Aminoacylase and methods of use thereof

Abstract

The present invention relates to an isolated polypeptide having aminoacylase activity and comprising an amino acid sequence having over its entire length at least 87 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. The invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding such an aminoacylase, to a plasmid vector comprising said nucleic acid molecule, to a recombinant host cell comprising the isolated nucleic acid molecule or the vector and to methods to produce said aminoacylase. The invention also covers the use of the aminoacylase according to the invention for the N-acylation of amino acids or salts thereof as well as methods to produce N-acyl amino acids or salts thereof using the aminoacylase. Furthermore, the invention relates to the obtained N-acyl amino acids, compositions comprising them and the use of the produced N-acyl amino acids in a cosmetic product, home care product or an industrial and / or institutional product.

Description

[0001] AMINOACYLASE AND METHODS OF USE THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to an isolated polypeptide having aminoacylase activity and comprising an amino acid sequence having over its entire length at least 87 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. The invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding such an aminoacylase, to a plasmid vector comprising said nucleic acid molecule, to a recombinant host cell comprising the isolated nucleic acid molecule or the vector and to methods to produce said aminoacylase. The invention also covers the use of the aminoacylase according to the invention for the N-acylation of amino acids or salts thereof as well as methods to produce N-acyl amino acids or salts thereof using the aminoacylase. Furthermore, the invention relates to the obtained N-acyl amino acids, compositions and products comprising them and the use of the obtained N-acyl amino acids in a cosmetic product, home care product or an industrial and / or institutional product.

[0004] BACKGROUND OF THE INVENTION

[0005] Acylamino acids and the structurally related acyl peptides are desirable ingredients for cosmetics, household and cleaning agents and show promise for use in textile technology, as they have low irritation potential and excellent dermatological compatibility. Further, they can be produced from renewable raw materials and thus are also promising in view of sustainability and environmental considerations. Some acylamino acids are already commercially available, either as mild surfactants (e.g. Plantapon® ACG from BASF (C12 / C14 acyl glutamate)) or anionic emulsifiers (e.g. Eumulgin® SG from BASF (C18 acyl glutamate)).

[0006] Besides glutamate, other amino acids, such as glycine, aspartate or lysine may also be used as polar head groups in such acylamino acids, for example for cosmetic applications. Other acyl amino acid compounds, such as acyltyrosine, are marketed as pharmaceutically active ingredients (e.g. Tyrostan® from Sinerga). Good surfactant properties were also found for lauroyl leucine and lauroyl phenylalanine, which were found to have good wetting properties and good foamability. Lauroyl arginine (e.g. AMISAFE® AL-01 from Ajinomoto) is used as a mild surfactant and hair conditioning agent.

[0007] All these products are easily degraded (typically hydrolyzed) on the skin, in the mouth, and, especially, by the microflora under the armpit (e.g. by C. striatum). This process can be used for the controlled release of nourishing, pharmaceutically active (e.g., antimicrobial) amino acids or peptides. In addition to the above applications, the use of acylamino acids as corrosion inhibitors for metals has been proposed (DE 102010062807 A1). Other suggested applications of acylamino acids include, for example, the preparation of chiral micelles, the use as chelating agents or the use of cysteine for the synthesis of acylcysteine-based gemini surfactants.

[0008] The chemical synthesis of acylamino acids in aqueous solvent mixtures based on the Schotten- Baumann reaction is well known. Following this procedure, acylamino acids and structurally related surfactants (acyl peptides, acyltaurine, acylsarcosine) can be obtained. Solvent-free syntheses could be realized, for example, by adding polyols (US6060613A) or by conducting the reaction in a strongly alkaline medium (WO 2002057217 A2). However, the presence of water always leads to the undesired hydrolysis of the acyl chlorides in a side reaction which lowers the final yield of the products.

[0009] Furthermore, despite various known optimizations of this process, it still requires acyl chlorides as the starting material, which have - compared to the direct synthesis from fatty acids and amino acids - considerable drawbacks as regards their sustainability. Acid chlorides are typically produced by chlorination with thionyl chloride or phosphorus chlorides, which leads to the stoichiometric formation of sulfurous or phosphorous acid. Furthermore, the reaction with acid chlorides generates HCI in stoichiometric amounts, which must be captured via the dosage of a corresponding stoichiometric amount of base. In sum, the acid chloride route requires two additional reaction steps and leads to significant amounts of inorganic waste. In view of the general trend towards more green, sustainable and environmentally friendly methods, these processes thus have significant drawbacks.

[0010] While an acid chloride-free route for the synthesis of acyl sarcosinates has previously been reported by BASF (US5856538A), the described process suffers from the necessity to react amino acid salts under fairly harsh conditions with nearly stoichiometric amounts of methylate. Although other amino acids are claimed as suitable, no concrete examples are given.

[0011] As another alternative, enzymes, such as proteases, lipases, and aminoacylases, have been proposed and used for the enzymatic synthesis of acylamino acids. All these enzymes belong to the hydrolase class of enzymes with their preferred reaction being the catalysis of the hydrolysis of acylamino acids. Consequently, almost all of the enzymes described so far are not suitable for technical synthesis, as it requires high yields and conversion rates. Another drawback is that lipases and proteases require the use of solvents and show only low product yields with very long reaction times. International patent publication WO 1999024599 A1 (Henkel) describes an enzymatic process for N-acylation of amino acids and protein hydrolysates in which lipases or proteases were used as catalysts. However, both types of enzymes have in the meantime been shown to be less suitable than aminoacylases.

[0012] It was shown that synthesis with proteases only works in immobilized form with the addition of solvents with a poor to moderate yield and requires very long reaction times.

[0013] If lipases are used, the formation of esters is strongly favored so that the alpha-amino group is hardly recognized as a substrate and with lysine, almost exclusively the primary amine of the side chain was found to be acylated. The synthesis with lipases typically also requires organic solvent systems, so that with both, lipases and proteases, sustainable solvent-free synthesis is not possible. For these reasons, lipases are also a suboptimal choice for the synthesis of N-acyl amino acids.

[0014] While the use of aminoacylases appears more promising, many of the aminoacylases known to date favor the hydrolysis of acetylamino acids and thus are not suitable for the synthesis of acylamino acids.

[0015] In a large comparative study including various lipases, proteases and acylases or aminoacylases, the acylase from pig kidney has previously been identified as the most suitable enzyme for the synthesis of acylamino acids (Wada et al. (2002), JAOCS 79, 41-46). However, with this acylase, high yields could only be achieved with low fatty acid concentrations and a high molar excess of amino acid (approximately 100-fold excess). For this reason, said enzyme is not suitable for the technical use. Although the enzyme was later cloned, heterologously expressed and genetically optimized, the synthesis performance of the enzyme could not be increased to such an extent that an efficient acylamino acid synthesis with high yields became possible: only yields of 1-2 mg / 50 ml culture (without co-expression with chaperons), or 3-4 mg / 50 ml culture (with coexpression with chaperones TF or GroEL-GroES) were obtained. Further, this enzyme tends to form inclusion bodies in E. coli and is sensitive to oxidation and temperature changes.

[0016] Some recently identified aminoacylases from Streptomyces ambofaciens, Streptomyces mobaraensis and Burkholderia sp., were found suitable to produce acylamino acids in solvent- free reaction systems. However, functional heterologous expression of these enzymes was either not possible at all or only in poorly active or inactive form, e.g. S. mobaraensis enzyme (Koreishi et al. (2006), Journal of agricultural and food chemistry 54, 72-8; Koreishi et al. (2009) Bioscience, biotechnology, and biochemistry 73, 1940-7) or S. lavendulae Penicillin V acylase (Torres- Bacete et al. (2015), Applied and Environmental Microbiology 81 , 1225-1233). These aminoacylases were therefore only available from the wildtype strain following a complex multi-step purification protocol which still yielded only small amounts of the active enzyme. For these reasons, said enzymes are similarly unsuited for the technical production of acylamino acids.

[0017] It was further reported that various aminoacylases with different amino acid specificities have been isolated from Streptomyces mobaraensis that could be successfully cloned and heterologously expressed. Two of these enzymes included an epsilon-specific lysine acylase, which has been patented together with Ajinomoto (US7888079B2), and an acylase for which hydrolytic activity could be demonstrated. However, the aminoacylase with the broadest synthesis spectrum (Koreishi et al. (2006), supra) has not been cloned and heterologously expressed. While a Penicillin V acylase from Streptomyces mobaraensis has been cloned and heterologously expressed in S. lividans its synthetic activity for acylamino acids was not investigated.

[0018] Another epsilon-specific lysine acylase was isolated from Streptomyces ambofaciens, cloned but not functionally expressed.

[0019] Bourkaib et al.(Bourkaib et al. (2020), Enzyme and Microbial Technology 137, 10953) reported the characterization of aminoacylase mixtures with broad amino acid substrate spectrum but failed to disclose the gene sequences thereof.

[0020] Recently, a novel aminoacylase from the organism Burkholderia sp. strain LP5_18 was isolated (Takakura & Asano (2019), Bioscience, biotechnology, and biochemistry 83, 1964-1973). This aminoacylase showed high synthesis performance for N acyl-amino acids with lauroyl-residues, high thermal and pH stability over a broad range of pH values. The gene of this aminoacylase was cloned and sequenced, however, recombinant expression in E. coli was not successful. Said enzyme therefore must be obtained from the wild type strain using a complex 5-step purification process under cooling. Using this laborious process, from 800 ml culture medium only 0.1 mg enzyme were isolated.

