Thermostable subtilisin variants and uses thereof

Abstract

The present invention relates to a subtilisin BPN'variant or a homolog thereof comprising a new mutation compared to a subtilisin BPN 'as shown in SEQ ID NO: 3 or a homologous sequence thereof. This mutation may occur at an amino acid position selected from the group consisting of H17W, S18K, N25G, V30I, S63G, T71V, V72I, S204N, E251L, N43K or S87D.

Description

Technical Field

[0001] The present invention relates to an enzyme referred to as peptistabiligase, which is a peptiligase and is characterized by having multiple single mutations and their combinations compared to peptiligases known in the art, thereby having higher enzyme stability against external stress factors while its ligation activity is not less effective than these enzymes. The present invention also relates to the use of such enzymes in peptide synthesis methods, and methods for catalyzing the coupling of peptide fragments by such enzymes. Background Art

[0002] Methods for synthesizing peptides are well known in the art. Relatively short peptides can be chemically synthesized in solution by a stepwise method or by a highly optimized solid-phase peptide synthesis method. However, peptides longer than 10 - 15 amino acids are often difficult to synthesize due to side reactions, making purification also difficult. Therefore, such peptides are often synthesized by a combination of solid-phase synthesis of side-chain protected peptide fragments and their subsequent chemical condensation in solution. The main drawback of chemical side-chain protected peptide fragment coupling is the racemization that occurs after activation of the C-terminal amino acid residue of the acyl donor. In contrast, no racemization is observed in enzyme-catalyzed peptide coupling reactions. Another advantage of enzyme-catalyzed peptide synthesis compared to chemical peptide synthesis is the absence of side reactions on side-chain functional groups. The combined application of chemically synthesized peptides with enzymatic coupling is referred to as chemo-enzymatic peptide synthesis.

[0003] Chemo-enzymatic peptide synthesis can include the enzymatic coupling of multiple peptide fragments that have been separately synthesized by using chemical synthesis (in solution and / or in the solid phase), fermentation, or by a combination of chemical and enzymatic coupling steps.

[0004] Enzymes that are very suitable for enzymatic coupling are the peptiligases described in WO2016 / 056913.

[0005] The development of these enzymes originated from subtilisin BPN', which is a serine protease (SEQ ID NO:2) of 275 amino acids secreted by the soil bacterium Bacillus amyloliquefaciens.

[0006] Wells et al. (US5,403,737) found that when two mutations, namely S221C and P225A, were introduced, the active site of this subtilisin BPN' was greatly altered, and compared with wild-type subtilisin BPN', the application of this enzyme could obtain a synthesis-to-hydrolysis ratio (S / H ratio) that was 500 times higher. This enzyme can now be used for the coupling of peptides in aqueous solutions. This enzyme is called subtiligase. In further experiments, Wells et al. introduced five additional mutations into this subtiligase, namely M50F, N76D, N109S, K213R, and N218S, to make the enzyme more stable (Proc. Natl. Acad. Sci. USA, 1994, 91, 12544). This new mutant, now called stabiligase, showed moderately higher resistance to treatment with sodium dodecyl sulfate and guanidine hydrochloride, but hydrolysis was still the main side reaction.

[0007] In addition, subtilisin B' has also been developed through mutagenesis engineering. Bryan (US6,541,234) described the benefits of deleting amino acids 75 to 83 (also called the Ca 2+ binding site), but some additional mutations had to be introduced to stabilize the resulting enzyme. Mutations found at the P5S, D41A, and K43N or K43R positions led to a measurable but not large increase in stability. Mutations found at positions 2, 3, 73, and 206 significantly increased the half-life of the mutant relative to the parental subtilisin. The most stable mutations were Q2K, S3C, A73L, and Q206C.

[0008] When used for peptide synthesis in an aqueous environment, problems that have long remained unresolved for enzymes such as subtiligase or stabiligase due to their excessive hydrolysis activity have been solved only by the peptide ligase provided in WO2016 / 056913. The peptide ligase can catalyze highly efficient peptide coupling in water and has a very high S / H ratio.

