Aminoacylase and its method of use

Abstract

The present invention relates to an isolated polypeptide comprising an amino acid sequence having aminoacylase activity and having at least 87% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 over its entire length. The present invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding aminoacylase, a plasmid vector comprising the nucleic acid molecule, a recombinant host cell comprising the isolated nucleic acid molecule or vector, and a method for producing the aminoacylase. The present invention also encompasses the use of the aminoacylase according to the present invention for N-acylation of amino acids or salts thereof, and a method for producing N-acylamino acids or salts thereof using the aminoacylase. Furthermore, the present invention relates to the obtained N-acylamino acids, compositions comprising the same, and the use of the produced N-acylamino acids in cosmetic products, home care products, or professional products.

Description

Technical Field

[0001] The present invention relates to an isolated polypeptide having aminoacylase activity and comprising an amino acid sequence having at least 87% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 over its entire length. The present invention further relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding aminoacylase or the like, a plasmid vector comprising the nucleic acid molecule, a recombinant host cell comprising the isolated nucleic acid molecule or vector, and a method for producing the aminoacylase. The present invention also encompasses the use of the aminoacylase according to the present invention for the N-acylation of an amino acid or its salt, and a method for producing an N-acyl amino acid or its salt using the aminoacylase. Furthermore, the present invention relates to the obtained N-acyl amino acid, a composition and a product containing the same, and the use of the obtained N-acyl amino acid in a cosmetic product, a home care product or an industrial product.

Background Art

[0002] Acyl amino acids and structurally related acyl peptides are desirable components in cosmetics, household and cleaning detergents because they are less likely to cause irritation and have excellent dermatological compatibility, and their use in textile technology is promising. Furthermore, they can be produced from renewable raw materials and are thus also promising in terms of sustainability and environmental considerations. Some acyl amino acids are already commercially available as either mild surfactants (e.g., Plantapon® ACG (C12 / C14 acyl glutamate) from BASF) or anionic emulsifiers (e.g., Eumulgin® SG (C18 acyl glutamate) from BASF).

[0003] In addition to glutamates, other amino acids such as glycine, aspartate, or lysine can also be used as polar head groups of such acyl amino acids, for example in cosmetic applications. Other acyl amino acid compounds, such as acyltyrosine, are commercially available as pharmaceutically active ingredients (e.g., Tyrostan® from Sinerga). Good surfactant properties have also been found in lauroyl leucine and lauroyl phenylalanine, which have been found to possess good wetting and foaming properties. Lauroyl arginine (e.g., AMISAFE® AL-01 from Ajinomoto) is used as a mild surfactant and hair conditioning agent.

[0004] All of these products are readily broken down (typically hydrolyzed) on the skin and in the mouth, particularly by the microflora of the armpits (e.g., by C. striatum). This process can be used for the controlled release of nutrient-rich, pharmaceutically active (e.g., antimicrobial) amino acids or peptides.

[0005] In addition to the uses described above, the use of acyl amino acids as metal corrosion inhibitors has been proposed (German Patent Application Publication No. 102010062807 A1). Other proposed uses of acyl amino acids include, for example, the preparation of chiral micelles, their use as chelating agents, or the use of cysteine ​​for the synthesis of acylcysteine-based gemini surfactants.

[0006] The chemical synthesis of acyl amino acids in aqueous solvent mixtures based on the Schotten-Baumann reaction is well known. Following this procedure yields acyl amino acids and structurally related surfactants (acyl peptides, acyl taurines, acyl sarcosines). Solvent-free synthesis has been achieved, for example, by adding polyols (U.S. Patent No. 6060613A) or by carrying out the reaction in a strongly alkaline medium (International Publication No. 2002057217 A2). However, the presence of water always leads to undesirable hydrolysis of acyl chlorides in side reactions, which reduces the final yield of the product.

[0007] Furthermore, despite various known optimizations for this method, it still requires acyl chlorides as starting materials, which is a major drawback in terms of sustainability compared to direct synthesis from fatty acids and amino acids. Acid chlorides are typically produced by chlorination with thionyl chloride or phosphorus chloride, leading to the stoichiometric formation of sulfites or phosphorous acids. Moreover, the reaction with acid chlorides generates stoichiometric amounts of HCl, which must be collected by adding a corresponding stoichiometric amount of base. In short, the acid chloride pathway requires two additional reaction steps and leads to a large amount of inorganic waste. Given the general trend towards greener, more sustainable, and environmentally friendly methods, these methods thus have serious drawbacks.

[0008] A synthesis route for acyl sarcosinates that does not use acid chlorides was previously reported by BASF (U.S. Patent No. 5,856,538A), but the described method has the drawback of requiring the reaction of an amino acid salt with a nearly stoichiometric amount of methylate under fairly harsh conditions. Although it is claimed that other amino acids are also suitable, no specific examples are provided.

[0009] As an alternative, enzymes such as proteases, lipases, and aminoacylases have been proposed and used in the enzymatic synthesis of acyl amino acids. All of these enzymes belong to the hydrolase enzyme class, in which their preferred reaction is the catalysis of acyl amino acid hydrolysis. Consequently, almost all of the enzymes described so far are unsuitable for the technical synthesis required for high yields and conversion rates. Another drawback is that lipases and proteases require the use of solvents and exhibit very long reaction times and low product yields.

[0010] International Publication No. 1999024599 A1 (Henkel) describes enzymatic methods for the N-acylation of amino acids and protein hydrolysates, in which lipases or proteases were used as catalysts. However, it has been shown that neither type of enzyme is particularly suitable compared to aminoacylases.

[0011] Protease synthesis is successful only when carried out in an immobilized form with the addition of a solvent, but the yield is low to moderate and requires an extremely long reaction time.

[0012] When lipases are used, the α-amino group is rarely recognized as a substrate because it works very favorably for ester formation. In lysine, it has been found that the primary amine in the side chain is acylated almost without exception. Since lipase synthesis also typically requires an organic solvent system, sustainable solvent-free synthesis is not possible with either lipases or proteases. For these reasons, lipases are also a suboptimal choice for the synthesis of N-acyl amino acids.

[0013] While the use of aminoacylases appears more promising, most aminoacylases known to date are favored for the hydrolysis of acetyl amino acids and are therefore unsuitable for the synthesis of acyl amino acids.

[0014] In large-scale comparative studies involving various lipases, proteases, and acylases or aminoacylases, acylase from porcine kidney has been identified as the most suitable enzyme for acylamino acid synthesis (Wada et al. (2002), JAOCS 79, 41-46). However, high yields with this acylase could only be achieved with low fatty acid concentrations and a significantly molar excess of amino acids (approximately 100-fold excess). Therefore, this enzyme is not suitable for technical use. Although this enzyme was later cloned, heterologously expressed, and genetically optimized, its synthetic performance could not be improved to a level that would enable efficient acylamino acid synthesis with high yields: yields of only 1-2 mg / 50 ml culture (without co-expression with chaperone) or 3-4 mg / 50 ml culture (with co-expression with chaperone TF or GroEL-GroES) were obtained. Furthermore, this enzyme tends to form inclusion bodies in Escherichia coli (E. coli) and is sensitive to oxidation and temperature changes.

[0015] Recently identified aminoacylases from Streptomyces ambofaciens, Streptomyces mobaraensis, and some species of the genus Burkholderia have been found to be suitable for the production of acyl amino acids in solvent-free reaction systems. However, functional heterologous expression of enzymes such as 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) was either completely impossible or only resulted in low-activity or inactive forms. Consequently, these aminoacylases could only be obtained from wild-type strains through complex multi-step purification protocols, and even then, only small amounts of active enzyme were produced. For these reasons, the aforementioned enzymes are also unsuitable for the technical production of acyl amino acids.

[0016] Furthermore, it has been reported that various aminoacylases with different amino acid specificities were successfully isolated from Streptomyces mobaraensis, and that cloning and heterologous expression were successful. Two of these enzymes included ε-specific lysine acylase, patented together with Ajinomoto (U.S. Patent No. 7888079B2), and an acylase whose hydrolytic activity could be demonstrated. However, the aminoacylase with the broadest synthetic range (Koreishi et al. (2006), cited above) could not be cloned and heterologously expressed. Penicillin V acylase from Streptomyces mobaraensis was cloned and heterologously expressed in S. lividans, but its acyl amino acid synthesis activity has not been investigated.