[0021] The above shows that chemical synthesis of acylamino acids suffers from sustainability issues as it requires the use of chlorinated fatty acids. While enzymatic methods may be more sustainable, these presently suffer from the lack of efficient biocatalysts and reaction systems. There is thus still need in the art for methods that allow the efficient and sustainable production of acylamino acids on an industrial scale, in particular an enzyme that is suitable for the enzymatic production of acylamino acids directly from amino acids and fatty acids to provide a sustainable synthesis method for the production of surfactants.

[0022] SUMMARY OF THE INVENTION

[0023] The present invention meets this need in that the inventors identified a novel aminoacylase from Paraburkolderia monticola DSM 100849 (PmAcy) that surprisingly was found to be able to produce N-acylamino acids directly from fatty acids and amino acids in good yields and in solvent- free reaction systems while at the same time being recombinantly obtainable from (heterologous) microbial expression systems in sufficiently high yields and activities.

[0024] Thus, in a first aspect, the present invention relates to an isolated polypeptide having aminoacylase activity and comprising an amino acid sequence having over its entire length at least 87 % sequence identity to the amino acid sequence set forth in SEQ ID NO:1 .

[0025] In another aspect, the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide according to the invention. In various embodiments, the nucleotide sequence comprises or consists of the sequence set forth in SEQ ID NO: 2. Said isolated nucleic acid molecule or nucleotide sequence may be comprised in a vector or said isolated nucleic acid molecule may be a vector, such as a plasmid vector.

[0026] The invention further relates to a recombinant host cell comprising the nucleic acid molecule or the vector according to the invention.

[0027] A still further aspect of the invention relates to a method for the production of the polypeptide having aminoacylase activity according to the invention, wherein the method comprises the steps of

[0028] (a) introducing a nucleic acid molecule or a vector as described above into a suitable host cell, wherein the nucleic acid molecule or vector comprises a nucleotide sequence that encodes the polypeptide having aminoacylase activity according to the invention; and

[0029] (b) cultivating the host cell in a culture medium under conditions that allow expression of the polypeptide.

[0030] In a still other aspect, the invention relates to a method for the production of N-acyl amino acids or salts thereof, the method comprising reacting at least one amino acid or salt thereof with at least one carboxylic acid, such as a fatty acid, or salt thereof in the presence of the polypeptide having aminoacylase activity according to the invention under conditions that allow the production of N-acyl amino acids or salts thereof.

[0031] In still another aspect, the invention relates to the use of the polypeptide having aminoacylase activity according to the invention for the N-acylation of at least one amino acid or salt thereof.

[0032] In a still further aspect, the invention also relates to the use of the polypeptide having aminoacylase activity according to the present invention for the synthesis of N-acyl amino acids or salts thereof from at least one amino acid or salt thereof and at least one carboxylic acid, such as a fatty acid, or salt thereof.

[0033] In still another aspect, the invention is also directed to the N-acyl amino acids obtainable according to the inventive methods as well as compositions and products containing them and their use in such compositions and products.

[0034] These and other aspects, embodiments, features, and advantages of the invention become apparent to the person skilled in the art from the following detailed description, claims and figures. Each feature from one aspect of the invention can be used in any other aspect of the invention. Furthermore, the examples contained herein are intended to describe and illustrate the invention, but not to restrict it. In particular, the invention is not limited to these examples.

[0035] BRIEF DESCRIPTION OF THE FIGURES

[0036] Fig. 1 shows the pH dependency of the (hydrolysis) activity of PmAcy (conditions: 3 mM lauroylalanine, 30 °C reaction temperature, 2 pg / ml PmAcy NTag, 200 pl reaction volume, 4 pM ZnCh, measurement via ninhydrin detection, sampling interval of 1 min und 4-10 min reaction time; the concentration of the buffer of the substrate solution was 50 mM; the reactions were carried out in triplicates).

[0037] Fig. 2 shows the pH dependency of PmAcy stability for 1 h (left) and 24 h (right) (conditions of incubation: The concentration of the buffer of the incubation solution was 100 mM with an enzyme concentration of 240 pg / ml; the incubation temperature was 30 °C, reaction conditions: 15 mM lauroylalanine in 200 mM borate pH 9.0, 30 °C reaction temperature, 12 pg / ml PmAcy NTag, 200 pl reaction volume, 10 pM ZnC , measurement via ninhydrin detection, sampling interval of 1 min und 4-10 min reaction time; the reactions were carried out in triplicates). Fig. 3 shows the temperature dependency of PmAcy activity (conditions: 3 mM lauroylalanine in 50 mM Tris-HCI pH 8.0, 30 °C reaction temperature, 2 pg / ml PmAcy NTag, 200 pl reaction volume, 4 pM ZnCh, measurement via ninhydrin detection, sampling interval of 1 min und 1-6 min reaction time; the reactions were carried out in triplicates; measurement via ninhydrin detection of the released alanine).

[0038] Fig. 4 shows the melting curve of PmAcy in thermal shift assay (for the assay, 30 pl protein solution (0,16 mg / ml) and 5 pl 50x SYPRO Orange were mixed, in 100 mM Tris-HCI pH 8,0; it was measured in the qPCR device qTower3G (Analytik Jena); in the program, the samples were heated from 25 - 95 °C, in 2 °C-steps with holding times of 120 s each at a heating rate of 4.4 °C / s; excited at 535 nm, measured at 580 nm).

[0039] Fig. 5 shows the temperature dependent PmAcy stability (incubation conditions: in a volume of 200 pl, 60 pg / ml PmAcy was incubated in 100 mM Tris-HCI. The buffer solution was adjusted to pH 8 at the respective temperature (20 - 90 °C); reaction conditions: 15 mM lauroyl alanine in 100 mM borate pH 9.0, 30 °C reaction temperature, 3 pg / ml PmAcy NTag, 200 pl reaction volume, 2,5 pM ZnCh, measurement via ninhydrin detection of the released alanine, sampling interval of 1 min und 4-10 min reaction time; the reactions were carried out in triplicates).

[0040] Fig. 6 shows a general reaction scheme of the Schotten-Baumann reaction with lauroylchlorid (reaction conditions: 70 mmol amino acid (1 eq.), 175 mmol NaOH (2.5 eq.; for aspartic acid and glutamic acid 245 mmol, 3.5 eq.), 84 mmol carboxylic acid chloride (1.2 eq.), 64 ml distilled water, 35 mL acetone, 4 h, 0 °C; isolation of the product was carried out by precipitation with 5 M HCI solution at pH = 1 , filtration und cleaning of the filter cake with distilled water and petroleum ether. The obtained product was analyzed by HPLC-ELSD und HPLC-MS).

[0041] Fig. 7 shows the reaction scheme of the acylation of phenylalanine and lauric acid by PmAcy.

[0042] Fig. 8 shows lauroyl arginine synthesis over time [min] at different temperatures (reaction conditions: 100 mM lauric acid (added as sodium laurate), 200 mM arginine, pH 9 in sodium borate buffer).

[0043] Fig. 9 shows lauroyl arginine synthesis at different substrate concentrations (reaction conditions: 50-400 mM sodium laurate, 50-400 mM arginine, pH 9 in sodium borate buffer). Fig. 10 shows lauroyl arginine synthesis in a buffer-free system over time (reaction conditions: 100 mM sodium laurate, 100 mM or 200 mM arginine, pH has been kept constant at pH 9 over 24 h via NaOH dosing).

[0044] DETAILED DESCRIPTION

[0045] The terms used herein have, unless explicitly stated otherwise, the meanings as commonly understood in the art.

[0046] “At least one” as used herein, means one or more, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or more of the referenced species. Similarly, “one or more”, as used herein, relates to at least one and comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or more. In connection with a given species, the term does not relate to the total number of molecules, but rather to the type of species. “At least one amino acid”, for example, thus means that one type of amino acid or two or more different types of amino acids may be present. In connection with amounts, the term relates to the total amount of the referenced species. In case of amino acids, for example, this means that the given amount is the total amount of all amino acids (types) in the composition.

[0047] Numeric values specified without decimal places here refer to the full value specified with one decimal place, i.e., for example, 99 % means 99.0 %, unless otherwise defined.

[0048] The terms “about”, “approximately” or “approx.”, in connection with a numerical value, refer to a variance of ± 10 %, preferably ± 5 %, with respect to the given numerical value.

[0049] All percentages given herein in relation to compositions or formulations relate to weight % (wt.- %) relative to the total weight of the respective composition or formula, if not explicitly stated otherwise. Numeric ranges specified in the format “from x to y” include the specified values. If multiple preferred numeric ranges are specified in this format, it is understood that all ranges created by combining the different endpoints are also included.

[0050] "Isolated" as used herein in relation to a molecule means that said molecule has been at least partially separated from other molecules it naturally associates with or other cellular components. "Isolated" may mean that the molecule has been purified to separate it from other molecules and components, such as other proteins and nucleic acids and cellular debris.

[0051] “Nucleic acid" as used herein includes all natural forms of nucleic acids, such as DNA and RNA. Preferably, the nucleic acid molecules of the invention are DNA. The term "peptide" is used throughout the specification to designate a polymer of amino acid residues connected to each other by peptide bonds. A peptide according to the present invention may have 2-100 amino acid residues. The terms "protein" and "polypeptide" are used interchangeably throughout the specification to designate a polymer of amino acid residues connected to each other by peptide bonds. A protein or polypeptide according to the present invention has preferably 100 or more amino acid residues.