[0009] In addition, since this enzyme does not require special recognition groups, it can be widely applied to the technique of traceless peptide ligation. In WO2018 / 212658, a peptide ligase having one or more specific mutations in the penultimate pocket near the coupling site, namely in the S2' pocket and / or the S2 pocket, was further disclosed, thereby broadening the peptide substrate range and improving the coupling efficiency.

[0010] In WO2019170895 and WO2019170918, methods for the enzymatic synthesis of liraglutide and semaglutide by coupling specific peptide fragments catalyzed by specific peptide ligases were illustrated.

[0011] Peptide ligases with one or more specific mutations are further described in WO2022171667, which mutations can increase the reaction rate and coupling efficiency of the catalytic reaction, or increase the selectivity of the enzyme.

[0012] These enzymes and peptide coupling techniques are particularly suitable for the manufacture of peptide drugs, such as glucagon, dasiglucagon, glepaglutide, elsiglutide, calcitonin, thymosin, lixisenatide, teriparatide, exenatide, liraglutide, semaglutide, teduglutide, and bivalirudin.

[0013] Particularly for peptides longer than 30 amino acids, compared to chemical coupling reactions, it has been found that using this enzymatic fragment condensation strategy can improve the crude purity and overall yield.

[0014] However, there is still a need to provide additional peptide ligases that can be used for the enzymatic synthesis of peptides by fragment coupling or cyclization, which, compared to the peptide ligases known in the prior art, need to maintain the high coupling reaction rate and coupling reaction efficiency of the peptide ligases described in the prior art while further better resisting the denatured state caused by unfolding. The unfolding of proteins is usually accompanied by a loss of activity and can be caused by various factors, such as the presence of cosolvents, changes in pH value, or freezing and / or heating processes. Especially in biotechnological applications, more stable enzymes are needed, which can be used at higher reaction temperatures, thereby achieving higher reaction rates and higher maximum solubilities, and thus obtaining faster reactions and increased space-time yields. In addition, an enzyme that is not easily denatured by common processing-related factors such as freeze-thaw cycles is needed, which has higher storage and shelf-life stability, simplifying the routine handling of the enzyme.

[0015] Therefore, there is still a need to improve the stability of the enzyme against some external stress factors, such as heat stress.

[0016] Especially when applied in an industrial environment, enzyme stability is crucial for achieving a robust process. Enzyme degradation leads to loss of activity and thus lower yields. Sometimes, yields are also limited due to peptide fragments and / or limited solubility of the enzyme. This solubility can be enhanced by adding cosolvents and / or chaotropes (such as urea and guanidine hydrochloride), and thus such conditions become more important in industrial applications. If the starting materials and the solubility or stability of the enzymatic coupling reaction products are better, the overall yield of the reaction process may be higher. Therefore, an enzyme with stronger stress stability is needed, which can tolerate a wider range and higher concentrations of cosolvents. Therefore, there is still a need to improve the stability of enzymes against external stress factors such as increased salt concentration and organic cosolvents.

[0017] Another need in the art is such a sufficiently robust peptide-stabilizing ligase that can be immobilized on a solid surface, thereby enabling more efficient recycling of the enzyme, increasing the turnover number of the catalyst, and at the same time reducing the conversion cost.

[0018] In addition, there is still a need to find efficient manufacturing methods for the enzymatic synthesis of pharmacologically significant peptides and suitable for scaling up to provide industrial conventional amounts.

[0019] The present invention solves or at least alleviates the above problems by providing new peptide ligases, characterized by having the new mutations as described below. An efficient method for peptide synthesis using these enzymes is also disclosed. Summary of the Invention

[0020] The present application has found that by providing new stable enzymes having one or more specific mutations, all or at least some of the above-discussed problems can be overcome or at least alleviated. These enzymes will be referred to as peptistabiligases, which are characterized by having one or more specific mutations at several positions compared to the peptide ligase that is a homolog of SEQ ID NO:3. Compared to the peptide ligase that is a homolog of SEQ ID NO:3 shown in SEQ ID NO:2, the peptide ligases of the homologs of SEQ ID NO:3 all show a deletion of amino acids at positions 75 - 83, an amino acid mutation at position S221 (the mutation is S221C or selenocysteine at S221, preferably S221C), and an amino acid mutation at position P225 (the mutation is preferably P225A or P225N, most preferably P225N). The peptistabiligase according to the present invention is further characterized by 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 as mutations relative to the peptide ligase shown in SEQ ID NO:3.

