E or Q;

[0198] xxv. the amino acid at position 240 is not Y, preferably the amino acid at position 240 is F;

[0199] xxvi. the amino acid at position 289 is not D, preferably the amino acid at position 289 is S or G; xxvii. the amino acid at position 291 is not G, preferably the amino acid at position 291 is E or W;

[0200] xxviii. the amino acid at position 317 is not N, preferably the amino acid at position 317 is T;

[0201] xxix. the amino acid at position 320 is not N, preferably the amino acid at position 320 is E or G;

[0202] xxx. the amino acid at position 321 is not L, preferably the amino acid at position 321 is F.

[0203] The amino acid sequence of the variant protein lipase in the process for hydrolysing a racemic substrate of formula (II) in an enantioselective manner to isolate an enantiomerically enriched or pure compound of formula (I) can be distinguished from the amino acid sequence of SEQ ID NO. 1 by at least one of the following modifications:

[0204] i. the amino acid at position 44 is W or Y;

[0205] ii. the amino acid at position 54 is F;

[0206] iii. the amino acid at position 55 is R;

[0207] iv. the amino acid at position 109 is H;

[0208] v. the amino acid at position 110 is T or V;

[0209] vi. the amino acid at position 117 is F;

[0210] vii. the amino acid at position 122 is Q or R;

[0211] viii. the amino acid at position 160 is E;

[0212] ix. the amino acid at position 216 is K;

[0213] x. the amino acid at position 236 is T;

[0214] xi. the amino acid at position 238 is K or E;

[0215] xii. the amino acid at position 240 is F.

[0216] Preferably, the amino acid sequence of the variant protease lipase in the process for hydrolysing a racemic substrate of formula (II) in an enantioselective manner to isolate an enantiomerically enriched or pure compound of formula (I) differs from the amino acid sequence of SEQ ID NO. 1 in that the amino acid at position 109 is H.

[0217] The amino acid sequence of the variant protease lipase in the process for hydrolysing a racemic substrate of formula (II) in an enantioselective manner to isolate an enantiomerically enriched or pure compound of formula (I) can differ from the amino acid sequence of SEQ ID NO. 1 in at least two, further preferably at least three, even further preferably at least four, particularly preferably at least five modifications selected from:

[0218] i. the amino acid at position 57 is not N, preferably the amino acid is P;

[0219] ii. the amino acid at position 109 is not G, preferably the amino acid is H;

[0220] iii. the amino acid at position 122 is not K, preferably the amino acid is R;

[0221] iv. the amino acid at position 212 is not A, preferably the amino acid is P;

[0222] v. the amino acid at position 234 is not S, preferably the amino acid is K;

[0223] vi. the amino acid at position 289 is not D, preferably the amino acid is G.

[0224] Preferably, the amino acid sequence of the variant differs from the amino acid sequence of SEQ ID NO. 1 in the following modifications:

[0225] i. the amino acid at position 57 is P;

[0226] ii. the amino acid at position 109 is H;

[0227] iii. the amino acid at position 212 is P;

[0228] iv. the amino acid at position 234 is K; and

[0229] v. the amino acid at position 289 is G.

[0230] Further preferably, the amino acid sequence of the variant differs from the amino acid sequence of SEQ ID NO. 1 in the following modifications:

[0231] i. the amino acid at position 57 is P;

[0232] ii. the amino acid at position 109 is H;

[0233] iii. the amino acid at position 122 is R;

[0234] iv. the amino acid at position 212 is P;

[0235] v. the amino acid at position 234 is K; and

[0236] vi. the amino acid at position 289 is G.

[0237] The amino acid sequence of the variant protein lipase in the process for hydrolysing a racemic substrate of formula (II) in an enantioselective manner to isolate an enantiomerically enriched or pure compound of formula (I) can differ from the amino acid sequence of SEQ ID NO. 1 in that:

[0238] - 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;

[0239] - and additionally 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, the amino acid at position 240 is F.

[0240] The protein according to the application encoding the lipase which can be used in a process for the hydrolysis of a racemic substrate of formula (II) in an enantioselective manner to separate an enantiomerically enriched or pure compound of formula (I) is preferably according to SEQ ID No. 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.