[0052] The term "an N-terminal fragment" relates to a peptide or protein sequence which is in comparison to a reference peptide or protein sequence C-terminally truncated, such that a contiguous amino acid polymer starting from the N-terminus of the peptide or protein remains.

[0053] The term "a C-terminal fragment" relates to a peptide or protein sequence which is in comparison to a reference peptide or protein sequence N-terminally truncated, such that a contiguous amino acid polymer starting from the C-terminus of the peptide or protein remains.

[0054] The term "fusion protein" as used herein concerns two or more peptides and proteins which are N- or C-terminally connected to each other, typically by peptide bonds, including via an amino acid / peptide linker sequence. Such fusion proteins may be encoded by two or more nucleic acid sequences which are operably fused to each other.

[0055] Generally, the skilled person understands that for putting the present invention into practice any nucleotide sequence described herein may comprise an additional start and / or stop codon or that a start and / or stop codon included in any of the sequences described herein may be deleted, depending on the nucleic acid construct used. The skilled person will base this decision, e.g., on whether a nucleic acid sequence comprised in the nucleic acid molecule of the present invention is to be translated and / or is to be translated as a fusion protein. In various embodiments, in particular if a tag sequence is added to the N-terminus of the amino acid sequence of the invention, a start codon encoding for an N-terminal M may be added.

[0056] The present invention relates to an isolated polypeptide having aminoacylase activity and comprising an amino acid sequence that has over its entire length at least 87 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 .

[0057] In various embodiments, the amino acid sequence has at least 88 %, at least 89 % or at least 90 %, preferably at least 91 %, at least 92 %, at least 93 % or at least 94 %, more preferably at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:1 over its entire length. In various embodiments, the amino acid sequence is of the same length as the sequence set forth in SEQ ID NO:1. In other embodiments, it is a shortened fragment thereof that may be obtainable by deletions / truncations. Such truncated versions are also referred to herein as functional fragments and are further defined below.

[0058] In especially preferred embodiments, the polypeptide essentially consists of or consists of an amino acid sequence having at least 87 %, preferably at least 88 %, at least 89 % or at least 90 %, more preferably at least 91 %, at least 92 %, at least 93 % or at least 94 %, more preferably at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO:1 over its entire length.

[0059] Determination of the sequence identity of nucleic acid or amino acid sequences can be done by a sequence alignment based on well-established and commonly used BLAST algorithms (See, e.g. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410, and Altschul, Stephan F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Hheng Zhang, Webb Miller, and David J. Lipman (1997): "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs"; Nucleic Acids Res., 25, S.3389-3402). Such an alignment is based on aligning similar nucleotide or amino acid sequences stretches with each other. Another algorithm known in the art for said purpose is the FASTA algorithm. Alignments, in particular multiple sequence comparisons, are typically done by using computer programs. Commonly used are the Clustal series (See, e.g., Chenna etal. (2003): Multiple sequence alignment with the Clustal series of programs. Nucleic Acid Research 31, 3497- 3500), T-Coffee (See, e.g., Notredame et al. (2000): T-Coffee: A novel method for multiple sequence alignments. J. Mol. Biol. 302, 205-217) or programs based on these known programs or algorithms.

[0060] Also possible are sequence alignments using the computer program Vector NTI® Suite 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA, USA) with the set standard parameters, with the AlignX module for sequence comparisons being based on the ClustalW. If not indicated otherwise, the sequence identity is determined using the BLAST algorithm.

[0061] Such a comparison also allows determination of the similarity of the compared sequences. Said similarity is typically expressed in percent identity, i.e. the portion of identical nucleotides / amino acids at the same or corresponding (in an alignment) sequence positions relative to the total number of the aligned nucleotides / amino acids. For example, if in an alignment 90 amino acids of a 100 amino acid long query sequence are identical to the amino acids in corresponding positions of a template sequence, the sequence identity is 90%. The broader term “homology” additionally considers conserved amino acid substitutions, i.e. amino acids that are similar regarding their chemical properties, since those typically have similar chemical properties in a protein. Accordingly, such homology can be expressed in percent homology.

[0062] If not indicated otherwise, sequence identity and sequence homology relate to the entire length of the aligned sequence. Specifically, the term that “the amino acid sequence over its entire length” has a given sequence identity to SEQ ID NO:1 means that the amino acid sequence comprises a continuous amino acid sequence that has said sequence identity to the sequence set forth in SEQ ID NO:1. In various embodiments, the subject amino acid sequence may have the given sequence identity also to the entire length of SEQ ID NO:1.

[0063] While the amino acid sequence may correspond to a continuous amino acid stretch of the amino acid sequence set forth in SEQ ID NO:1 having the indicated length, it is similarly possible that the amino acid sequence corresponds to discontinuous stretches of the amino acid sequence set forth in SEQ ID NO:1, for example if it corresponds to stretches of SEQ ID NO:1 with certain amino acids or amino acid sequences being deleted therefrom. The amino acid sequence may thus be derived from the amino acid sequence set forth in SEQ ID NO:1 by any one or more of an N-terminal truncation, a C-terminal truncation or a deletion of one or more amino acids, in particular as described above. Any such shortened variants of the amino acid sequence of SEQ ID NO:1 are covered by the term “fragment”. The term “functional fragment” additionally implies that the respective remaining polypeptide sequence retains its catalytic activity, as defined herein below. Insofar the present polypeptides comprise functional fragments of the amino acid sequence set forth in SEQ ID NO:1, said fragments are preferably N- and / or C-terminally truncated fragments, optionally lacking 1-50 or 1-40 or 1-30 or 1-20 or 1-10 amino acids from one or both ends.

[0064] In various embodiments, the polypeptide according to the invention can have the wild type sequence of the aminoacylase enzyme from Paraburkolderia monticola DSM 100849 (PmAcy) or may be a variant thereof.

[0065] “Having aminoacylase activity”, as used herein, means that the polypeptide has enzymatic activity and can catalyze the reaction of amino acids or salts thereof and carboxylic acids or salts thereof to yield acylamino acids or salts thereof and / or the hydrolysis of acylamino acids into the respective reactants. In various embodiments, said term means that the polypeptide has at least 50% of the enzymatic activity of the full length sequence of SEQ ID NO:1 under identical conditions that allow aminoacylase activity, preferably at least 60, at least 70, at least 80, at least 90 or at least 100 % activity of the aminoacylase of SEQ ID NO:1. In various embodiments, having aminoacylase activity means that the polypeptide is in particular capable to catalyze the reaction of (alpha) amino acids and carboxylic acids, preferably fatty acids, or their respective salts, to N-acylamino acids or salts thereof. It may be preferred that acylation occurs on the alpha-amino group if the amino acid comprises a side chain amino group.

[0066] The term “wild type” refers to the entire cell as well as to individual native genes, nucleotide sequences, proteins / enzymes or amino acid sequences that occurs naturally in nature. The term “wild type” therefore does not include cells, genes, nucleotide sequences, proteins, enzymes or amino acid sequences whose sequences have been at least partially modified using recombinant / genetic engineering methods.

[0067] “Variant” as used herein, refers to naturally or artificially produced variation of a native enzyme that has a modified amino acid sequence relative to the reference form.

[0068] “Modified" or "modification" in relation to a nucleotide or amino acid sequence or a nucleic acid or protein / enzyme means that the corresponding sequence is modified from the naturally occurring sequence (wild type) so that it is distinguishable from it. In various embodiments, the modification lies in the fact that the sequence is mutated, e.g. by substitution, deletion or insertion. In various other embodiments, the native amino acid sequence can be modified by attaching an affinity tag to the naturally occurring sequence, such as a His-tag or a Strep-tag, to allow isolation and purification of the enzyme after production in the host organism.

[0069] In various embodiments, the polypeptide of the invention comprises, in addition to the amino acid sequence that has at least 87% sequence identity to SEQ ID NO:1 at least one other amino acid sequence that may be located N-terminally or C-terminally to said sequence and is typically fused thereto, i.e. connected by a peptide bond. Said additional amino acid sequence may be a functional sequence, such as an affinity tag that allows isolation and purification or the like or may allow detection. Such a functional amino acid sequence may be connected to amino acid sequence encoding the enzymatically active part of the polypeptide by a suitable linker sequence. Such a linker sequence may, in various embodiments include a protease recognition site to allow cleavage of the tag from the enzymatically active portion of the molecule, if desired.

[0070] In various embodiments, the polypeptide according to the invention comprises an N- or C-terminal affinity tag, for example a His-Tag (6xHis-tag) ora Strep-Tag, preferably an N-terminal Strep-Tag. Such Strep-Tags are known and may consist of the amino acid sequence WSHPQFEK (SEQ ID NO:3).

[0071] The term “N-terminus” or “N-terminal” means in the context of the present invention the end of the amino acid chain that has a free amino acid group.

[0072] The term “C-terminus” or “C-terminal” means in the context of the present invention the end of the amino acid chain that has a free carboxyl group.

[0073] In various embodiments, the polypeptide of the invention comprises a Strep-Tag that is directly attached to the N-terminus of the amino acid sequence having aminoacylase activity. In various other embodiments, it is connected to the amino acid sequence of the aminoacylase via a short linker sequence. In preferred embodiments, said linker sequence comprises or consists of 1 to 5 amino acids, for example the linker sequence may comprise or consist of two amino acids, such as SG.