[0017] Another ε-specific lysine acylase was isolated and cloned from Streptomyces ambofaciens, but it was not functionally expressed.

[0018] Bourkaib et al. (Bourkaib et al. (2020), Enzyme and Microbial Technology 137, 10953) reported on the characterization of an aminoacylase mixture with a broad amino acid substrate range, but they have not been able to disclose its gene sequence.

[0019] Recently, a novel aminoacylase was isolated from the Burkholderia sp. strain LP5_18 (Takakura & Asano (2019), Bioscience, Biotechnology, and Biochemistry 83, 1964-1973). This aminoacylase exhibited high synthesis performance for N-acyl amino acids containing lauroyl residues, high thermal stability, and pH stability over a wide range of pH values. The gene for this aminoacylase was cloned and sequenced, but recombinant expression in Escherichia coli (E. coli) was unsuccessful. Therefore, the enzyme must be obtained from the wild-type strain using a complex five-step purification method under cold conditions. Even with this laborious method, only 0.1 mg of the enzyme was isolated from 800 ml of culture medium. [Overview of the Initiative] [Problems that the invention aims to solve]

[0020] The above demonstrates that the chemical synthesis of acyl amino acids has a weakness in terms of sustainability because it requires the use of chlorinated fatty acids. Enzymatic methods can be said to be more sustainable, but they currently suffer from the weakness of lacking efficient biocatalysts and reaction systems. Therefore, in this field, there is still a need for methods that enable the efficient and sustainable production of acyl amino acids on an industrial scale, and more specifically, for enzymes suitable for enzymatically producing acyl amino acids directly from amino acids and fatty acids, which would provide a sustainable synthesis method for the production of surfactants. [Means for solving the problem]

[0021] The present invention addresses this need in that the inventors have identified a novel aminoacylase (PmAcy) from Paraburkolderia monticola DSM 100849, which surprisingly can directly generate N-acyl amino acids from fatty acids and amino acids in a solvent-free reaction system in good yields and at the same time is obtainable in sufficiently high yields and activities from a recombinant (heterologous) microbial expression system.

[0022] Thus, in a first aspect, the present invention relates to an isolated polypeptide comprising an amino acid sequence having at least 87% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 over its entire length and having aminoacylase activity.

[0023] In another aspect, the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide according to the present invention. In various embodiments, the nucleotide sequence comprises or consists of the sequence shown in SEQ ID NO: 2. The isolated nucleic acid molecule or nucleotide sequence may be comprised in a vector, or the isolated nucleic acid molecule may be a vector, such as a plasmid vector.

[0024] The present invention further relates to a recombinant host cell comprising a nucleic acid molecule or vector according to the present invention.

[0025] Yet a further aspect of the present invention is a method for producing a polypeptide having aminoacylase activity according to the present invention, (a) introducing a nucleic acid molecule or vector as described above into a suitable host cell, wherein the nucleic acid molecule or vector comprises a nucleotide sequence encoding a polypeptide having aminoacylase activity according to the present invention; and (b) culturing the host cell in a culture medium under conditions that allow for the expression of the polypeptide comprising the method.

[0026] In still another aspect, the present invention relates to a method for producing an N-acyl amino acid or a salt thereof, which comprises reacting at least one amino acid or a salt thereof with at least one carboxylic acid such as a fatty acid or a salt thereof in the presence of a polypeptide having aminoacylase activity according to the present invention under conditions enabling the production of the N-acyl amino acid or a salt thereof.

[0027] In yet another aspect, the present invention relates to the use of a polypeptide having aminoacylase activity according to the present invention for the N-acylation of at least one amino acid or a salt thereof.

[0028] In still further aspect, the present invention also relates to the use of a polypeptide having aminoacylase activity according to the present invention for the synthesis of an N-acyl amino acid or a salt thereof from at least one amino acid or a salt thereof and at least one carboxylic acid such as a fatty acid or a salt thereof.

[0029] In yet another aspect, the present invention also relates to an N-acyl amino acid obtainable by the method of the present invention, and a composition and a product containing the same, and its use in such a composition and a product.

[0030] These and other aspects, embodiments, features, and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, claims, and drawings. Each feature from one aspect of the present invention can be used in any other aspect of the present invention. Further, the examples included in this specification are intended to explain and illustrate the present invention, rather than to limit it. Specifically, the present invention is not limited to these examples.

Brief Description of the Drawings

[0031] [Figure 1]This shows the pH dependence of the (hydrolysis) activity of PmAcy (conditions: 3 mM lauroyl alanine, reaction temperature 30°C, 2 μg / ml PmAcy NTag, reaction volume 200 μl, 4 μM ZnCl2, measurement by ninhydrin detection method, sample collection interval 1 minute, and reaction time 4-10 minutes; the concentration of the substrate solution buffer was 50 mM; the reaction was carried out in triplicates). [Figure 2] The pH dependence of PmAcy stability over 1 hour (left) and 24 hours (right) is shown (Incubation conditions: The concentration of the buffer solution in the incubation solution was 100 mM with an enzyme concentration of 240 μg / ml; the incubation temperature was 30°C; the reaction conditions were as follows: 200 mM borate, 15 mM lauroyl alanine in pH 9.0, reaction temperature of 30°C, 12 μg / ml PmAcy NTag, reaction volume of 200 μl, 10 μM ZnCl2, measurement by ninhydrin detection method, sample collection interval of 1 minute, and reaction time of 4-10 minutes; the reaction was carried out in triplicates). [Figure 3] This shows the temperature dependence of PmAcy activity (conditions: 3 mM lauroyl alanine in 50 mM Tris-HCl, pH 8.0, reaction temperature 30°C, 2 μg / ml PmAcy NTag, reaction volume 200 μl, 4 μM ZnCl2, measurement by ninhydrin detection method, 1 minute sample collection interval and 1-6 minute reaction time; reaction was performed in triplicate; free alanine measured by ninhydrin detection method). [Figure 4] The melting curve of PmAcy in a thermal shift assay is shown (for this assay, 30 μl of protein solution (0.16 mg / ml) and 5 μl of 50×SYPRO Orange were mixed in 100 mM Tris-HCl, pH 8.0; this was measured using a qPCR instrument qTower3G (Analytik Jena); the program heated the sample from 25 to 95°C in increments of 2°C at a heating rate of 4.4°C / second, with a retention time of 120 seconds; excitation was performed at 535 nm and measurement was performed at 580 nm). [Figure 5]This demonstrates the temperature-dependent stability of PmAcy (Incubation conditions: 60 μg / ml PmAcy was incubated in 100 mM Tris-HCl in a volume of 200 μl. The buffer solution was adjusted to pH 8 at each temperature (20-90°C); Reaction conditions: 15 mM lauroyl alanine in 100 mM borate, pH 9.0, reaction temperature of 30°C, 3 μg / ml PmAcy NTag, reaction volume of 200 μl, 2.5 μM ZnCl2, measurement of free alanine by ninhydrin detection, sample collection interval of 1 minute, and reaction time of 4-10 minutes; The reaction was carried out in triplicates). [Figure 6] The general reaction scheme for the Schotten-Baumann reaction with lauroyl chloride is shown (reaction conditions: 70 mmol of amino acid (1 equivalent), 175 mmol of NaOH (2.5 equivalents; 245 mmol and 3.5 equivalents for aspartic acid and glutamic acid), 84 mmol of carboxylate chloride (1.2 equivalents), 64 ml of distilled water, 35 ml of acetone, 4 hours, 0°C; isolation of the product was performed by precipitation with a 5 M HCl solution at pH=1, filtration, and washing of the filtration cake with distilled water and petroleum ether. The obtained product was analyzed by HPLC-ELSD and HPLC-MS). [Figure 7] The reaction scheme for the acylation of phenylalanine and lauric acid by PmAcy is shown. [Figure 8] This shows the synthesis of lauroylarginine over time [minutes] at different temperatures (reaction conditions: 100 mM lauric acid (added as sodium laurate), 200 mM arginine, in pH 9 sodium borate buffer). [Figure 9] This shows the synthesis of lauroylarginine at different substrate concentrations (reaction conditions: 50-400 mM sodium laurate, 50-400 mM arginine, pH 9 sodium borate buffer). [Figure 10] This shows the synthesis of lauroylarginine in a buffer-free system over time (reaction conditions: 100 mM sodium laurate, 100 mM or 200 mM arginine; pH was kept constant at pH 9 for 24 hours by adding NaOH). [Modes for carrying out the invention]

[0032] Unless otherwise explicitly stated, terms used herein have the meanings commonly understood in the art.