[0241] A further embodiment of the present application is the use of a protein according to the first aspect of the present application for the stereoselective hydrolysis of racemic methyl 3-hydroxy-2-methylene-butyrate to separate methyl (3S)-3-hydroxy-2-methylene-butyrate.

[0242] "Enantiomerically enriched" means herein that one of the two enantiomers is present in the composition in a higher amount than the other enantiomer, preferably at least 60% of one enantiomer is present in the composition, more preferably at least 65% of one enantiomer is present in the composition, 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.

[0243] "Enantiomerically nearly pure" means herein 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 97.0 %, even more preferably one of the two enantiomers is present in the composition in an amount of at least 98.0 %, particularly preferably one of the two enantiomers is present in the composition in an amount of at least 98.5 %, most preferably one of the two enantiomers is present in the composition in an amount of at least 99.0 %, or especially preferably one of the two enantiomers is present in the composition in an amount of at least 99.5 %.

[0244] A third aspect of the present application relates to a process for the enantiomeric enrichment of a compound of formula (I)

[0245]

[0246] which comprises reacting a compound of formula (II) with a compound of formula (IV)

[0247]

[0248] and in particular

[0249]

[0250] wherein R 1 and R 2 are as defined above, and R 3 is a substituted or unsubstituted (n)-alkyl or isoalkyl group.

[0251] "Anhydrous conditions" means a reaction system comprising at most 3 % water in the liquid phase.

[0252] The reaction is preferably carried out in an organic solvent, such as hexane or n-heptane, or without any further solvent.

[0253] R 3 may preferably be a linear C 1-16 residue, in particular a methyl group or an unsubstituted and saturated C8-C 12 residue. The compound (IV) can in particular be vinyl acetate or vinyl laurate.

[0254] The lipase is preferably immobilized on a solid carrier during the action. Immobilization leads to an increased stability of the enzyme in non-aqueous solutions.

[0255] The reaction is preferably carried out at a temperature of 20-40°C, preferably 25-35°C. Such a temperature range allows for optimal enzymatic reactivity.

[0256] The reaction is preferably carried out for at least 1 h, further preferably at least 3 h, even further preferably at least 5 h.

[0257] The lipase is preferably recycled after the reaction has taken place. This allows for designing the process in a cost-saving manner. Thus, the lipase can be used multiple times for the reaction according to the third aspect or for additional reactions.

[0258] The compound of formula (I) is preferably isolated directly from the reaction mixture or from the supernatant obtained after pouring the reaction mixture by distillation. The lipase is preferably retained in the remaining reaction mixture and can be reused for another reaction.

[0259] The lipase is preferably a CALB lipase. CALB is a non-specific lipase derived from Candida antarctica B, which was first described in 1994 (Uppenberg J, Patkar S, Bergfors T, Jones TA (1994) J Mol Biol 235(2): 790-792).

[0260] Furthermore, the lipase is preferably immobilized on a hydrophobic carrier, such as an acrylic resin. Commercial versions of CALB are, for example, 435, which can be used to carry out the reaction. DETAILED DESCRIPTION

[0261] The polypeptide (enzyme, i.e. lipase) and the process according to the present application allow for an efficient enantiomeric enrichment of the compound of formula (I)

[0262]

[0263] It was found that the introduction of certain amino acid modifications into the protein variants according to the present application increases the activity of the lipase, in particular with respect to its substrate specificity, which means that these further modified lipase variants are better suited for the production of enantiomerically enriched or almost pure products compared to known lipase variants. This refers in particular to the substrate (1-1). Enantiomerically pure or at least enriched (3S)-3-hydroxy-2-methylene-butyric acid methyl ester

[0264]

[0265] It is the S-enantiomer of racemic 3-hydroxy-2-methylene-butyric acid methyl ester and is an important intermediate in the corresponding synthesis of the compounds described in WO 2018 / 228985. The lipase variant of the present invention selectively catalyzes the hydrolysis of racemic 3-hydroxy-2-methylene-butyric acid methyl ester to (3R)-3-hydroxy-2-methylene-butyric acid, leaving the unhydrolyzed (3S)-3-hydroxy-2-methylene-butyric acid methyl ester.

[0266] The terms used herein are known to the person skilled in the art. Otherwise, the following definitions apply:

[0267] For the purposes of the present application, the term "alkyl" includes saturated hydrocarbon residues, which can be branched or straight-chain and which are unsubstituted or at least monosubstituted. Examples of suitable alkyl residues which can be unsubstituted or 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.

