Thermostable subtilisin variants and their use

Abstract

The invention relates to a subtilisin BPN' variant or homologue thereof, comprising new mutations compared to subtilisin BPN' represented by SEQ ID NO: 3 or a homologue sequence thereof. Such mutations may occur at amino acid positions selected from the group consisting of H17W, S18K, N25G, V30I, S63G, T71V, V72I, S204N, E251L, N43K or S87D.

Description

[0001] Thermostable Subtilisin variants and their use

[0002] Field of the invention

[0003] The invention relates to an enzyme, referred to as peptistabiligase, which is a peptiligase, which is characterized by single mutations as well as combinations thereof resulting in a higher enzyme stability towards external stress factors as compared to peptiligases described in the art, while not being less efficient in their ligating activity than these enzymes. The invention further relates to the use of such enzymes in methods for peptide synthesis and to a method wherein such enzyme catalyses the coupling of peptide fragments.

[0004] Background art

[0005] Processes for synthesizing peptides are generally known in the art. Relatively short peptides can be chemically synthesized in a stepwise fashion in solution or by solid phase peptide synthesis via highly optimized processes. However, peptides longer than 10-15 amino acids are often difficult to synthesize due to side reactions and, consequently, purification is troublesome. Therefore, such peptides are often synthesized by a combination of solid phase synthesis of side-chain protected peptide fragments which are subsequently chemically condensed in solution. The major drawback of chemical side-chain protected peptide fragment coupling is the racemisation that occurs upon activation of the acyl donor C-terminal amino acid residue. In contrast, in enzyme-catalysed peptide coupling reactions racemisation is not observed. Another advantage of enzyme-catalysed peptide synthesis over chemical peptide synthesis is the absence of side reactions on the side-chain functionalities. The combined use of chemically synthesized peptides which are enzymatically coupled is referred to as chemo-enzymatic peptide synthesis.

[0006] Chemo-enzymatic peptide synthesis may comprise the enzymatic coupling of peptide fragments which have individually been synthesized using chemical synthesis (either in solution and / or solid phase), fermentation, or by a combination of chemical and enzymatic coupling steps.

[0007] Enzymes which are well suited for the enzymatic coupling are the peptiligases described in WO 2016 / 056913.

[0008] These were originally developed starting from subtilisin BPN’, which is a 275 amino acid serine protease secreted from the soil bacterium Bacillus amyloliquefaciens (SEQ ID NO: 2).

[0009] It was found by Wells et al. (US 5,403,737) that the introduction of two mutations, namely S221 C and P225A altered the active site of such subtilisin BPN’ so significantly that the use of the enzyme resulted in a 500-fold increased synthesis over hydrolysis ratio (S / H ratio) as compared to wild-type subtilisin BPN’. This enzyme could now be used for the coupling of peptides in aqueous solution. This enzyme was called subtiligase. In further experiments Wells et al. introduced five additional mutations to the subtiligase, i.e. M50F, N76D, N109S, K213R and N218S, to make the enzyme more stable (Proc. Natl. Acad. Sci. USA, 1994, 91 , 12544). The new mutant, now called stabiligase, appeared moderately more resistant to a treatment with sodium dodecasulphate and guanidinium hydrochloride, but hydrolysis was still a major side reaction.

[0010] Also, the subtilisin B’ proteases were developed further by mutational engineering. Bryan (US 6,541 ,234) described the benefits of the deletion of amino acids 75 to 83, also referred to as Ca2+ binding site, but had to stabilize the resulting enzyme by introducing a number of additional mutations. Mutations found at positions P5S, D41 A, and K43N or K43R resulted in measurable but modest increases in stability. Mutations found at positions 2, 3, 73, and 206 significantly increased the half-life of the mutant relative to the parent subtilisin. The most stabilizing mutations were Q2K, S3C, A73L, and Q206C.

[0011] The remaining problem of the undesirably high hydrolytic activity of enzymes like subtiligase or stabiligase when used for peptide synthesis in an aqueous environment was only solved by providing the peptiligases of WO 2016 / 056913. The peptiligases are considered to catalyze efficient peptide coupling in water with a significantly high S / H ratio.

[0012] Furthermore, as the enzymes do not require a particular recognition motif, they can be broadly applied in traceless peptide ligation technology. In WO 2018 / 212658 further peptiligases were disclosed with one or more specific mutations in the penultimate pockets to the coupling site, i.e. in the S2’ pocket and / or in the S2 pocket, resulting in a broader peptide substrate scope and improved coupling efficiency.

[0013] In WO2019170895 and in W02019170918 examples were given for methods for enzymatically synthesising liraglutide and semaglutide by specific peptide fragment coupling, catalysed by specific peptiligases.

[0014] In WO2022171667 further peptiligases were described with one or more specific mutations improving the reaction rate and coupling efficiency of the catalysed reaction or improving the enzymes selectivity.

[0015] These enzymes and the peptide coupling technology are particularly useful in the manufacture of peptide drugs such as, for example, glucagon, dasiglucagon, glepaglutide, elsiglutide, calcitonin, thymosin, lixisenatide, teriparatide, exenatide, liraglutide, semaglutide, teduglutide and bivalirudin.

[0016] Especially for peptides with a length exceeding about 30 amino acids, the crude purity and overall yield were found to be improved when using such an enzymatic fragment condensation strategy as compared to the chemical coupling reaction.

[0017] Nevertheless, there remains a need to provide further peptiligases that can be used in the enzymatic synthesis of peptides by fragment coupling or cyclization, which are better at resisting the unfolding to a denatured state than the peptiligases known in the art, while maintaining the high coupling reaction rate and the coupling reaction efficiency of the peptiligases described in the art. Unfolding of a protein is usually accompanied by a loss of activity and can be caused by several drivers, such as the presence of cosolvents, a pH shift, or freezing and / or heating processes. Especially in biotechnological applications, there is a need for a more stable enzyme, which can be used at higher reaction temperatures, thereby allowing higher reaction rates and higher maximum solubilities, resulting in faster reactions and increased spacetime yields. There is also a need for an enzyme that is less susceptible to denaturation due to common handling related factors such as freeze-thaw-cycles, with an increased storage and shelf-life stability, making the general handling of the enzyme easier.

[0018] Therefore, there remains a need to improve the enzyme stability towards a number of external stress factors, such as heat stress.

[0019] Especially when used in an industrial setting, enzyme stability is pivotal for a robust process. Enzyme degradation would result in loss of activity and therefore a low yield. Sometimes the yield is limited due to the limited solubility of the peptide fragments and / orthe enzyme. This solubility can be increased by addition of co-solvents and / or chaotropic agents, such as, urea and guanidinium chloride, therefore such conditions become more important in industrial applications. A better solubility or stability of the starting materials, as well as of the product of the enzymatic coupling reaction might result in higher overall yield of the reaction process. Accordingly there is a need for a more stress-stable enzyme which tolerates a larger range and higher concentrations of co-solvents. Therefore, there remains a need to improve the enzyme stability towards the external stress factors of increased salt concentration and organic co-solvents.

[0020] Another need in the art is such a peptistabiligase that is robust enough to get immobilized to a solid surface, thereby allowing the enzyme to be re-cycled much more efficiently increasing the turnover number of the catalyst as well as decreasing the transformation cost.

[0021] There also remains a need to find efficient manufacturing methods for enzymatically synthesising peptides of pharmacological interest, that are suitable for up-scaling to a level that provides industry common amounts.

[0022] The present invention solves, or at least lessens, the described problems, by providing new peptiligases which are characterized by new mutations, as described below. It also discloses efficient methods of peptide synthesis by using these enzymes.

[0023] Summary of the invention

[0024] It has now been found that all or at least some of the above discussed problems can be overcome or at least lessened by providing new stable enzymes with one or more specific mutations. These enzymes will be referred to as peptistabiligases which are characterized by one or more specific mutations in several positions compared to the peptiligases which are homologues of SEQ ID NO: 3. Compared to subtilisin BPN’ as presented by SEQ ID NO: 2, the peptiligases which are homologues of SEQ ID NO: 3, all show a deletion of the amino acids at positions 75-83, a mutation at the amino acid position S221 , the mutation being S221 C or S221 selenocysteine, preferably S221 C, and a mutation at the amino acid position P225, the mutation preferably being P225A or P225N, most preferably P225N. The peptistabiligase according to the invention is further characterized by comprising at least one mutation selected from the group consisting of H17W, S18K, N25G, V30I, S63G, S204N, E251 L, N43K and S87D, wherein the amino acid mutations are defined with reference to the peptiligase represented by SEQ ID NO: 3.

[0025] The numbering of the positions is consistent in all the peptiligases described herein and is the same as in the subtilisin BPN’ presented by SEQ ID NO: 2 by simply maintaining the numbers as if the deleted amino acids (75-83) would still be present.

[0026] Accordingly, in a first aspect the present invention provides a peptistabiligase, having at least 80% sequence identity to the peptiligase represented by SEQ ID NO: 3, characterized by comprising a deletion of the amino acids at positions 75-83 compared to BPN’; and having a cysteine or selenocysteine, preferably cysteine, at position 221 ; and having an amino acid which is an alanine or asparagine, preferably asparagine at position 225; and comprising at least one mutation selected from the group consisting of H17W, S18K, N25G, V30I, S63G, S204N, E251 L, N43K and S87D; wherein the amino acid mutations are defined with reference to the peptiligase represented by SEQ ID NO: 3.

[0027] A preferred embodiment is a peptistabiligase according to the invention, further comprising at least one additional mutation selected from T71 V and V72I.

[0028] Another preferred embodiment of the invention is a peptistabiligase according to the invention, comprising both the mutations N43K and S87D.

[0029] It is preferred that in the enzyme according to the invention some positions remain unchanged compared to peptiligase SEQ ID NO: 3. Preferably the peptistabiligases have the following amino acids at one or more, preferably at 11 positions selected from the group consisting of K2, C3, S5, A9, L31 , F50, LA73, A169, P188, C206, G212, A254 and E271.