[0074] In various embodiments, the polypeptide of the invention comprises the amino acid sequence that has 87% sequence identity to SEQ ID NO:1 and a Strep-Tag of the amino acid sequence of SEQ ID NO:3 that is attached to the N-terminus of the aminoacylase sequence via the linker SG and additionally comprises an N-terminal residue M. Such a construct that also comprises the full- length sequence of SEQ ID NO: 1 is set forth in SEQ ID NO:4.

[0075] In various embodiments, the aminoacylase according to the invention comprises or consists of the wild type sequence from Paraburkolderia monticola DSM 100849 as set forth in SEQ ID NO: 1 . It is further preferred that the native amino acid sequence is modified with an affinity tag as described above and, optionally, with a linker sequence as described above that is preferably N- terminally attached to the amino acid sequence.

[0076] In a further aspect, the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide according to the invention. The encoded polypeptide may be any of those described above and may, in various embodiments, comprise, in addition to the amino acid sequence defined herein that has aminoacylase activity other amino acid sequences that encode further (poly)peptides, for example (poly)peptide tags that are used or useful for detection or purification and linker sequences. As the nucleic acid molecule encodes the entire polypeptide, the different amino acid sequences combined therein can be expressed as a single fusion protein. Since, due to the degeneracy of the genetic code, a certain amino acid sequence can be encoded by several different nucleic acids, all nucleotide sequences which can encode the polypeptides as described herein, are encompassed by the present invention. The person skilled in the art is able to determine these nucleotide sequences without any doubts, since defined amino acids can be assigned to individual codons despite the degeneracy of the genetic code. Therefore, starting from an amino acid sequence, the person skilled in the art can easily determine nucleic acids encoding for this amino acid sequence. In the nucleotide sequences according to the invention, one or more codons can be replaced by synonymous (i.e. coding for the same amino acid) codons relative to the wild-type or starting sequence. This is particularly useful for heterologous expression since each organism has a fixed codon usage, so that in a given organism a certain codon may be translated less efficiently than a synonymous codon encoding the same amino acid. Exchanging codons such that translation is optimized in a given host organism (based on its codon usage) without changing the encoded polypeptide sequence is referred to as “codon optimization”.

[0077] In various embodiments, the nucleotide sequence according to the invention is codon-optimized for the host organism, in which it is to be expressed. Said host organism may be any suitable host organism, but may, in preferred embodiments, be Escherichia coli (E. coli).

[0078] Such codon-optimized nucleotide sequence may have the nucleotide sequence as set forth in SEQ ID NO:2.

[0079] In certain embodiments, the above defined nucleic acid molecules may be comprised in a vector, for example a cloning or expression vector. Generally, the nucleic acid molecules of the invention can also be part of a vector or any other kind of cloning vehicle, including, but not limited to a plasmid, a phagemid, a phage, a baculovirus, a cosmid, or an artificial chromosome. Generally, a nucleic acid molecule disclosed in this application may be "operably linked" to a regulatory sequence (or regulatory sequences) to allow expression of this nucleic acid molecule.

[0080] Such cloning vehicles can include, besides the regulatory sequences described above and a nucleic acid sequence of the present invention, replication and control sequences derived from a species compatible with the host cell that is used for expression as well as selection markers conferring a selectable phenotype on transformed or transfected cells. Large numbers of suitable cloning vectors are known in the art and are commercially available.

[0081] In certain embodiments the nucleic acid molecules disclosed herein are comprised in a cloning vector. In some embodiments the nucleic acid molecules disclosed herein are comprised in an expression vector. The vectors may comprise regulatory elements for replication and selection markers. In certain embodiments, the selection marker may be selected from the group consisting of genes conferring ampicillin, kanamycin, chloramphenicol, tetracycline, blasticidin, spectinomycin, gentamicin, hygromycin, and zeocin resistance. In various other embodiments, the selection may be carried out using antibiotic-free systems, for example by using toxin / antitoxin systems, cer sequence, triclosan, auxotrophies or the like. Suitable methods are known to those skilled in the art.

[0082] The above-described nucleic acid molecule of the present invention, if integrated in a vector, must be integrated such that the polypeptide can be expressed. Therefore, a vector of the present invention comprises sequence elements which contain information regarding to transcriptional and / or translational regulation, and such sequences are "operably linked" to the nucleotide sequence encoding the polypeptide. An operable linkage in this context is a linkage in which the regulatory sequence elements and the sequence to be expressed are connected in a way that enables gene expression. The precise nature of the regulatory regions necessary for gene expression may vary among species, but in general these regions comprise a promoter which, in prokaryotes, contains both the promoter per se, i.e. DNA elements directing the initiation of transcription, as well as DNA elements which, when transcribed into RNA, will signal the initiation of translation. Such promoter regions normally include 5' non-coding sequences involved in initiation of transcription and translation, such as the -35 / - 10 boxes and the Shine-Dalgarno element in prokaryotes or the TATA box, CAAT sequences, and 5'-capping elements in eukaryotes. These regions can also include enhancer or repressor elements as well as translated signal and leader sequences for targeting the native polypeptide to a specific compartment of a host cell.

[0083] In addition, the 3' non-coding sequences may contain regulatory elements involved in transcriptional termination, polyadenylation or the like. If, however, these termination sequences are not satisfactory functional in a particular host cell, then they may be substituted with signals functional in that cell.

[0084] In various embodiments, a vector comprising a nucleic acid molecule of the invention can therefore comprise a regulatory sequence, preferably a promoter sequence. In certain embodiments, the promoter is identical or homologous to promoter sequences of the host genome. In such cases endogenous polymerases may be capable to transcribe the nucleic acid molecule sequence comprised in the vector. In various embodiments, the promoter is selected from the group of weak, intermediate and strong promoters, preferably from weak to intermediate promoters. In another embodiment, a vector comprising a nucleic acid molecule of the present invention comprises a promoter sequence and a transcriptional termination sequence. Suitable promoters for prokaryotic expression are, for example, the araBAD promoter, the tet-promoter, the lacllV5 promoter, the CMV promo tor, the EF1 alpha promotor, the AOX1 promotor, the tac promotor, the T7 promoter, or the lac promotor. Furthermore, a nucleic acid molecule of the invention can comprise transcriptional regulatory elements, e.g., repressor elements, which allow regulated transcription and translation of coding sequences comprised in the nucleic acid molecule. Repressor element may be selected from the group consisting of the Lac-, AraC-, or MalR- repressor.

[0085] The vector may be effective for prokaryotic or eukaryotic protein expression. In particular, the nucleic acid molecules of the present invention may be comprised in a vector for prokaryotic protein expression. Such vector sequences are constructed such that a sequence of interest can easily be inserted using techniques well known to those skilled in the art. In certain embodiments, the vector is selected from the group consisting of a pET-vector, such as pET28a, a pBAD-vector, a pK184-vector, a pMONO-vector, a pSELECT-vector, pSELECT-Tag-vector, a pVITRO-vector, a pVIVO-vector, a pORF-vector, a pBLAST-vector, a pUO-vector, a pDUO-vector, a pZERO- vector, a pDeNy-vector, a pDRIVE-vector, a pDRIVE-SEAP-vector, a HaloTag®Fusion-vector, a pTARGET™-vector, a Flexi®-vector, a pDEST-vector, a pHIL-vector, a pPIC-vector, a pMET- vector, a pPink-vector, a pLP -vector, a pTOPO-vector, a pBud-vector, a pCEP-vector, a pCMV- vector, a pDisplay-vector, a pEF-vector, a pFL-vector, a pFRT-vector, a pFastBac-vector, a pGAPZ-vector, a plZ / V5-vector, a p3S-vector, a plAR-vector, pSEC, pMS, a pSU2726-vector, a pLenti6 -vector, a pMIB-vector, a pOG-vector, a pOpti-vector, a pREP4-vector, a pRSET-vector, a p SCREEN- vector, a pSecTag-vector, a pTEFI -vector, a pTracer- vector, a pT rc-vector, a pUB6-vector, a pVAXI-vector, a pYC2-vector, a pYES2-vector, a pZeo-vector, a pcDNA-vector, a pFLAG-vector, a pTAC-vector, a pT7-vector, a gateway®-vector, a pQE-vector, a pLEXY-vector, a pRNA-vector, a pPK-vector, a pUMVC-vector, a pLIVE-vector, a pCRUZ-vector, a Duet-vector, and other vectors or derivatives thereof.

[0086] The vectors of the present invention may be chosen from the group consisting of high, medium and low copy vectors.

[0087] The above described vectors of the present invention may be used for the transformation or transfection of a host cell in order to achieve expression of a polypeptide which is encoded by an above described nucleic acid molecule and comprised in the vector DNA. Thus, in a further aspect, the present invention also relates to a host cell comprising a vector or nucleic acid molecule as disclosed herein. The terms “host organism” and “host cell” are used interchangeably in the context of the present invention.

[0088] Also contemplated herein are host cells, which comprise a nucleic acid molecule as described herein integrated into their genomes. The skilled person is aware of suitable methods for achieving the nucleic acid molecule integration. For example, the molecule may be delivered into the host cells by means of liposome transfer or viral infection and afterwards the nucleic acid molecule may be integrated into the host genome by means of homologous recombination. In certain embodiments, the nucleic acid molecule is integrated at a site in the host genome, which mediates transcription of the peptide or protein of the invention encoded by the nucleic acid molecule. In various embodiments, the nucleic acid molecule further comprises elements which mediate transcription of the nucleic acid molecule once the molecule is integrated into the host genome and / or which serve as selection markers.