[0033] When used herein, “at least one” means one or more of the species being referenced, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more. Similarly, when used herein, “one or more” means at least one and includes 1, 2, 3, 4, 5, 6, 7, 8, 9 or more. In relation to a given species, this term relates to the types of species rather than the total number of molecules. Thus, “at least one amino acid” means, for example, that there may be one type of amino acid or two or more different amino acids. In relation to quantity, this term relates to the total amount of the species being referenced. For example, in the case of amino acids, this means that a given quantity is the total amount of all amino acids (types) present in the composition.

[0034] In this specification, numbers specified without decimal places refer to the complete value specified with one decimal place unless otherwise defined; for example, 99% means 99.0%.

[0035] The terms "approximately," "about," or "approx." refer to a variation of ±10%, preferably ±5%, from a given numerical value when used in relation to numerical values.

[0036] All percentages presented herein with respect to compositions or formulations, unless otherwise explicitly stated, are weight percent (wt.-%) of the total weight of each composition or formula. Numerical ranges specified in the form "x~y" include the specified values. If multiple preferred numerical ranges are specified in this form, it is understood that all ranges created by combining different endpoints are also included.

[0037] When used herein in reference to a molecule, “isolated” means that the molecule has been separated, at least partially, from other molecules or other cellular components to which it is naturally bound. “Isolated” may also mean that the molecule has been separated from other molecules and components, such as other proteins and nucleic acids, as well as cellular debris, by purification.

[0038] As used herein, "nucleic acid" includes all natural forms of nucleic acids, such as DNA and RNA. Preferably, the nucleic acid molecule of the present invention is DNA.

[0039] Throughout this specification, the term "peptide" refers to a polymer of amino acid residues linked together by peptide bonds. The peptides according to the present invention may have 2 to 100 amino acid residues. Throughout this specification, the terms "protein" and "polypeptide" are used synonymously to refer to a polymer of amino acid residues linked together by peptide bonds. The proteins or polypeptides according to the present invention preferably have 100 or more amino acid residues.

[0040] The term "N-terminal fragment" refers to a peptide or protein sequence in which the C-terminal side is truncated compared to a reference peptide or protein sequence, and therefore a continuous amino acid polymer remains, starting from its N-terminus.

[0041] The term "C-terminal fragment" refers to a peptide or protein sequence in which the N-terminal side is truncated compared to a reference peptide or protein sequence, and therefore a continuous amino acid polymer remains, starting from its C-terminus.

[0042] The term "fusion protein," as used herein, refers to two or more peptides and proteins that are typically linked to each other by a peptide bond at their N-terminus or C-terminus, including via an amino acid / peptide linker sequence. Such a fusion protein may be encoded by two or more nucleic acid sequences that are operably fused to each other.

[0043] In general, those skilled in the art will understand that, in carrying out the present invention, depending on the nucleic acid construct used, any nucleotide sequence described herein may include additional start and / or stop codons, or any start and / or stop codons contained in any of the sequences described herein may be deleted. Those skilled in the art will make this decision, for example, based on whether the nucleic acid sequence contained in the nucleic acid molecule of the present invention will be translated and / or translated as a fusion protein. In various embodiments, particularly when a tag sequence is added to the N-terminus of the amino acid sequence of the present invention, a start codon encoding the N-terminal M may be added.

[0044] The present invention relates to an isolated polypeptide having aminoacylase activity and comprising an amino acid sequence having at least 87% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 over its entire length.

[0045] 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 with the amino acid sequence shown in SEQ ID NO: 1 over its entire length. In various embodiments, the amino acid sequence is the same length as the sequence shown in SEQ ID NO: 1. In other embodiments, this is a shortened fragment that may be available by deletion / truncation. Such a truncated version is also referred to herein as a functional fragment and is further defined below.

[0046] In a particularly preferred embodiment, the polypeptide essentially consists of or comprises an amino acid sequence having sequence identity of 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% across the entire length of the amino acid sequence shown in SEQ ID NO: 1.

[0047] The determination of sequence identity of nucleic acids or amino acid sequences can be performed by sequence alignment based on the well-established and commonly used BLAST algorithm (see, for example, Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (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, pp. 3389-3402). Such alignment is based on aligning similar sequences of nucleotides or amino acids with each other. Another algorithm known in the art for the aforementioned purposes is the FASTA algorithm. Alignment, in particular multiple sequence comparison, is typically performed using computer programs. Commonly used programs include the Clustal series (see, e.g., Chenna et al. (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.

[0048] Furthermore, sequence alignment is also possible using the Vector NTI® Suite 10.3 (Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA, USA), a computer program based on ClustalW with the AlignX module for sequence comparison, with standard parameter settings. Unless otherwise specified, sequence identity is determined using the BLAST algorithm.

[0049] Such comparisons also allow for the determination of the similarity of the sequences being compared. This similarity is typically expressed as percentage identity, i.e., the proportion of identical nucleotides / amino acids at the same or corresponding sequence positions (in the alignment) relative to the total number of nucleotides / amino acids aligned. For example, if 90 amino acids in a 100-amino acid query sequence are identical to amino acids at corresponding positions in the template sequence during the alignment, the sequence identity is 90%. The broader term "homology" also considers conserved amino acid substitutions, i.e., amino acids that are similar in terms of their chemical properties, because they typically have similar chemical properties in proteins. Thus, such homology can be expressed as percentage homology.

[0050] Unless otherwise specified, sequence identity and sequence homology refer to the entire length of the aligned sequence. Specifically, the term “the amino acid sequence over its entire length” having a given sequence identity with SEQ ID NO: 1 means that the amino acid sequence includes a contiguous amino acid sequence having the sequence identity shown in SEQ ID NO: 1. In various embodiments, the amino acid sequence of the subject may also have a given sequence identity with the entire length of SEQ ID NO: 1.

[0051] The amino acid sequence may correspond to a continuous sequence of amino acids in the amino acid sequence shown in SEQ ID NO: 1 having the indicated length, but it may also correspond to a discontinuous sequence of amino acids in the amino acid sequence shown in SEQ ID NO: 1 if the amino acid sequence corresponds to a sequence of SEQ ID NO: 1 from which certain amino acids or amino acid sequences are deleted. Thus, the amino acid sequence may be obtained from the amino acid sequence shown in SEQ ID NO: 1 by one or more of the N-terminal truncation, C-terminal truncation, or deletion of one or more amino acids, as described in detail above. Such shortened variants of the amino acid sequence of SEQ ID NO: 1 are encompassed by the term “fragment”. The term “functional fragment” also implies that each remaining polypeptide sequence retains its catalytic activity, as defined below herein. Insofar as the polypeptide contains a functional fragment of the amino acid sequence shown in SEQ ID NO: 1, the fragment is preferably an N-terminal and / or C-terminal truncated fragment, optionally lacking 1 to 50, 1 to 40, 1 to 30, 1 to 20, or 1 to 10 amino acids from one or both ends.

[0052] In various embodiments, the polypeptide according to the present invention may have a wild-type sequence of aminoacylase enzyme (PmAcy) from Paraburkolderia monticola DSM 100849, or a variant thereof.

[0053] When used herein, "having aminoacylase activity" means that a polypeptide has enzymatic activity and can catalyze the reaction of an amino acid or a salt thereof with a carboxylic acid or a salt thereof to produce the hydrolysis of the acyl amino acid to an acyl amino acid or a salt thereof and / or their respective reactants. In various embodiments, the term means that under the same conditions that enable aminoacylase activity, the polypeptide has at least 50% of the enzymatic activity of the full-length sequence of SEQ ID NO: 1, preferably at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the aminoacylase of SEQ ID NO: 1.

[0054] In various embodiments, having aminoacylase activity means that the polypeptide has the ability to catalyze the reaction of (α) amino acids and carboxylic acids, preferably fatty acids, or salts thereof, to N-acyl amino acids or salts thereof. If the amino acid contains a side-chain amino group, it may be said that acylation at the α-amino group is preferable.

[0055] The term "wild-type" refers to the entire cell, as well as individual naturally occurring genes, nucleotide sequences, proteins / enzymes, or amino acid sequences found in nature. Therefore, the term "wild-type" does not include cells, genes, nucleotide sequences, proteins, enzymes, or amino acid sequences that have been at least partially modified using recombinant / genetic engineering methods.