[0030] The peptistabiligase according to the invention has catalytic activity with respect to the formation of a peptide bond (coupling activity). Such activity is also referred to as “ligase activity”.

[0031] Obviously, catalytic sites responsible for certain activity should not be mutated. Other parts of the enzyme that should not be mutated are conserved regions that can be identified by multiple sequence alignments of various homologs (e.g. clustal Omega, (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ) or Geneious alignment (https: / / www.geneious.com / features / sequence-alignment / ).

[0032] A person of average skill in the art will recognize those (catalytic or conserved) sites which should not be altered to not jeopardize the enzymes functionality without undue burden.

[0033] The peptistabiligase according to the invention shows an increased stress tolerance as compared to the peptiligases, not carrying the characterizing mutations mentioned above. It shows an increased thermostability, i.e. an increased stability towards temperature increases. The enzyme’s melting temperature is increased compared to enzymes not having the mutations according to the invention. Accordingly, the invention provides enzymes which have a higher thermotolerance compared to the other peptiligases, while maintaining the high coupling reaction rate and the coupling reaction efficiency of the peptiligase.

[0034] The peptistabiligase also shows an increased stability towards the presence of salts, especially chaotropic agents, such as, for example, guanidinium chloride, and towards the presence of organic cosolvents, compared to known peptiligases, while maintaining the high coupling reaction rate and the coupling reaction efficiency of the peptiligase.

[0035] In a second aspect, the present invention provides a method for enzymatically synthesizing a peptide, wherein the coupling of two peptide fragments is carried out in an aqueous solution, and wherein the coupling is catalysed by a peptistabiligase, as described above.

[0036] Accordingly, in a second embodiment, the present invention provides a method for enzymatically synthesizing a peptide, comprising a step of coupling (a) a peptide C-terminal ester or thioester with (b) a peptide nucleophile having an N-terminal unprotected amine, wherein the coupling is performed in an aqueous solution, and wherein the coupling is catalyzed by a peptistabiligase having at least 80% sequence identity to the peptiligase represented by SEQ ID NO: 3, comprising a deletion of the amino acids at positions 75-83 compared to BPN’ (SEQ ID NO: 2); and having a cysteine or selenocysteine, preferably cysteine, at position 221 ; and having an amino acid which is an alanine or asparagine, preferably asparagine at position 225; and wherein the peptistabiligase comprises at least one mutation selected from the group consisting of H17W, S18K, N25G, V30I, S63G, S204N, E251 L, N43K and S87D; and preferably one mutation selected from T71 V and V72I; wherein the amino acid mutations are defined with reference to a peptiligase represented by SEQ ID NO: 3.

[0037] Detailed description of the invention

[0038] As used herein, the term “reaction" refers to reactions taking place under catalysis by subtilisin BPN’ variants with ligase activity, especially by peptiligases. Specifically, the terms “coupling reaction” or “coupling” refer to the formation of an amide bond (or peptide bond) between an amino group and a carboxylic group. This may involve two molecules (intermolecular coupling reaction) or one molecule (intramolecular coupling reaction).

[0039] As used herein, the term “(thio)ester” is a shortened form for the phrase “ester or thioester”.

[0040] As used herein, the term “enzyme stability” refers to the ability of the enzyme to resist high temperatures, chaotropic agents and / or organic co-solvents. An enzyme with increased stability is an enzyme which has a higher melting temperature in comparison to other similar enzymes. Whether the higher stability results from a specific mutation can be determined when compared to the reference enzyme, i.e. the reference enzyme not having these mutations.

[0041] As used herein, the term “Tmapp" refers to the apparent melting temperature of an enzyme. This refers to the temperature at which half of the enzyme is in the unfolded state.

[0042] As used herein, the term “peptide” refers to any sequence composed of two or more amino acids linked one to the other linearly by amide bonds. A peptide is usually formed by alpha-amino acids, although a peptide may comprise other amino acids, such as one or more beta-amino acids and / or one or more gamma-amino acids. Any peptide is defined by its specific sequence of amino acids. Peptides are distinguished from proteins by their shorter length, although the cut-off number of amino acids for differentiating a peptide from a protein varies in the art. Typically, a peptide comprises from 2 to 500, more typically from 2 to 200 or from 2 to 100 amino acids. Preferably, the peptide comprises at least 10 amino acids, more preferably at least 15 amino acids. Furthermore, the peptide is not more than 200, preferably not more than 100, most preferably not more than 50 amino acids long. Accordingly, most preferably the length of a peptide is between 10 and 50 amino acids.

[0043] A peptide may contain proteinogenic amino acids and / or non-proteinogenic amino acids. The proteinogenic amino acids are the alpha-amino acids with L-configuration that are encoded by the genetic code. Non-proteinogenic amino acids are non-natural amino acids, such as D-amino acids, L- or D- phenylglycine, DOPA (3,4-dihydroxy-L-phenylalanine), beta-amino acids, 4-fluoro-phenylalanine, alpha- aminoisobutiryc acid (Aib), other C-alpha-alkylated amino acids and selenocysteine (Sec, U), which is an amino acid whose structure corresponds to cysteine but with a selenium replacing the sulphur atom. A peptide may be linear, branched or cyclic, wherein a branched peptide has at least two interconnected amino acid sequences.

[0044] A peptide may be a biologically active peptide. Preferred examples of biologically active peptides include glucagon, glp-1 , glp-2 and their analogues, such as dasiglucagon, exenatide, liraglutide, semaglutide, lixisenatide, teduglutide, glepaglutide, dulaglutide, and elsiglutide; thymosin-alpha-1 , thymosin-alpha-1 analogues, teriparatide, salmon calcitonin, bivalirudin, and peptides comprising the sequence of any of these and at least one further amino acid.

[0045] As used herein, the term “cyclic peptide” refers to a peptide with a ring structure, wherein such peptide results from the formation of an amide bond between the terminal alpha-amino group and the terminal alpha-carboxylic group of an amino acid sequence, also referred to as “cyclization reaction”. In particular, such amino acid sequence has at least 12 amino acids.

[0046] As used herein, the term “peptide bond” refers to the amide bond between (i) the amino group of one amino acid and (ii) the carboxyl group of another amino acid. In particular, a peptide bond may be between the alpha-amino group of one alpha-amino acid and the alpha-carboxyl group of another alpha-amino acid.

[0047] As used herein when referring to proteins or enzymes, the term "mutated" or “mutation” means that at least one amino acid in the wild-type or naturally occurring protein or enzyme 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 includes, for example, site-directed mutagenesis by means of PCR or via oligonucleotide-mediated mutagenesis as described, for instance, in Siloto et al. (2012) Site saturation mutagenesis: Methods and applications in protein engineering, Biocatalysis and Agricultural Biotechnology 1 181-189. The term "mutated" or “mutation” as used herein when referring to nucleic acids or genes means that at least one nucleotide in the nucleic acid sequence, 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 or quantitatively altered function or resulting in a “knock-out” of that nucleic acid, which refers to a nucleic acid that no longer encodes a protein with the functionality it used to encode before it was mutated.

[0048] In the present disclosure, a mutation (of an amino acid) is described by using the single letter amino acid code of the amino acid that is substituted, followed by the number designating where in the protein amino acid sequence the substitution is made. This number is the amino acid position of the reference amino acid sequence, often referred to as the wild type. Thus, for the mutated amino acid sequence it is the amino acid position corresponding to the position with that number in the respective reference enzyme. Depending on the context, the reference enzyme referred to herein can be the subtilisin BPN’ prime (SEQ ID NO 2) or a peptiligase according to any of SEQ ID NO 3 to SEQ ID NO: 6. The relevant reference enzyme is indicated. Due to one or more other mutations at a lower position (additions, insertions, deletions, etc.) the actual position in the mutant is not necessarily identical, however in the embodiments of this invention they are. The skilled person will be able to determine the corresponding positions using a generally known alignment technique, such as NEEDLE. The number is then followed by the single letter code of the amino acid that replaces the reference amino acid therein. For example, F189W denotes the substitution of phenylalanine (F) at the position 189 with tryptophan (W). X is used to indicate that any proteinogenic amino acid other than the amino acid which is to be substituted may be present at this position. For example, F189X denotes the substitution of phenylalanine at the position 189 with any other proteinogenic amino acid.

[0049] As used herein, the term “ligase” refers to an enzyme having catalytic activity in the coupling of two peptides by catalysing the formation of a peptide bond between the C-terminus of a first peptide and the N- terminus of another peptide. Such activity is also referred to as “ligase activity”. This activity may also be referred to as “cyclase activity” in case that such enzyme has catalytic activity in the formation of an intramolecular peptide bond between the C-terminus and the N-terminus of the same peptidic molecule. Therefore, the same enzyme can have ligase and / or cyclase activity. An enzyme may be characterized as having ligase activity when its synthesis over hydrolysis ratio is above 1 . This S / H ratio can be determined by HPLC analysis of the respective amounts.

[0050] A ligase typically has an S / H ratio larger than 1 , preferably 2 or more, in particular 5 or more in the used reaction medium, in particular in a reaction medium comprising water, more in particular an aqueous medium, also referred to as aqueous solution. The upper value of this quotient is not critical; it may be, for instance, 100 or less.

[0051] According to the invention, a peptistabiligase is an enzyme, having ligase and / or cyclase activity and having a sequence identity of at least 80 %, preferably at least 85 %, more preferably at least 90 %; even more preferably the sequence identity is at least 95 %, at least 96 %, at least 97 %, at least 98 % or at least 99 % with the respective reference enzyme. According to the invention the preferred reference enzyme is the peptiligase represented by SEQ ID NO 3. The percent identity is determined according to the NEEDLE EMBOSS method as outlined below. Evidently, the percent identity will be less than 100 %. The percent identity will depend on the number of mutations and the length of the peptide (enzyme) with which the homologue is compared.