[0089] In certain embodiments, the nucleic acid molecule of the present invention is transcribed by a polymerase natively encoded in the host genome. In various embodiments, the nucleic acid molecule is transcribed by an RNA-polymerase which is non-native to the host genome. In such embodiments, the nucleic acid molecule of the present invention may further comprise a sequence encoding for a polymerase and / or the host genome may be engineered, or the host cell may be infected to comprise a nucleic acid sequence encoding for an exogenous polymerase. The host cell may be specifically chosen as a host cell capable of expressing the gene. In addition, or otherwise, in order to produce the polypeptide of the invention, the nucleic acid coding for it can be genetically engineered for expression in a suitable system. Transformation can be performed using standard techniques (Sambrook, J. et al. (2001), Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0090] Host organisms comprising such a vector for recombinant expression of the polypeptide as described herein form also part of the present invention. The host organism is preferably a prokaryotic or eukaryotic organism, in particular a bacteria, yeast or unicellular fungi. Exemplary host organisms are those of the genera Aspergillus, Corynebacterium, Brevi bacterium, Bacillus, Acinetobacter, Alcaligenes, Actinobacillus, Anaerobiospirillum, Basfia, Wollinella, Fibrobacter, Ruminococcus, Mannheimia, Lactobacillus, Lactococcus, Paracoccus, Lactococcus, Candida, Pichia (also called Komagataella), Hansenula, Kluveromyces, Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methylobacterium, Ralstonia, Pseudomonas, Rhodospirillum, Rhodobacter, Burkholderia, Clostridium or Cupriavidus, with Aspergillus nidulans, Aspergillus niger, Alcaligenes latus, Bacillus megaterium, Bacillus subtilis, Brevi bacterium flavum, Brevifermentbacterium lactofermentum, Escherichia coli, Basfia succiniciproducens, Wollinella succinogenes, Fibrobacter succinogenes, Ruminococcus flavefaciens, Anaerobiospirillum succiniciproducens, Mannheimia succiniciproducens, Actinobacillus succinogenes, Saccharomyces cerevisiae, Kluveromyces lactis, Kluyveromyces marxianus, Candida blankii, Candida rugosa, Corynebacterium glutamicum, Corynebacterium efficiens, Zymonomas mobilis, Yarrowia lipolytica, Methylobacterium extorquens, Flansenula polymorpha, Ralstonia eutropha, Rhodobacter sphaeroides, Paracoccus versutus, Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Pichia pastoris (also called Komagataella phaffii), Thermoanaerobacter kivui, Acetobacterium woodii, Acetoanaerobium notera, Clostridium aceticum, Butyribacterium methylotrophicum, Clostridium acetobutylicum, Clostridium saccharoperbutylacetonicum, Clostridium beijerinckii, Clostridium butyricum, Moorella thermoacetica, Eubacterium limosum, Peptostreptococcus productus, Clostridium ljungdahlii, Clostridium carboxidivorans, Clostridium scatalogenes, Rhodospirillum rubrum, Burkholderia thailandensis and Pseudomonas putida being especially suitable.

[0091] In various embodiments, the host cells are selected from the group consisting of gram-positive and gram-negative bacteria. In some embodiments, the host cell is a gram-negative bacterium, such as E. coli. In certain embodiments, the host cell is E. coli, in particular E. coli BL21 (DE3) or other E. coli K12 or E. coli B834 derivatives. In various embodiments, the host cell is, for example but without limitation, selected from E.coli BL21 (DE3), E. coli BL21 , E. coli K12, E. coli BLR, E. coli BL21 Al, E. coli BL21 pLysS, E. coli XL1 and E. coli DH5a. Further suitable E. coli strains include, but are not limited to DH1 , DH5a, DM1 , HB101, JMIOI-110, Rosetta (DE3) pLysS, SURE, TOP10, XLI-Blue, XL2-Blue and XLIO-Blue strains.

[0092] The transformed host cells are cultured under conditions suitable for expression of the nucleotide sequence encoding the polypeptide of the invention. In certain embodiments, the cells are cultured under conditions suitable for expression of the nucleotide sequence encoding a polypeptide of the invention.

[0093] For producing the recombinant polypeptide described herein, a vector of the invention can be introduced into a suitable host organism by means of recombinant DNA technology (as already outlined above). For this purpose, the host cell is first transformed with a vector comprising a nucleic acid molecule according to the present invention using established standard methods (Sambrook, J. et al. (2001), supra). The host cell is then cultured under conditions, which allow expression of the heterologous DNA and thus the synthesis of the corresponding polypeptide. Subsequently, the polypeptide is recovered either from the cell or from the cultivation medium. For expression of the polypeptides of the present invention several suitable protocols are known to the skilled person. The expression of a recombinant polypeptide of the present invention may be achieved by the following method comprising the steps of: (a) introducing a nucleic acid molecule or vector of the invention into a host cell, wherein the nucleic acid molecule or vector encodes the recombinant polypeptide; and (b) cultivating the host cell in a culture medium under conditions that allow expression of the recombinant polypeptide. The expression may be heterologous expression in that the host cells does not naturally express the polypeptide.

[0094] Step (a) may be carried out by using suitable transformation and transfection techniques known to those skilled in the art. These techniques are usually selected based on the type of host cell into which the nucleic acid is to be introduced. In some embodiments, the transformation may be achieved using electroporation or heat shock treatment of the host cell.

[0095] In various embodiments, in step (a) of the method, a nucleotide sequence encoding for at least one chaperone, preferably the chaperone GroEL / ES, is also introduced into the host organism. Said nucleotide sequence may be comprised in a second nucleic acid molecule. In preferred embodiments, the chaperone nucleotide sequence is introduced to the host organism via a second vector, in particular via the vector pGro7 encoding the chaperone GroE ES.

[0096] The chaperone, in particular the chaperone GroEL / ES, serves to improve the production of the polypeptide of the invention in the host cells. Accordingly, in such embodiments, the cultivation conditions of step (b) further allow the co-expression of the at least one chaperone. If in the following reference is made to the expression of the polypeptide of the invention, it is understood that if a chaperone is co-expressed, said disclosure similarly applies to expression of the chaperone.

[0097] Step (b) may include a cultivation step that allows growth of the host cells. Alternatively, such step allowing growth of the host cells and a step that allows expression of the polypeptide, and optionally the chaperone, may be performed separately in that the cells are first cultivated such that they grow to a desired density and then they are cultivated under conditions that allow expression of the polypeptide. The expression step can however still allow growth of the cells.

[0098] Generally, any known culture medium suitable for growth of the selected host may be employed in this method. In various embodiments, the medium is a rich medium or a minimal medium. Also contemplated herein is a method, wherein the steps of growing the cells and expressing the peptide or protein comprise the use of different media. For example, the growth step may be performed using a rich medium which is replaced by a minimal medium in the expression step. In certain cases, the medium is selected from the group consisting of LB medium, TB medium, 2YT medium, synthetical medium and minimal medium.

[0099] The method may further include a step of recovering the expressed polypeptide. The polypeptide may be recovered from the growth medium, if it is secreted, or from the cells or both. The recovery / isolation of the polypeptide may include various purification steps.

[0100] For example, the isolation may include disrupting or breaking open the cells (cell lysis) and, subsequently, separating the cell debris from the polypeptide by, e.g. centrifugation, filtration, sedimentation or a combination thereof. The thus isolated polypeptide, typically in the supernatant, may then be further purified, for example via chromatographic methods such as for example affinity chromatography, ion exchange chromatography, reverse phase chromatography, size exclusion chromatography, and combinations thereof. In various embodiments, purification may include a first affinity chromatography optionally followed by further purification steps (e.g. size exclusion chromatography). In the context of the present invention, any suitable cultivation method, isolation method, separation method or purification method known to the person skilled in the art can be used.

[0101] In one aspect, the invention is therefore directed to a method for expression of the (recombinant) polypeptide of the invention using the above-described nucleic acid molecules and may comprise the steps of:

[0102] (a) introducing a nucleic acid molecule or a vector as described above into a suitable host cell, wherein the nucleic acid molecule or vector comprises a nucleotide sequence that encodes the recombinant polypeptide; and

[0103] (b) cultivating the host cell in a culture medium under conditions that allow expression of the recombinant polypeptide.

[0104] Said method may further comprise the steps of isolating and, optionally, purifying the thus produced polypeptide, for example using the above-described techniques.

[0105] In a further aspect, the present invention relates to the use of a vector or nucleic acid molecule as disclosed herein for the expression of a recombinant polypeptide. In some embodiments, the vector is used for the expression and optionally secretion of a recombinant polypeptide according to the invention. The expression or expression and secretion may be achieved using the method described herein. The terms "heterologous" or "recombinant" are used herein to indicate that the corresponding molecule or nucleotide sequence is not naturally occurring in the host organism. The heterologous or recombinant expression of one or more nucleotide sequence(s) in a host organism thus means that this host organism does not contain or express this nucleotide sequence(s) under natural conditions. As a result, heterologous or recombinant proteins can be produced in the host organism which would not be produced in the host organism under natural conditions. The nucleotide sequence introduced into the host organism can be a wild-type sequence from another organism and / or a modified sequence.