[0268] Unless defined elsewhere, the term "aryl" alone or in combination with other terms is to be understood as monocyclic or polycyclic, preferably monocyclic or bicyclic, aromatic hydrocarbon radicals having preferably 6, 10 or 14 carbon atoms. The aryl group can be unsubstituted or monosubstituted or polysubstituted, which can have identical or different substituents.

[0269] Sequence Description

[0270] Throughout the application, the abbreviations for nucleotides and amino acids are used in accordance with the following IUPAC code:

[0271] Table 1

[0272] IUPAC nucleotide codes Base A Adenine C Cytosine G Guanine T (or U) Thymine (or Uracil) R A or G Y C or T S G or C W A or T K G or T M A or C B C or G or T D A or G or T H A or C or T V A or C or G N Any base - Gap

[0273] In order to distinguish between amino acids and nucleotides, the capital nucleotide code abbreviations given in the above table are written in lower case letters in this table.

[0274] Table 2

[0275] IUPAC amino acid codes Three letter codes Amino acid A Ala Alanine C Cys Cysteine D Asp Aspartic acid E Glu Glutamic acid F Phe Phenylalanine G Gly Glycine H His Histidine I Ile Isoleucine K Lys Lysine L Leu Leucine M Met Methionine N Asn Asparagine P Pro Proline Q Gln Glutamine R Arg Arginine S Ser Serine T Thr Threonine V Val Valine W Trp Tryptophan Y Tyr Tyrosine

[0276] The codon usage herein follows the so-called "universal genetic code" according to the following table, wherein "t" is replaced by "u" in the ribonucleic acid (RNA) sequence.

[0277] Table 3

[0278]

[0279]

[0280]

[0281] The sequence listing associated with this application is filed in electronic form and is hereby incorporated by reference in its entirety into the specification. "PRT" stands for "protein" and "NUC" stands for "nucleic acid".

[0282] Table 4a: Lipase variants according to the first aspect of the application (SEQ ID No. 7-169) and further lipases (SEQ ID No. 1-6)

[0283]

[0284]

[0285]

[0286]

[0287]

[0288] Table 4b: Further lipase variants according to the application (proteins SEQ ID No. 171-239). The variants comprise the backbone mutations N57P, K122R, A212P, S234K, D289G and G109H (SEQ ID No. 171, 172), unless (i) the mutation "R122K" is indicated in addition, which means that the K122R mutation has been back-mutated so that position 122 carries a K again, or (ii) the mutation "R122Q" is indicated in addition, which means that the K122R mutation has been further mutated so that position 122 carries a Q.

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295] Examples

[0296] The following examples illustrate the application without limiting it.

[0297] Starting materials and protocols

[0298] Analytical grade chemicals and ready-to-use kits were purchased from common suppliers such as Sigma Aldrich, Acros Organics, Fisher Scientific, Qiagen or Stratagene. 435 purchased from Sigma Aldrich.

[0299] Example 1

[0300] Cloning of lipase variants:

[0301] The nucleic acid sequence of SEQ ID No. 1 or a variant thereof was cloned into a pKA81 based expression vector. The genetic elements were incorporated into the vector by methods known in the art. To express different lipase variants, the vector was introduced into electrocompetent E. coli W3110 cells.

[0302] Variant library:

[0303] Substitutions of nucleotides in the wild type sequence or sequences derived therefrom were made to allow for amino acid substitutions. This exchange can be achieved by various methods of molecular biology. One method to substitute nucleic acids is site-directed mutagenesis, which can result in mutation of one or more sites in the amino acid sequence. Methods for site-directed mutagenesis are state of the art and documented in the literature (e.g. Directed Mutagenesis: A Practical Approach, 1991, Edited by M.J. McPHERSON, IRL PRESS) and can be purchased as ready-to-use kits (e.g. QUIKCHANGE® lightening mutagenesis kit from Qiagen or Stratagene). After insertion of the mutation in the gene sequence and cultivation in a suitable E. coli cloning strain, the obtained plasmid was transformed into E. coli W3110. TM lightening mutagenesis kit) purchasing. After insertion of the mutation in the gene sequence and cultivation in a suitable E. coli cloning strain, the obtained plasmid was transformed into E. coli W3110.

