WO2025058511A1 - Acyltransferase-catalysed modification of peptides - Google Patents

Abstract

The invention relates to a method for modifying a polypeptide, comprising the coupling of the polypeptide which polypeptide provides an acyl functionality for the coupling, and a nucleophile, which nucleophile provides a nucleophilic functionality for the coupling, wherein a reaction mixture is provided comprising an extended polypeptide having an extension at said acyl functionality of said polypeptide, which extension is an enzymatically cleavable amino acid unit or enzymatically cleavable peptide, the reaction mixture further comprising the nucleophile and a acyltransferase; the extended polypeptide is subjected to a cleavage reaction catalysed by the acyltransferase whereby the extension is cleaved from said polypeptide and the polypeptide from which the extension is cleaved off is coupled via said acyl functionality to the nucleophilic amino functionality of the nucleophile, which coupling is catalysed by the acyltransferase, which acyltransferase is a subtilisin variant or homologue thereof. The invention further relates to an acyltransferase.

Description

Title: Acyltransferase-catalysed modification of peptides

The invention relates to a method for modifying a polypeptide, comprising the coupling of a polypeptide providing an acyl functionality and a nucleophile. The invention further relates to an enzyme having acyltransferase activity.

Polypeptides have, amongst others, important applications as pharmaceutical agents, prodrugs, nutritional ingredients and cosmetic ingredients. Methods for obtaining various polypeptides are well known in the art. E.g, polypeptides can be isolated from a natural source (if occurring in nature), produced by fermentation, including recombinant fermentation, produced by chemical synthesis or produced (chemo-)enzymatically.

Polypeptides, e.g. proteins, of interest include polypeptides that consist of a linear peptide chain, branched polypeptides, cyclic polypeptides and polypeptide conjugates with one or more further moieties, e.g. another polypeptide, a carbohydrate, a lipid, a polynucleotide, a hydrocarbon etc.. Continuing efforts are made in the art to modify polypeptides with other peptides or other molecules, in particular to increase shelf life, improve bioavailability, enhance pharmacokinetic properties and the like. Examples of polypeptide properties of which it can be desired to be altered include solubility in a solvent of interest, permeability through tissue of a living being, in-vivo half live extension or targeting a tumour cell with a toxic pay load.

Modification of a polypeptide, such as conjugation or functionalisation is most commonly accomplished via chemical, chemo-enzymatic or enzymatic means and takes place between one or more polypeptides or between a polypeptide and another molecule.

Chemical modification tends to be non-selective and may be prone to racemisation reactions.