[0052] 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 the commonly used His tags or other tags used, for instance, for purification, signaling, solubilization and localization purposes, are not to be considered in the determination of the percent identity. 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 carried out over a length of at least 200 amino acids of the sequences being compared.

[0053] 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.

[0054] 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 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.

[0055] The percent identity as herein defined is obtainable from NEEDLE and is labelled in the output of the program as “longest identity”.

[0056] In particular, the invention provides an isolated enzyme. Herein the term “isolated” means that it was isolated from the organism wherein it has been expressed - typically a recombinant organism, if it has been produced in an organism - or from the reaction medium in which it has been synthesized.

[0057] In particular, an enzyme according to the present invention is considered isolated either in the crude form or substantially purified by any suitable technique such as, for example, the single-step purification method disclosed in Smith and Johnson, Gene 67:31-40 (1988).

[0058] An enzyme according to the present invention can be provided in at least substantially pure form, wherein the term “substantially pure enzyme” refers to an enzyme of at least 75 wt. %, preferably of more than 80 wt. % purity. The enzyme may also be provided in a mixture with one or more other components, e.g. in the form of a stock solution, preferably in an aqueous buffer solution.

[0059] An enzyme according to the present invention may comprise a terminal His tag, preferably a 6-His tag.

[0060] The degree of resistance towards melting at increasing temperatures, is considered an indicator of enzyme stability. The stability or robustness of the enzyme can be determined by identifying the melting temperature Tmappof the enzyme. This may be analysed by using an assay, also referred to as thermofluor assay, which uses a fluorescent dye binding to hydrophobic regions of the enzyme which get exposed during thermal denaturation. By slowly increasing the temperature while measuring the fluorescence signal the Tmappof a variant can be determined. The higher the detected Tmapp, the more stable the enzyme. The assay used in the experimental section is described in Lavinder et al. (2009) J.AM.CHEM.SOC. 2009, 131 , 3794-3795, wherein the method is referred to as HTTS (high-throughput thermal screening) method.

[0061] The amino acid sequence of subtilisin BPN’ is given in SEQ ID NO: 2 (mature form). The nucleic acid sequence encoding for subtilisin BPN’ amino acids -107 to 275 is given in SEQ ID NO: 1 .

[0062] The amino acid sequence of a typical peptiligase, which is a suitable reference enzyme is given in SEQ ID NO: 3. It shows a subtilisin BPN’ variant, with deletion of the amino acids corresponding to positions 75-83 (so called Ca2+binding loop), with the S221 mutation being a S221 C and P225 mutation being a P225N and having 14 more mutations being Q2K, S3C, P5S, S9A, 131 L, K43N, M50F, A73L, G169A, S188P, Q206C, N212G, T254A and Q271 E, wherein the amino acid mutations are defined with reference to BPN’ represented by SEQ ID NO: 2.

[0063] Surprisingly, it was found that in relation to peptiligases such as the one presented by SEQ ID NO 3, or those known from WO2016 / 06913, any single additional mutation selected from the group consisting of H17W, S18K, N25G, V30I, S63G, T71V, V72I, S204N, E251 L, N43K and S87D, significantly improves the enzyme stability. Combining some of these mutations results in even further improvements of the enzyme stability.

[0064] Accordingly, the present invention provides a peptistabiligase, having at least 80% sequence identity to the peptiligase represented by SEQ ID NO: 3, characterized by comprising a deletion of the amino acids at positions 75-83 compared to BPN’; and having a cysteine or selenocysteine, preferably cysteine, at position 221 ; and having an amino acid which is an alanine or asparagine, preferably asparagine at position 225; and comprising at least one mutation selected from the group consisting of H17W, S18K, N25G, V30I, S63G, S204N, E251 L, N43K and S87D; wherein the amino acid mutations are defined with reference to the peptiligase represented by SEQ ID NO: 3.

[0065] It is preferred that the peptistabiligase has at least 90% sequence identity, and even more preferably 93% sequence identity to the peptiligase represented by SEQ ID NO: 3.

[0066] It is a preferred embodiment wherein the peptistabiligase further comprises at least one additional mutation selected from T71V and V72I.

[0067] In a further preferred embodiment, the peptistabiligase further comprises both mutations N43K and S87D.

[0068] The amino acid sequences of peptistabiligases according to the invention therefore differ from the SEQ ID NO:3 by at least one stabilizing mutation selected from the group consisting of H17W, S18K, N25G, V30I, S63G, S204N, E251 L, N43K and S87D with reference to SEQ ID NO 3.

[0069] Surprisingly, it was also found, that when combining two or three or four or five or six mutations of H17W, S18K, N25G, V30I, S63G, T71V, V72I, S204N, E251 L, N43K or S87D, a cumulative effect was observed.

[0070] Thus, in preferred embodiments, the peptistabiligase according to the present invention comprises one of the following:

[0071] - one mutation selected from the group consisting of H17W, S18K, N25G, V30I, S63G, T71 V, V72I, S204N, E251 L, N43K, S87D; or preferably

[0072] - two, three or four mutations selected from the group consisting of H17W, S18K, N25G, V30I, S63G, T71 V, S204N, E251 L and the combination of N43K and S87D; or selected from the group consisting of H17W, S18K, N25G, V30I, S63G, V72I, S204N, E251 L and the combination of N43K and S87D; or more preferably

[0073] - five mutations selected from the group consisting of S18K, N25G, S63G, T71V, S204N, E251 L and the combination of N43K and S87D; or from the group consisting of S18K, N25G, S63G, V72I, S204N, E251 L and the combination of N43K and S87D; or even more preferably

[0074] - six mutations, wherein four mutations are selected from the group consisting of S18K, N25G, S63G, T71 V and S204N; or from the group consisting of S18K, N25G, S63G, V72I, S204N and E251 L; and further two mutations are N43K and S87D;

[0075] The peptistabiligase according to the invention preferably has the same amino acids as peptiligase SEQ ID NO: 3 in one position, preferably at two or three, more preferably at four or five or six positions, even more preferably at seven or eight or nine positions, but most preferably at 10 or 11 positions selected from the group consisting of K2, C3, S5, A9, L31 , F50, L73, A169, P188, C206, G212, A254 and E271 ; more preferably from the group consisting of K2, S5, A9, L31 , F50, L73, A169, P188, G212, A254 and E271 .

[0076] In a preferred embodiment the peptistabiligase maintains at least eleven of these positions unaltered compared to those of SEQ ID NO: 3. In comparison to the subtilisin BPN’ wildtype of SEQ ID NO:2 these amino acids are referred to as mutations Q2K, S3C, P5S, S9A, 131 L, M50F, A73L, G169A, S188P, Q206C, N212G, T254A and Q271 E.

[0077] In a preferred embodiment, the peptistabiligase according to the invention further comprises one or more mutations at amino acid positions selected from the group consisting of S33, N62, E156, G166, Y217, N218 and F189, such mutation being preferably selected from the group consisting of S33T, N62A, N62R, N62K, E156S, E156N, E156K, E156R, G166S, G166E, G166D, Y217L, Y217H, Y217R, N218S, N218D and F189W.

[0078] In another preferred embodiment, peptistabiligase according to the invention further comprises at least one mutation at the amino acid positions M222 and Y217, or pairs of mutations, wherein the single mutation is preferably selected from the group consisting of M222P, M222G, M222H, Y217H, Y217G, Y217F, Y217L, and Y217R, and wherein the pairs of mutations are preferably selected from the group consisting of M222P and Y217H; M222P and Y217G; M222G and Y217F; M222G and Y217G; M222G and Y217L; M222H and Y217R and, M22G and Y217R. These mutations do not necessarily add to the stability of the preferred enzymes, but are responsible for an enzyme’s coupling specificity and an improved yield.

[0079] In one embodiment the invention provides a peptistabiligase which has an increased melting temperature (Tmapp') compared to a peptiligase. The Tmappis increased at least 0.5 degree centigrade, more preferably 1 degree centigrade, even more preferably 2 degrees centigrade and even more preferably 3 degrees centigrade.

[0080] The peptistabiligase according to the invention preferably is immobilized to a solid support. There are several methodologies for water reactions based on entrapment, encapsulation, covalent binding and cross linking. The technology of choice is determined by the peptistabiligase of choice and the substrates used in the reaction.

[0081] The enzymes of the present invention are generally produced by recombinant methods, preferably by expression of a subtilisin BPN’ DNA which has been mutated such that upon expression it results in a subtilisin BPN’ variant, referred to as peptistabiligase according to the invention which is enzymatically active.

[0082] Accordingly, the invention further provides a recombinant method for preparing the enzyme according to the invention, said method comprising the steps of: a) providing a recombinant host cell functionally expressing a gene encoding the enzyme, e.g. bacterial cells such as E. coli or Bacillus; b) culturing said host cell under conditions which provide for the expression of the enzymatically active enzyme; and c) recovering the expressed enzyme from said microbial host.

[0083] The invention further provides a recombinant polynucleotide comprising a sequence which encodes for an enzyme according to the invention.

[0084] The invention further provides a host cell, comprising a polynucleotide according to the invention, which polynucleotide is capable of expressing the enzyme.

[0085] The enzyme according to the invention shows an increased stability towards chaotropic agents such as, for example, guanidinium chloride as compared to other known peptiligases. The enzyme variants according to the invention show much higher retained ligating activity in the presence of different concentrations of guanidinium chloride, compared to enzyme variants not having the specified mutations. The activity of the enzymes according to the invention is retained in at least 0.5 M more GnCI, more preferably 1 M more GnCI, even more preferably 2 M more GnCI, while the reference enzyme's activity declined significantly.