[0304] The transformed cells were tested in appropriate biotransformation reactions to determine product yield and selectivity. Appropriate biotransformation reactions are described below, see Example 2. Sequence verification was performed as known in the art.

[0305] Glycerol stocks of E. coli cultures transformed with the respective expression plasmids were prepared by adding one volume of a 40% glycerol solution to one volume of E. coli culture.

[0306] For isolation of single bacterial colonies, an appropriate dilution of the E. coli culture was inoculated onto LB-agar plates containing the appropriate concentration of kanamycin and incubated at 37°C until multiple single colonies were obtained.

[0307] Example 2

[0308] Culture:

[0309] For the preparation of pre-cultures, sterile 2 ml 96-deep well plates (Eppendorf, Hamburg, Germany) were used and mixed with 590 μΐ of TB medium (50 μg / ml kanamycin) and 10 μΐ of a glycerol stock filled with the respective E. coli strain. Alternatively, cell material from agar plate colonies was used to inoculate 590 μΐ of TB medium (50 μg / ml kanamycin). The pre-cultures were incubated at 37°C and 250 rpm for 17 h.

[0310] For the production of the main cultures, sterile 2 ml 96-deep well plates containing 510 μΐ of TB medium (50 mg / L kanamycin) were used. The expression cultures were inoculated with 30 μΐ of the pre-cultures and incubated at 37°C and 250 rpm. After 4 h of incubation, the expression of the enzyme was induced by the addition of 60 μΐ of IPTG (10 mM IPTG diluted in expression medium supplemented with 50 mg / L kanamycin). The expression cultures were then incubated at 28°C and 250 rpm for 20 h.

[0311] The cells were harvested by centrifugation at 4°C and 2500 x g for 15 min. The culture supernatant was discarded and the cell pellet was suspended in 200 μΐ of PBS. The cells were then lyophilized for 24 h and stored at 4°C until use.

[0312] Bioconversion, chiral resolution and analysis:

[0313] The chiral resolution of (3S)-3-hydroxy-2-methylene-butyric acid methyl ester was performed in microtiter plates with lyophilized supernatant. For the chiral resolution, 100 μΐ of racemic 3-hydroxy-2-methylene-butyric acid methyl ester, 105 μΐ of MTBE (methyl tert-butyl ether) and 45 μΐ of ultrapure water (containing 416.6 g / L KHCO3) were used per well. Each plate was sealed and incubated at 45°C in a shaker at 300 rpm for 6 h. The reaction was then stopped by the addition of 22.5 μΐ of a 20% H2SO4 solution. For further extraction, 1 mL of MTBE was added per well in a deep well plate. The plate was shaken for 10 min at room temperature and then centrifuged at 2500 x g for 10 min. Then 10 μΐ of the organic phase was transferred into a 96-well PCR plate containing 100 μΐ of MTBE and analyzed by HPLC.

[0314] HPLC analysis method

[0315] The samples were analyzed using HPLC in the following settings:

[0316] Instrument: Agilent Technologies 1290 Infinity II; Column: Lux cellulose-2, 100 x 4.6 mm, 3 pm; Eluent A: pre-mixed heptane (+0.05% formic acid); Eluent B: ethanol; Flow rate: isocratic (90% eluent A / 10% eluent B), flow rate: 0.8 mL / min; Temperature: 25 °C; Sample injection volume: 1 pL; Detection: absorption at 210 nm.

[0317] Racemic 3-hydroxy-2-methylene-butyric acid methyl ester and racemic 3-hydroxy-2- methylene-butyric acid (prepared according to the method for the preparation of the reference biotransformation sample) were used as reference material and quantification standard. Appropriate dilutions of the standard were used in order to be able to quantify the used substrate and the resulting product using the standard line. The substrate conversion and product formation of the analyzed samples were analyzed. The evaluation and comparison between the individual samples was done by determining the substrate and product ee [%].

[0318] Example 3: Enzymatic hydrolysis of racemic 3-hydroxy-2-methylene-butyric acid methyl ester to (3R)-3-hydroxy-2-methylene-butyric acid

[0319] The cultivation, biotransformation and HPLC analysis were performed as described in Example 2. During cultivation, the glycerol culture was used for inoculation. The activity and selectivity results of the enzyme variants with single point mutations can be found in Tables 5 and 6 below. The activity and selectivity results of the enzyme variants with combined mutations can be found in Tables 7 and 8. The activity and selectivity results of the enzyme variants with additional combined mutations can be found in Tables 9 and 10.