Chemo-enzymatic polypeptide modifications combine the advantages of the flexibility of chemical methods and regio- and stereoselectivity of the enzymatic reactions. Several examples have been described in literature not only for polypeptides (Morgan et al., Chem. Soc. Rev., 2022, 51, 4121-4145 - but also for glycans (Qiang Chao et al., Front. Chem., 16 June 2020) and (other) natural products (Kaspar and Schallmey, Current Opinion in Biotechnology Volume 77, October 2022, 102759). Milczek (Chem. Rev. 2018, 118, p119-141) provided an extensive review of developments in enzymatic processes to deliver functionalised proteins. Milczek refers to the modest selectivity of chemical methods and emphasises the need for complementary enzymatic methodology to complement the existing toolbox of conjugation strategies. Table 1 provides an overview of three different types of modification (crosslinking, ligation and glycosylation) with a reference to known biocatalysts. Table 2 lists known modifications by reaction class. Chapter 4 discusses pharmaceutical applications of enzymatic protein conjugation. Amongst others, the enzymatic production of antibody-drug conjugates (ADC’s) is discussed. Further, transpeptidation with Sortase A is discussed. Sortase A recognises selectively a specific C-terminal signalling motif (LPXTG) and cleaves selectively the T-G bond. Sortase transpeptidation reversibility problems are addressed by removing the product from the reaction medium or by including an irreversible N- gly coupling partner providing by-products that cannot actively participate in catalysis. Sortase catalysed PEGylation of a protein with a mass of up to 20kDa is mentioned in particular. Besides reversibility of reaction problems experienced with Sortase, its applicability is specifically to cleaving and coupling a (peptide with) glycine at the C-terminal end and the presence of the C-terminal LPXTG motif in the product that is obtained. This motif is an epitope also found in bacterial peptides and can cause an undesired immune response in humans. Besides, the need for this motif when using a Sortase is a limitation to the diversity of polypeptides that can be modified using a Sortase-catalysed reaction. Other promising applications that are mentioned are PEGylation in the presence of a transglutaminase and tyrosinase catalysed functionalisation of human serum albumin conjugates for drug delivery. In these applications similar drawbacks as with Sortase can occur; e.g. with respect to the specific recognition motif. Also, in a peptide comprising several glutamine residues, several tyrosine residues or both, multiple modifications may take place, which is detrimental to selectivity. In another review, Lujuan Xu et al (Angew. Chem. Int. Ed.2021, 60, p13757-13777), discuss contemporary approaches for site-selective dual functionalisation of proteins. They discuss chemical methods and genetic engineering. In chapter 4.3 it is discussed that dual modification can be done using two distinct enzymes, each recognising a different tag (a peptide sequence) of the protein to be modified. Further, commonly used peptide tags and their respective enzymes for enzymatic functionalisation of proteins are mentioned in Table 2. Thus, as illustrated by the above cited documents, (modified) polypeptides form a highly interesting class of bioactive compounds, showing a high molecular diversity and having a wide spectrum of activity that may e.g. be used for various pharmaceutical applications or as a research or development tool, e.g. in the search for pharmaceutically useful polypeptides with improved biological properties such as improved in vivo half-lives, efficacy at lower doses, reduced frequency of administration or which may be suitable for new therapeutic applications. The documents also illustrates that there is an ongoing need for alternative synthesis methodology, because of challenges and limitations in the preparation with known methods. Webb and co-workers (Chem. Soc. Rev., 2022, 51, p4121-4145) concludes that “the sortases and the peptidyl asparaginyl ligases such as Butelase and OaAEP1 show the greatest promise for future applications in protein engineering. While ligases generated from proteases such as subtiligase have potential, their requirement for ester and thioester substrates and low specificity makes their application in protein engineering more challenging.” This especially applies to modifications at the C-terminus. It is an object of the present invention to provide a novel method for preparing a modified (derivatised, conjugated, functionalised) polypeptide. There is a need for alternative methods in general, in particular in order to broaden the palette of tools for making specific (modified) polypeptides. In particular, it is an object to modify a polypeptide in order to change its application window, preferably to improve a therapeutic window. In particular, it is an object of the invention to provide a method addressing one or more of the problems encountered with other methods, such as a limitation in applicability or other drawback described elsewhere herein. In particular, it is an object of the present invention to develop an enzymatic method that is traceless. It is further an object to provide a novel enzyme, suitable for use in catalysing polypeptide modifications at a C-terminal end, catalysing polypeptide synthesis, or other enzyme applications. One or more further objectives, may be derived from the disclosure herein below. It has now surprisingly been found possible to effectively couple a nucleophile to a C-terminal end of a polypeptide acyl donor enzymatically, making use of a subtilisin variant or homologue, without needing to chemically activate the C-terminal end of the polypeptide with an ester or thioester functionality, also in a reaction medium comprising a substantial amount of water, even in a fully aqueous reaction medium. This has been found possible by extending the polypeptide with one or more amino acid units and contacting the extended polypeptide with the nucleophile in the presence of a modified subtilisin having a specific catalytic activity. Accordingly, the present invention relates to a method for modifying a polypeptide, comprising reacting of a (i) polypeptide (which is the polypeptide to be modified), which polypeptide provides an acyl functionality for the coupling, and (ii) a nucleophile, which nucleophile provides a nucleophilic functionality for the reaction (typically an amino, hydroxyl amine or hydrazine functionality), wherein a reaction mixture is provided comprising an extended polypeptide having an extension at said acyl functionality of said polypeptide, which extension is an amino acid unit or peptide which amino acid unit or peptide is enzymatically cleavable from the extended polypeptide, the reaction mixture further comprising the nucleophile and an enzyme catalysing the modification, wherein the extended polypeptide is subjected to a cleavage reaction catalysed by the enzyme whereby the extension is cleaved from said polypeptide (whereby the acyl functionality becomes available for the reaction) and said polypeptide (from which the extension has been cleaved off) reacts via said acyl functionality with the nucleophilic functionality of the nucleophile, which coupling is catalysed by the enzyme; and wherein the enzyme is a subtilisin BPN’ variant or homologue thereof, with the proviso that the enzyme has an active site, in particular a catalytic triad, wherein serine is conserved. Typically, the enzyme is a subtilisin BPN’ variant or homologue thereof comprising a mutation compared to subtilisin BPN’ represented by SEQUENCE ID NO: 1 or a homologue sequence thereof. Advantageously at least a mutation is present at the amino acid position corresponding to P225 of SEQUENCE ID NO: 1. The enzyme catalysing the coupling typically has acyltransferase activity (hereafter abbreviated as acyltransferase). Said active side, in particular catalytic triad typically contributes to the acyltransferase activity. In an embodiment, the acyltransferase activity is or comprises a transpeptidase activity (hereinafter abbreviated as transpeptidase). In accordance with the invention the acyl transferase catalyse the cleavage of an amide bond of a peptide (i.e. the extended polypeptide) and the subsequent coupling of a nucleophile. The nucleophile typically has a nucleophile functionality (participating in the reaction with the acyl functionality of the polypeptide) selected from amino, hydrazine and hydroxylamine functionalities. The coupling with an amino group results in an amide (-(C=O)-(NH)-) between the remainder polypeptide and nucleophile; the coupling with a hydrazine functionality results in a hydrazide functionality (-(C=O)-(NH)-NH-) between the remainder polypeptide and nucleophile; and the coupling with a hydroxylamine results in a hydrazone (-(C=O)-(NH)-O-) between the remainder polypeptide and nucleophile. Without being bound by theory, it is thought that the amide bond between the polypeptide and the extension is cleaved, whilst the carbonyl (acyl functionality) at the polypeptide C-terminal end (at which the extension was previously bound) forms an acyl-enzyme complex with the acyltransferase; next said nucleophile is coupled to the acyl functionality of the polypeptide, resulting in a modified polypeptide. The modification can be a derivatisation, functionalisation, conjugation or a ligation. Further, the invention relates to an enzyme having acyltransferase (e.g. transpeptidase) activity (herein after abbreviated as ‘acyltransferase’), which acyltransferase is a subtilisin BPN’ variant or homologue thereof, comprising at least one mutation compared to subtilisin BPN’ represented by SEQUENCE ID NO: 1 or a homologue sequence thereof, which at least one mutation is a mutation at the amino acid position corresponding to P225 of SEQUENCE ID NO: 1, with the proviso that the acyltransferase has an active site having a conserved serine, in particular a conserved serine at the amino acid position corresponding to S221 of SEQUENCE ID NO: 1. An enzyme according to the invention typically has acyltransferase activity without needing to chemically activate the C-terminal end of the polypeptide with an ester or thioester functionality, also in a reaction medium comprising a substantial amount of water, even in a fully aqueous reaction medium. Thus, the enzyme according to the invention typically has acyl transferase activity with respect to peptide substrate that is free of a(n activating) C-terminal ester group or C-terminal thioester group. The enzyme typically has acyl transferase activity with respect to a peptide substrate (extended with an amino acid tag or peptide tag) that has a free C-terminal carboxylic acid or a C- terminal amide. Herewith the invention also provides a conceptually distinct approach from methods that make use of chemo-enzymatic activation with (thio)ester groups to couple a nucleophile at a C-terminal end of a peptide substrate in the presence of a ligase, like WO2016/056913 describes for the coupling of two peptide fragments. Further, the invention relates to a nucleic acid encoding the acyltransferase according to the invention. Further, the invention relates to an expression vector, comprising a nucleic acid encoding the acyltransferase according to the invention. Further, the invention relates to a host cell comprising the expression vector comprising the nucleic acid encoding the acyltransferase according to the invention. Such host cell is capable of producing the acyltransferase according to the invention. Typically, the enzyme according to the invention is isolated (such as isolated from the organism in which it has been produced). In principle it is possible that the enzyme is naturally occurring. Good results have been achieved with an enzyme that is not naturally occurring. The enzyme has usually been obtained from a genetically modified organism, wherein the enzyme is (recombinantly) expressed. Further, the invention relates to the use of an enzyme according to the invention as a catalyst, in particular as a catalyst, catalysing the enzymatic modification of a polypeptide, more in particular as an acyltransferase. A method respectively enzyme according to the invention has been found particularly suitable to selectively change an acyl functionality (or a specific amide) of a donor polypeptide (with an amino acid or peptide extension), in particular at a C-terminus, with a different functional group, such as a hydrazine, or to attach another nucleophilic compound selectively to a C-terminal end of the polypeptide. Herewith it is possible to alter relevant properties of the polypeptides e.g. pharmacokinetic properties in a selective manner. The invention herewith provides a new tool to modify polypeptides, with compounds of which it may be expected per se that they can be used to impart a certain effect on a polypeptide’s properties, if coupled to the peptide. Changing the acyl functionality can also be useful for secondary chemical ligation methods. E.g., a hydrazide can be used for chemical ligation with a ketone or aldehyde. Further, side-chain protection or protection of a terminal end of which it is not desired that it participate in the reaction is possible but not generally needed. The method, enzyme, respectively use according to the invention further offers a remarkable flexibility in recognition motif and tags that can be employed in a method for modifying a polypeptide. This not only makes the invention useful for a broad range of polypeptides to be modified and types of modifications, but also can avoid the presence of a potentially allergenic epitopes at the C-terminal side of a modified polypeptide, e.g. as with a transpeptidase like Sortase. For instance, the invention allows the preparation of a modified insulin having the natural amino acid sequence without remains of a recognition motif that is not part of natural insulin. Analogously other pharmaceutical polypeptides can be modified in accordance with the invention without introducing allergenic epitopes. It is in particular surprising that an acyltransferase, e.g. a transpeptidase, can be used effectively in the production of a variety of modified polypeptides. Further, it is surprising that a subtilisin BPN’ variant or homologue thereof with the position corresponding to S221 being conserved is effective in catalysing the formation of an amide bond, also in an aqueous environment. After all this position is part of the active site of the enzyme (the catalytic triad formed by the amino acid positions corresponding to D32, H64, S221), see e.g. Bryan (Biochemica et Biophysica Acta 1543 (2000) p 203-222) . The serine has been shown to be crucial for hydrolytic reactions and – according to the prior art - should be changed to obtain an enzyme for the reverse hydrolytic reaction (the coupling). The inventors have surprisingly found that reverse hydrolytic reactions can be performed whilst the active site serine is conserved. The term “or” as used herein is defined as “and/or” unless it is specified otherwise or it follows from the context that it means ‘either ….or…’ . The term “a” or “an” as used herein is defined as “at least one” unless it is specified otherwise or it follows from the context that it should refer to the singular only. When referring to a noun (e.g. a compound, an additive, etc.) in the singular, the plural is meant to be included, unless it follows from the context that it should refer to the singular only. For the purpose of clarity and a concise description, features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described. The term “pH” is used herein for the apparent pH, i.e. the pH as measured with a standard, calibrated pH electrode. For the purpose of this invention, with “peptide” is meant any molecular structure at least composed of two or more amino acids joined by an amide bond. Thus, peptides are generally amides at least conceptually composed of two or more amino carboxylic acid molecules (i.e. amino acids) by formation of a covalent bond from the carbonyl carbon of one to the nitrogen atom of another with formal loss of water. A polypeptide is a peptide composed at least conceptually of three or more amino acids. A moiety in the peptide that at least conceptually is the remains from an amino acid once it has formed the peptide is generally known in the art as an “ amino acid residue” or “ amino acid unit” of the peptide, although colloquially one may also state that a peptide comprises amino acids, when actually meaning to state a peptide comprises amino acid residues. The term “peptide” includes peptide conjugates, a conjugate of a peptide of at least two amino acids and another molecule, for instance a carbohydrate (forming a glycopeptide), a nucleotide construct (conjugate of the peptide and a nucleotide, an oligonucleotide or a polynucleotide) or a lipid (forming a lipopeptide) or another moiety, e.g. a stationary phase. Peptides usually comprise a backbone comprising a sequence of two or more alpha-amino acid units, between which a peptide bond is present; however, the backbone of peptides may comprise one or more other amino acids, such as one or more amino acids selected from the group consisting of beta-amino acids, gamma-amino acids, delta-amino acids and epsilon-amino acids. A polypeptide to be modified in accordance with the invention generally comprises at least 4 amino acid residues, in particular at least 4 alpha- amino acid units, between which a peptide bond is present. Peptides used as a polypeptide providing the acyl function or peptides used as a nucleophile can, but do not need to, essentially consist of a peptide chain, can, but do not need to, essentially consist of a peptide chain and protective groups. Peptides used as a polypeptide providing the acyl function or peptides used as a nucleophile can have a secondary structure, such as an alpha-helix or beta-sheet. Peptides used as a polypeptide providing the acyl function or peptides used as a nucleophile can have a tertiary structure. As is generally known, these are generally formed by multiple interactions, among others hydrogen bonding, hydrophobic interactions, van der Waals interactions, ionic interactions and disulphide bonds. Disulphide bonds are typically bonds between two cysteine units (formed by oxidation). Thus, two amino acids in a same peptide chain (amino acid sequence) can be covalently bound, also if they are not adjacent amino acids in the amino acid sequence. Also, a disulphide bond between a first cysteine of a first peptide chain and a second cysteine of a second peptide chain, which may have the same or a different amino acid sequence, can be formed to form a peptide. Such peptide comprises more than one peptide chain. An example of a peptide composed of more than one peptide chain, wherein the different chains are bound via a disulphide bond is insulin. Other bonds to join different peptide chains are generally known in the art. Typically, polypeptides - which term includes oligopeptides, proteins and chimeric peptides - comprise up to about 50000 amino acid, in particular up to 35000 amino acid units, up to 20000, up to 5000 amino acid units, up to 500 amino acid units, up to 200 amino acid units or up to 50 amino acid units. For the purpose of this invention, with “peptide bond” is meant the amide bond between an amine group of a first amino acid and a carboxyl group of a second amino acid. The peptide bond can be a bond between the alpha -amino terminus of a first alpha-amino acid and the alpha-carboxyl terminus of a second alpha-amino acid (also referred to in the art as an eupeptidic bond) or an isopeptidic bond, i.e. a peptide bond formed between an amine group of a first amino acid and a carboxyl group of a second amino acid, wherein at least one of said groups is not the alpha-group. E.g. a side chain of a peptide is generally bound to the backbone via an isopeptidic bond. The term “amino acid” encompasses both proteinogenic and non- proteinogenic amino acid. Proteinogenic amino acids are the amino acids that are encoded by the genetic code. Proteinogenic amino acids include: alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), serine (Ser), threonine (Thr), methionine (Met), cysteine (Cys), asparagine (Asn), glutamine (Gln), tyrosine (Tyr), tryptophan (Trp), glycine (Gly), aspartic acid (Asp), glutamic acid (Glu), histidine (His), lysine (Lys), arginine (Arg), proline (Pro) and phenylalanine (Phe). Selenocysteine (Sec, U) is an amino acid, of which the structure corresponds to cysteine, with the proviso that it contains a selenium instead of a sulphur atom. Proteinogenic amino acids are the L-stereoisomers of said amino acids (except for glycine, which does not have a stereo-isomeric form). Generally, at least a number of the amino acid units forming the polypeptide providing the acyl functionality are proteinogenic amino acids, in particular at least the amino acid residue at the P1 position, at the P4 position and preferably also at the P2 and/or P3 position. In an advantageous embodiment, the (extended) polypeptide providing the acyl function is made fermentatively, in which case usually all amino acid residues of the (extended) polypeptide are proteinogenic. Fermentative production of the (extended) polypeptide or of a peptide nucleophile can be done based on methodology known per se, e.g. making use of bacteria, yeasts, archaea, algae, mammalian cells or other cell cultures, wherein the (extended) polypeptide is recombinantly expressed. The fermentative production can be done aerobically, anaerobically or under oxygen limited conditions. When an enzyme is mentioned herein with reference to an enzyme class (EC) between brackets, the enzyme class is a class wherein the enzyme is classified or may be classified, on the basis of the Enzyme Nomenclature provided by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB), which nomenclature may e.g. be found at https://iubmb.qmul.ac.uk/. Other suitable enzymes that have not (yet) been classified in a specified class but may be classified as such, are meant to be included. The term "mutated", “mutation” or “mutant” as used herein regarding proteins or polypeptides – in particular enzymes such as acyltransferases - means that at least one amino acid in the wild-type or naturally occurring protein or polypeptide sequence has been replaced with a different amino acid, inserted into, appended to, or deleted from the sequence via mutagenesis of nucleic acids encoding these amino acids. Mutagenesis is a well-known method in the art, and includes, for example, site-directed mutagenesis by means of PCR or via oligonucleotide-mediated mutagenesis as described in Sambrook et al., Molecular Cloning-A Laboratory Manual, 2nd ed., Vol. 1-3 (1989). The term "mutated" or “mutation” as used herein regarding genes means that at least one nucleotide in the nucleic acid sequence of that gene or a regulatory sequence thereof, has been replaced with a different nucleotide, has been inserted into, has been appended to, or has been deleted from the sequence via mutagenesis, resulting in the transcription of a protein sequence with a qualitatively of quantitatively altered function or resulting in the knock-out of that gene. Schechter and Berger.( Biochem Biophys Res Commun. 1967 Apr 20;27(2):157-62.)) defined that the active site residues in proteases) are composed of contiguous pockets termed subsites. Each subsite pocket binds to a corresponding residue in the peptide substrate sequence, referred to here as the sequence position. According to this definition, amino acid residues in the substrate sequence are consecutively numbered outward from the cleavage sites as ...-P4-P3- P2-P1-P1'-P2'-P3'-P4'-... (the scissile bond is located between the P1 and P1' positions), while the subsites (pockets) in the active site are correspondingly labelled as...