[0056] As used herein, "mutant" refers to a variant of an undenatured enzyme, either naturally occurring or artificially produced, having a modified amino acid sequence compared to the reference form.

[0057] With respect to nucleotides or amino acid sequences, or nucleic acids or proteins / enzymes, "modified" or "modified" means that the corresponding sequence has been modified to be distinguishable from the naturally occurring sequence (wild type). In various embodiments, modification is the fact that the sequence has been mutated, for example, by substitution, deletion, or insertion. In various other embodiments, a natural amino acid sequence may be modified by adding affinity tags, such as His tags or Strep tags, to the naturally occurring sequence, which enable the isolation and purification of the enzyme after production in a host organism.

[0058] In various embodiments, the polypeptide of the present invention comprises, in addition to an amino acid sequence having at least 87% sequence identity with SEQ ID NO: 1, at least one other amino acid sequence, which may be located at the N-terminal or C-terminal end of the aforementioned sequence, and is typically fused to it, i.e., linked by a peptide bond. The additional amino acid sequence may be a functional sequence, such as an affinity tag, that enables isolation and purification, or detection. Such a functional amino acid sequence may be linked by a linker sequence suitable for the amino acid sequence encoding the enzymatically active portion of the polypeptide. Such a linker sequence may optionally include, in various embodiments, a protease recognition site that enables cleavage of the tag from the enzymatically active portion of the molecule.

[0059] In various embodiments, the polypeptide according to the present invention includes an N-terminal or C-terminal affinity tag, such as a His tag (6xHis tag) or a Strep tag, preferably an N-terminal Strep tag.

[0060] Such Strep tags are publicly known and can consist of the amino acid sequence WSHPQFEK (SEQ ID NO: 3).

[0061] In the context of this invention, the term "N-terminus" or "N-terminal side" refers to the end of an amino acid chain that has a free amino acid group.

[0062] In the context of this invention, the term "C-terminus" or "C-terminal side" refers to the end of an amino acid chain that has a free carboxyl group.

[0063] In various embodiments, the polypeptide of the present invention includes a Strep tag directly attached to the N-terminus of an amino acid sequence having aminoacylase activity. In various other embodiments, this is linked to the amino acid sequence of the aminoacylase via a short linker sequence. In a preferred embodiment, the linker sequence contains or consists of 1 to 5 amino acids, for example, the linker sequence may contain or consist of 2 amino acids, such as SG.

[0064] In various embodiments, the polypeptide of the present invention comprises an amino acid sequence having 87% sequence identity with SEQ ID NO: 1, and a Strep tag of the amino acid sequence of SEQ ID NO: 3, which is attached to the N-terminus of the aminoacylase sequence via linker SG, and also comprises an N-terminal residue M. Such a construct, which also includes the full-length sequence of SEQ ID NO: 1, is shown in SEQ ID NO: 4.

[0065] In various embodiments, the aminoacylase according to the present invention comprises or consists of a wild-type sequence from Paraburkolderia monticola DSM 100849 as shown in SEQ ID NO: 1. Furthermore, this natural amino acid sequence is modified to have the affinity tag described above and optionally to have the linker sequence described above, preferably attached to the amino acid sequence at its N-terminus.

[0066] In a further embodiment, the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide according to the present invention. The encoded polypeptide may be any of those described above, and in various embodiments, in addition to the amino acid sequence defined herein having aminoacylase activity, it may also include other amino acid sequences encoding further (poly)peptides, such as (poly)peptide tags and linker sequences used or useful for detection or purification. As the nucleic acid molecule encodes an entire polypeptide, the various different amino acid sequences combined therein can be expressed as a single fusion protein.

[0067] Due to the degeneracy of the genetic code, certain amino acid sequences can be encoded by several different nucleic acids; therefore, all nucleotide sequences capable of encoding polypeptides as described herein are included in the present invention. Since defined amino acids can be assigned to individual codons despite the degeneracy of the genetic code, those skilled in the art can determine these nucleotide sequences without any doubt. Thus, those skilled in the art can easily determine the nucleic acid encoding a given amino acid sequence, starting from that amino acid sequence. In the nucleotide sequences according to the present invention, one or more codons can be replaced with synonymous codons (i.e., codons encoding the same amino acid) with respect to the wild type or starting sequence. This is particularly useful for heterologous expression because there is a fixed codon usage in each organism, such that certain codons may have lower translation efficiency compared to synonymous codons encoding the same amino acid in a given organism. Replacing codons without changing the encoded polypeptide sequence so that translation in a given host organism is optimized (based on its codon usage) is called "codon optimization."

[0068] In various embodiments, the nucleotide sequences according to the present invention are codon-optimized for the host organism in which they are expressed. The host organism may be any suitable host organism, but in preferred embodiments it may be Escherichia coli (E. coli).

[0069] Such a codon-optimized nucleotide sequence may have the nucleotide sequence shown in Sequence ID No. 2.

[0070] In certain embodiments, the nucleic acid molecules defined above may be contained within a vector, such as a cloning vector or an expression vector. Generally, the nucleic acid molecules of the present invention may also be part of a vector, or any other type of cloning medium, including, but not limited to, plasmids, phagemids, phages, baculoviruses, cosmids, or artificial chromosomes. Generally, the nucleic acid molecules disclosed herein may be "operably ligated" to a regulatory sequence (or a set of regulatory sequences) to enable the expression of the nucleic acid molecule.

[0071] Such cloning vectors may include, in addition to the regulatory sequences and nucleic acid sequences of the present invention described above, replication and regulatory sequences derived from a species compatible with the host cell used for expression, and selection markers that confer a selectable phenotype to transformed or transfected cells. Numerous suitable cloning vectors are known and commercially available in the art.

[0072] In certain embodiments, the nucleic acid molecules disclosed herein are contained in a cloning vector. In some embodiments, the nucleic acid molecules disclosed herein are contained in an expression vector. The vector may contain replication regulatory elements and selection markers. In certain embodiments, the selection marker may be selected from the group consisting of genes conferring resistance to ampicillin, kanamycin, chloramphenicol, tetracycline, blastosidine, spectinomycin, gentamicin, hygromycin, and zeosin. In various other embodiments, selection may be performed using an antibiotic-free system, for example, by using a toxin / antitoxin system, cer sequences, triclosan, nutritional requirements, etc. Preferred methods are known to those skilled in the art.

[0073] When the nucleic acid molecules of the present invention described above are incorporated into a vector, they must be incorporated in such a way that the polypeptide can be expressed. Accordingly, the vector of the present invention includes a sequence element containing information regarding transcription and / or translation regulation, and such sequence is "operably linked" to a nucleotide sequence encoding a polypeptide. In this context, an operable linkage is a linkage such that the regulatory sequence element and the sequence to be expressed are linked in a manner that enables gene expression. The exact nature of the regulatory regions required for gene expression may differ between species, but generally these regions include a promoter, which in prokaryotes includes both the promoter itself, i.e., the DNA element that directs the initiation of transcription, and the DNA element that, when transcribed to RNA, will signal the initiation of translation. Such promoter regions typically include 5' non-coding sequences involved in the initiation of transcription and translation, such as the -35 / -10 box and Shine-Dalgarno element in prokaryotes, or the TATA box, CAAT sequence, and 5'-capping element in eukaryotes. These regions may also include enhancer or repressor elements, as well as translated signal and leader sequences for targeting the native polypeptide to specific compartments of the host cell.

[0074] In addition, the 3' non-coding sequence may contain regulatory elements involved in transcription termination, polyadenylation, and other processes. However, if these termination sequences do not function satisfactorily in a particular host cell, they may be replaced with signals that function in that cell.

[0075] In various embodiments, the vectors containing the nucleic acid molecules of the present invention may include regulatory sequences, preferably promoter sequences. In certain embodiments, the promoter is identical or homologous to the promoter sequence of the host genome. In such cases, an endogenous polymerase may have the ability to transcribe the nucleic acid molecular sequence contained 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.

[0076] In another embodiment, the vector comprising the nucleic acid molecule of the present invention comprises a promoter sequence and a transcription termination sequence. Suitable promoters for prokaryotic expression include, for example, the araBAD promoter, tet- promoter, lacUV5 promoter, CMV promoter, EF1α promoter, AOX1 promoter, tac promoter, T7 promoter, or lac promoter. Furthermore, the nucleic acid molecule of the present invention may include transcriptional regulatory elements, such as repressor elements, that enable regulation of the transcription and translation of the coding sequence contained in the nucleic acid molecule. The repressor elements may be selected from the group consisting of Lac-, AraC-, or MalR- repressors.