[0320] The high selectivity and high activity of the individual enzyme variants were decisive for the efficient conversion of racemic 3-hydroxy-2-methylene-butyric acid methyl ester to (3R)-3-hydroxy-2-methylene-butyric acid to produce almost enantiomerically pure product in high yield.

[0321] The enzyme selectivity ee [%] 3-hydroxy-2-methylene-butyrate is defined by the difference of the molar fraction of (3R)-3-hydroxy-2-methylene-butyrate and (3S)-3-hydroxy-2- methylene-butyrate divided by the sum of the molar fraction of (3R)-3-hydroxy-2- methylene-butyrate and (3S)-3-hydroxy-2-methylene-butyrate.

[0322]

[0323] The enzyme activity ee [%] of the molar fraction conversion of the substrate (3R) 3-hydroxy-2-methylene-butyric acid methyl ester is described by the difference between the molar fractions of (3S) 3-hydroxy-2-methylene-butyric acid methyl ester and (3R) 3-hydroxy-2-methylene-butyric acid methyl ester divided by the sum of (3S) 3-hydroxy-2-methylene-butyric acid methyl ester and methyl (3R) 3-hydroxy-2-methylene-butyric acid ester.

[0324]

[0325] The lipase of SEQ ID No. 1 shows 100% selectivity and 100% activity.

[0326] Table 5: Lipase variants showing a relative improvement of the enzyme activity compared to the lipase of SEQ ID No. 1. The relative improvement of the enzyme activity is defined as the quotient of the enantiomeric excess ee [%] of the respective variant and the enantiomeric excess ee [%] of the reference lipase (SEQ ID No. 1) in percent. The substrate ee [%] of the lipase of SEQ ID No. 1 is 9.8.

[0327]

[0328] Table 6: Lipase variants showing a relative improvement of the enzyme selectivity compared to the reference lipase of SEQ ID No. 1. The relative improvement of the enzyme selectivity is defined as the quotient of the ee [%] of 3-hydroxy-2-methylene-butyric acid of the respective variant and the ee [%] of 3-hydroxy-2-methylene-butyric acid of the reference lipase of SEQ ID No. 1 in percent. The product ee [%] of the reference lipase is 75.8.

[0329]

[0330]

[0331] Table 7: Lipase variants showing a relative improvement of the enzyme activity compared to the reference lipase of SEQ ID No. 1. The relative improvement of the enzyme activity is defined as the quotient of the enantiomeric excess ee [%] of the respective variant and the enantiomeric excess ee [%] of the reference lipase (SEQ ID No. 1) in percent.

[0332]

[0333] Table 8: Lipase variants showing a 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 of the ee [%] of 3-hydroxy-2-methylene-butyric acid of the respective variant and the ee [%] of 3-hydroxy-2-methylene-butyric acid of the reference lipase of SEQ ID No. 1, expressed in percent.

[0334]

[0335] Example 4:

[0336] The variants based on the scaffold mutations N57P, K122R, A212P, S234K, D289G and G109H have been supplemented with further mutations in the sequence as described in the examples above and tested for enzyme activity and selectivity as described in example 3.

[0337] Table 9: Lipase variants showing a relative improvement in enzyme activity compared to the reference lipase of SEQ ID No. 171. The relative improvement in enzyme activity is defined as the quotient of the enantiomeric excess ee [%] of the respective variant and the enantiomeric excess ee [%] of the reference lipase (SEQ ID No. 171), expressed in percent. All lipase variants in the table carry additionally the mutations N57P, K122R, A212P, S234K, D289G, G109H.

[0338]

[0339]

[0340] Table 10: Lipase variants showing a 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 of the ee [%] of 3-hydroxy-2-methylene-butyric acid of the respective variant and the ee [%] of 3-hydroxy-2-methylene-butyric acid of the reference lipase of SEQ ID No. 171, expressed in percent. All lipase variants in the table carry additionally the mutations N57P, K122R, A212P, S234K, D289G, G109H.