-S4-S3-S2-S1-S1'-S2'-S3'-S4'. It should be noted that not all proteases have all of said subsites. E.g. an S3’ and/or an S4’ pocket may be absent. The inventor realised that this not only applies to the N-terminus and C-terminus of peptides to be coupled, but that it is also possible with the acyltransferase used in accordance with the invention to couple other nucleophiles to an acyl functionality (at the C-terminus) of the polypeptide to be modified. For the purpose of this invention, with “S1, S2, S3 and S4 pocket” is meant the amino acids of a protease (in particular an acyltransferase) which interact with the amino acids of a peptide acyl donor. The amino acid (1^st amino acid; P1) at which the acyl functionality participating in the formation of the peptide bond interacts with the amino acids in the S1 pocket of the protease. The penultimate amino acid (2^nd amino acid counted from the acyl functionality; P2) of the acyl donor peptide interacts with the amino acids in the S2 pocket of the protease, the third amino acid (P3) with the S3 and the fourth amino acid (P4) with the S4 pocket. The S1-S4 binding pockets of a protease are defined by several amino acids which can be distant in the primary structure of the protease, but are close in the three dimensional space. For the purpose of this invention, with S1’ and S2’ pockets are meant the amino acids of a protease (having peptide cyclase activity) which interact with the amino acids near the functionality of the nucleophile participating in the coupling. In case of a peptide nucleophile, the amino acid (P1’) at which the amine participating in the coupling is present interacts with the amino acids in the S1’ pocket of the protease. The amino acid (P2’) adjacent to P1’ interacts with the amino acids in the S2’ pocket of the protease. The S1’ and S2’ binding pockets of a protease are defined by several amino acids which can be distant in the primary structure of the protease, but are close in the three dimensional space. Homologues typically are peptides or enzymes having an intended function in common with the peptide or enzyme, of which it is a homologue, such as being capable of catalysing the same reaction, in particular an enzymatic reaction (cleavage and coupling) of a method according to the invention. Thus, a homologue acyltransferase in accordance with the invention comprising at least one mutation compared to subtilisin BPN’ represented by SEQUENCE ID NO 1 or compared to an acyltransferase in accordance with the invention comprising another sequence of an acyltransferase in accordance with the invention, such as SEQUENCE ID NO, 2, 4, 5, 6, 7, 8 or 9, has acyltransferase activity allowing catalysis of the modification of the polypeptide in a method according to the invention. The term homologue is generally further defined by a certain level of similarity of its sequence compared to the enzyme it is a homologue of. Such level of similarity is referred to herein as “percent identity” or “sequence identity”, which terms are used interchangeably herein. The sequence identity of a homologue peptide, more in particular an enzyme, such as an acyltransferase in accordance with the invention, is usually at least 50 %, preferably at least 60 %, more preferably at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % with the peptide, in particular enzyme, with which the homologue peptide or enzyme is compared. A acyltransferase according to the invention thus usually has a sequence identity of at least 50 %, preferably at least 60 %, more preferably at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % with an acyltransferase comprising an amino acid sequence represented by SEQUENCE ID NO 1, 2, 4, 5, 6, 7, 8 or 9. The percent identity is determined according to the NEEDLE EMBOSS method as outlined below. The percent identity will be less than 100 %. The percent identity depends on the number of mutations and the length of the peptide (enzyme) with which the homologue is compared. For the purpose of this invention, it is defined herein that in order to determine the percent identity of two amino acid sequences, the complete mature sequences are aligned for optimal comparison purposes such that similar regions are aligned. Any sequence elongations (either at N or C-terminus), such as tags (e.g. His-tags), for instance, for purification, signaling, solubilization and localization purposes, are not be considered in the determination of the percent identity. In order to optimize the alignment between the two sequences, gaps may be introduced in any of the two sequences that are compared. The alignment used to determine a sequence identity % value is typically carried out over a length of at least 200 amino acids of the sequences being compared. A comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, for instance the Needleman-Wunsch algorithm (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol.48(3), pp 443-453), which has been implemented in the computer program NEEDLE. The NEEDLE program from the EMBOSS package is used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A., Trends in Genetics 16, (6) pp 276—277) for the calculation of percent identity. For protein sequences, EBLOSUM62 should be used for the substitution matrix. The parameters used for alignment of amino acid sequences have to be set as a gap-open penalty of 10 and a gap extension penalty of 0.5. The homology or percent identity between the two aligned sequences is calculated as follows: the number of corresponding positions in the alignment showing an identical amino acid in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. The percent identity as herein defined is obtainable from NEEDLE and is labelled in the output of the program as “identity”. The term “analogue” of a peptide is used herein in particular for peptides that are structural analogues and/or functional analogues of said peptide. Functional analogues have a same in vivo target (e.g. the same target receptor on a cell membrane); structural analogues have a high similarity in amino acid sequence. Functional analogues of a peptide may have a relatively low amino acid sequence identity, e.g. of about 50 % or less over the full amino acid sequence, yet a high sequence identity (and thus a high structural similarity) with the peptide of which they are an analogue in a segment of the amino acid sequence, such as near the N-terminal part or near the C- terminal part. In particular, a structural analogue comprises an amino acid sequence that has at least 80 %, preferably at least 85 %, more preferably at least 90 % sequence identity, even more preferably at least 95 % sequence identity with the amino acid sequence of the peptide of which a peptide is an analogue. The term “analogue” is used herein when referring to the target peptide of the coupling reactions. The term “variant” or “mutant” of an enzyme is used herein for enzymes that are structural analogues of an enzyme, having at least one mutation or mutated amino acid relative to such enzyme. In the context of this application, the term "about" means in particular a deviation of 10 % or less from the given value, more in particular 5 % or less, even more in particular 3 % or less. When referring to a compound of which stereoisomers exist, the compound may be any of such stereoisomers or a mixture thereof. Thus, when referred to, e.g., an amino acid of which enantiomers exist, the amino acid may be the L-enantiomer, the D-enantiomer or a mixture thereof. In case a natural stereoisomer exists, the compound is preferably a natural stereoisomer. The acyl transferase is typically a subtilisin variant (EC 3.4.21.62) or a homologue thereof. Particularly good results have been achieved with a variant of subtilisin BPN’; subtilisin BPN’ is a subtilisin from B. amyloliquefaciens. When referred to subtilisin BPN’ in the present disclosure, the wild type sequence as described is SEQUENCE ID NO 1 is meant (SUBT_BACAM Subtilisin BPN' Bacillus amyloliquefaciens mature 1 to 275). Subtilisin variants with acyl transferase activity having a conserved serine in the catalytic triad and a mutation at the position corresponding to P225 are minted herein as ‘peptilisins’. Advantageously, all three amino acids in the catalytic triad are conserved. SEQUENCE ID NO 2 shows a sequence of a preferred acyltransferase in accordance with the invention, wherein a number of positions that can advantageously be mutated are identified with an ‘X’, wherein X stands for any proteinogenic amino acid. SEQUENCE ID NO 3 shows a specific example of a DNA sequence encoding an example of an acyl transferase (peptilisin comprising SEQUENCE ID NO 4) in accordance with the invention with which good results have been achieved. Further good results have been achieved with an acyltransferase in accordance with the invention comprising an amino acid sequence according to SEQUENCE ID NO 5. Further good results have been achieved with an acyltransferase in accordance with the invention comprising an amino acid sequence according to SEQUENCE ID NO 6. Further good results have been achieved with an acyltransferase in accordance with the invention comprising an amino acid sequence according to SEQUENCE ID NO 7. Further good results have been achieved with an acyltransferase in accordance with the invention comprising an amino acid sequence according to SEQUENCE ID NO 8. Further good results have been achieved with an acyltransferase in accordance with the invention comprising an amino acid sequence according to SEQUENCE ID NO 9. Accordingly, in a particularly preferred embodiment the acyltransferase comprises an amino acid sequence of SEQUENCE ID NO 2, SEQUENCE ID NO 4,SEQUENCE ID NO 5, SEQUENCE ID NO 6, SEQUENCE ID NO 7, SEQUENCE ID NO 8 or SEQUENCE ID NO 9 wherein ‘X’ stands for any proteinogenic amino acid, or is a homologue thereof, wherein the position corresponding to S221 of subtilisin BPN’ is conserved and wherein the position corresponding to P225 of subtilisin BPN’ preferably is mutated, in particular substituted. The mutation at the position corresponding to P225 is usually selected from the group of amino acid positions corresponding to P225G, P225C, P225S, P225T, P225I, P225V, P225M, P225N, P225A, P225L and P225Y. As illustrated in Example 6, these are shown to result in a high yield compared to P225. In a particularly preferred embodiment, the mutation at the position corresponding to P225 is selected from the group of amino acid positions corresponding to P225G, P225C, P225S, P225T, P225I, P225V, P225M, P225N and P225A. In particular, good results, have been achieved with an acyltransferase having a mutation selected from the group of amino acid positions corresponding to P225C, P225N, P225S, P225T and P225G. Further, in particular good results have been achieved with an acyltransferase, wherein the mutation at the position corresponding to P225 is P225A. In a specific embodiment, the position corresponding to P225 of the acyltransferase is not mutated. Of such acyltransferase, the production rate has been found relatively fast, yet total yield is relatively low due to a relatively high hydrolytic activity. In addition to said mutation at the position corresponding to P225, the acyltransferase preferably comprises one or more further mutations. In particular for stabilising the acyltransferase it is preferred that the acyltransferase comprises 1-14, in particular 6-13, more in particular 8-11 mutations selected from the group of mutations at an amino acid position corresponding to Q2, S3, P5, S9, I31, K43, M50, A73, G169, S188, Q206, N212, T254 and Q271 of subtilisin BPN’. It is particularly preferred that one or more of said mutations, more preferably at least six of said mutations, more preferably at least eight of said mutations, in particular at least ten or at least twelve of said mutations are selected from the group of positions corresponding to Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, G169A, S188P, Q206C, N212G, T254A and Q271E. Good results are obtained with E156, G166 or both non-mutated. However, a mutation of the position corresponding to E156, G166 or both - such as E156S, G166S or both - can be present, e.g. to further contribute to a change for the S1 pocket of the enzyme. It is further in particular preferred that the acyltransferase’s calcium binding domain is deleted. This domain corresponds to the amino acids 75-83 of SEQUENCE ID NO: 1. Alternatively the calcium binding domain can be inactivated at least partially, e.g. by deletion of a part of the domain or by one or more mutations having a negative effect on the calcium binding activity. The deletion or at least partial inactivation is in particular preferred for a high enzyme stability. However, a good acyltransferase activity can be obtained when the calcium binding domain is included, e.g. as illustrated in the Examples for acyltransferases comprising a sequence according to SEQUENCE ID NO: 6 or 7. One or more further optional mutations can in particular be present at one or more of the pockets (S1, S2, S3 or S4) of the acyltransferase; The acyltransferase may additionally or alternatively have one or more mutations at one or more of the pockets (typically at least S1’ or S2’, further in particular S3’) capable of recognising a moiety (amino acid residue or other moiety) of the nucleophile or the extension of the acyl donor; for instance, the position corresponding to may be mutated. For instance, one or more of the amino acid positions corresponding to the following positions of subtilisin BPN’ may be mutated: E156, G166 (S1 pocket), S33, N62 (S2 pocket), I107 (P4 pocket), M222, Y217 (S1’pocket), F189, N218 (S2’ pocket); or L96, D99, A223, S224. Particularly preferred mutations are shown in Sequence ID NO 4 and Sequence ID NO 5. Thus, one or more, in particular three or more, more in particular five or more mutations compared to subtilisin BPN’ are preferably chosen from the mutations shown in Sequence ID NO 4 or Sequence ID NO 5. In Sequence ID NO 4 and Sequence ID NO 5 the position corresponding to 225 (marked X) preferably is a mutation compared to the wild-type (P), such as specified elsewhere in the present disclosure. Considerations for further mutations can for instance be based on Bryan (Biochemica et Biophysica Acta 1543 (2000) p 203-222), with the proviso that the serine of the catalytic triad is conserved (S at position corresponding to 221 of subtilisin BPN’) and that the position corresponding to P225 is preferably mutated. The polypeptide providing the acyl functionality usually comprises a sequence of amino acid residues, which are recognisable by an active subsite (pocket) of the acyltransferase. The extension of the polypeptide is typically recognised by the acyltransferase; the N-terminal amino acid residue (P1’) of the extension by the S1’ pocket; the penultimate amino acid residue of the extension (P2’) - if present - by S2’; if present the third (P3’) amino acid residue from the N- terminal end of the extension by S3’. Recognition allows the extension to be cleaved off; upon which cleavage a complex is formed between the acyltransferase and the remaining polypeptide (the polypeptide without extension). Typically, the acyltransferase comprises pockets recognising amino acid residues at and near the acyl functionality that is to be modified (typically a C-terminus of the remaining polypeptide) of the remaining polypeptide, which enables subsequent coupling of a nucleophile to the remaining polypeptide. The acyltransferase usually comprises four pockets (typical of subtilisins) for such recognition: S4, S3, S2 and S1. S1 typically recognises the first amino acid residue, i.e. the residue that provides the acyl functionality forming a bond with the nucleophile (P1); S2 the second amino acid residue (P2) next to P1; S3 the third amino acid residue (P3) next to P2 and S4 the fourth amino acid residue (P4), next to P3. Remarkably, the nature of P4-P3- P2-P1 is not particularly critical. The polypeptide’s amino acid residues to be recognised by the active site of the acyltransferase are preferably proteinogenic amino acids, since this usually facilitates effective/efficient catalysis by the acyltransferase. Further, this allows microbiological (fermentative) production of the segment, e.g. making use of recombinant technology. Usually, the acyl functionality of the polypeptide to which the nucleophile is to be coupled (the polypeptide to be modified) is at the C-terminal amino acid residue (P1) of the polypeptide without the extension. The polypeptide that is to be modified in accordance with the invention has an extension (tag) of one or more amino acid residues at the carboxylic acid functionality that is to participate in the coupling reaction. The extension is usually bound to the acyl functionality of the polypeptide to be modified via the alpha-amine functionality of the amino acid (if the extension is an amino acid unit) or via the N-terminus of the peptide (if the extension is a peptide). If the extension is a peptide, it is usually a linear peptide, preferably a dipeptide or a tripeptide, more preferably a tripeptide. The use of a tripeptide tag has been found to outperform an amino acid tag in terms of net production rate, e.g. when comparing a modification of insulin wherein an amino acid tag (threonine) is cleaved off with insulin glargine (extended with two arginine units compared to insulin), wherein the tripeptide thr-arg-arg is cleaved off. A longer extension usually does not provide an improved synthesis, but may be present; e.g. a His-tag or the like can be present that has been used for upstream processing (e.g. isolation) of the first polypeptide segment. The amino acid(s) forming the extension is (are) preferably proteinogenic, since this usually facilitates effective/efficient catalysis by the acyltransferase. Further, this allows microbiological (fermentative) production of the segment, e.g. making use of recombinant technology. Particularly advantageous peptidic extensions for a fast reaction have been found to be extensions of which the N-terminal amino acid residue (P1’) is Asn or Ser. Good results have also been achieved with extensions wherein the P1’ amino acid residue is Thr. Ala and Asp at the P1’ position of the extension have also been found to be suitable tags. Thus, the extended polypeptide providing the acyl functionality can in particular schematically be represented by the following structure R1-P4-P3-P2-P1- [AA]n. R1 is the optionally present remainder of the first polypeptide segment. It can be or comprise a further amino acid residue or further peptide structure (linear, branched, cyclic, conjugate); it can be another moiety, such as a moiety described when discussing (poly)peptides in general (above) or a moiety (Rx ) of the nucleophile (below); e.g. it can be or comprise a sugar moiety, a lipid moiety, a label; or a combination of a peptide structure and other moiety. Each [AA] independently represents an amino acid residue of the extension and n is at least 1, preferably 1, 2 or 3. Usually the number of amino acid residues n of the extension is 12 or less, in particular 9 or less, more in particular 5 or less. The nucleophile can generally be represented by the general formula R2-NH₂. R2 can in principle be any moiety as long as the amine functionality is reactive with the acyl functionality of the polypeptide to which the nucleophile is to be coupled. Suitable nucleophiles can in particular be selected from the group consisting of hydrazine, organic compounds comprising a hydrazine functionality (Rx-NH-NH₂, i.e. R2 = Rx-NH), hydroxyl amine and organic compounds comprising a hydroxylamine functionality (Rx-O-NH₂, i.e. R2 = R3-O-), ammonia and organic compounds comprising an amino group (Rx-NH₂, i.e. R2 = Rx). The amino or hydrazine or hydroxyl amine functionality participating in the enzymatic coupling advantageously is at an extremity of the nucleophile, e.g. at the C1 position of a carbon chain or at the N-terminus of a peptide nucleophile. The group Rx can consist of hydrogen and carbon or comprise one or more heteroatoms, in particular one or more heteroatoms selected from S, O, N, P and halogens. The group R3-O is an example of a group Rx with a heteroatom (O). The amine or hydrazine functionality of the nucleophile can be the only functional group or the only nucleophilic functional group. Rx can comprise one or more additional functionalities, such as one or more carboxyl groups, one or more hydroxyl groups, one or more phosphate groups, one or more sulphate groups etc. Preferably, the Rx is free of amino or hydrazine functionalities capable of acting as the nucleophile in order to avoid undesired side-reactions. The organic compound (Rx, when Rx is not hydrogen) comprising an amino or hydrazine or hydroxyl amine function has at least 1 carbon atom, in particular at least 2, at least 3, at least 4, at least 5 or at least 6 carbon atoms. In an embodiment wherein the organic compound (Rx) comprising an amino or hydrazine function is not a peptide, it usually has 40 or less carbon atoms, in particular 25 or less, more in particular 15 carbon atoms or less or 10 carbon atoms or less. In particular, the group Rx can be an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl. In an advantageous embodiment, the nucleophile is hydrazine; hydroxylamine; an amino acid; a peptide; a carbohydrate (such as an monosaccharide, oligosaccharide, polysaccharide (of which polysialic acid is an example) provided with a hydrazine, hydroxylamine or amino group (allowing the derivatisation of the polypeptide into a glycopeptide); a lipid (such as a fatty acid, a glyceride) provided with a hydrazine, hydroxylamine or amino group (allowing the derivatisation of the polypeptide into a lipopeptide); a nucleotide, oligonucleotide or polynucleotide provided with a hydrazine, hydroxylamine or amino functionality; a steroid provided with a hydrazine, hydroxylamine or amino functionality; a polyalkylene glycol, provided with a hydrazine, hydroxylamine or amino functionality. In an embodiment, the nucleophile provides an imaging agent (e.g. radioactive, fluorescent, luminescent, phosphorescent, chromogenic), a radio-therapeutic agent, a toxin, a chelating agent or another non-peptidic agent or a non-peptidic biologically active moiety to the polypeptide to be modified. In a specific embodiment, the nucleophile is a peptide or an amino acid or an amino acid amide. In such embodiment, a polypeptide product of interest can be formed from two peptides or from a peptide (the polypeptide providing the acyl functionality) and an amino acid (the nucleophile), which can have been made by known techniques, e.g. chemically or microbiologically. This allows the enzymatic synthesis of polypeptides, by coupling two polypeptide segments via an peptide bond without needing to activate the C-terminal end of polypeptide segment providing the acyl functionality with an ester or thioester. Usually, the amine functionality of the peptide nucleophile to which the first polypeptide segment is to be coupled is the N-terminal amine of the peptide nucleophile. In an advantageous embodiment a non-proteinogenic amino acid or a peptide comprising one or more non-proteinogenic amino acids is used as the nucleophile. This allows, the addition of one or more non-proteinogenic (e.g. non-natural) amino acids at the C-terminal side of a fermentatively produced polypeptides. Such amino acids, which are not genetically encoded, can generally not be included fermentatively in a polypeptide. It also allows the coupling of an amino acid C-terminal amide; the product is protected against exoprotease activity. Good results have also been achieved with hydrazine (H2N-NH₂) as the nucleophile, whereby a functionalised polypeptide is obtained wherein at the C- terminus of the polypeptide a hydrazide -(C=O)-NH-NH₂ functionality is formed. This can be used for further ligation of larger polypeptide constructs or for the synthesis of (thio)esters or for direct ligation with a ketone or aldehyde, thereby further adding to the palette of tools for making specific polypeptides. Using an organic amine as a nucleophile can be very useful to alter physical-chemical properties of a polypeptide, whereby e.g. solubility in a solvent, release from a controlled release device, uptake of the polypeptide by an organism can be altered. For instance, an alkylamine or other hydrophobic organic amine as the nucleophile is useful to provide a polypeptide that is more hydrophobic, compared to the polypeptide that is to be modified. Good results have for instance been achieved with ethylamine. Hydrophilic amines (e.g. glycosamines, glycosaminoglucans) may be used to provide a more hydrophilic polypeptide. Of the organic amines, an alkynyl amine, in particular propargylamine, has been found to be a highly reactive nucleophile in a method according to the invention, allowing to functionalise a peptide with a triple carbon-carbon bond (CH≡C-). Further, particularly good results have been achieved with functionalisation with allyl amine. Hydroxyl amine has also been found to be a nucleophile that can be coupled very well in accordance with the invention. It can be used as the nucleophile to obtain a functionalised polypeptide wherein at C-terminus of the first polypeptide segment a -(C=O)-NH-OH functionality is formed. This can be used for further reactions e.g. by hydrazone ligation, thereby further adding to the palette of tools for making specific polypeptides. Further, good results have been achieved with an alkanolamine as the nucleophile in a method according to the invention, in particular ethanolamine, but also another alkanolamine may be used, such as propanolamine or butanolamine. Lipid compounds provided with an amino or hydrazine group can be used as nucleophile to decrease solubility in an aqueous environment or to increase solubility in a lipophilic environment. Polyalkylene glycols, such as polyethylene glycols (PEG), provided with an amino or hydrazine group can be used as nucleophile to increase solubility or in vivo half-life in an aqueous environment, e.g. blood plasma. The coupling of the polypeptide having the acyl functionality and the nucleophile in a method according to the invention is typically performed in a fluid comprising water. Preferably the reaction is performed in a buffered fluid. The water content usually is 10-100 vol %, based on total liquids, preferably 50 vol. % or more, preferably 70 vol. % or more, in particular 80 vol. % or more, more in particular 90 vol. % or more. The term ‘aqueous’ is used for media at least substantially consisting of water. In principle, any buffer is suitable. Good buffers are known to a person skilled in the art. See for instance David Sheehan in Physical Biochemistry, 2^nd Ed. Wiley-VCH Verlag GmbH, Weinheim 2009; http://www.sigmaaldrich.com/life- science/core-bioreagents/biological-buffers/learning-center/buffer-calculator.html. The pH of the buffer for the enzymatic modification of the polypeptide can be determined based on the information disclosed herein and common general knowledge. The pH is usually in the range of about 5 to about 11. The pH is preferably about 5 or more, in particular about 6 or more, more in particular at least about 7. A desired pH is usually 10 or less, preferably 9 or less. Usually the optimal pH for the enzymatic coupling is in the range of about 7 to about 9. Suitable ratios of the amounts of polypeptide to be modified and the nucleophile in the reaction mixture can be determined based on the information disclosed herein and common general knowledge. In principle a stoichiometric ratio may be considered. Usually or a molar excess of nucleophile is advantageous to improve intended product yield or net formation rate of intended product. Usually a molar ratio nucleophile functionality to acyl functionality of 10:1 or more, in particular of at least about 100:1, more in particular of at least about 1000:1 is employed for an efficient product formation rate. The upper limit for the molar ratio nucleophile functionality is generally not critical. In practice, the molar ratio nucleophile functionality to acyl functionality is 100000 or less, in particular 25000 or less, e.g. 10000 or less or 5000 or less. In the method of the invention, it may be advantageous to add additives to the fluid wherein the reaction is carried out to improve the solubility of the peptide fragments or to improve the reaction yield. Such additives may be a salt or an organic molecule, for instance guanidinium hydrochloride, urea, sodium dodecasulphate or Tween. The reaction may be carried out in a fully aqueous liquid or in a mixture of water and a water miscable co-solvent such as N,N-dimethylformamide (DMF), N- methyl-pyrrolidinone (NMP), N,N-dimethylacetamide (DMA), dimethylsulphoxide (DMSO), acetonitrile, an ether, such as tetrahydrofuran (THF), 2-methyl- tetrahydrofuran (Me-THF) or 1,2-dimethoxyethane, or a (halogenated) alcohol, such as methanol, ethanol, isopropanol, tert-butanol, 2,2,2-trifluoroethanol (TFE), 1,1,1,3,3,3-hexafluoroisopropanol, or a mixture of these organic solvents. Depending on the stability of the acyltransferase and the solubility of the substrates, the amount of co-solvent preferably is 50 vol% or less, more preferably 30 vol% or less, in particular 20 vol% or less, more in particular 10 vol% or less. In principle the temperature during the enzymatic coupling is not critical, as long as a temperature is chosen at which the acyltransferase and substrates to be used show sufficient activity and stability. Such a temperature can be routinely determined. Generally, the temperature may be at least -10 °C, in particular at least 0 °C or at least 10 °C. Generally, the temperature may be 70°C or less, in particular 60°C or less or 50 °C or less. Optimal temperature conditions can easily be identified for a specific enzyme for a specific coupling by a person skilled in the art through routine experimentation based on common general knowledge and the information disclosed herein. In general, the temperature advantageously is in the range of 20- 50°C.