[0086] Therefore, in another embodiment of the invention, the invention provides a method for enzymatically synthesizing a peptide, comprising a step of coupling (a) a peptide C-terminal ester or thioester with (b) a peptide nucleophile having an N-terminal unprotected amine, wherein the coupling is performed in an aqueous solution, and wherein the coupling is catalysed by a peptistabiligase according to the invention; having at least 80% sequence identity to the peptiligase represented by SEQ ID NO: 3, comprising a deletion of the amino acids at positions 75-83, compared to BPN’ (SEQ ID NO: 2); and having an amino acid which is a cysteine or selenocysteine, preferably cysteine, at position 221 ; and having an amino acid which is an alanine or asparagine, preferably asparagine at position 225; and wherein the peptistabiligase comprises at least one mutation selected from the group consisting of H17W, S18K, N25G, V30I, S63G, S204N, E251 L, N43K and S87D, and preferably at least one mutation selected from T71 V and V72I; and wherein the amino acid mutations are defined with reference to the peptiligase represented by SEQ ID NO: 3.

[0087] It is preferred that a peptistabiligase is used which has the amino acids of peptiligase SEQ ID NO: 3 at one or more, preferably at 11 , positions selected from the group consisting of K2, C3, S5, A9, L31 , F50, L73, A169, P188, C206, G212, A254 and E271.

[0088] It is preferred that the peptistabiligase used in said method has at least 90% sequence identity, and even more preferably 93% sequence identity to the peptiligase represented by SEQ ID NO: 3.

[0089] The use of an enzyme according to the invention extends beyond the catalysis of the cyclization reaction of a peptide and / or the coupling of a peptide C-terminal (thio)ester to a peptide nucleophile, such as described above. The peptistabiligase may be used in the formation of an amide bond, other than a peptide bond, although its use related to a peptide bond is particularly preferred.

[0090] In the method according to the invention, the reaction is typically performed in an aqueous solution, preferably, comprising a buffer. The aqueous solution may also contain organic solvents or chaotropic agents. Suitable solvents are water miscible co-solvents.

[0091] It may be advantageous to add additives to the aqueous solution in order to improve the solubility of the peptide fragments orto improve the reaction yield. Such additives, also referred to as chaotropic agents may be salts or organic molecules. Common chaotropic agents include guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, sodium dodecyl sulfate, thiourea, polysorbate, and urea.

[0092] Accordingly it is preferred that in the method according to the invention the aqueous solution comprises a chaotropic agent. Preferably the chaotropic agent is selected from the group consisting of guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, sodium dodecyl sulfate, thiourea, polysorbate (also known as Tween®) and urea, most preferably the chaotropic agent is guadinium chloride. A suitable concentration range of guadinium chloride in the aqueous solution is 1 to 5 M. This allows coupling reactions of fragments which otherwise do not dissolve. In a preferred embodiment the aqueous solution therefore comprises guanidinium chloride at a concentration range of 1-5 M, preferably 2-4 M.

[0093] The peptistabiligases according to the invention also show an increased stability compared to peptiligases towards the presence of organic co-solvents such as DMF, DMSO or acetonitrile in the reaction medium. Therefore, in the method according to the invention, the aqueous solution may comprise organic solvents. It is preferred that such one or more organic solvents are selected from the list consisting of N,N- dimethylformamide (DMF), N-methyl-pyrrolidinone (NMP), N-ethyl-pyrrolidone (NEP), N,N- dimethylacetamide (DMA), dimethylsulphoxide (DMSO), acetonitrile (ACN), ether, such as tetrahydrofuran (THF), 2-methyl-tetrahydrofuran (Me-THF), 1 ,2-dimethoxyethane, an alcohol, such as methanol, ethanol, isopropanol, tert-butanol, 2,2,2-trifluoroethanol (TFE), and 1 ,1 ,1 ,3,3,3-hexafluoroisopropanol.

[0094] Depending on the stability of the peptiligase and the solubility of the peptide substrates, the amount of co-solvent is adjusted. If the solubility of the peptide fragments or the coupled peptide is low up to 50% of the aqueous solution may be an organic solvent. Many peptiligases known from the art become less active or inactive at such concentrations, but the enzymes according to the invention are surprisingly resistant towards presence of such high amounts of organic solvents. The enzyme variants according to the invention show much higher retained activity rates in the presence of increasing concentrations of these organic co-solvents, as exemplified in the examples, than peptiligases, such as exemplified in SEQ ID NO 3.. The activity is retained in at least 10 vol% more co-solvent compared to the reference enzyme, more preferably 15 vol% more co-solvent, even more preferably 20 vol% more co-solvent.

[0095] Accordingly, a method according to the invention is preferred wherein the aqueous solution comprises organic solvents at a concentration range of 20-50%, preferably of 30-50%, most preferably at 40-50%.

[0096] In principle the temperature during the coupling or cyclization reaction of peptiligases is not critical, as long as a temperature is chosen at which the enzyme to be used shows sufficient activity and stability. Such a temperature can be routinely determined. Generally, the temperature during the coupling or cyclization reactions is in the range of 20-50°C. However, whereas common peptiligases tend to denature at higher temperatures, the enzymes according to the invention tolerate higher temperatures. Therefore, according to the invention methods are preferred which are performed at temperatures of 20° to 70°C, more preferably at 30° to 70°C, most preferably at 35° to 70°C.

[0097] The peptide C-terminal ester or thioester (commonly referred to herein as (thio)ester) typically used was an activated (thio)ester, i.e. it contains a carboxy ester or carboxy thioester group that can take part in the reaction. In principle, any substituted or unsubstituted alkyl or any substituted or unsubstituted aryl (thio)ester can be used. Typical examples of (thio)esters which can take part in the reaction are methyl-, ethyl-, propyl-, isopropyl-, phenyl-, benzyl- (such as p-carboxy-benzyl-), 2,2,2-trichloroethyl-, 2,2,2- trifluoroethyl-, cyanomethyl- and carboxamidomethyl-(thio)esters (OCam (thio)esters).

[0098] It has been described in detail e.g. in WO2022171667 which esters work particularly well, how these may be synthesized and / or protected.

[0099] In a preferred embodiment the synthesized peptide in the method according to the invention is selected from the group consisting of liraglutide, semaglutide, glucagon, dasiglucagon, glepaglutide, elsiglutide, dulaglutide, thymosin, thymosin-alpha-1 , thymosin-alpha-1 analogues, calcitonin, lixisenatide, teriparatide, exenatide, bivalirudin or teduglutide. In one embodiment the invention provides a method for enzymatically synthesizing teduglutide, comprising a step of coupling (a) a peptide C-terminal ester His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met- Asn-Thr-lle-Leu-Asp-Asn-Leu-ester (SEQ ID NO 9) with (b) a peptide nucleophile having an N-terminal unprotected amine, Ala-Ala-Arg-Asp-Phe-lle-Asn-Trp-Leu-lle-GIn-Thr-Lys-lle-Thr-Asp (SEQ ID NO 10), wherein the coupling is performed in an aqueous solution, and wherein the coupling is catalysed by a peptistabiligase according to the invention.

[0100] In a preferred embodiment the aqueous solution comprises guanidinium chloride at a concentration of 4M. Preferably the peptistabiligase of this embodiment comprises the mutations, S18K, N25G, N43K, S87D, S63G, T71 V, V72I and S204N wherein the amino acid mutations are defined with reference to the peptiligase represented by SEQ ID NO: 3.

[0101] Preferably the peptistabiligases according to the invention are used for producing therapeutic peptides by enzymatic coupling as described herein, in an industrial setting, where high yields of peptide are needed, and the processes need to be efficient. The surprising robustness of these peptistabiligase allows for a good recovery rate of these enzymes after the reaction completed, while still providing for high coupling reaction rates and high coupling reaction efficiency.

[0102] The invention will now be illustrated by the following examples.

[0103] ABBREVIATIONS

[0104] SPPS Solid Phase Peptide Synthesis

[0105] CTC 2-chloro-trityl chloride

[0106] AEEA 2-[2-(2-aminoethoxy)ethoxy]acetyl

[0107] Cbz Benzyloxycarbonyl

[0108] For Formyl

[0109] Fmoc 9-Fluorenyl methoxycarbonyl

[0110] Boo Tert-butyloxycarbonyl

[0111] Smoc 2.7-disulfo-9-fluorenylmethoxycarbonyl

[0112] Ac Acetyl

[0113] PhAc Phenacetyl

[0114] Trt Trityl (triphenylmethyl) tBu Tert-butyl

[0115] Pbf 2.2.4.6.7-Pentamethyl-dihydrobenzofuran-5-sulfonyl eq Equivalent h hour / s min minute / s

[0116] HPLC High Performance Liquid Chromatography

[0117] DIPEA N,N-Diisopropylethyl amine

[0118] TFA Trifluoroacetic acid

[0119] TIS Triisopropylsilane AC2O Acetic anhydride

[0120] DMF N, N-Dimethylformamide

[0121] DMA N, N-Dimethylacetamide

[0122] DCM Dichloromethane

[0123] THF Tetrahydrofuran

[0124] NMP N-Methyl-2-pyrrolidinone

[0125] MTBE Methyl-tert-butylether

[0126] MeOH Methanol

[0127] DCC N, N ‘-D icy clohexy Icarbod 11 m ide

[0128] EDC N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide

[0129] HOBt 1 -Hydroxybenzotriazole

[0130] HOAt 1 -Hydroxy-7-azabenzotriazole

[0131] TCEP tris(2-carboxyethyl)phosphine

[0132] Tricine N-(2-Hydroxy-1 ,1-bis(hydroxymethyl)ethyl)glycine

[0133] OCam ester carboxamidomethyl ester

[0134] EXAMPLES

[0135] Production of enzymes (or enzyme variants) (for use) according to the invention

[0136] Mutagenesis, Cloning and Expression

[0137] The reference enzyme denoted as SEQ ID NO 4: Ptl-10 is a subtilisin BPN’ variant with deletion of the amino acids corresponding to positions 75-83 and having mutations Q2K, S3C, P5S, S9A, 131 L, K43N, M50F, A73L, A75-83, E156N, G166D, G169A, S188P, F189W, Q206C, N212G, Y217R, N218S, S221 C, M222G, P225N, T254A and Q271 E, plus a 6-His tag. The gene coding for Ptl-10 with a His-tag was cloned into a pUB-110 based E. coll - B. subtilis shuttle vector (i.e. pBS42 or pBES).