[0106] In a further aspect, the invention relates to a method for the production of N-acyl amino acids or salts thereof, the method comprising reacting at least one amino acid or salt thereof with at least one carboxylic acid or salt thereof in the presence of the polypeptide having aminoacylase activity according to the invention under suitable conditions to produce N-acyl amino acids or salts thereof.

[0107] In various embodiments, the polypeptide having aminoacylase activity is used in amounts of 0.00001 to 10 wt.-% (active protein), preferably 0.00001 to 1 wt.-% (active protein), such as 0.0001 to 0.1 wt.-% (active protein) or 0.0005 to 0.05 wt.-% (active protein), based on the total weight of the reaction mixture.

[0108] Protein concentration can be determined by known methods, for example, the BCA (bicinchoninic acid; 2,2’-biquinolyl-4,4’-dicarboxylic acid) method or the Biuret method (Gornall et al., 1948, J. Biol. Chem., 177:751-766). Determination of active protein content can be accomplished by titration of the active sites using a suitable irreversible inhibitor and determination of the residual activity (Bender et al., 1966, J. Am. Chem. Soc. 88, 24:5890-5913).

[0109] In various embodiments, the enzymatically active polypeptide is added in purified form or in form of an enzyme composition that contains said polypeptide together with other components. In various embodiments, the enzyme formulation can be lyophilized.

[0110] The amino acid used can be any amino acid, including proteinogenic amino acids as well as non- proteinogenic amino acids. The 20 proteinogenic amino acids are arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In various embodiments, the amino acid is alanine, glycine, arginine, aspartic acid, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, valine, proline, serine or tyrosine, preferably alanine, isoleucine, methionine, leucine, valine, proline, glycine, glutamine, phenylalanine, lysine, serine or tyrosine, in particular isoleucine, valine, proline, methionine, leucine, arginine or phenylalanine. Most preferably, the amino acid is arginine or phenylalanine.

[0111] Non-proteinogenic amino acids include, without limitation, taurine, sarcosine, 2,6-diaminopimelic acid, beta-alanin, ornithin, homoserine, homoproline, N-methyltaurine, norvaline, norleucin, citrulline, hydroxyproline and the like.

[0112] The amino acids may be L- or D-amino acids. In various embodiments, L-amino acids are preferred.

[0113] In various embodiments, peptides can be used instead of or in addition to monomeric amino acids, for example di- or tripeptides or even longer peptides.

[0114] In various embodiments, it is also possible to use modified amino acids and peptides that do not occur naturally.

[0115] In a specific embodiment, the amino acid is selected from the group consisting of: arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, taurine, sarcosine, 2,6-diaminopimelic acid, beta-alanin, ornithin, homoserine, homoproline, N- methyltaurine, norvaline, norleucin, citrulline, hydroxyproline and the salts thereof.

[0116] In various embodiments, the carboxylic acid used for acylation is a fatty acid. Suitable fatty acids include, but are not limited to unsaturated or saturated, linear or branched fatty acids, for example C6-C24 or C8-C22 or C8-C18 fatty acids, or salts thereof. In various embodiments, the fatty acids used are linear, C8-C22, for example C8 to C18, unsaturated or saturated fatty acids or salts thereof, such as caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, linolenic acid, or linoleic acid or salts thereof. In specific embodiments, lauric acid, myristic acid, linoleic acid, linolenic acid or oleic acid or the respective salts are used, preferably lauric acid or myristic acid or a salt thereof, in particular lauric acid or a salt thereof. The fatty acid may also be mixtures of fatty acids, such as cocoyl fatty acids, which predominantly comprise C12-C14 fatty acids.

[0117] The salts of the amino acids and / or carboxylic acids may be any suitable salts, including the alkaline and alkaline earth metal salts, such as sodium, potassium, magnesium, and calcium. Preferably the counterion is sodium or potassium. In various embodiments, the produced N-acyl amino acids are N-lauroyl amino acids, N-myristoyl amino acids, N-palmitoyl amino acids, N-oleoyl amino acids, N-linoleoyl amino acids, N-linolenoyl amino acid or N-stearoyl amino acids or salts thereof, preferably lauroyl amino acids, myristoyl amino acids or palmitoyl amino acids or salts thereof, more preferably lauroyl amino acids or a salt thereof. In various embodiments, the produced N-acyl amino acids may be derived from mixtures of fatty acids, such as any two or more of the above, including cocoyl fatty acids.

[0118] In various embodiments, the method can further comprise an isolation step to isolate and / or purify the N-acyl amino acids or salts thereof. The isolation step can be any suitable isolation method known to the person skilled in the art. Preferably, the isolation step is a precipitation step via pH variation (e.g. acidification) or cold precipitation, i.e. lowering the temperatures.. In various embodiments, a purification step may include, for example, a filtration step and / or another suitable washing step.

[0119] Preferably, the method is carried out in an aqueous reaction system such as water or a buffer system. In preferred embodiments, the method is carried out in an organic solvent-free reaction system. Preferably the reaction is carried out in water or a buffer system without the addition of further organic solvents. In various embodiments, the buffer system is citrate, acetate, MES, Tris- HCI, borate, or phosphate, without being limited thereto.

[0120] In various embodiments, the reaction is carried out at pH 4 to 13, preferably pH 7 to 13, more preferably pH 8 to 12. The temperature may be in the range from 40 to about 80 °C, preferably 45 to 75 °C.

[0121] In various embodiments, the amino acid or salt thereof and the carboxylic acid, such as a fatty acid, or salt thereof are used in a molar ratio of 1 :100 to 100:1 , preferably 1:50 to 50:1 , more preferably 1 :10 to 10:1, more preferably 1 :5 to 5:1 , more preferably 1 :3 to 3:1, most preferably 1 :2 to 2.5:1, in particular in a molar ratio of about 2:1 (amino acid:carboxylic acid).

[0122] In a still further aspect, the invention relates to the use of the polypeptide having aminoacylase activity according to the invention for the N-acylation of at least one amino acid or salt thereof.

[0123] In another aspect, the invention also relates to the use of the polypeptide having aminoacylase activity according to the present invention for the synthesis of N-acyl amino acids or salts thereof from at least one amino acid or salt thereof and at least one carboxylic acid, such as a fatty acid, or salt thereof. In said reactions, N-acylation is catalyzed by the polypeptide having aminoacylase activity according to the invention.

[0124] In a further aspect, the invention therefore relates to N-acylamino acids obtainable by the method according to the invention.

[0125] The N-acylamino acids or salts thereof thus obtained can be used in cosmetic products, home care products such as detergents, dishwashing agents and cleaning agents, and in l&l (industrial and institutional) products for example in the sanitization or food industry. Preferably, the produced N-acyl amino acids or salts thereof can be used as skin-friendly surfactants, in particular if the acyl moiety is 10 to 14 carbon atoms in length, or as emulsifiers, in particular if the acyl moiety is more than 14 carbon atoms in length, such as C18-fatty acids. Preferred applications include cosmetic products, (laundry) detergents, dishwashing agents and cleaning compositions, in particular cosmetic products such as hair and body care products including baby shampoos.

[0126] In various embodiments, the N-acyl amino acids or salts thereof are used in these compositions in amounts of 0.5 to 40 wt.-%, based on the total weight of the composition.

[0127] Thus, in another aspect, the invention relates to compositions comprising the N-acyl amino acids according to the invention, wherein the composition optionally is a cosmetic product, home care product or an industrial and / or institutional product.

[0128] Furthermore, the invention relates to the use of the N-acyl amino acids according to the invention in a cosmetic product, home care product or an industrial and / or institutional product.

[0129] All embodiments and examples described herein for the polypeptide, the nucleic acid molecule, the vector, the host cell or the obtained N-acyl amino acids according to the invention, also apply to the methods, uses, compositions and products disclosed herein and vice versa.

[0130] Other embodiments are within the following non-limiting examples.

[0131] EXAMPLES

[0132] Example 1 : Cloning and expression

[0133] The protein sequence of PmAcy with attached Strep-tag (PmAcy N Tag, SEQ ID NO:4) was translated reversely, the obtained DNA sequence codon-optimized for E. coli and was commercially synthesized as a synthetic DNA strand. Using polymerase chain reaction, Bsal restriction sites were added to both ends of the strand, so that it was possible to clone the gene via Golden Gate-cloning into the vector pET28a. pET28a is an expression plasmid for the T7 system. The expression was carried out in E. coli BL21 (DE3) cells further comprising the plasmid pGro7. The strain is able to express the target gene of plasmid pET28a after induction with lactose or IPTG. Plasmid pGro7 comprises a sequence encoding the chaperone GroEL / ES and serves to improve the production of the aminoacylase.

[0134] Example 2: Purification

[0135] Purification was carried out via affinity chromatography using the Strep-tag system. The Strep- tag (SEQ ID NO:3) was attached to the N-terminus of the aminoacylase using the dipeptide linker sequence SG. Using this system, the aminoacylase PmAcy N Tag (SEQ ID NO:4) could be purified in a single chromatography step. Purity and mass of the protein (monomer) were analyzed and verified by SDS page and MALDI-TOF.

[0136] Example 3: pH-dependent stability and activity of Pm / Xcy

[0137] Pm / cy N Tag was found to be active in the alkaline range with a pH-optimum at pH 12.0. The enzyme was found to be stable over a broad pH range and to not show a significant activity loss after incubation for 24 h at pH 12 and 30 °C. At pH 13, the enzyme is stable for 1 h at 30 °C and after 24 hours the residual activity is still 30 % (see Figs. 1 and 2).