EXAMPLES Enzyme preparation Acyltransferase variants were prepared either by gene synthesis at GenScript or by QuikChange site-directed mutagenesis. Expression and enzyme purification were performed as described in literature (T. Nuijens, et al. Adv Synth Catal, 358, 2016). As expression host B. subtilis KO7-S (ID: 1S145) from the Bacillus Genetic Stock Center was used. The enzyme concentration and purity were determined using a nanophotometer (Westburg, Implen) and sodium dodecyl sulphate–polyacrylamide gel electrophoresis. The obtained aqueous enzyme solutions were uses as such for the acyltransferase (e.g. transpeptidation) reactions. Enzymes according to the invention are referred to as peptilisins. Materials and Methods Chemicals for SPPS and analytics were obtained from several commercial sources. Rink-amide resin (0.67 mmol/g) and Fmoc-AAx-OH building blocks were purchased from GL-Biochem, Dichloromethane, dimethylformamide, N,N′- Diisopropylcarbodiimide, piperidine, acetic anhydride, diisopropyl ether, trifluoroacetic acid (peptide grade), trifluoroacetic acid (LC-MS grade), formic acid (LC-MS grade) were purchased from Biosolve. OxymaPure was purchased from Iris- Biotech. N,N-Diisopropylethylamine was puchased from TCI-Chemicals. Triisopropylsilane and methanesulfonic acid were purchased from Sigma Aldrich. Ultrapure waters (milliQ) was obtained from a MilliQ Direct-Q 3. Peptides were manually synthesized using standard solid phase synthesis techniques. Analytical HPLC was performed on an Agilent 110 series liquid chromatography system. A reversed-phased column (Aeris WIDEPORE, 3.6 µm, XB-C8, 150x4.6 mm or Kinetex, EVO, 5 µm, C18, 100 Å, 150x4.6 mm) was used at 40 C° with a flow of 1.5 mL/min and a mobile phase containing water/FTA (99.95/0.05 Vol%) and acetonitrile/TFA (99.95/0.05 Vol%). An optimal gradient for analysis was determined for each experiment separately. Example 1: Peptilisin (SEQUENCE ID NO 5) activity in presence of hydrazine A reaction was performed using peptilisin (SEQUENCE ID NO 5, elongated with a His-tag (HHHHHH) at the C-terminal end; X225N) as acyltransferase and hydrazine as nucleophile. 0.77 mg of the extended polypeptide Ac-Asp-Phe-Ser-Lys-Leu-Ala-Leu-Arg-NH₂ (N-terminal Asp is acetylated (Ac), Arg- NH₂ at C-terminal side is part of the tag) was dissolved in 61 µL tricine buffer (1 M, pH 8.5). The pH was adjusted to 8.5 (using 3M NaOH) and the stock solution was set to a total volume 305 µL with milliQ water. From this stock solution, 50 µL was added to an HPLC-vial and 50 µL of a 4 M hydrazine solution in milliQ (pH 8,5 adjusted with 3M HCl) was added. The reaction was started by addition of 3.1 µL enzyme solution to the vial containing peptide starting material and hydrazine. The final peptilisin solution contained a concentration of 1 mM Ac-Asp-Phe-Ser- Lys-Leu-Ala-Leu-Arg-NH₂ , 0.1 mg/mL peptilisin, 100 mM tricine buffer and 2 M hydrazine (pH 8,5). The reactions were incubated at 22 °C and analysed by LC-MS by direct injection of 5 µL per measurement. The starting material (extended polypeptide), hydrolysis (Ac-Asp-Phe-Ser-Lys-Leu-OH) and product (Ac-Asp-Phe-Ser-Lys-Leu- NH-NH₂) peaks were integrated.