[0341]

[0342]

[0343] Example 5: Enzymatic hydrolysis of different esters

[0344] In addition to 3-hydroxy-2-methylene-butyric acid methyl ester (a), the corresponding tert-butyl ester (b), isobutyl ester (c) and isopropyl ester (d) were also tested for enzymatic hydrolysis under suitable conditions (1 mL range, 5 g / L ester compound, 5 g / L lyophilisate of the enzyme of SEQ ID No. 1, 100 mM KPI buffer pH 8):

[0345]

[0346] All esters (a) to (d) allowed efficient enantiomeric resolution, with the (R)-esters being preferentially hydrolyzed:

[0347]

[0348]

[0349] * compared to zero time point control

[0350] Example 6: Alternative solvents

[0351] Several different solvents have been tested for chiral resolution of 3-hydroxy-2- methylene-butyric acid methyl ester. Reaction conditions: 100 mL total volume, 300 g / L of racemic 3-hydroxy-2-BCS methylene-butyric acid methyl ester, 20 g / L of spray-dried SEQ ID No. 1 lipase, 70% solvent, 0.54 equivalents of KHCO3, pH 8.5 (titrated with 40% w / v K2CO3), 45 °C, 6 h. In particular, MTBE, CPME, MIBK and toluene allowed high enantiomeric excess of the hydrolysis product (3R)-3-hydroxy-2-methylene-butyric acid (ee).

[0352]

[0353] Example 7: Further examples of enzymatic hydrolysis of racemic 3-hydroxy-2- methylene-butyric acid methyl ester to (3R)-3-hydroxy-2-methylene-butyric acid

[0354] In addition to the lipase of SEQ ID No. 1, three further related variants have been tested, namely a variant with mutations Q295V and K298A (SEQ ID No. 3); and a variant with mutations V33I and I254S (SEQ ID No. 5); and a variant with mutations T188S, I254S, P302L and Q304E (SEQ ID No. 241).

[0355] Cultivation, biotransformation and HPLC analysis were performed as described in Example 2. Enzymatic selectivity ee [%] was defined as described in Example 3 for 3-hydroxy-2-methylene-butyric acid ester from the difference of (3R)-3-hydroxy-2-methylene-butyric acid and (3S)-3-hydroxy-2-methylene-butyric acid divided by the sum of (3R)-3-hydroxy-2-methylene-butyric acid and (3S)-3-hydroxy-2-methylene-butyric acid. Enzymatic activity ee [%] for the substrate (3R) 3-hydroxy-2-methylene-butyric acid methyl ester conversion was described by the difference between (3S) 3-hydroxy-2-methylene-butyric acid methyl ester and (3R) 3-hydroxy-2-methylene-butyric acid methyl ester divided by the sum of (3S) 3-hydroxy-2-methylene-butyric acid methyl ester and (3R) 3-hydroxy-2-methylene-butyric acid methyl ester. The reference lipase of SEQ ID No. 1 showed 100% selectivity and 100% activity.

[0356] Table 11: Comparative lipase variants showing similar enzymatic activity profile compared to the reference lipase of SEQ ID No. 1. The relative difference in enzymatic activity was defined as the quotient of the enantiomeric excess ee [%] of the respective variant and the enantiomeric excess ee [%] of the reference lipase (SEQ ID No. 1) in percent.

[0357]

[0358] Table 12: Comparative lipase variants showing similar enzymatic selectivity compared to the reference lipase of SEQ ID No. 1. The relative difference in enzymatic selectivity was defined as the quotient of the ee [%] of 3-hydroxy-2-methylene-butyric acid of the respective variant and the ee [%] of 3-hydroxy-2-methylene-butyric acid of the reference lipase of SEQ ID No. 1 in percent.

[0359]

[0360] As shown in Tables 11 and 12, the compared lipases have a worse lipase activity and do not improve the selectivity for the discussed substrates.