[0138] The corresponding amino acid sequence is numbered according to the subtilisin BPN’ numbering scheme. Amino acids -107 to -1 comprise the signal sequence, the pre sequence and a pro sequence which are cleaved off upon full maturation. Amino acids 1-275 comprise the mature enzyme which exhibits the full catalytic activity. To enable a fast and efficient purification after amino acid 275 a C-terminal His-tag is attached. Because of the removal of the calcium binding site, Ptl-10 contains a deletion of 9 amino acids compared to subtilisin BPN’ comprising the amino acids corresponding to L75, N76, N77, S78, I79, G80, V81 , L82 and G83 in subtilisin BPN’. To maintain the subtilisin BPN’ numbering for Ptl-10 (and for the enzymes of the invention) the numbering leaps from 74 to 84. In the shuttle vector, the expression of the gene is under the control of aprE promoter. The resulting plasmid pBES-Ptl-10 HIS was propagated in E. coll TOP10 and transformed into B. subtilis BH13 with the gene deletions as stated in the table below. How to generate such a B.subtilis BH13 strain is disclosed in detail in Halmschlag (2020). Tailored poly-y- glutamic acid production with Bacillus subtilis 168. PhD Thesis. Rheinisch-Westfalische Technische Hochschule Aachen, Germany. Using pBES- Ptl-10 HIS as the template, mutagenesis was carried out by the MISO method (L.A. Mitchell at al, ACS Synth Biol 2013,:473-7). Alternatively, other methods for site directed mutagenesis known in the art may be used. Alternatively, plasmid was synthesized by Ranomics Inc., CAN.

[0139] Production and purification of synthetic subtilisin BPN’ variants which carry a His-tag:

[0140] A single microbial colony of B. subtilis containing a plasmid with the subtilisin variant gene of interest was inoculated in 5mL LB with kanamycin (10 pg / mL) at 37°C in a shaking incubator. To the 30 mL Terrific Broth supplemented with antibiotic (kanamycin 10 pg / mL) and amino acids (100 mg / L Trp, 100 mg / L Met and 100 mg / L Lys) 0.6 mL of the overnight culture was added. The cells were grown 48h at 37°C in a shaking incubator (200 rpm). The cells were harvested by centrifugation (30 min, 4,000 rpm, 4°C). The medium (30 mL) was decanted and for His-tag purification PureCube Co-NTA Agarose XL resin (2.5 ml, Cube Biotech) was added to a plastic column cartridge. The resin was first washed with 20 mL demineralized water and equilibrated with 20 mL of buffer A. The crude medium with resin was incubated 1 hour at orbital shaker at 4°C. After incubation the resin was washed with 100 mL buffer A. The enzyme was eluted with 15 mL buffer B (25 mM Tricine, pH 7.5, 0.5 M NaCI, 0.5 M imidazole). The elute was further incubated 30 min with 6 mM TCEP (tris(2-carboxyethyl)phosphine) and concentrated on a Vivaspin® unit (20ml, 10kDa MW cut-off) by centrifugation (30 min, 4000 rpm, 4°C) and the buffer was exchanged to 125 mM Tricine, 0.5M NaCI, pH 7.5 in three washing / concentrating steps (15 ml buffer, 10 min, 4, 000 rpm, 4°C). Alternatively, potassium phosphate buffer can be used.

[0141] The purity was determined by SDS-PAGE and densitometric analysis (BioRad GS-900). Enzyme concentration was determined by measuring absorbance at 280 nm using Nanodrop (Thermo Scientific) with 1 Abs=1 mg / ml. The obtained aqueous solution (125 mM Tricine, 0.5M NaCI, pH 7.5) containing about 0.1-2 mg / ml of the obtained enzyme. The solution with corrected enzyme concentration (mg / ml*purity) was used as such for the coupling and cyclization reaction.

[0142] For a detailed description regarding the production and purification of synthetic subtilisin BPN’ variants reference is made to WO 2016 / 056913 and WO 2018 / 212658.

[0143] Enzymatic fragment coupling examples

[0144] Materials and methods

[0145] Unless stated otherwise, chemicals were obtained from commercial sources and used without further purification. Analytical HPLC was performed on an HP1090 Liquid Chromatograph, using a reversed-phase column (Phenomenex, C18, 5 pm particle size, 150 x 4.6 mm) at 40°C. UV detection was performed at 220 nm using a UV-VIS 204 Linear spectrometer. The gradient program was: 0-25 min linear gradient ramp from 5% to 98% eluent B and from 25.1-30 min 5% eluent B (eluent A: 0.5 mL / L methane sulfonic acid (MSA) in H2O, eluent B 0.5 mL / L MSA in acetonitrile). The flow was 1 mL / min from 0-25.1 min and 2 mL / min from 25.2-29.8 min, then back to 1 mL / min until stop at 30 min. Injection volumes were 20 pL. Preparative HPLC was performed on a Varian PrepStar system using a stationary-phase column (Pursuit XRs, C18, 10 pm particle size, 500 x 41 .4 mm). LC-MS was performed on an Agilent 1200 series Liquid Chromatograph, using a reversed-phase column (Phenomenex, C18, 5 pm particle size, 150 x 4.6 mm) at 40°C. UV detection and gradient program were as described for analytical HPLC. The molecular weights were determined using an Agilent 6130 quadrupole LC / MS system. Protocol 1: Peptide-OCam-Leu-OH esters preparation

[0146] 1 g of Fmoc-Leu-Wang resin (with a loading of 0.72 mmol / g) was washed with DCM (2 x 2 min, 10 mL) and DMF (2 x 2 min, 10 mL) and Fmoc-deprotected using piperidine / DMF (1 / 4, v / v, 2 x 8 min, 10 mL). After washing with DMF (2 x 2 min, 10 mL), DCM (2 x 2 min, 10 mL) and DMF (2 x 2 min, 10 mL), iodoacetic acid (4 eq) was coupled to the resin using DCC (4 eq) and HOAt (4 eq) in DCM (45 min, 10 mL). After washing with DMF (2 x 2 min, 10 mL), DCM (2 x 2 min, 10 mL) and THF (2 x 2 min, 10 mL), the resin was loaded with a Fmoc-protected amino acid using 4 eq. Fmoc-XXX-OH and 10 eq. DIPEA in DMF / THF (1 / 1 , v / v, 10 mL) at 50°C for 20h. Here and in other parts of this disclosure ‘XXX’ stands for one amino acid (variable depending on the target peptide, as indicated in the examples below).

[0147] After washing with DMF (2 x 2 min, 10 mL), DCM (2 x 2 min, 10 mL) and DMF (2 x 2 min, 10 mL), standard SPPS protocols were followed to elongate the peptide. Cleavage from the resin and side-chain deprotection was performed using a mixture of TFA, TIS and water (95 / 2.5 / 2.5, v / v / v, 15 mL) for 120 min. The crude peptide was precipitated using MTBE / n-heptanes (1 / 1 , v / v, 50 mL). The precipitated peptide was collected by centrifugation and washed twice with MTBE / n-heptanes (1 / 1 , v / v, 50 mL) followed by lyophilization from acetonitrile / water (1 / 1 , v / v, 50 mL).

[0148] Protocol 2: C-terminal amide peptide nucleophiles preparation

[0149] 1 g of Rink resin (4-((2,4-dimethoxyphenyl)(Fmoc-amino)methyl)phenoxyalkyl linker, with a loading of 0.64 mmol / g) was washed with DCM (2 x 2 min, 10 mL) and DMF (2 x 2 min, 10 mL) and Fmoc- deprotected using piperidine / DMF (1 / 4, v / v, 2 x 8 min, 10 mL). Standard SPPS protocols were followed to elongate the peptide. Cleavage from the resin and side-chain deprotection was performed using a mixture of TFA / TIS / water (95 / 2.5 / 2.5, v / v / v, 15 mL) for 120 min. The crude peptide was precipitated using MTBE / n- heptanes (1 / 1 , v / v, 50 mL). The precipitated peptide was collected by centrifugation, washed twice with MTBE / n-heptanes (1 / 1 , v / v, 50 mL) and dried in vacuo. Prior to enzymatic ligation, the crude peptides were purified by preparative HPLC followed by lyophilization of the pure fractions.

[0150] Protocol 3: C-terminal amide peptide nucleophiles preparation

[0151] A preloaded Wang resin (Fmoc-Xxx-Wang-resin with a loading of 0,3 mmol / g) was washed with DCM (2 x 2 min, 10 mL) and DMF (2 x 2 min, 10 mL) and Fmoc-deprotected using piperidine / DMF (1 / 4, v / v, 2 x 8 min, 10 mL). Standard SPPS protocols were followed to elongate the peptide. Cleavage from the resin and side-chain deprotection was performed using a mixture of TFA / TIS / water (95 / 2.5 / 2.5, v / v / v, 15 mL) for 120 min. The crude peptide was precipitated using MTBE / n-heptanes (1 / 1 , v / v, 50 mL). The precipitated peptide was collected by centrifugation, washed twice with MTBE / n-heptanes (1 / 1 , v / v, 50 mL) and dried in vacuo. Prior to enzymatic ligation, the crude peptides were purified by preparative HPLC followed by lyophilization of the pure fractions.