Conclusion Peptilisin comprising the amino acid sequence of SEQUENCE ID NO: 5 can be used for efficient catalysis of an acyltransferase reaction of an acyl donor peptide with a nucleophile. Example 2: Peptilisin (SEQUENCE ID NO: 4) activity in presence of hydrazine A reaction was performed as described above changing the type of peptilisin to SEQUENCE ID NO 4 (X225N mutation; elongated with a His-tag (HHHHHH) at the C-terminal end) and peptide substrate to Ac-Glu-Ile-Thr-Thr- Lys-Asp-Leu-Lys- NH₂. The final peptilisin solution contained a concentration of 1 mM Ac-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-NH₂, 0.1 mg/mL peptilisin, 100 mM tricine buffer and 2 M hydrazine (pH 8,5). The reactions were incubated at 22 °C and analysed by LC-MS by direct injection of 5 µL per measurement. The starting material, hydrolysis side product (Ac-Glu-Ile-Thr-Thr-Lys-OH) and product (Ac- Glu-Ile-Thr-Thr-Lys-NH-NH₂) peaks were integrated.

Conclusion Acyltransferases (peptilisins) in accordance with the invention having different sequences (example 1 SEQUENCE ID NO: 5 and example 2 SEQUENCE ID NO: 4) can be used for an efficient acyltransferase catalysed reaction. Acyl donor substrates that are totally different in P1, P2, P3 and P4 position, as compared to example 1, can be used for the acyl transfer reaction. Example 3: Peptilisin (SEQUENCE ID NO: 4) activity in the presence of different P1’ peptide sequences The reaction of Example 2 was repeated resulting in the final reaction mixture containing 1 mM Ac-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-NH₂, 0.1 mg/mL peptilisin (SEQUENCE ID NO: 4 + His tag; X225N) in 100 mM tricine buffer and 2 M hydrazine (pH 8.5). In an identical manner several different peptides were investigated as starting material (extended polypeptide) using the sequence Ac- Glu-Ile-Thr-Thr-Lys-Xxx-Leu-Lys-NH₂ (wherein Xxx = Asp, Asn, Thr, Ser or Ala). After 15 min a 30 µL aliquot of the reaction mixture was drawn, quenched with 60 µL methane sulphonic acid (MSA)/water/MeCN (4/48/48 Vol.%) and analysed by LC-MS. The absorbance at 220 nm of starting material, peptide hydrazide product (Ac-Glu-Ile-Thr-Thr-Lys-NH-NH₂ ) and hydrolysed peptide by- product were integrated. The area % product is defined as the amount of peptide hydrazide product divided by the total amount of starting material, product and hydrolysed peptide.