[0138] Example 4: Temperature-dependent stability and activity of P / nAcy

[0139] The aminoacylase PmAcy is a thermophilic enzyme and has a temperature optimum at 70 °C. At 80 °C, the activity slightly decreases (see Fig. 3).

[0140] Thermal stability was first tested via thermal shift assay. In this assay, fluorescence of the dye SYPRO Orange increases, if the protein is denatured thermally, since the dye binds to hydrophobic side chains. Thermal denaturation at 78 °C was detected. Due to a generally hydrophobic surface of the protein, the fluorescence was already high at the start and first decreased with increasing temperatures. Excitation of the dye was at 535 nm and fluorescence measured at 580 nm (see Fig. 4).

[0141] To detect the thermal stability of PmAcy over a longer period of time, the enzyme was incubated for up to 4 days at 20 to 90 °C and the residual activity was measured at defined time points via a hydrolysis assay. The results are shown in Fig. 5. The enzyme is stable up to 80 °C for more than one hour without a loss of activity. After one day of incubation at 70 °C, there is a residual activity of 82 %. After 4 days at 70 °C, 38 % hydrolysis activity was observed. After 4 days at 50 °C, the hydrolysis activity was still 81 %. PmAcy was found to be highly heat stable, which facilitates its technical use, for example in longer syntheses at 50 °C. Example 5: Synthesis of reference compounds by Schotten-Baumann reaction

[0142] Reference compounds were synthesized by Schotten-Baumann reaction using the 20 proteinogenic amino acids and lauroyl chloride in an alkaline water / acetone solvent system. Some amino acids (alanine, glutamine) were additionally acylated with palmitoyl chloride (see Fig. 6).

[0143] Table 1 : Synthesis of reference compounds with indication of the isolated yield (%1.

[0144] The reference compounds could be obtained in good to very good yields. Only the isolation of lauroyl histidine, lauroyl proline and lauroyl threonine did not result in target product or only low amounts of the target product.

[0145] Example 6: Hydrolytic cleavage of lauroyl amino acids with PmAcy

[0146] The hydrolysis spectrum shows a preference for lauroyl amino acids with short hydrophobic side chains. Substrates that were best converted were lauroyl alanine, lauroyl isoleucine, lauroyl methionine and lauroyl valine. Good conversion was shown with palmityl alanine, lauroyl glutamine, palmityl glutamine, lauroyl phenylalanine, lauroyl serine and lauroyl tyrosine. Furthermore, activity with lauroyl leucine was observed. However, the enzyme shows only low activity for acetyl amino acids.

[0147] Table 2: Substrate spectrum of PmAcy (reaction conditions: 15 mM substrate in 100 mM borate pH 9.0, 50 °C reaction temperature, 6 - 60 pg / ml PmAcy NTag (SEQ ID NO:4), 200 pl reaction volume, 50 pM ZnCh, measurement via ninhydrin detection of the released amino acid, sampling interval of 1 min and a reaction time of 3 to 4 min. The reactions were carried out in triplicates; (remark: the temperature was increased to 50 °C to completely dissolve the substrates). Example 7: Comparison of hydrolytic cleavage of acetyl- and palmityl amino acids

[0148] PmAcy NTag also cleaves palmitoyl amino acids, however, with decreased activity relative to the corresponding lauroylamino acids (see Table 2). Activity with acetyl amino acids is not relevant, since these are barely hydrolyzed. Hence, the enzyme shows a clear preference for longer-chain fatty acids, this being a preferred characteristic for the synthesis of amino acid-based surfactants.

[0149] Example 8: Time-dependent acylation of phenylalanine and lauric acid by PmAcy

[0150] The conversion of amino acids by the aminoacylase was analyzed first with 200 mM phenylalanine und 100 mM lauric acid. The reaction was carried out in basic borate buffer (pH = 9) with 10 % ethanol for solution mediation and 10 pl (12 pg, 0.6 U) PmAcy N Tag (SEQ ID NO:4), the complete reaction volume was 1 ml_ (Fig. 7). The reaction had a yield of up to 40 % (24 h, 50 °C, 250 rpm) as determined by analysis of the chromatograms and comparison to reference spectra of phenylalanine and N-lauroyl phenylalanine classically produced via Schotten-Baumann reaction.

[0151] Example 9: Examination of the amino acid substrate spectra at various pH values

[0152] Subsequently, the amino acid substrate spectra were analyzed by using the proteinogenic amino acids. Because of the higher solubility in aqueous media, sodium laurate was used instead of lauric acid. Thus, addition of ethanol was not necessary. The reactions were carried out at pH 8, 10, and 12. The results were as shown in Table 3 below.

[0153] Table 3: Amino acid spectrum for the formation of N-lauroyl amino acids with PmAcy at different pH values.

[0154] (-: low activity (0% < A < 5%); O: medium activity (5% < A < 20%); +: high activity (20% < A < 39%); ++: very high activity (40% < A); reaction conditions: 100 mM sodium laurate, 200 mM amino acid, 1 mL 50 mM TRIS-Puffer (pH = 8) / borate-buffer (pH = 10) / phosphate-buffer (pH = 12), 10 pl (12 pg, 0.6 U) purified PmAcy, 0.5 mM ZnCh, 50 °C, 250 rpm. Sampling was carried out after 10 min., 1 h und 24 h reaction time, respectively. For that, 10 pl of the reaction mixture were taken and mixed with 990 pl of boiling acetonitrile. Subsequently, the mixture was incubated for 20 min at 100 °C, 400 rpm, for enzyme inactivation. The sample substance was diluted with distilled water in a ratio of 1 :1 and analyzed via HPLC-ELSD. The activity was assigned by comparing the areas obtained. The implementation was carried out in duplicates.

[0155] Especially for phenylalanine, good conversions at all three alkaline pH values were observed. Also for isoleucine, lysine, valine and methionine, moderate conversions at all tested pH values were obtained. Arginine already showed conversion after short reaction times of only 10 minutes.

[0156] Example 10: Fatty acid substrate spectrum of PmAcy

[0157] Additionally, the spectrum of the accepted fatty acids was tested in more detail. For that, model amino acids phenylalanine and arginine were used. The amino acids were mixed with the fatty acids or the sodium salts thereof, respectively. The results were as shown in Table 4 below.

[0158] Table 4: Fatty acid spectrum for the formation of N-acyl arginine and -phenylalanine with PmAcy (reaction conditions: 100 mM fatty acid, 200 mM amino acid, 50 mM TRIS-buffer (pH = 8), 10 pL (12 pg, 0.6 U) PmAcy, 0.5 mM ZnCh, 1 ml total volume, 50 °C, 250 rpm. a) 0.5 ml total volume, 40 pl (48 pg, 2.4 U) PmAcy. Sampling was carried out after 10 min, 1 h and 24 h reaction time, respectively. For that, 10 pl of the reaction mixture were taken und were mixed with 990 pl boiling acetonitrile, subsequently, the mixture was incubated for 20 min at 100 °C, 400 rpm, for enzyme inactivation. The sample substance was diluted with distilled water in a ratio of 1 :1 and analyzed via HPLC-ELSD. The implementation was carried out in duplicates).

[0159] The synthesis was successful for lauric acid and oleic acid as well as for the corresponding sodium salts already at 12 pg enzyme / ml reaction volume. For the remaining fatty acids and salts thereof, no significant conversions could be observed. By increasing the amount of enzyme and simultaneous reduction of the reaction volume to 0.5 ml (final enzyme concentration of 96 pg / ml), a conversion of caprylic acid, palmitic acid and stearic acid as well as of sodium stearate to acyl arginine could be observed. For phenylalanine, the reaction was only successful under analog reaction conditions for palmitic acid and sodium stearate.

[0160] Example 11 : Product isolation and purification of lauroyl phenylalanine

[0161] The model amino acid phenylalanine was used for a first test of product isolation from the reaction mixture (reaction conditions: 100 mM sodium laurate, 200 mM phenylalanine, 50 mM TRIS-buffer (pH = 8), 50 pL (60 pg, 3.0 U) PmAcy, 0,5 mM ZnCh, 5 ml total volume, 50 °C, 250 rpm; 5 M HCI were slowly added to the reaction mixture until a pH value of 1 was reached; the precipitated solid was separated from the superior solution by centrifugation and decantation and then dried under vacuum). It was possible to precipitate the acylamino acid as a pure substance without impurities of the remaining amino acids in the reaction mixture by addition of 5 M hydrochloric acid at a pH of 1. The product was further filtered and washed with petroleum ether and water. The chromatogram of the obtained product showed a yield of 47 % of theory and the identity was confirmed by mass spectroscopy and1H-NMR-spectroscopy of the product.

[0162] Example 12: pH-optimization of PmAcy synthesis conditions for lauroyl arginine synthesis For a condensation reaction of 100 mM lauric acid (added as sodium laurate) and 200 mM arginine, reaction conditions were optimized regarding the pH value. It was shown that in particular a synthesis reaction at pH 9 using sodium borate buffer showed a yield of more than 90 %. Due to the different ionic properties of amino acids caused by their side groups, optimal pH ranges vary from pH 8 to 12 for acylamino acid synthesis with PmAcy.