[0152] Protocol 4: N-Acetyl-protected peptide activated ester preparation

[0153] After SPPS of the desired sequence according to one of the protocols 1 or 2, the resin bound peptide was Fmoc-deprotected using piperidine / DMF (1 / 4, v / v, 2 x 8 min, 10 mL). The resin was washed with DMF (2 x 2 min, 10 mL), DCM (2 x 2 min, 10 mL) and DMF (2 x 2 min, 10 mL) and the peptide / V-terminal amine function was acetylated using a mixture of AC2O (10 vol%), DIPEA (5 vol%), HOBt (0.2 wt%) in DMF (2 x 10 min, 10 mL). The resin was washed with DMF (3 x 2 min, 10 mL) and DCM (3 x 2 min, 10 mL). Cleavage from the resin and side-chain deprotection was performed using a mixture of TFA / TIS / water (95 / 2.5 / 2.5, v / v / v, 15 mL) for 120 min. The crude peptide was precipitated using MTBE / n-heptanes (1 / 1 , v / v, 50 mL). The precipitated peptide was collected by centrifugation, washed twice with MTBE / n-heptanes (1 / 1 , v / v, 50 mL) and dried in vacuo. Prior to enzymatic ligation, the crude peptides were purified by preparative HPLC followed by lyophilization of the pure fractions.

[0154] Analogously, N-Phenacetyl-protected peptide activated esters are also prepared, by using PhAc2O instead of AC2O.

[0155] Peptiligases, which were used in the experimental section as reference enzymes are described in detail below:

[0156] Reference enzyme Ptl-10, a thymoligase GM0400, is a subtilisin BPN’ variant (not according to the invention), comprising a deletion of the amino acids at positions 75-83 compared to BPN’ (SEQ ID NO: 2); and having a cysteine at position 221 ; and having an asparagine at position 225; having further mutations with reference to SEQ ID NO: 2 as follows: Q2K, S3C, P5S, S9A, 131 L, K43N, M50F, A73L, E156N, G166D, G169A, S188P, F189W, Q206C, N212G, Y217R, N218S, M222G, T254A and Q271 E, plus a 6-His tag. This reference enzyme has the amino acids K2, C3, S5, A9, L31 , N43, F50, L73, A169, P188, C206, G212, A254 and E271 and differs from SEQ ID NO: 3 by providing the following mutations E156N, G166D, F189W, Y217R, N218S und M222G, which are of no relevance to the stability. It is represented by SEQ ID NO: 4.

[0157] Reference enzyme Ptl-02, an omniligase EU GMO 79, is a subtilisin BPN’ variant (not according to the invention), comprising a deletion of the amino acids at positions 75-83 compared to BPN’ (SEQ ID NO: 2); and having a cysteine at position 221 ; and having an asparagine at position 225; having further mutations with reference to SEQ ID NO: 2 as follows: Q2K, S3C, P5S, S9A, 131 L, K43N, M50F, A73L, 1107V, E156S, G166S, G169A, S188P, F189W, Q206C, N212G, Y217H, N218S, M222P, T254A and Q271 E, plus a 6-His tag. This reference enzyme has the amino acids K2, C3, S5, A9, L31 , N43, F50, L73, A169, P188, C206, G212, A254 and E271 and differs from SEQ ID NO: 3 by providing the following mutations 1107V, E156S, G166S, F189W, Y217H, N218S and M222P, which are of no relevance to the stability. It is represented by SEQ ID NO: 5.

[0158] Reference enzyme Ptl-84 is a subtilisin BPN’ variant (not according to the invention), comprising a deletion of the amino acids at positions 75-83 compared to BPN’ (SEQ ID NO: 2); and having a cysteine at position 221 ; and having an asparagine at position 225; having further mutations with reference to SEQ ID NO: 2 as follows: Q2K, S3C, P5S, S9A, 131 L, K43N, M50F, N62A, A73L, E156N, G166E, G169A, S188P, F189W, Q206C, N212G, Y217H, N218D, M222P, T254A, and Q271 E, plus a 6-His tag .This reference enzyme has the amino acids K2, C3, S5, A9, L31 , N43, F50, L73, A169, P188, C206, G212, A254 and E271 and differs from SEQ ID NO: 3 by providing the following mutations E156N, G166E, F189W, Y217H, N218D and M222P. It is represented by SEQ ID NO: 6.

[0159] Sequences of peptistabiligases, i.e. enzymes according to the invention, differ from SEQ ID NO: 3 in having at least one of the following mutations H17W, S18K, N25G, V30I, S63G, S204N, E251 L, N43K and S87D, preferably they have also one or both of the additional mutations T71V and V72I. Examples thereof are presented as SEQ ID NO 7 and SEQ ID NO 8.

[0160] Example 1 : Stabilized variants of Ptl-1O, according to sequence SEQ ID NO: 4.

[0161] The melting temperatures of reference enzyme Ptl-1O (in the table refered to as Wild-type) and variants with a single stability mutation were determined using a dye-binding thermal shift screen, as described in detail in Lavinder et al. (2009) J.AM.CHEM.SOC. 2009, 131 , 3794-3795, wherein the method is referred to as HTTS (high-throughput thermal screening) method. 19 pL enzyme stock were mixed with 1 pL Sypro Orange (1000x stock). The Tmapp(herein referred to as Tm) of different mutants with a positive effect on the melting temperature are reported in the table below. The table shows average values of duplicate measurements in this experiment.

[0162] Clearly, each of these single mutations H17W, S18K, N25G, V30I, S63G, T71V, V72I, S204N, E251 L, N43K and S87D has a positive effect on enzyme stability, indicated by an increased melting temperature Tm.

[0163] Example 2: Combining positive mutations on Ptl-10, according to sequence SEQ ID NO: 4 resulting in peptistabiligases according to the invention.

[0164] Several of the positive single mutations from Example 1 were subsequently added to the reference enzyme. The melting temperatures were determined using the same assay as described above for which 19 pL enzyme stock solution was mixed with 1 pL Sypro Orange (1000x stock). Melting temperatures per variant are reported in table below. In the first column Tmappvalues (“Tm”) are disclosed collected from a variety of performed experiments. In the second column Tmappvalues from one single experiment are disclosed, the close resemblance of these values demonstrates that it is a reproducible effect.

[0165] Combining several of the single mutations resulted in further increased melting temperatures. Subsequent addition of N43K, S87D, S18K, S63G, T71 V, V72I, N25G, S204N resulted in a thermostable enzyme variant with 9°C increased melting temperature.

[0166] Example 3: Combining 8 of these identified mutations on other enzyme backbones, according to sequence SEQ ID NO: 5 and variants of sequence SEQ ID NO: 6.

[0167] The melting temperatures of several reference enzymes (such as Ptl-10; Ptl-02 + A9S + L31 I + S156E + G212N +C3S +C206Q; or Ptl-84 + L96I + N156K + E166D + C3S + C206Q; or Ptl-84 + P222H + H217R + N156K + E166D + L96I + D99R + S224V+ C3S + C206Q; or Ptl 10) and their corresponding stabilized variants according to the invention, were determined using the same assay as described above for which 19 pL enzyme stock solution was mixed with 1 pL Sypro Orange (1000x stock). Melting temperatures per variant are reported in the table below.

[0168] Reference Enzyme: Ptl-10 (SEQ ID NO: 4)

[0169] Reference Enzyme: Ptl-02 + A9S + L31 I + S156E + G212N +C3S +C206Q according to SEQ ID NO: 5

[0170] (mutations with reference to SEQ ID NO: 5)

[0171] Reference Enzyme: Ptl-02 +C3S +C206Q according to SEQ ID NO: 5 (mutations with reference to SEQ

[0172] ID NO: 5)

[0173] Reference Enzyme: Ptl-84 + L96I + N156K + E166D + C3S + C206Q (mutations with reference to SEQ

[0174] ID NO: 6) Reference Enzyme: Ptl-84 + P222H + H217R + N156K + E166D + L96I + D99R + S224V + C3S +

[0175] C206Q (mutations with reference to SEQ ID NO: 6) Clearly, addition of thermostable mutations N43K + S87D + S18K + S63G + T71 V + V72I + N25G + S204N increased the melting temperature, and thereby the robustness and stress resistance of different enzyme variants.

[0176] Example 4: Increased stability of a thermostable variant of Ptl-10 against chaotropic reagents compared to Ptl-10 (according to SEQ ID NO: 4) (herein used as reference enzyme) in the synthesis of thymosin alpha 1 . Enzyme activities of a reference enzyme Ptl-10 and a thermostable variant (Ptl-10 + C3S + N43K + S87D + S18K + S63G + T71V + V72I + N25G + S204N + C206Q (mutations given with reference to SEQ ID NO: 4)) were determined for different guanidinium chloride (GnCI) concentrations in reaction buffer. The reaction was prepared by combining 1 pL TCEP (100mg / mL; pH 8.0), 72 pL incubation buffer (82 mM Tricine + X M GnCI, pH 8.5, wherein X = 0, 1 .5, 2.9, 4.4 and 5.8), and 6.5 pL enzyme stock solution (0.06 mg / mL). The enzyme was incubated in this (chaotropic denaturation) mixture for 30 minutes at room temperature. The reaction was started by adding 25 pL thymosin alpha 1 peptide mix (0.7 mg Ac-Ser-Asp-Ala-Ala-Val-Asp- Thr-Ser-Ser-Glu-lle-Thr-Thr-Lys-OCam-Leu-OH + 1.0 mg H-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu- Glu-Ala-Glu-Asn-OH in 50mM Tricine; pH 8.0). After 30 minutes the amount of product formed was analyzed using HPLC-MS (integration of starting material acyl donor, hydrolyzed ester and ligation product) and the percentage of thymosin alpha 1 product (that resulted from ligating the two fragments) is reported in the table below as % conversion.