Conclusion Acyltransferases (peptilisins) in accordance with the invention can recognise a variety of substrates in the P1’ position in the starting material (P1’ in the extended polypeptide is the N-terminal amino acid of the tag, which is cleaved off). Some substrates react faster than others. Asn and Ser are preferred in the P1’ position. Example 4: Peptilisin (SEQUENCE ID NO: 4) activity in the presence of different P4 peptide sequences The reaction of Example 3 was repeated but with half the amount of enzyme resulting in a final reaction mixture containing 1 mM Ac-Glu-Ile-Thr-Thr- Lys-Asn-Leu-Lys-NH₂ , 0.05 mg/mL peptilisin (SEQUENCE ID NO 4 + His tag; X225N) in 100 mM tricine buffer and 2 M hydrazine (pH 8,5). In an identical manner several different peptides were investigated using the sequence Ac-Glu- Xxx-Thr-Thr-Lys-Asn-Leu-Lys-NH₂ (wherein Xxx = Leu, Val, Phe or Tyr). After 15 min a 30 µL aliquot of the reaction mixture was drawn, quenched with 60 µL MSA/water/MeCN (4/48/48 Vol.%) and analysed by LC-MS. The absorbance at 220 nm of starting material, peptide hydrazide product and hydrolysed peptide by-product were integrated. The area % product is defined as the amount of peptide hydrazide product divided by the total amount of starting material, product and hydrolysed peptide.

Conclusion Acyltransferases (peptilisins) in accordance with the invention can recognise a variety of substrates in the P4 position. Some substrates react faster than others. Example 5: Peptilisin (SEQUENCE ID NO 4) activity in the presence of different P3’ peptide sequences The reaction of Example 4 was repeated resulting in a final reaction mixture containing 1 mM Ac-Glu-Tyr-Thr-Thr-Lys-Asn-Leu-Lys-NH₂ , 0.05 mg/mL peptilisin (SEQUENCE ID NO 4 + His tag; X225N) in 100 mM tricine buffer and 2 M hydrazine (pH 8,5). In an identical manner several different peptides were investigated using the sequence Ac-Glu-Tyr-Thr-Thr-Lys-Asn-Leu-Xxx-NH₂ (wherein Xxx = Arg, His, or Glu). After 15 min a 30 µL aliquot of the reaction mixture was drawn, quenched with 60 µL MSA/water/MeCN (4/48/48 Vol.%) and analysed by LC-MS. The absorbance at 220 nm of starting material, peptide hydrazide product and hydrolysed peptide by-product were integrated. The area % product is defined as the amount of peptide hydrazide product divided by the total amount of starting material, product and hydrolysed peptide.

Conclusion Acyltransferases (peptilisins) in accordance with the invention can recognise a variety of substrates in the P3’ position. Some substrates react faster than others. Example 6: Mapping of the P225X (wherein X = any amino acid) position of peptilisin (SEQUENCE ID NO 5) for substrate acyl transfer. To map the substrate acyltransfer reaction to peptide hydrazide, the following standard reactions were performed on each P225X variant (SEQUENCE ID NO 5, with X any of the 20 canonical amino acids). Per variant, 0.48 mg of peptide Ac-Asp-Phe-Ser-Lys-Leu-Ala-Leu-Arg-NH₂ was dissolved in 100 µL Hydrazine solution (4M, pH 8.5) to a final concentration of 4 mM. 20 µL of 1 M tricine buffer (1M, pH 8.5) was added and the pH was corrected to 8.5 with 3 M NaOH solution. milliQ and subsequently enzyme P225X variant were added in such a ratio that the final hydrolase solution volume was 200 µL with 2 mM peptide, 2 M hydrazine, 100 mM tricine and 0.1 mg/mL enzyme. After 120 min a 30 µL aliquot of the reaction mixture was drawn, quenched with 60 µL MSA/water (4/96 Vol.%) and analysed by LC-MS. The absorbance at 220 nm of starting material, peptide hydrazide product and hydrolysed peptide by-product were integrated. The area % of product after 120 min is summarized in the table below.

Conclusion The P225X position has a large effect on transpeptidation efficiency. Mutations P225X with small canonical amino acids (P225A, P225G, P225S, P225C, P225N and P225T) and some hydrophobic amino acids (P225M, P225I, P225L, P225Y and P225V) have a positive effect on area% of product, compared to P225. Example 7: Peptilisin (SEQUENCE ID NO 5) activity in the presence of Glargine A reaction was performed using peptilisin (SEQUENCE ID NO 5+His tag; X225N) as acyltransferase and hydrazine as nucleophile.1 mg of Glargine was dissolved in 900 µL tricine buffer (1 M, pH 8.5). The Glargine was not acetylated at the N-terminus, i.e. it had a free amino group at the N-terminus. The extended Glargine had a carboxylic acid at the C-terminus. The pH was adjusted to 8.5 and the total stock solution was set to a total volume 1 mL with milliQ water. From this stock solution, 50 µL was added to an HPLC-vial and 50 µL of a 4 M hydrazine solution in milliQ (pH 8,5 adjusted with 3M HCl) was added. The reaction was started by addition of 5 µL enzyme solution to the vial containing starting material and hydrazine. The final peptilisin solution contained a concentration of 0.5 mg/mL Glargine , 0.1 mg/mL peptilisin, 500 mM tricine buffer and 2 M hydrazine (pH 8,5). The reactions were incubated at 22 °C. After 3 and 6 hours a 30 µL aliquot of the reaction mixture was drawn, quenched with 60 µL MSA/water (4/96 Vol.%) and analysed by LC-MS. The absorbance at 220 nm of starting material, glargine hydrazide product and hydrolysed glargine by-product were integrated. The area % product is defined as the amount of glargine hydrazide product divided by the total amount of starting material, product and hydrolysed glargine. After 6 hours, there was 30% of glargine hydrazide product formation, 15% hydrolysed glargine by-product and still 55% of starting material. Conclusion Peptilisin comprising the amino acid sequence of SEQUENCE ID NO: 5 can be used for efficient catalysis of an acyltransferase reaction of an acyl donor protein with a nucleophile. Example 8: Acyl transfer reactions using different nucleophiles A screening of different nucleophiles was performed for the acyltransferase reaction with peptilisin. Per reaction, 0.50 mg of peptide Ac-Glu-Ile- Thr-Thr-Lys-Asn-Leu-Lys-NH₂ was dissolved in 200 µL of 3.5 M nucleophile solution in water. The pH was corrected with 12 M HCl between 10.5-10.9. Subsequently, Peptilisin (SEQUENCE ID NO: 4) was added to a final concentration of 0.01 mg/mL in a sample containing 2.48 mM substrate and 3.47 M nucleophile. After 180 min a 30 µL aliquot of the reaction mixture was drawn, quenched with 60 µL MSA/water (4/96 Vol.%) and analyzed by LC-MS. The absorbance at 220 nm of starting material, synthesis product and hydrolyzed peptide by-product were integrated. The area % of product after 180 min is summarized in the table below.

Conclusion Clearly, many different nucleophiles can be used in the acyltransferase reaction using peptilisin. Example 9: Peptilisin (SEQUENCE ID NO 4) acyl transfer using acyl donors without N-terminal protection and varying length of the cleavage tag. To map the substrate acyl transfer reaction to peptide hydrazide, the following standard reactions were performed with the peptilisin variant of SEQUENCE ID NO 4. The following peptides were dissolved in 100 µL Hydrazine solution (4M, pH 8.5) to a final concentration of 4 mM. H-Phe-Tyr-Thr-Pro-Lys-Asn-Leu-Lys-NH₂ (Pep1) H-Phe-Tyr-Thr-Pro-Lys-Asn-Leu-NH₂ (Pep2) H-Phe-Tyr-Thr-Pro-Lys-Asn-NH₂ (Pep3) H-Phe-Tyr-Thr-Pro-Lys-Asn-Leu-Lys-OH (Pep4) H-Phe-Tyr-Thr-Pro-Lys-Asn-Leu-OH (Pep5) The amino acid residues that are underlined and in bold are the extensions (tags) that are to be cleaved off. Thus, the extensions varied in length from 1 to 3 amino acid residues. 20 µL of 1 M tricine buffer (1M, pH 8.5) was added and the pH was corrected to 8.5 with a 3 M NaOH solution. MilliQ and subsequently the wildtype enzyme were added in such a ratio that the final hydrolase solution volume was 200 µL with 2.5 mM peptide, 2 M hydrazine, 100 mM tricine and 0.01 mg/mL enzyme. After 120 min a 20 µL aliquot of the reaction mixture was drawn, quenched with 60 µL MSA/water (4/96 Vol.%) and analyzed by LC-MS. The absorbance at 220 nm of starting material, peptide hydrazide product (H-Phe-Tyr- Thr-Pro-Lys-NH-NH₂) and hydrolyzed peptide by-product (H-Phe-Tyr-Thr-Pro-Lys- OH) were integrated. The area % of product after 120 min is summarized in the table below.