[0077] The vector may be effective for prokaryotic or eukaryotic protein expression. More specifically, the nucleic acid molecule of the present invention may be included in a vector for prokaryotic protein expression. Such a vector sequence is constructed so that the target sequence can be easily inserted using techniques well known to those skilled in the art. In certain embodiments, the vector is a pET vector such as pET28a, pBAD vector, pK184 vector, pMONO vector, pSELECT vector, pSELECT-Tag vector, pVITRO vector, pVIVO vector, pORF vector, pBLAST vector, pUO vector, pDUO vector, pZERO vector, pDeNy vector, pDRIVE vector, pDRIVE-SEAP vector, HaloTag® fusion vector, pTARGET® vector, Flexi® vector, pDEST vector, pHIL vector, pPIC vector, pMET vector, pPink vector, pLP vector, pTOPO vector, pBud vector, pCEP vector, pCMV vector, pDisplay vector, pEF vector, pFL vector, pFRT vector, pFast Selected from the group consisting of Bac vector, pGAPZ vector, pIZ / V5 vector, p3S vector, pIAR vector, pSEC, pMS, pSU2726 vector, pLenti6 vector, pMIB vector, pOG vector, pOpti vector, pREP4 vector, pRSET vector, pSCREEN vector, pSecTag vector, pTEFl vector, pTracer vector, pTrc vector, pUB6 vector, pVAXl vector, pYC2 vector, pYES2 vector, pZeo vector, pcDNA vector, pFLAG vector, pTAC vector, pT7 vector, gateway® vector, pQE vector, pLEXY vector, pRNA vector, pPK vector, pUMVC vector, pLIVE vector, pCRUZ vector, Duet vector, and other vectors or derivatives thereof.

[0078] The vectors of the present invention may be selected from the group consisting of high copy number, medium copy number, and low copy number vectors.

[0079] The vectors of the present invention described above can be used for the transformation or transfection of host cells to achieve the expression of polypeptides encoded by the nucleic acid molecules and contained in the vector DNA. Thus, in further embodiments, the present invention also relates to host cells containing vectors or nucleic acid molecules as disclosed herein. The terms “host organism” and “host cell” are used synonymously in connection with the present invention.

[0080] Furthermore, this specification also considers host cells containing nucleic acid molecules as described herein, which are incorporated into their genome. Those skilled in the art know suitable methods for achieving the incorporation of nucleic acid molecules. For example, the molecules may be delivered to the host cell using liposome transfer or viral infection, and then the nucleic acid molecules may be incorporated into the host genome using homologous recombination. In certain embodiments, the nucleic acid molecule is incorporated at a site in the host genome, which mediates the transcription of the peptide or protein of the present invention encoded by the nucleic acid molecule. In various embodiments, the nucleic acid molecule further includes elements that, once incorporated into the host genome, act as elements and / or selection markers that mediate the transcription of the nucleic acid molecule.

[0081] In certain embodiments, the nucleic acid molecule of the present invention is transcribed by a polymerase naturally encoded in the host genome. In various embodiments, the nucleic acid molecule is transcribed by an RNA polymerase not naturally occurring in the host genome. In such embodiments, the nucleic acid molecule of the present invention may further contain a sequence encoding a polymerase, and / or the host genome may be manipulated to contain a nucleic acid sequence encoding an exogenous polymerase, or host cells may be infected. The host cells may be specifically selected as host cells capable of expressing the gene. In addition, or otherwise, the nucleic acid encoding the polypeptide of the present invention may be genetically engineered for expression in a suitable system in order to produce the polypeptide. Transformation can be carried out using standard techniques (Sambrook, J. et al. (2001), Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0082] A host organism containing such a vector for recombinant expression of polypeptides as described herein also constitutes part of the present invention. The host organism is preferably a prokaryote or a eukaryote, more particularly a bacterium, yeast, or unicellular fungus. Exemplary host organisms include the genera Aspergillus, Corynebacterium, Brevibacterium, Bacillus, Acinetobacter, Alcaligenes, Actinobacillus, Anaerobiospirillum, Basfia, Wollinella, Fibrobacter, Ruminococcus, Mannheimia, Lactobacillus, Lactococcus, Paracoccus, and Lactococcus. These include species belonging to the genera Candida, Pichia (also known as Komagataella), Hansenula, Kluveromyces, Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methylobacterium, Ralstonia, Pseudomonas, Rhodospirillum, Rhodobacter, Burkholderia, Clostridium, or Cupriavidus, as well as Aspergillus nidurans. Aspergillus niger, Alcaligenes latus, Bacillus megatheriummegaterium), Bacillus subtilis, Brevibacterium flavum, Brevifermentbacterium lactofermentum, Escherichia coli, Basfia succiniciproducens, Wollinella succinogenes, Fibrobacter succinogenes, Ruminococcus flavefaciens, Anaerobiospirillum succiniciproducens, Mannheimia succiniciproducens, Actinobacillus succiniciproducens succinogenes, Saccharomyces cerevisiae, Kluveromyces lactis, Kluyveromyces marxianus, Candida blankii, Candida rugosa, Corynebacterium glutamicum, Corynebacterium efficiens, Zymonomas mobilis, Yarrowia lipolytica, Methylobacterium extorquens, Flansenula polymorpha Polymorpha), Ralstonia eutropha, Rhodobacter sphaeroides, Paracoccus bellus* Pseudomonas aeruginosa*, *Acinetobacter calcoaceticus*, *Pichia pastoris* (also known as *Komagataella phaffii*), *Thermoanaerobacter kivui*, *Acetobacterium woodii*, *Acetoanaerobium notera*, *Clostridium aceticum*, *Butyribacterium methylotrophicum*, *Clostridium acetobutylicum*, *Clostridium saccharoperbutylacetonicum* *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* Putida is particularly preferred.

[0083] In various embodiments, the host cell is 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 Escherichia coli (E. coli). In specific embodiments, the host cell is Escherichia coli, more specifically Escherichia coli BL21(DE3) or other Escherichia coli K12 or Escherichia coli B834 derivatives. In various embodiments, the host cell is selected from, for example, without limitation, Escherichia coli BL21(DE3), Escherichia coli BL21, Escherichia coli K12, Escherichia coli BLR, Escherichia coli BL21 AI, Escherichia coli BL21 pLysS, Escherichia coli XL1, and Escherichia coli DH5a. Further suitable Escherichia coli (E. coli) strains include, but are not limited to, DH1, DH5a, DM1, HB101, JMlOl-110, Rosetta(DE3)pLysS, SURE, TOP10, XLi-Blue, XL2-Blue, and XLIO-Blue.

[0084] Transformed host cells are cultured under conditions suitable for the expression of the nucleotide sequence encoding the polypeptide of the present invention. In certain embodiments, cells are cultured under conditions suitable for the expression of the nucleotide sequence encoding the polypeptide of the present invention.

[0085] For the production of the recombinant polypeptides described herein, the vector of the present invention can be introduced into a suitable host organism using recombinant DNA technology (as outlined above). For this purpose, first, host cells are transformed with a vector containing the nucleic acid molecule according to the present invention using an established standard method (Sambrook, J. et al. (2001), cited above). Next, the host cells are cultured under conditions that allow for the expression of heterologous DNA and, consequently, the synthesis of the corresponding polypeptide. Subsequently, the polypeptide is recovered from either the cells or the culture medium.

[0086] Certain preferred protocols for the expression of the polypeptide of the present invention are known to those skilled in the art. The expression of the recombinant polypeptide of the present invention can be achieved by the following method, namely, a method comprising the steps of (a) introducing a nucleic acid molecule or vector of the present invention into a host cell, wherein the nucleic acid molecule or vector encodes the recombinant polypeptide; and (b) culturing the host cell in a culture medium under conditions that enable the expression of the recombinant polypeptide. This expression may be heterologous in that the host cell does not naturally express the polypeptide.

[0087] Step (a) may be carried out using suitable transformation and transfection techniques known to those skilled in the art. These techniques are typically selected based on the type of host cell into which the nucleic acid will be introduced. In some embodiments, transformation may be achieved using electroporation or heat shock treatment of the host cell.