[0177] Clearly, the thermostable variant shows increased retained activity compared to the reference enzyme Ptl- 10 when incubated with the chaotropic reagent guanidinium chloride (GnCI). At high concentrations all of the reference enzyme Ptl-10 is denatured, and no activity is observed while the stabilized variant still forms the desired product. Example 5: Stability of a thermostable variant of Ptl-10 against several organic co-solvents in the synthesis of thymosin alpha 1

[0178] Enzyme activity of a thermostable variant (Ptl-10 + C3S + N43K + S87D + S18K + S63G + T71 V + V72I + N25G + S204N + C206Q (mutations given with reference to SEQ ID NO: 4)) was determined in the presence of different co-solvents (DMSO, DMF and ACN) and different concentrations thereof in a reaction buffer solution. The reaction was prepared by combining 1 pL TCEP (100 mg / mL; pH 8.0), 72 pL incubation buffer (82 mM Tricine pH 8.5, containing 0, 14.5%, 29.0%, 43.5%, 58.1 % and 72.6% co-solvent), and 6.5 pL enzyme stock solution (0.06 mg / mL). The enzyme was incubated in this denaturing organic co-solvent mixture for 30 minutes at room temperature. The reaction was started by adding 25 pL thymosin alpha 1 peptide mix (0.7 mg Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-lle-Thr-Thr-Lys-OCam-Leu-OH + 1.0 mg H-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-OH in 50mM Tricine; pH 8.0). After 30 minutes the amount of product formed was analyzed using HPLC-MS (integration of starting material acyl donor, hydrolyzed ester and ligation product) and the percentage of thymosin alpha 1 product (that resulted from ligating the two fragments) is reported in the table below as % conversion.

[0179] The thermostable variant characterized by the mutations C3S + N43K + S87D + S18K + S63G + T71 V + V72I + N25G + S204N + C206Q shows retained activity even at higher concentrations of different cosolvents. Example 6: Stability of a thermostabile variant of Ptl-02 against several organic co-solvents and chaotropic reagents in a ligation reaction (of a hexapeptide and a tripeptide) Enzyme activity of a thermostable variant (Ptl-02 + C3S + N43K + S87D + S18K + S63G + T71V + V72I + N25G + S204N + C206Q +A9S +L31 I +S156E +G212N (mutations given with reference to SEQ ID NO: 5)) was determined for different co-solvents (DMSO, DMF and ACN) and chaotropic reagents in reaction buffer. The incubation mixture was prepared by combining 2.5 pL TCEP (100 mg / mL; pH 8.0), 72 pL incubation buffer (82 mM Tricine pH 8.5 and different concentrations of GnCI or co-solvent), 3.75 pL milliQ water and 1.25 pL enzyme stock solution (0.1 mg / mL). The enzyme was incubated in this denaturing organic cosolvent or chaotropic reagent mixture for 30 minutes at room temperature. The reaction was started by adding 25 pL peptide mix (10 mM Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-OH + 15 mM H-Ala-Leu-Arg-NH2 in milliQ water). After 15 minutes the amount of product formed was analyzed using HPLC-MS (integration of starting material acyl donor, hydrolyzed ester and ligation product) and the percentage of ligation product (Ac-Asp-Phe-Ser-Lys-Leu-Ala-Leu-Arg-NH2) is reported in the table below.

[0180] The thermostable variant of Ptl-02 specified above showed good activity against chaotropic reagents and organic co-solvents. The stability mutations according to the invention can be added to different enzyme backbones still showing the stabilizing effect.

[0181] When the experiment was performed with the reference enzyme Ptl-02 (SEQ ID NO: 5) the % conversion values decreased by 90% from a reaction in 1 M GnCI to 2 M GnCI, and again by 90% when the guanidinium chloride was increased further from 2 M GnCL to 3 M GnCI. The % conversion values only decreased by 25% and 50% respectively in the stabilized variant, described above. The inventors did repeat the experiment for another thermostable variant (Ptl-02 + C3S + N43K + S87D + S18K + S63G + T71V + V72I + N25G + S204N + C206Q (mutations given with reference to SEQ ID NO: 5)) differeing from the one specified above by not carrying the four mutations A9S, L311, S156E and G212N and observed the very same trend, a much more stable performance (% conversion remained higher) than with the reference enzymes (variants of BPN’ not carrying these mutations).

[0182] Example 7: Synthesis of teduglutide using chaotropic reagents with a thermostabile variant of Ptl-10 compared to the same reaction performed with the reference enzyme Ptl-10 (SEQ ID NO: 4)

[0183] Synthesis of teduglutide using reference enzyme Ptl-10 or a thermostable variant (Ptl 10 + C3S + N43K + S87D + S18K + S63G + T71 V + V72I + N25G + S204N + C206Q) was investigated with and without GnCI. Forthe reaction, 2 mg H-His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-lle-Leu-Asp-Asn-Leu-OCam- Leu-OH (SEQ ID NO: 9) and 1 mg H-Ala-Ala-Arg-Asp-Phe-lle-Asn-Trp-Leu-lle-GIn-Thr-Lys-lle-Thr-Asp-OH (SEQ ID NO: 10) were dissolved / suspended in 66.6 pL 300mM Tricine (pH 8.5) and 133.3 pL milliQ water or 133.3 pL 6M GnCI (pH 7.7). To the peptide mix, 2 pL TCEP (100 mg / mL; pH 8.0) was added and the pH was adjusted to 8.0 using an aqueous 3 M NaOH solution. The reaction was started upon addition of 5 pg enzyme. After 30 minutes the amount of product formed was analyzed using HPLC-MS (integration of starting material acyl donor, hydrolyzed ester and ligation product) and the percentage of teduglutide product is reported in the table below as % conversion. Due to the very poor solubility of the teduglutide fragments in aqueous buffer, no product is formed in the reactions without Guanidinium Chloride present. When the fragments are solubilized using 4 M GnCI the refernce enzyme Ptl-10 is denatured and no product is formed. However, using the stable variant the ligation reaction proceeds smoothly in 4 M GnCI.

[0184] SEQUENCES

[0185] SEQ ID NO: 1 : wild type gene encoding for subtilisin BPN' amino acids -107 to 275

[0186] ENA|K02496|K02496.1 B. Subtilisin BPN' Bacillus amyloliquefaciens

[0187] GTGAGAGGCAAAAAAGTATGGATCAGTTTGCTGTTTGCTTTAGCGTTAATCTTTACGATGGCGTTCGGCAGCACA

[0188] TCCTCTGCCCAGGCGGCAGGGAAATCAAACGGGGAAAAGAAATATATTGTCGGGTTTAAACAGACAATGAGCAC

[0189] GATGAGCGCCGCTAAGAAGAAAGATGTCATTTCTGAAAAAGGCGGGAAAGTGCAAAAGCAATTCAAATATGTAG

[0190] ACGCAGCTTCAGCTACATTAAACGAAAAAGCTGTAAAAGAATTGAAAAAAGACCCGAGCGTCGCTTACGTTGAAG

[0191] AAGATCACGTAGCACATGCGTACGCGCAGTCCGTGCCTTACGGCGTATCACAAATTAAAGCCCCTGCTCTGCAC

[0192] TCTCAAGGCTACACTGGATCAAATGTTAAAGTAGCGGTTATCGACAGCGGTATCGATTCTTCTCATCCTGATTTA

[0193] AAGGTAGCAGGCGGAGCCAGCATGGTTCCTTCTGAAACAAATCCTTTCCAAGACAACAACTCTCACGGAACTCA

[0194] CGTTGCCGGCACAGTTGCGGCTCTTAATAACTCAATCGGTGTATTAGGCGTTGCGCCAAGCGCATCACTTTACG

[0195] CTGTAAAAGTTCTCGGTGCTGACGGTTCCGGCCAATACAGCTGGATCATTAACGGAATCGAGTGGGCGATCGCA

[0196] AACAATATGGACGTTATTAACATGAGCCTCGGCGGACCTTCTGGTTCTGCTGCTTTAAAAGCGGCAGTTGATAAA

[0197] GCCGTTGCATCCGGCGTCGTAGTCGTTGCGGCAGCCGGTAACGAAGGCACTTCCGGCAGCTCAAGCACAGTG

[0198] GGCTACCCTGGTAAATACCCTTCTGTCATTGCAGTAGGCGCTGTTGACAGCAGCAACCAAAGAGCATCTTTCTC

[0199] AAGCGTAGGACCTGAGCTTGATGTCATGGCACCTGGCGTATCTATCCAAAGCACGCTTCCTGGAAACAAATACG

[0200] GGGCGTACAACGGTACGTCAATGGCATCTCCGCACGTTGCCGGAGCGGCTGCTTTGATTCTTTCTAAGCACCC

[0201] GAACTGGACAAACACTCAAGTCCGCAGCAGTTTAGAAAACACCACTACAAAACTTGGTGATTCTTTCTACTATGG

[0202] AAAAGGGCTGATCAACGTACAGGCGGCAGCTCAGTAA

[0203] SEQ ID NO: 2: wild type subtilisin BPN' (mature)

[0204] >SUBT_BACAM Subtilisin BPN' Bacillus amyloliquefaciens mature 1 to 275

[0205] AQSVPYGVSQ IKAPALHSQG YTGSNVKVAV IDSGIDSSHP DLKVAGGASM VPSETNPFQD 60

[0206] NNSHGTHVAG TVAALNNSIGVLGVAPSASLYAVKVLGADG SGQYSWIINGIEWAIANNMDVINMSLGGPS 130

[0207] GSAALKAAVDKAVASGWWAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFS 190

[0208] SVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPNWTNTQVRSSL 250

[0209] ENTTTKLGDSFYYGKGLI NVQAAAQ

[0210] SEQ ID NO: 3: shows the sequence of a peptiligase, i.e. subtilisin BPN' variant with a deletion of the amino acids corresponding to positions 75-83 (so called Ca2+binding loop), with the S221 mutation (denoted as S221C) and P225 mutation (denoted as P225N) and a number of mutations as compared to SEQ ID NO:2, resulting in the following amino acids K2, C3, S5, A9, L31 , N43, F50, L73, A169, P188, C206, G212, A254 and E271

[0211] AKCVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQD 60

[0212] NNSHGTHVAGTVLA-VAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPS 130(-9)

[0213] GSAALKAAVDKAVASGWWAAAGNEGTSGSSSTVGYPAKYPSVIAVGAVDSSNQRAPFS 190(-9)

[0214] SVGPELDVMAPGVSICSTLPGGKYGAYNGTCMASNHVAGAAALILSKHPNWTNTQVRSSL 250(-9) ENTATKLGDSFYYGKGLI NVEAAAQ

[0215] SEQ ID NO 4: reference enzyme Ptl-1O thymoligase GM0400. subtilisin BPN' variant having the deletion at A75-83, the mutation S221C and P225N, having amino acids K2, 03, S5, A9, L31, N43, F50, L73, A169, P188, C206, G212, A254, and E271; having mutations E156N, G166D, F189W, Y217R, N218S, M222G, plus a 6-His tag.