Conclusion The substrate does not require an N-terminal protecting group for synthesis of the C-terminal peptide hydrazide and is comparable to the peptide containing N-terminal protecting group. The C-terminal functionalization can be either a carboxylic acid or an amide, without having influence on the acyl transfer by the peptilisin enzyme. The cleavage tag can vary in length, although slower for a single amino acids the acyl transfer is still feasible. Example 10: Acyl transfer of peptilisin including calcium binding domain (SEQUENCE ID NO 6) containing different mutations at P225X (wherein X = A, P, C). To map the substrate acyltransfer reaction to peptide hydrazide, the following standard reactions were performed on each P225X variant (SEQUENCE ID NO 6, with X corresponding to either alanine, proline or cysteine). Per variant, 0.49 mg of peptide Ac-Asp-Phe-Ser-Lys-Leu-Ala-Leu-Arg-NH₂ was dissolved in 100 µL Hydrazine solution (4M, pH 8.5) to a final concentration of 4 mM. 20 µL of 1 M tricine buffer (1M, pH 8.5) was added and the pH was corrected to 8.5 with 3 M NaOH solution. MilliQ and subsequently enzyme N225X variants were added in such a ratio that the final hydrolase solution volume was 200 µL with 2.5 mM peptide, 2 M hydrazine, 100 mM tricine and 0.01 mg/mL enzyme. After 120 min a 20 µL aliquot of the reaction mixture was drawn, quenched with 60 µL MSA/water (4/96 Vol.%) and analyzed by LC-MS. The absorbance at 220 nm of starting material, peptide hydrazide product and hydrolyzed peptide by-product were integrated. The area % of product after 30 min is summarized in the table below.

Conclusion The acyl transfer reaction is efficient also using peptilisin without calcium domain deletion and the P225X position has an effect on transpeptidation efficiency. Example 11: Mapping of the P225X (wherein X = A, C, G, P) position of peptilisin (SEQUENCE ID NO 7) for substrate acyl transfer. To map the substrate acyltransfer reaction to peptide hydrazide, the following standard reactions were performed on each P225X variant (SEQUENCE ID NO 7, with X corresponding to either alanine, cysteine, glycine, or proline). Per variant, 0.49 mg of peptide Ac-Glu-Ile-Thr-Thr-Lys-Asn-Leu-Lys-NH₂ was dissolved in 100 µL Hydrazine solution (4M, pH 8.5) to a final concentration of 4 mM.20 µL of 1 M tricine buffer (1M, pH 8.5) was added and the pH was corrected to 8.5 with 3 M NaOH solution. MilliQ and subsequently enzyme P225X variants were added in such a ratio that the final hydrolase solution volume was 200 µL with 2.5 mM peptide, 2 M hydrazine, 100 mM tricine and 0.01 mg/mL enzyme. After 15 min a 20 µL aliquot of the reaction mixture was drawn, quenched with 60 µL MSA/water (4/96 Vol.%) and analyzed by LC-MS. The absorbance at 220 nm of starting material, peptide hydrazide product and hydrolyzed peptide by-product were integrated. The area % of product after 30 min is summarized in the table below.

Conclusion The acyl transfer reaction is efficient also using peptilisin without calcium domain deletion variants and the P225X position has an effect on transpeptidation efficiency. Per variant, different amino acids at P225 can be optimal. Example 12: Two P225 variants of peptilisin (SEQUENCE ID NO 8) for substrate acyl transfer. To map the substrate acyltransfer reaction to peptide hydrazide, the following standard reactions were performed on P225P and P225N variant (SEQUENCE ID NO 8, with X corresponding to either P or N; six mutations in total compared to subtilisin BPN’). Per variant, 0.49 mg of peptide Ac-Glu-Ile-Thr- Thr-Lys-Asn-Leu-Lys-NH2 was dissolved in 100 µL Hydrazine solution (4M, pH 8.5) to a final concentration of 4 mM. 20 µL of 1 M tricine buffer (1M, pH 8.5) was added and the pH was corrected to 8.5 with 3 M NaOH solution. milliQ and subsequently enzyme variants were added in such a ratio that the final hydrolase solution volume was 200 µL with 2.5 mM peptide, 2 M hydrazine, 100 mM tricine and 0.01 mg/mL enzyme. After 30 min a 20 µL aliquot of the reaction mixture was drawn, quenched with 60 µL MSA/water (4/96 Vol.%) and analysed by LC-MS. The absorbance at 220 nm of starting material, peptide hydrazide product and hydrolysed peptide by-product were integrated. The area % of product after 30 min is summarized in the table below.

Conclusion The P225 position has an effect on transpeptidation efficiency. Changing the P225 position to an asparagine has a positive effect on area% of product, Example 13 Two P225 variants of peptilisin (SEQUENCE ID NO 9) for substrate acyl transfer. To map the substrate acyltransfer reaction to peptide hydrazide, the following standard reactions were performed on P225A and P225N variant (SEQUENCE ID NO 9, with X being A or N). Per variant, 0.49 mg of peptide Ac- Glu-Ile-Thr-Thr-Lys-Asn-Leu-Lys-NH2 was dissolved in 100 µL Hydrazine solution (4M, pH 8.5) to a final concentration of 4 mM. 20 µL of 1 M tricine buffer (1M, pH 8.5) was added and the pH was corrected to 8.5 with 3 M NaOH solution. milliQ and subsequently enzyme variants were added in such a ratio that the final hydrolase solution volume was 200 µL with 2.5 mM peptide, 2 M hydrazine, 100 mM tricine and 0.01 mg/mL enzyme. After 30 min a 20 µL aliquot of the reaction mixture was drawn, quenched with 60 µL MSA/water (4/96 Vol.%) and analysed by LC-MS. The absorbance at 220 nm of starting material, peptide hydrazide product and hydrolysed peptide by-product were integrated. The area % of product after 30 min is summarised in the table below.

Conclusion The modification of the P225 position has an effect on transpeptidation efficiency, without needing further modifications compared to subtilisin BPN’. Amino acids Ala and Asn at the P225 position leads to efficient acyl transferases. Sequences SEQUENCE ID NO 1: subtilisin BPN’: SUBT_BACAM Subtilisin BPN' Bacillus amyloliquefaciens mature 1 to 275 AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSE TNPFQDNNSHGTHVAGTVAALNNSIGVLGVAPSASLYAVKVLGADGSGQYSWII NGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGTSGS SSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYG AYNGTSMASPHVAGAAALILSKHPNWTNTQVRSSLENTTTKLGDSFYYGKGLI NVQAAAQ SEQUENCE ID NO: 2 subtilisin BPN’ variant with preferred mutation positions (X) compared to SEQ ID NO 1 (provided S221 is conserved and at the 225 position X is not P) AXXVXYGVXQIKAPALHSQGYTGSNVKVAVXDXGIDSSHPDLXVAGGASXVPSE TNPFQDNXSHGTHVAGTVXAVAPSASLYAVKVXGAXGSGQYSWXINGIEWAIAN NMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNXGTSGSSSTVXYPXK YPSVIAVGAVDSSNQRAXXSSVGPELDVMAPGVSIXSTLPGXKYGAXXGTSXXX XHVAGAAALILSKHPNWTNTQVRSSLENTXTKLGDSFYYGKGLINVXAAAQ SEQUENCE ID NO 3: Peptilisin variant (DNA-sequence, mature) 1 gcgaagtgcg tgtcttacgg cgtagcgcaa attaaagccc ctgctctgca cgcttcacgc acagaatgcc gcatcgcgtt taatttcggg gacgagacgt 51 ctctcaaggc tacactggat caaatgttaa agtagcagtt cttgacagcg gagagttccg atgtgaccta gtttacaatt tcatcgtcaa gaactgtcgc 101 gtatcgattc ttctcatcct gatttaaacg tagcaggcgg agccagcttc catagctaag aagagtagga ctaaatttgc atcgtccgcc tcggtcgaag 151 gttccttctg aaacaaatcc tttccaagac aacaactctc acggaactca caaggaagac tttgtttagg aaaggttctg ttgttgagag tgccttgagt 201 cgttgccggc acagttttgg ctgttgcgcc aagcgcatca ctttacgctg gcaacggccg tgtcaaaacc gacaacgcgg ttcgcgtagt gaaatgcgac 251 taaaagttct cggtgctgac ggttccggcc aatacagctg gattattaac attttcaaga gccacgactg ccaaggccgg ttatgtcgac ctaataattg 301 ggaatcgagt gggcgatcgc aaacaatatg gacgttatta acatgagcct ccttagctca cccgctagcg tttgttatac ctgcaataat tgtactcgga 351 cggcggacct tctggttctg ctgctttaaa agcggcagtt gataaagccg gccgcctgga agaccaagac gacgaaattt tcgccgtcaa ctatttcggc 401 ttgcatccgg cgtcgtagtc gttgcggcag ccggtaacaa tggcacttcc aacgtaggcc gcagcatcag caacgccgtc ggccattgtt accgtgaagg 451 ggcagctcaa gcacagtgga ttaccctgct aaataccctt ctgtcattgc ccgtcgagtt cgtgtcacct aatgggacga tttatgggaa gacagtaacg 501 agtaggcgct gttgacagca gcaaccaaag agcaccgtgg tcaagcgtag tcatccgcga caactgtcgt cgttggtttc tcgtggcacc agttcgcatc 551 gacctgagct tgatgtcatg gcacctggcg tatctatctg tagcacgctt ctggactcga actacagtac cgtggaccgc atagatagac atcgtgcgaa 601 cctggaggca aatacggggc gagatctggt acgtcaggcg catctaatca ggacctccgt ttatgccccg ctctagacca tgcagtccgc gtagattagt 651 cgttgccgga gcggctgctt tgattctttc taagcacccg aactggacaa gcaacggcct cgccgacgaa actaagaaag attcgtgggc ttgacctgtt 701 acactcaagt ccgcagcagt ttagaaaaca ccgctacaaa acttggtgat tgtgagttca ggcgtcgtca aatcttttgt ggcgatgttt tgaaccacta 751 tctttctact atggaaaagg gctgatcaac gtagaagcag cagctcagca agaaagatga taccttttcc cgactagttg catcttcgtc gtcgagtcgt 801 ccaccaccac caccactaa ggtggtggtg gtggtgatt SEQUENCE ID NO 4. Peptilisin variant PL-01 (amino acid-sequence) AKCVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASF VPSETNPFQDNNSHGTHVAGTVLAVAPSASLYAVKVLGADGSGQYSWIIN GIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNNGTS GSSSTVDYPAKYPSVIAVGAVDSSNQRAPWSSVGPELDVMAPGVSICSTL PGGKYGARSGTSGASXHVAGAAALILSKHPNWTNTQVRSSLENTATKLGD SFYYGKGLINVEAAAQ SEQUENCE ID NO 5. Peptilisin variant PL-02 (amino acid-sequence) AKCVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSE TNPFQDNNSHGTHVAGTVLAVAPSASLYAVKVLGADGSGQYSWVINGIEWAIA NNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNSGTSGSSSTVSYPA KYPSVIAVGAVDSSNQRAPWSSVGPELDVMAPGVSICSTLPGGKYGAHSGTSPA SXHVAGAAALILSKHPNWTNTQVRSSLENTATKLGDSFYYGKGLINVEAAAQ SEQUENCE ID NO 6. Peptilisin variant PL-02 including calcium binding domain (amino acid-sequence) AKCVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSE TNPFQDNNSHGTHVAGTVAALNNSIGVLAVAPSASLYAVKVLGADGSGQYSWV INGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNSGTSG SSSTVSYPAKYPSVIAVGAVDSSNQRAPWSSVGPELDVMAPGVSICSTLPGGKYG AHSGTSPASXHVAGAAALILSKHPNWTNTQVRSSLENTATKLGDSFYYGKGLIN VEAAAQ SEQUENCE ID NO 7. Peptilisin variant PL-01 including calcium binding domain (amino acid-sequence) AKCVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSE TNPFQDNNSHGTHVAGTVAALNNSIGVLAVAPSASLYAVKVLGADGSGQYSWII NGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNNGTSGS SSTVDYPAKYPSVIAVGAVDSSNQRAPWSSVGPELDVMAPGVSICSTLPGGKYG ARSGTSGASXHVAGAAALILSKHPNWTNTQVRSSLENTATKLGDSFYYGKGLIN VEAAAQ SEQUENCE ID NO 8. Peptilisin variant PL-03 (amino acid-sequence) AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASFVPSET NPFQDNNSHGTHVAGTVAALDNSIGVLGVAPSASLYAVKVLGADGSGQYSWIIS GIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGTSGSS STVGYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIQSTLPGNRYGA YSGTSMASXHVAGAAALILSKHPNWTNTQVRSSLENTTTKLGDSFYYGKGLINV QAAAQ SEQUENCE ID NO 9. Peptilisin variant PL-04 (amino acid-sequence) AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSE TNPFQDNNSHGTHVAGTVAALNNSIGVLGVAPSASLYAVKVLGADGSGQYSWII NGIEWAIANNMDVINMSLGGPSGSAALKAAVDKAVASGVVVVAAAGNEGTSGS SSTVGYPGKYPSVIAVGAVDSSNQRASFSSVGPELDVMAPGVSIQSTLPGNKYG AYNGTSMASXHVAGAAALILSKHPNWTNTQVRSSLENTTTKLGDSFYYGKGLI NVQAAAQ