[0163] Table 5: pH optimization

[0164] 8 Tris-HCI 61

[0165] 9 Sodium borate 95

[0166] 10 Sodium borate 21

[0167] Example 13: Temperature-optimization of PmAcy synthesis conditions for lauroyl arginine synthesis

[0168] For a condensation reaction of 100 mM lauric acid (added as sodium laurate) and 200 mM arginine, reaction conditions were optimized regarding the temperature at a pH value of 9 in sodium borate buffer. Time-conversion-curves have been measured in the temperature range of 35-65°C. It has been shown that the best synthesis values have been obtained at the lowest temperature. After 24 hours, more than 90 % yield of lauroyl arginine have been obtained at a temperature range up to 50 °C (see Fig. 8).

[0169] Example 14: Analysis of substrate concentrations for the PmAcy-catalyzed synthesis of lauroyl arginine

[0170] Synthesis of lauroyl arginine was examined in the concentration range of 50 mM to 400 mM sodium laurate and 50 mM to 400 mM arginine at pH 9 using sodium borate buffer. Conversions were achieved for all concentration ratios, so that a synthesis reaction is possible with substrate concentrations up to 400 mM sodium laurate and 400 mM arginine. Due to substrate solubility, higher concentrations can only be reached by means of dosing or technical stirrer (see Fig. 9).

[0171] Example 15: Synthesis of lauroyl arginine in a buffer-free system

[0172] The use of borate buffer may have certain disadvantages with respect to use of chemicals. For that reason, lauroyl arginine synthesis with PmAcy in a buffer-free system was examined. The pH value was kept constant at pH 9 for 24 h via NaOH dosing. For substrate concentrations of sodium laurate / arginine 100 mM / 100 mM and 100 mM / 200mM, time-conversion-curves showed a continuous formation of lauroyl arginine (see Fig. 10).

[0173] The inventors surprisingly identified a novel aminoacylase from Paraburkolderia monticola DSM 100849 (PmAcy) (SEQ ID NO:1) that could be functionally expressed in microbial expression systems and could therefore be produced in comparably large amounts. Using shaking flask expression with co-expression of chaperon GroEL / ES, 40 mg target enzyme I 800 ml culture could be obtained. This is a major advantage over other known aminoacylases, such as the aminoacylase from the organism Burkholderia sp. strain LP5_18 (Takakura & Asano, supra) that could not be heterologously expressed and was only obtained in yields of about 0.1 mg / 800 ml culture. Hence, a particular advantage of the aminoacylase of Paraburkholderia monticola is the heterologous expressibility in microbial host strains in comparably high yields, which provides for easy technical availability of the enzyme.

[0174] After heterologous expression, PmAcy could be obtained by its N-terminally appended Strep-Tagsequence in a one-step purification by affinity chromatography. In contrast, the aminoacylase from the organism Burkholderia sp. strain LP5_18 could only be obtained from the wild type strain by an elaborated 5-step purification process under cooling. Therefore, another advantage of the novel enzyme is its easy availability.

[0175] PmAcy was found to have a high temperature- and pH stability. After 24 hours of incubation at 70 °C, residual activity was still 82 %. After 4 days at 70 °C, hydrolysis activity remained 38 %, and at 50 °C 81 %. The enzyme is stable in the range of pH 4 to pH 13 over 60 min and no loss of activity was detected. The enzyme was stable over 24 h in a pH range of 4 to 12 and at pH 13 30 % residual activity was measured after 24 h. These high stabilities make the enzyme particularly suitable for technical use.

[0176] Furthermore, the enzyme shows a broad substrate spectrum for the hydrolysis of lauroyl amino acids with respect to the amino acid moieties and further hydrolyzes palmitoyl amino acids with a relative activity of 36 to 50 % based on the respective lauroyl amino acids. In contrast, the related aminoacylase from Burkholderia sp. strain LP5_18 showed only very low hydrolytic activity for long-chain fatty acids (4 % residual activity with stearoyl amino acids).

[0177] PmAcy was found to have synthetic activity and thus to allow synthesis of acylamino acids using lauric acid as well as oleic acid as starting materials. The enzyme was found to have a broad substrate spectrum with respect to the amino acid used and synthesis of acylamino acids could be carried out with Ala, Arg, Asn, Gin, His, He, Leu, Lys, Met, Phe and Vai. In contrast, the aminoacylase from Burkholderia sp. strain LP5_18 could not produce acylamino acids from Met, lie, Leu, Asn and His.

[0178] It was found that the product can be readily isolated from the reaction mixture via precipitation e.g. by pH variation (e.g. lowering by acidification) or via cold precipitation (i.e. lowering the temperature) and can be purified by washing. No side products were detected by chromatographic analysis. Thus, the reaction products can be obtained from the aqueous reaction mixture via two easy and technically easily scalable processes with high purity. In summary, it was found that the aminoacylase from Paraburkolderia monticola is superior to previously described acylamino acids regarding the synthesis of acylamino acids, since it combines all necessary features, which are needed for the technical synthesis: - broad substrate spectrum with respect to accepted amino acids substrate spectrum with respect to accepted fatty acids (preferably chain lengths of C12 (surfactants) and C18 (emulsifiers) are accepted) high synthesis performance at high substrate concentrations and low substrate excess of one reaction partner - good producibility via functional expression in microbial expression systems easy one-step purification by affinity chromatography high process stability (especially temperature- and pH stability).

Claims

CLAIMS1. An isolated polypeptide having aminoacylase activity and comprising an amino acid sequence having over its entire length at least 87 % sequence identity to the amino acid sequence set forth in SEQ ID NO:1.

2. The polypeptide of claim 1, wherein the amino acid sequence has over its entire length at least 90 %, preferably at least 91 %, at least 92 %, at least 93 % or at least 94 %, more preferably at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 .

3. The polypeptide of claim 1 or 2, wherein the polypeptide further comprises an affinity tag, preferably an N-terminal Strep-Tag, wherein the polypeptide optionally has the amino acid sequence set forth in SEQ ID NO:4.

4. An isolated nucleic acid molecule comprising a nucleotide sequence encoding for the polypeptide according to any one of claims 1 to 3.

5. The isolated nucleic acid molecule according to claim 4, wherein the nucleotide sequence encoding for the polypeptide according to any one of claims 1 to 3 is codon-optimized for expression in Escherichia coli, wherein the nucleotide sequence preferably comprises or consists of the nucleotide sequence set forth in SEQ ID NO:2.

6. A plasmid vector comprising the nucleic acid molecule according to claim 4 or 5.

7. A recombinant host cell comprising the isolated nucleic acid molecule according to claim 4 or 5 or the plasmid vector according to claim 6.

8. A method for the production of the polypeptide having aminoacylase activity according to any one of claims 1 to 3, comprising the steps of(a) introducing a nucleic acid molecule or a vector according to any one of claims 4 to 6 into a suitable host cell; and(b) cultivating the host cell in a culture medium under conditions that allow expression of the polypeptide.

9. The method according to claim 8, whereini) in step (a) a further nucleotide sequence encoding for at least one chaperone, preferably the chaperone GroEL / ES, is introduced into the same host cell, and wherein the cultivation conditions of step (b) allow the co-expression of the at least one chaperone; and / or ii) the host cell is E. coli.

10. A method for the production of N-acyl amino acids or salts thereof, the method comprising reacting at least one amino acid or salt thereof with at least one carboxylic acid or salt thereof, preferably fatty acid or salt thereof, in the presence of the polypeptide according to any one of claims 1 to 3 under conditions that allow the production of N-acyl amino acids or salts thereof.

11. The method according to claim 10, wherein i) the amino acid is selected from the group consisting of: arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, taurine, sarcosine, 2,6-diaminopimelic acid, beta-alanin, ornithin, homoserine, homoproline, N-methyltaurine, norvaline, norleucin, citrulline, hydroxy proline and the salts thereof, preferably arginine or phenylalanine; and / or ii) the carboxylic acid is a linear or branched, unsaturated or saturated fatty acid or a salt thereof or a mixture of two or more fatty acids or salts thereof, preferably a linear C8-C22 unsaturated or saturated fatty acid or a salt thereof or a mixture of two or more fatty acids or salts thereof, more preferably caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, linolenic acid, or linoleic acid or a salt thereof or a mixture thereof, more preferably a linear Cs-Cis unsaturated or saturated fatty acid or a salt thereof or a mixture of two or more fatty acids or salts thereof, such as lauric acid, myristic acid, linoleic acid, linolenic acid or oleic acid or a salt thereof or a mixture thereof, most preferably lauric acid or myristic acid or a salts thereof or a mixture thereof; and / or iii) the method is carried out a) at pH 4 to 13, preferably pH 7 to 13, more preferably pH 8 to 12; and / or b) at a temperature of 40 to 80 °C, preferably 45 to 75 °C.

12. N-acyl amino acids obtainable according to the method of claim 10 or 11.

13. Composition comprising the N-acyl amino acids of claim 12, wherein the composition optionally is a cosmetic product, home care product or an industrial and / or institutional product.

14. Use of the N-acyl amino acids of claim 12 in a cosmetic product, home care product or an industrial and / or institutional product.

15. Use of the polypeptide having aminoacylase activity according to any one of claims 1 to 3 for the N-acylation of at least one amino acid or salt thereof or for the synthesis of N-acyl amino acids or salts thereof from at least one amino acid or salt thereof and at least one carboxylic acid, such as a fatty acid, or salt thereof.

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