[0216] AKCVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQD 60

[0217] NNSHGTHVAG TVLAVAPSAS LYAVKVLGAD GSGQYSWIIN GIEWAIANNMDVINMSLGGPS 130(-9)

[0218] GSAALKAAVDKAVASGWWAAAGNNGTSGSSSTVDYPAKYPSVIAVGAVDSSNQRAPWS 190(-9)

[0219] SVGPELDVMAPGVSICSTLPGGKYGARSGTCGASNHVAGAAALILSKHPNWTNTQVRSSL 250(-9)

[0220] ENTATKLGDSFYYGKGLINVEAAAQHHHHHH

[0221] SEQ ID NO 5: reference enzyme Ptl-02 omniligase EU GMO 79. subtilisin BPN' variant having the deletion at A75-83, the mutation S221C and P225N, having amino acids K2, 03, S5, A9, L31, N43, F50, L73, A169, P188, 0206, G212, A254, and E271; having mutations 1107V, E156S, G166S, F189W, Y217H, N218S, M222P, plus a 6-His tag.

[0222] AKCVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQD 60

[0223] NNSHGTHVAGTVLA-VAPSASLYAVKVLGADGSGQYSWVINGIEWAIANNMDVINMSLGGPS 130(-9)

[0224] GSAALKAAVDKAVASGWWAAAGNSGTSGSSSTVSYPAKYPSVIAVGAVDSSNQRAPWS 190(-9)

[0225] SVGPELDVMAPGVSICSTLPGGKYGAHSGTCPASNHVAGAAALILSKHPNWTNTQVRSSL 250(-9)

[0226] ENTATKLGDSFYYGKGLINVEAAAQHHHHHH

[0227] SEQ ID NO 6: reference enzyme Ptl-84. subtilisin BPN' variant having the deletion at A75-83, the mutation S221C and P225N, having amino acids K2, 03, S5, A9, L31, N43, F50, L73, A169, P188, 0206, G212, A254, and E271; having mutations E156N.G166E, F189W, Y217H, N218D, M222P, plus a 6-His tag.

[0228] AKCVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQD 60

[0229] NASHGTHVAGTVLAVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPS 130(-9)

[0230] GSAALKAAVDKAVASGWWAAAGNNGTSGSSSTVEYPAKYPSVIAVGAVDSSNQRAPWS 190(-9)

[0231] SVGPELDVMAPGVSICSTLPGGKYGAHDGTCPASNHVAGAAALILSKHPNWTNTQVRSS 250(-9)

[0232] LENTATKLGDSFYYGKGLINVEAAAQHHHHHH

[0233] SEQ ID NO: 7 is an example of a peptistabiligase, as used in example 7, wherein S18K, N25G, N43K, S63G, T71V, V72I, S87D and S204N, are mutated.

[0234] AKSVSYGVAQIKAPALHKQGYTGSGVKVAVLDSGIDSSHPDLKVAGGASFVPSETNPFQD 60

[0235] NNGHGTHVAGVILA-VAPDASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPS 130(-9)

[0236] GSAALKAAVDKAVASGWWAAAGNNGTSGSSSTVDYPAKYPSVIAVGAVDSSNQRAPWS 190(-9)

[0237] SVGPELDVMAPGVNIQSTLPGGKYGARSGTCGASNHVAGAAALILSKHPNWTNTQVRSSL 250(-9)

[0238] ENTATKLGDSFYYGKGLINVEAAAQHHHHHH SEQ ID NO: 8 is another example of a peptistabiligase wherein S18K, N25G, N43K, S63G, T71V, V72I, S87D and

[0239] S204N, are mutated

[0240] AKCVSYGVAQIKAPALHKQGYTGSGVKVAVLDSGIDSSHPDLKVAGGASFVPSETNPFQD 60 NNGHGTHVAGVILA-VAPDASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPS 130(-9)

[0241] GSAALKAAVDKAVASGWWAAAGNNGTSGSSSTVDYPAKYPSVIAVGAVDSSNQRAPWS 190(-9)

[0242] SVGPELDVMAPGVNICSTLPGGKYGARSGTCGASNHVAGAAALILSKHPNWTNTQVRSSL 250(-9)

[0243] ENTATKLGDSFYYGKGLINVEAAAQHHHHHH SEQ ID NO: 9 provides the amino acid sequence of the first teduglutide fragment of example 7

[0244] His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-lle-Leu-Asp-Asn-Leu-OCam-Leu-OH

[0245] SEQ ID NO: 10 provides the amino acid sequence of the second teduglutide fragment of example 7

[0246] Ala-Ala-Arg-Asp-Phe-lle-Asn-Trp-Leu-lle-GIn-Thr-Lys-lle-Thr-Asp

Claims

Claims:

1. A peptistabiligase, having at least 80% sequence identity to the peptiligase represented by SEQ ID NO: 3, characterized by comprising a deletion of the amino acids at positions 75-83 compared to BPN'; and having a cysteine or selenocysteine, preferably cysteine, at position 221; and having an amino acid which is an alanine or asparagine, preferably asparagine at position 225; and comprising at least one mutation selected from the group consisting of H17W, S18K, N25G, V30I, S63G, S204N, E251L, N43K and S87D; wherein the amino acid mutations are defined with reference to the peptiligase represented by SEQ ID NO: 3.

2. The peptistabiligase according to claim 1 having at least 90% sequence identity to the peptiligase represented by SEQ ID NO:

33. The peptistabiligase according to to any of the preceding claims further comprising at least one additional mutation selected from T71V and V72I.

4. The peptistabiligase according to any of the preceding claims, comprising both mutations N43K and S87D.

5. The peptistabiligase according to any of the preceding claims comprising two, three or four mutations selected from the group consisting of H17W, S18K, N25G, V30I, S63G, T71V, S204N, E251L and the combination of N43K and S87D; or selected from the group consisting of H17W, S18K, N25G, V30I, S63G, V72I, S204N, E251L and the combination of N43K and S87D.

6. The peptistabiligase according to any of claims 1 to 4, comprising five mutations selected from the group consisting of S18K, N25G, S63G, T71V, S204N, E251L and the combination of N43K and S87D; or from the group consisting of S18K, N25G, S63G, V72I, S204N, E251L and the combination of N43K and S87D.

7. The peptistabiligase according to any of claims 1 to 4, comprising six mutations, wherein four mutations are selected from the group consisting of S18K, N25G, S63G, T71V and S204N; or from the group consisting of S18K, N25G, S63G, V72I, S204N and E251L; and further two mutations are N43K and S87D.

8. The peptistabiligase according to any of claims 1 to 4, comprising the mutations S18K, N25G, S63G, T71V, V72I and S204N; and N43K and S87D.

9. The peptistabiligase according to any of claims 1 to 8 characterized by having the amino acids of peptiligase SEQ ID NO: 3 at one or more, preferably 11, positions selected from the group consisting of K2, C3, S5, A9, L31, F50, L73, A169, P188, C206, G212, A254 and E271.

10. A peptistabiligase according to any of the preceding claims wherein said peptistabiligase is immobilized to a solid support.

11. A method for enzymatically synthesizing a peptide, comprising a step of coupling (a) a peptide C-terminal ester or thioester with (b) a peptide nucleophile having an N- terminal unprotected amine, wherein the coupling is performed in an aqueous solution, and wherein the coupling is catalyzed by a peptistabiligase having at least 80%, preferably at least 90% sequence identity to the peptiligase represented by SEQ ID NO: 3, comprising a deletion of the amino acids at positions 75-83 compared to BPN' (SEQ ID NO: 2); and having an amino acid which is a cysteine or selenocysteine, preferably cysteine, at position 221; and having an amino acid which is an alanine or asparagine, preferably asparagine at position 225; and wherein the peptistabiligase comprises at least one mutation selected from the group consisting of H17W, S18K, N25G, V30I, S63G, S204N, E251L, N43K and S87D; and preferably at least one mutation selected from T71V and V72I; and wherein the amino acid mutations are defined with reference to the peptiligase represented by SEQ ID NO: 3.

12. The method according to claim 10, wherein the peptistabiligase is further characterized by having the amino acids of peptiligase SEQ ID NO: 3 at one or more, preferably at 11, positions selected from the group consisting of K2, C3, S5, A9, L31, F50, L73, A169, P188, C206, G212, A254 and E271.

13. The method according to claim 10 or 11, wherein the aqueous solution comprises a chaotropic agent.

14. The method according to claim 12, wherein the chaotropic agent is selected from the group consisting of guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, sodium dodecyl sulfate, thiourea, polysorbate and urea.

15. The method according to claim 12, wherein the aqueous solution comprises guanidinium chloride.

16. The method according to claim 12, wherein the aqueous solution comprises guanidinium chloride at a concentration range of 1-5 M, preferably 2-4 M.

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