[0088] In various embodiments, step (a) of the method also introduces a nucleotide sequence encoding at least one chaperone, preferably chaperone GroEL / ES, into the host organism. The nucleotide sequence may be contained in a second nucleic acid molecule. In a preferred embodiment, the chaperone nucleotide sequence is introduced into the host organism by a second vector, more specifically, the vector pGro7 encoding chaperone GroEL / ES.

[0089] The chaperone, specifically the chaperone GroEL / ES, enhances the production of the polypeptide of the present invention in host cells. Therefore, in such embodiments, the culture conditions of step (b) further allow for the co-expression of at least one chaperone. Where the expression of the polypeptide of the present invention is referred to below, it should be understood that the foregoing disclosure also applies to the expression of the chaperone if the chaperone is co-expressed.

[0090] Step (b) may include a culture step that enables the growth of host cells. Alternatively, such a step that enables the growth of host cells, as well as a step that enables the expression of polypeptides and, optionally, chaperones, may be carried out separately, in which the cells are first cultured to grow to a desired density, and then cultured under conditions that enable polypeptide expression. However, the expression step may still enable cell growth.

[0091] In general, this method may utilize any known culture medium suitable for the growth of a host of choice. In various embodiments, the medium is either a nutrient-rich medium or a minimal medium. This specification also considers methods in which the steps of growing cells and expressing peptides or proteins are carried out using different media. For example, the growth step may be carried out using a nutrient-rich medium, which is then replaced with a minimal medium in the expression step. In some cases, the medium is selected from the group consisting of LB medium, TB medium, 2YT medium, synthetic medium, and minimal medium.

[0092] This 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.

[0093] For example, isolation may involve destroying or damaging cells (cell lysis), followed by separating cell debris from polypeptides by, for example, centrifugation, filtration, sedimentation, or a combination thereof. Thus, the isolated polypeptide, typically present in the supernatant, can then be further purified by, for example, chromatographic methods such as affinity chromatography, ion exchange chromatography, reverse-phase chromatography, size exclusion chromatography, and combinations thereof. In various embodiments, purification may include an initial affinity chromatography followed optionally by further purification steps (e.g., size exclusion chromatography). Any suitable culture, isolation, separation, or purification method known to those skilled in the art can be used in connection with the present invention.

[0094] In one embodiment, the present invention relates to a method for expressing the (recombinant) polypeptide of the present invention using the nucleic acid molecules described above. (a) the step of introducing a nucleic acid molecule or vector as described above into a suitable host cell, wherein the nucleic acid molecule or vector comprises a nucleotide sequence encoding a recombinant polypeptide; and (b) A step of culturing host cells in culture medium under conditions that enable the expression of recombinant polypeptides. It may include.

[0095] The method may further include the steps of isolation and, optionally, purifying the polypeptide thus produced, for example, using the technique described above.

[0096] In further embodiments, the present invention relates to the use of vectors or nucleic acid molecules as disclosed herein for the expression of recombinant polypeptides. In some embodiments, vectors are used for the expression and optionally secretion of recombinant polypeptides according to the present invention. Expression or expression and secretion may be achieved using the methods described herein.

[0097] The terms “heterogeneous” or “recombinant” are used herein to refer to the absence of a corresponding molecule or nucleotide sequence in the host organism in nature. Thus, heterogeneous or recombinant expression of one or more nucleotide sequences in a host organism means that, under natural conditions, the host organism does not contain or express that one or more nucleotide sequences. As a result, heterogeneous or recombinant proteins that would not be produced in the host organism under natural conditions can be produced. The nucleotide sequences introduced into the host organism may be wild-type sequences and / or modified sequences from another organism.

[0098] In a further embodiment, the present invention relates to a method for producing an N-acyl amino acid or a salt thereof, comprising reacting at least one amino acid or a salt thereof with at least one carboxylic acid or a salt thereof under conditions suitable for the production of an N-acyl amino acid or a salt thereof in the presence of a polypeptide having aminoacylase activity according to the present invention.

[0099] In various embodiments, the polypeptide having aminoacylase activity is used in an amount of 0.00001 to 10 wt.-% (active protein), preferably 0.00001 to 1 wt.-% (active protein), for example, 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.

[0100] Protein concentration can be determined by known methods, such as 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). The active protein content can be determined by measuring the titer of the active site and determining the residual activity using a suitable irreversible inhibitor (Bender et al., 1966, J. Am. Chem. Soc. 88, 24:5890-5913).

[0101] In various embodiments, the enzymatically active polypeptide is added in a purified form or in the form of an enzyme composition containing the polypeptide together with other components. In various embodiments, the enzyme formulation may be lyophilized.

[0102] The amino acids used may be any amino acids, including both proteogenic and non-proteogenic amino acids. The 20 proteogenic 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 acids are 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, more specifically isoleucine, valine, proline, methionine, leucine, arginine, or phenylalanine. Most preferably, the amino acid is arginine or phenylalanine.

[0103] Examples of non-proteinogenic amino acids include, without limitation, taurine, sarcosine, 2,6-diaminopimelic acid, β-alanine, ornithine, homoserine, homoproline, N-methyltaurine, norvaline, norleucine, citrulline, and hydroxyproline.

[0104] The amino acid may be L- or D-amino acid. In various embodiments, L-amino acids are preferred.

[0105] In various embodiments, peptides, such as dipeptides or tripeptides, or even longer-chain peptides, can be used instead of or in addition to monomeric amino acids.

[0106] In various embodiments, it is also possible to use modified amino acids and peptides that do not exist in nature.

[0107] In specific embodiments, 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, β-alanine, ornithine, homoserine, homoproline, N-methyltaurine, norvaline, norleucine, citrulline, hydroxyproline, and salts thereof.

[0108] 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, such as C6-C24, C8-C22, or C8-C18 fatty acids, or salts thereof. In various embodiments, the fatty acids used are linear C8-C22, for example, C8-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 salts thereof, preferably lauric acid or myristic acid or salts thereof, more specifically lauric acid or salts thereof. The fatty acids may also be mixtures of fatty acids, such as cocoyl fatty acids predominantly containing C12-C14 fatty acids.

[0109] The salts of amino acids and / or carboxylic acids may be any suitable salts, including alkali salts and alkaline earth metal salts such as sodium, potassium, magnesium, and calcium. Preferably, the counterion is sodium or potassium.

[0110] In various embodiments, the N-acyl amino acids produced are N-lauroyl amino acids, N-myristoyl amino acids, N-palmitoyl amino acids, N-oleoyl amino acids, N-linoleoyl amino acids, N-linolenoyl amino acids, 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 salts thereof. In various embodiments, the N-acyl amino acids produced may be derived from a mixture of fatty acids, including cocoyl fatty acids and any two or more of the above.

[0111] In various embodiments, the method may further include an isolation step of isolating and / or purifying an N-acyl amino acid or a salt thereof. The isolation step may be any suitable isolation method known to those skilled in the art. Preferably, the isolation step is a precipitation step by changing the pH (e.g., acidification) or a cold precipitation step, i.e., a step of lowering the temperature. In various embodiments, the purification step may include, for example, a filtration step and / or another suitable washing step.

[0112] Preferably, the method is carried out in an aqueous reaction system, such as water or a buffer system. In a preferred embodiment, the method is carried out in an organic solvent-free reaction system. Preferably, this reaction is carried out in water or a buffer system without the addition of further organic solvents. In various embodiments, the buffer system may be, but is not limited to, citrate, acetate, MES, tris-HCl, borate, or phosphate.

[0113] In various embodiments, the reaction is carried out at a pH of 4 to 13, preferably 7 to 13, and more preferably 8 to 12. The temperature may be in the range of 40 to about 80°C, preferably 45 to 75°C.

[0114] In various embodiments, amino acids or salts thereof and carboxylic acids such as fatty acids or salts 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, and more specifically, a molar ratio of about 2:1 (amino acids:carboxylic acids).

[0115] In a further embodiment, the present invention relates to the use of a polypeptide having aminoacylase activity according to the present invention for the N-acylation of at least one amino acid or a salt thereof.

[0116] In another embodiment, the present invention also relates to the use of polypeptides having aminoacylase activity according to the present invention for the synthesis of N-acylamino acids or salts thereof from at least one amino acid or salt thereof and at least one carboxylic acid or salt thereof, such as a fatty acid.