Claims

Claims 1. A method for modifying a polypeptide, comprising the coupling of the polypeptide which polypeptide provides an acyl functionality for the coupling, and a nucleophile, which nucleophile provides a nucleophilic functionality for the coupling, wherein a reaction mixture is provided comprising an extended polypeptide having an extension at said acyl functionality of said polypeptide, which extension is an enzymatically cleavable amino acid unit or enzymatically cleavable peptide, the reaction mixture further comprising the nucleophile and an acyltransferase; the extended polypeptide is subjected to a cleavage reaction catalysed by the acyltransferase whereby the extension is cleaved from said polypeptide and the polypeptide from which the extension is cleaved off is coupled via said acyl functionality to the nucleophilic amino functionality of the nucleophile, which coupling is catalysed by the acyltransferase; and the acyltransferase is a subtilisin variant or homologue thereof, with the proviso that the acyltransferase has an active site wherein serine is conserved.

2. The method according to claim 1, wherein the acyltransferase is a subtilisin variant or homologue thereof, wherein the conserved serine is a serine in a catalytic triad of the subtilisin variant or homologue thereof, the subtilisin variant or homologue thereof comprising a mutation compared to subtilisin BPN’ represented by SEQUENCE ID NO: 1 or compared to a homologue sequence thereof at the amino acid position corresponding to P225 of SEQUENCE ID NO: 1, which mutation preferably is selected from the group of amino acid positions corresponding to P225C, P225T, P225I, P225S, P225V, P225M, P225G, P225L, P225Y, P225N and P225A.

3. The method according to claim 2, wherein the mutation at the position corresponding to P225 is selected from the group of amino acid positions corresponding to P225C, P225T, P225I, P225S, P225A, P225G and P225N.

4. The method according to any of the preceding claims, wherein the nucleophile is selected from the group consisting of hydrazine, organic compounds comprising a hydrazine functionality, ammonia, hydroxyl amine, organic compounds comprising a hydroxylamine functionality and organic compounds comprising an amino group; preferably selected from the group consisting of hydrazine; hydroxylamine; amines; amino acids; peptides; carbohydrates (such as an monosaccharide, oligosaccharide, or polysaccharide) provided with a hydrazine, hydroxylamine or amino group; lipids (such as a fatty acid, a glyceride) provided with a hydrazine, hydroxylamine or amino group; nucleotides, oligonucleotides or polynucleotides, provided with a hydrazine, hydroxylamine or amino group; steroids provided with a hydrazine, hydroxylamine or amino group; polyalkylene glycols provided with a hydrazine, hydroxylamine or amino group.

5. The method according to any of the preceding claims, wherein the extension is at a C-terminal end of the extended polypeptide.

6. The method according to any of the preceding claims, wherein the polypeptide providing the acyl functionality comprises four or more amino acid residues, preferably 30 or more, more preferably 50 or more.

7. The method according to any of the preceding claims, wherein the polypeptide providing the acyl functionality is selected from the group of antibodies, antibody-fragments, nanobodies, peptide-based receptor ligands, albumins, biotins, growth factors, hormones, antimicrobial peptides, glycopeptides, lipopeptides, conjugates of a peptide and a nucleotide, conjugates of a peptide and a polyalkylene glycol (in particular polyethylene glycol), conjugates of a peptide and a fatty acid, conjugates of a peptide and a polysialic acid, conjugates of a peptide and a label (such as a fluorescent label, phosphorescent label, radioactive label, a dye), chimeric peptides and insulinotropic peptides (such as insulin, glargine).

8. The method according to any of the preceding claims, wherein the extended polypeptide providing the acyl functionality is prepared fermentatively, in particular expressed recombinantly.

9. The method according to any of the preceding claims, wherein the conserved serine of the acyltransferase is at the amino acid position corresponding to S221 of SEQUENCE ID NO: 1.

10. The method according to any of the preceding claims, wherein a calcium binding domain, corresponding to the amino acids 75-83 of SEQUENCE ID NO: 1, in the acyltransferase is absent or comprises one or more mutations whereby the calcium binding domain is at least partially inactivated.

11. The method according to any of the preceding claims, wherein the acyl transferase has 1-14, preferably 6-13, in particular 8-12, more in particular 10-12 mutations selected from the group of mutations at an amino acid position corresponding to Q2, S3, P5, S9, I31, K43, M50, A73, G169, S188, Q206, N212, T254 and Q271 of SEQUENCE ID NO 1, wherein preferably one or more of said mutations, more preferably at least six of said mutations, in particular at least eight, at least ten or at least twelve of said mutations are selected from the group of positions corresponding to Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, G169A, S188P, Q206C, N212G, T254A and Q271E.

12. The method according to any of the preceding claims, wherein the reaction mixture in which the hydrolysis reaction and the coupling are carried out is an aqueous reaction mixture comprising 50-100 wt.% of water, based on total liquid, preferably 70 - 100 wt. % water based on total liquid.

13. The method according to any of the preceding claims, wherein the acyltransferase has a sequence identity of at least 50 %, preferably at least 60 %, more preferably at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % with an acyltransferase comprising an amino acid sequence represented by SEQUENCE ID NO 1, 2, 4, 5, 6, 7, 8 or 9.

14. An acyltransferase, which acyltransferase is a subtilisin BPN’ variant or homologue thereof, comprising at least one mutation compared to subtilisin BPN’ represented by SEQUENCE ID NO: 1 or a homologue sequence thereof, which at least one mutation is a mutation at the amino acid position corresponding to P225 of SEQUENCE ID NO: 1, with the proviso that the acyltransferase has an active site having a conserved serine, in particular a conserved serine at the amino acid position corresponding to S221 of SEQUENCE ID NO: 1.

15. The acyltransferase according to claim 14, wherein the mutation at the amino acid position corresponding to P225 is selected from the group consisting of P225C, P225T, P225I, P225S, P225V, P225M, P225G, P225L, P225Y and P225N.

16. The acyltransferase according to claim 15, wherein the mutation at the amino acid position corresponding to P225 is selected from the group consisting of P225C, P225T, P225I, P225S, P225G and P225N.

17. The acyltransferase according to claim 14, 15 or 16, wherein a calcium binding domain, corresponding to the amino acids 75-83 of SEQUENCE ID NO: 1, is absent or comprises one or more mutations whereby the calcium binding domain is at least partially inactivated.

18. The acyltransferase according to claim 14, 15 or 16, wherein a calcium domain is present at a position corresponding to the amino acids 75-83 of SEQUENCE ID NO: 1.

19. The acyltransferase according to any of the claims 14-18, having a sequence identity of at least 50 %, preferably at least 60 %, more preferably at least 70 %, at least 80 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % with an acyltransferase comprising an amino acid sequence represented by SEQUENCE ID NO 1, 2, 4, 5, 6, 7, 8 or 9.

20. The acyltransferase according to any of the claims 14-19, wherein the acyltransferase has 1-14, in particular 6-13, more in particular 10-12 mutations selected from the group of mutations at an amino acid position corresponding to Q2, S3, P5, S9, I31, K43, M50, A73, G169, S188, Q206, N212, T254 and Q271 of SEQUENCE ID NO 1. 21 The acyltransferase according to claim 20, wherein one or more of said mutations, preferably at least six of said mutations, in particular at least eight, at least ten, or at least twelve of said mutations are selected from the group of positions corresponding to Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, G169A, S188P, Q206C, N212G, T254A and Q271E.