[0361] Example 8: Enzymatic hydrolysis of racemic 3-hydroxy-2-methylene-butyric acid methyl ester to (3R)-3-hydroxy-2-methylene-butyric acid

[0362] Example 1 : Enantioselective acylation of racemic 3-hydroxy-2-methylene-butyric acid methyl ester

[0363] Example 9: Enzymatic hydrolysis of racemic 3-hydroxy-2-methylene-butyric acid methyl ester to (3R)-3-hydroxy-2-methylene-butyric acid

[0364] Example 9: Enzymatic hydrolysis of racemic 3-hydroxy-2-methylene-butyric acid methyl ester to (3R)-3-hydroxy-2-methylene-butyric acid

[0365] Example 10: Enantioselective acylation of racemic 3-hydroxy-2-methylene-butyric acid methyl ester

[0366] A suspension of 1400 g of racemic 3-hydroxy-2-methylene-butyric acid methyl ester [10.62 mol, 98.7% purity] and 105 g of Novozyme 435 was heated to an internal temperature of 25°C. Using a metering pump, 1373 g of vinyl dodecanoate (5.95 mol, 98%) was added over 3 h under a vacuum of 50 mbar. Subsequently, the reaction mixture was heated to an internal temperature of 35°C and 50 mbar for an additional 8 h. Acetaldehyde was distilled off under vacuum. The suspension was then kept at 35°C and 50 mbar for an additional 8 hours. The reaction mixture was heated to a jacket temperature of 115°C under vacuum to distil (3S)-3-hydroxy-2-methylene-butyric acid methyl ester from the suspension. In this respect, the vacuum was gradually reduced to 3 mbar and the jacket temperature was increased to 135°C. The product was analysed using standard chiral HPLC methods. A chemical purity of >99% and an enantiomeric excess of >98% ee were reached. The isolated yield of (3S)-3-hydroxy-2-methylene-butyric acid methyl ester was 41%.

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. Its features are, The amino acid sequence of the protein variant differs from that of SEQ ID NO.1 at at least 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 either T or V; ix. The amino acid at position 111 is S; x. The amino acid at position 117 is either 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 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; The amino acid at position 216 is either 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; The amino acid at position 240 of xxv is F; xxvi. The amino acid at position 289 is either S or G; xxvii. The amino acid at position 291 is either E or W; xxviii. The amino acid at position 317 is T; xxix. The amino acid at position 320 is either E or G; The amino acid at position 321 of xxx is F.

2. The protein variant according to claim 1, characterized in that, The amino acid sequence of the protein variant differs from that of SEQ ID NO.1 in that it contains at least one of the following mutations: i. The amino acid at position 44 is either 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 either 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. The protein variant according to claim 1 or 2, characterized in that, The amino acid sequence of the protein variant differs from that of SEQ ID NO.1 in that it has at least the following mutation: the amino acid at position 109 is H.

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

5. A protein variant according to any one of the preceding claims, wherein the amino acid sequence of the protein variant differs from that of SEQ ID NO.1 in that it is selected from 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: 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. A protein variant according to any one of the preceding claims, wherein the amino acid sequence of the protein variant differs from that of SEQ ID NO.1 by the following modifications: 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 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. A protein variant according to any one of the preceding claims, wherein the protein variant carries SEQ ID No. 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, 13 The amino acid sequence shown is one of the following: 7, 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 lipase activity according to any one of the preceding claims.

9. The nucleic acid molecule according to claim 8, wherein the nucleic acid molecule encodes a protein having lipase activity, said nucleic acid molecule being selected from... a) Contains SEQ ID No. 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, 13 Nucleic acid molecules with the nucleic acid sequences shown in 8, 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) is a nucleic acid molecule that has at least 60%, preferably 70%, more preferably 80%, further preferably 90%, even more preferably 95%, even further preferably 96%, particularly preferably 97%, most preferably 98%, or especially preferably 99% identity with the nucleic acid sequence shown in a).

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

11. The recombinant nucleic acid molecule according to claim 10, wherein the recombinant nucleic acid molecule is a vector or 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 the protein according to any one of claims 1 to 7 in the stereoselective hydrolysis of racemic methyl 3-hydroxy-2-methylene-butyrate to (3R)-3-hydroxy-2-methylene-butyric acid.

14. A method for enantioselectively hydrolyzing a substrate of formula (II) into a compound of formula (III), comprising contacting the substrate with a protein variant according to any one of claims 1 to 7, Where R 1 and R 2 It is independently selected from substituted or unsubstituted (n)-alkyl, isoalkyl, aryl, alkyl-substituted or aryl-substituted aryl groups.

15. The method according to claim 14, used for enantioselectively hydrolyzing a substrate of formula (II-1) into a compound of formula (III-1), Further including compounds of the isolated form (I-1).

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

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