[0117] In the above reaction, N-acylation is catalyzed by a polypeptide having aminoacylase activity according to the present invention.

[0118] In a further embodiment, the present invention therefore relates to N-acyl amino acids obtainable by the method according to the present invention.

[0119] The N-acyl amino acids or salts thereof obtained in this way can be used in cosmetic products, home care products, such as detergents, dishwashing detergents and cleaning detergents, as well as in I&I (industrial) products, for example, in the hygiene or food industry. Preferably, the resulting N-acyl amino acids or salts thereof can be used as skin-friendly surfactants, particularly when the acyl group has a carbon atom length of 10 to 14, or, more specifically, as emulsifiers, such as C18 fatty acids, when the acyl group has a carbon atom length of more than 14. Preferred applications include cosmetic products, (laundry) detergents, dishwashing detergents and cleaning compositions, and more specifically, cosmetic products such as hair care and body care products, including baby shampoos.

[0120] In various embodiments, N-acyl amino acids or salts thereof are used in these compositions in an amount of 0.5 to 40 wt.-% based on the total weight of the composition.

[0121] Thus, in another embodiment, the present invention relates to a composition comprising an N-acyl amino acid according to the present invention, wherein the composition is optionally a cosmetic product, a home care product, or a professional product.

[0122] Furthermore, the present invention relates to the use of N-acyl amino acids according to the present invention in beauty products, home care products, or professional products.

[0123] All embodiments and examples described herein with respect to polypeptides, nucleic acid molecules, vectors, host cells, or obtained N-acyl amino acids according to the present invention also apply to the methods, uses, compositions, and products disclosed herein, and vice versa.

[0124] Other embodiments are found in the following non-limiting examples. [Examples]

[0125] Example 1: Cloning and Expression The protein sequence of PmAcy with a Strep tag (PmAcy N Tag, Sequence ID No. 4) was back-translated, and the resulting DNA sequence was codon-optimized for E. coli (E. coli) and commercially synthesized as a synthetic DNA strand. BsaI restriction sites were added to both ends of this strand using polymerase chain reaction, making it possible to clone the gene into the vector pET28a by golden gate cloning. pET28a is an expression plasmid for the T7 system. Expression was performed in E. coli (E. coli) BL21(DE3) cells further containing plasmid pGro7. This strain is capable of expressing the target gene of plasmid pET28a after induction with lactose or IPTG. Plasmid pGro7 contains a sequence encoding the chaperone GroEL / ES and functions to enhance aminoacylase production.

[0126] Example 2: Purification Purification was performed by 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 chromatographic step. The purity and mass of the protein (monomer) were analyzed and confirmed by SDS-Page and MALDI-TOF.

[0127] Example 3: pH-dependent stability and activity of PmAcy PmAcy N Tag was found to be active in the alkaline range, with an optimal pH of 12.0. This enzyme was stable over a wide pH range, showing no significant activity loss even after incubation at pH 12 and 30°C for 24 hours. At pH 13, the enzyme was stable at 30°C for 1 hour, and its residual activity remained at 30% after 24 hours (see Figures 1 and 2).

[0128] Example 4: Temperature-dependent stability and activity of PmAcy Aminoacylase PmAcy is a thermophilic enzyme with an optimal temperature of 70°C. At 80°C, its activity decreases slightly (see Figure 3).

[0129] First, thermal stability was tested using a thermal shift assay. Since the dye is bound to hydrophobic side chains, this assay indicates an increase in the fluorescence of the dye SYPRO Orange when the protein undergoes thermal denaturation. Thermal denaturation was detected at 78°C. Because the protein surface is generally hydrophobic, fluorescence was already high at the start and initially decreased with increasing temperature. Dye excitation occurred at 535 nm, and fluorescence was measured at 580 nm (see Figure 4).

[0130] To detect the thermal stability of PmAcy over longer periods, the enzyme was incubated at 20–90°C for up to 4 days, and residual activity was measured by a hydrolysis assay at specified time points. The results are shown in Figure 5. The enzyme remained stable for more than 2 hours without loss of activity up to 80°C. After incubation at 70°C for 1 day, 82% residual activity was observed. 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 have extremely high thermal stability, which facilitates its technical use in longer-term synthesis at, for example, 50°C.

[0131] Example 5: Synthesis of reference compound by Schotten-Baumann reaction The reference compound was synthesized by the Schotten-Baumann reaction using 20 proteogenic amino acids and lauroyl chloride in an alkaline water / acetone solvent system. Some amino acids (alanine, glutamine) were additionally acylated with palmitoyl chloride (see Figure 6).

[0132] [Table 1]

[0133] The reference compound could be obtained in good to very good yields. Only the isolation of lauroyl histidine, lauroyl proline, and lauroyl threonine yielded either no target product or only very low amounts of the target product.

[0134] Example 6: Hydrolytic cleavage of lauroyl amino acids by PmAcy In terms of the hydrolysis range, lauroyl amino acids with short hydrophobic side chains are preferred. The substrates that were best converted were lauroyl alanine, lauroyl isoleucine, lauroyl methionine, and lauroyl valine. Good conversion was also observed with palmityl alanine, lauroyl glutamine, palmityl glutamine, lauroyl phenylalanine, lauroyl serine, and lauroyl tyrosine. Furthermore, activity was observed with lauroyl leucine. However, this enzyme showed only low activity towards acetyl amino acids.

[0135] [Table 2]

[0136] Example 7: Comparison of hydrolytic cleavage of acetyl amino acids and palmityl amino acids PmAcy NTag also cleaves palmitoyl amino acids, however, its activity is lower compared to that of the corresponding lauroyl amino acids (see Table 2). Since acetyl amino acids are hardly hydrolyzed, its activity with acetyl amino acids is irrelevant. Consequently, this enzyme clearly exhibits a preference for long-chain fatty acids, which is a desirable characteristic for the synthesis of amino acid-based surfactants.

[0137] Example 8: Time-dependent acylation of phenylalanine and lauric acid by PmAcy The amino acid conversion by aminoacylase was first analyzed using 200 mM phenylalanine and 100 mM lauric acid. The reaction was carried out in a basic borate buffer (pH=9) containing 10% ethanol as a solution medium and 10 μl (12 μg, 0.6 U) PmAcy N Tag (SEQ ID NO: 4), with a total reaction volume of 1 mL (Figure 7). This reaction yielded a maximum of 40% (24 hours, 50°C, 250 rpm), as determined by chromatographic analysis and comparison with the reference spectra of phenylalanine and N-lauroylphenylalanine classically produced by the Schotten-Baumann reaction.

[0138] Example 9: Investigation of amino acid substrate range at various pH values Next, the amino acid substrate range was analyzed using proteogenic amino acids. Sodium laurate was used instead of lauric acid due to its high solubility in aqueous media. Therefore, the addition of ethanol was unnecessary. The reaction was carried out at pH 8, 10, and 12. The results are shown in Table 3 below.

[0139] [Table 3]

[0140] In particular, good conversion was observed for phenylalanine at all three alkaline pH values. Moderate conversion was also observed for isoleucine, lysine, valine, and methionine at all tested pH values. Arginine showed conversion after a short reaction time of only 10 minutes.

[0141] Example 10: Fatty acid substrate region of PmAcy In addition, the spectrum of acceptable fatty acids was examined in more detail. For this, the model amino acids phenylalanine and arginine were used. The amino acids were mixed with fatty acids or their sodium salts, respectively. The results are shown in Table 4 below.

[0142] [Table 4]

[0143] Lauric acid, oleic acid, and their corresponding sodium salts were successfully synthesized at a reaction volume of 12 μg of enzyme / ml. Significant conversion was not observed for the remaining fatty acids and their salts. By increasing the amount of enzyme while simultaneously reducing the reaction volume to 0.5 ml (final enzyme concentration 96 μg / ml), conversion of caprylic acid, palmitic acid, stearic acid, and sodium stearate to acylarginine was observed. Phenylalanine was successfully synthesized only under reaction conditions similar to those for palmitic acid and sodium stearate.

[0144] Example 11: Isolation and Purification of Lauroyl Phenylalanine Product For the initial test of product isolation from the reaction mixture, the model amino acid phenylalanine was used (reaction conditions: 100 mM sodium laurate, 200 mM phenylalanine, 50 mM Tris buffer (pH=8), 50 μL (60 μg, 3.0 U) PmAcy, 0.5 mM ZnCl2, 5 ml total volume, 50°C, 250 rpm; 5 M HCl was slowly added to the reaction mixture until the pH reached 1; the precipitated solid was separated from the upper layer of solution by centrifugation and decantation, and then dried under vacuum). By adding 5 M hydrochloric acid at pH 1, it was possible to precipitate the acyl amino acid as a pure substance free from impurities of the remaining amino acids in the reaction mixture. This product was further filtered and washed with petroleum ether and water. The chromatogram of the obtained product showed a theoretical yield of 47%, and the mass spectrum of the product and 1 Identity was confirmed by 1H-NMR spectroscopy.

[0145] Example 12: pH optimization of PmAcy synthesis conditions for lauroylarginine synthesis The reaction conditions for the condensation reaction of 100 mM lauric acid (added as sodium laurate) and 200 mM arginine were optimized with respect to pH. Specifically, it was shown that a yield of over 90% was observed in the synthesis reaction at pH 9 using sodium borate buffer. Since amino acids exhibit various ionic properties depending on their side groups, the optimal pH range for acyl amino acid synthesis by PmAcy varies from pH 8 to 12.

[0146] [Table 5]

[0147] Example 13: Temperature optimization of PmAcy synthesis conditions for lauroylarginine synthesis The reaction conditions for the condensation reaction of 100 mM lauric acid (added as sodium laurate) and 200 mM arginine were optimized with respect to temperature in a sodium borate buffer with a pH of 9. Time-conversion curves were measured in the temperature range of 35–65°C. It was shown that the best synthesis value was achieved at the lowest temperature. After 24 hours, a yield of over 90% lauroylarginine was achieved in the temperature range up to 50°C (see Figure 8).

[0148] Example 14: Analysis of substrate concentration for PmAcy-catalyzed synthesis of lauroylarginine For the synthesis of lauroylarginine, the concentration ranges of 50 mM to 400 mM sodium laurate and 50 mM to 400 mM arginine were investigated using sodium borate buffer at pH 9. Since conversion was achieved at all concentration ratios, it was determined that the synthesis reaction is possible at substrate concentrations of up to 400 mM sodium laurate and 400 mM arginine. Due to substrate solubility, high concentrations could only be achieved by injection or technical stirring (see Figure 9).

[0149] Example 15: Synthesis of lauroylarginine in a buffer-free system The use of borate buffers may be disadvantageous in some respects due to the use of chemicals. For this reason, we investigated the synthesis of lauroylarginine by PmAcy in a buffer-free system. The pH was kept constant at pH 9 for 24 hours by adding NaOH. Continuous formation of lauroylarginine was observed using time-conversion curves for substrate concentrations of sodium laurate / arginine at 100 mM / 100 mM and 100 mM / 200 mM (see Figure 10).

[0150] The inventors have unexpectedly identified a novel aminoacylase (PmAcy) (SEQ ID NO: 1) from Paraburkolderia monticola DSM 100849 that can be functionally expressed in a microbial expression system and therefore produced in relatively large quantities. Using shaking flask expression with co-expression of the chaperone GroEL / ES, 40 mg of target enzyme / 800 ml of culture medium was obtained. This is a significant advantage compared to other known aminoacylases, such as the aminoacylase from the Burkholderia sp. strain LP5_18 (Takakura & Asano, cited above), which could not be heterologously expressed and was only obtained in a yield of approximately 0.1 mg / 800 ml of culture medium. Furthermore, a special advantage of the aminoacylase from Paraburkholderia monticola is that it can be heterologously expressed in relatively high yields in microbial host strains, which contributes to the technical accessibility of this enzyme.

[0151] After heterologous expression, PmAcy could be obtained by a single-step purification using affinity chromatography due to the Strep tag sequence added to its N-terminus. In contrast, the aminoacylase from the Burkholderia sp. strain LP5_18 could only be obtained from the wild-type strain through a meticulous five-step purification process under cold conditions. Therefore, another advantage of this novel enzyme is its ease of acquisition.

[0152] PmAcy was found to possess high temperature and pH stability. After incubation at 70°C for 24 hours, 82% of the activity remained. After 4 days at 70°C, 38% of the hydrolytic activity remained, and at 50°C, 81% remained. The enzyme was stable for 60 minutes in the pH range of 4 to 13, with no loss of activity detected. The enzyme was stable for 24 hours in the pH range of 4 to 12, and 30% of the activity was measured after 24 hours at pH 13. Due to these high levels of stability, this enzyme is particularly suitable for technical use.

[0153] Furthermore, this enzyme exhibits a broad substrate range in terms of amino acid groups for the hydrolysis of lauroyl amino acids, and also hydrolyzes palmitoyl amino acids with relative activity of 36-50% relative to each lauroyl amino acid. In contrast, the related aminoacylase from the Burkholderia sp. strain LP5_18 showed extremely low hydrolytic activity for long-chain fatty acids (4% residual activity for stearoyl amino acids).

[0154] PmAcy was found to possess synthetic activity, enabling the synthesis of acyl amino acids using lauric acid and oleic acid as starting materials. This enzyme was found to have a broad substrate range in terms of the amino acids it can use, and was able to synthesize acyl amino acids from Ala, Arg, Asn, Gln, His, Ile, Leu, Lys, Met, Phe, and Val. In contrast, aminoacylase from the Burkholderia sp. strain LP5_18 was unable to produce acyl amino acids from Met, Ile, Leu, Asn, and His.

[0155] The product was found to be easily isolated from the reaction mixture by precipitation due to changes in pH (e.g., by lowering the pH by acidification) or by cold precipitation (i.e., by lowering the temperature), and could be purified by washing. No by-products were detected by chromatographic analysis. Therefore, the reaction product can be obtained in high purity from the aqueous reaction mixture by a simple, technically scalable two-step process.

[0156] In summary, aminoacylase from Paraburkolderia monticola is -Broad substrate range for acceptable amino acids - Substrate range for acceptable fatty acids (preferably those with chain lengths of C12 (surfactant) and C18 (emulsifier) ​​are acceptable) - High synthetic performance due to a single reaction partner with high substrate concentration and low substrate excess. - Excellent production capacity through functional expression in microbial expression systems - Easy one-step purification by affinity chromatography - High process stability (especially temperature and pH stability) Because it possesses all the essential characteristics required for such technical synthesis, it was found to be superior to previously described acyl amino acids in terms of acyl amino acid synthesis.

Claims

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

2. The polypeptide according to claim 1, wherein the amino acid sequence has 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 with the amino acid sequence shown in Sequence ID No. 1 over its entire length.

3. The polypeptide according to claim 1 or 2, further comprising an affinity tag, preferably an N-terminal stripe tag, and optionally having the amino acid sequence shown in SEQ ID NO:

4.

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

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

2.

6. A plasmid vector comprising the nucleic acid molecule described in claim 4 or 5.

7. Recombinant host cells comprising an isolated nucleic acid molecule according to claim 4 or 5 or a plasmid vector according to claim 6.

8. A method for producing a polypeptide having aminoacylase activity according to any one of claims 1 to 3, (a) the step of introducing a nucleic acid molecule or vector according to any one of claims 4 to 6 into a suitable host cell; and (b) A step of culturing the host cells in a culture medium under conditions that enable the expression of the polypeptide. A method that includes this.

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

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

11. 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, β-alanine, ornithine, homoserine, homoproline, N-methyltaurine, norvaline, norleucine, citrulline, hydroxyproline and their salts, preferably arginine or phenylalanine; and / or ii) The carboxylic acid is a linear, 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 C ８ ~C ２２ Unsaturated or saturated fatty acids or salts thereof, or mixtures 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 salts thereof or mixtures thereof, more preferably linear C ８ ~C １８ Unsaturated or saturated fatty acids or salts thereof, or mixtures of two or more fatty acids or salts thereof, such as lauric acid, myristic acid, linoleic acid, linolenic acid, or oleic acid, or salts thereof, or mixtures thereof, most preferably lauric acid or myristic acid, or salts thereof, or mixtures thereof; and / or iii) The above method, 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 The method according to claim 10, which is carried out.

12. An N-acyl amino acid available according to the method described in claim 10 or 11.

13. A composition comprising the N-acyl amino acid described in claim 12, wherein the composition is optionally a cosmetic product, a home care product, or a professional product.

14. Use of the N-acyl amino acid described in claim 12 in a beauty product, home care product, or professional product.

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

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