[0021] The position numbering is consistent among all the peptide ligases described herein and is the same as that in subtilisin BPN’ shown in SEQ ID NO:2, simply maintaining the numbers as if the missing amino acids (75 - 83) were still present.

[0022] Thus, in a first aspect, the present invention provides a peptide - stabilizing ligase that has at least 80% sequence identity with the peptide ligase shown in SEQ ID NO:3, characterized by

[0023] - compared with BPN’, containing a deletion of the amino acids at positions 75 - 83; and

[0024] - having a cysteine or selenocysteine at position 221, preferably cysteine; and

[0025] - having an amino acid that is alanine or asparagine at position 225, preferably asparagine; and

[0026] - containing at least one mutation selected from the group consisting of H17W, S18K, N25G, V30I, S63G, S204N, E251L, N43K, and S87D;

[0027] wherein the amino acid mutations are defined as mutations relative to the peptide ligase shown in SEQ ID NO:3.

[0028] A preferred embodiment is a peptide - stabilizing ligase according to the present invention, further comprising at least one additional mutation selected from T71V and V72I.

[0029] Another preferred embodiment of the present invention is a peptide - stabilizing ligase according to the present invention, containing two mutations N43K and S87D.

[0030] Preferably, some positions in the enzyme according to the present invention remain unchanged relative to the peptide ligase shown in SEQ ID NO:3. Preferably, the peptide - stabilizing ligase has the listed amino acids selected from the group consisting of K2, C3, S5, A9, L31, F50, LA73, A169, P188, C206, G212, A254, and E271 at one or more, preferably 11 positions.

[0031] The peptide - stabilizing ligase according to the present invention has catalytic activity (coupling activity) regarding peptide bond formation. This activity is also referred to as "ligase activity".

[0032] Obviously, the catalytic site responsible for a certain activity should not be mutated. Other parts of the enzyme that should not be mutated are conserved regions, which can be determined by multiple sequence alignment of various homologs (such as clustal Omega, (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ) or Geneious alignment (https: / / www.geneious.com / features / sequence-alignment / )).

[0033] A person of ordinary skill in the art can identify those (catalytic or conserved) sites without undue experimentation, and these sites should not be altered so as not to impair the functionality of the enzyme.

[0034] Compared with the peptide ligase not carrying the above characteristic mutations, the peptide-stable ligase of the present invention shows higher stress resistance. It shows higher thermal stability, that is, increased stability against temperature elevation. Compared with the enzyme without the mutation according to the present invention, the melting temperature of the enzyme is increased. Therefore, the enzyme provided by the present invention has higher heat resistance compared with other peptide ligases, while maintaining the high coupling reaction rate and coupling reaction efficiency of the peptide ligase.

[0035] Compared with known peptide ligases, the peptide-stable ligase shows increased stability to the presence of salts, especially chaotropic agents (such as, for example, guanidine hydrochloride), and to the presence of organic cosolvents, while maintaining the high coupling reaction rate and coupling reaction efficiency of the peptide ligase.

[0036] In a second aspect, the present invention provides a method for the enzymatic synthesis of peptides, wherein the coupling of two peptide fragments is carried out in an aqueous solution, and wherein the coupling is catalyzed by the above-mentioned peptide-stable ligase.

[0037] Accordingly, in a second embodiment, the present invention provides a method for the enzymatic synthesis of peptides, comprising the step of coupling (a) a peptide C-terminal ester or thioester with (b) a peptide nucleophile having an unprotected N-terminal amine, wherein the coupling is carried out in an aqueous solution and wherein the coupling is catalyzed by a peptide-stabilizing ligase having at least 80% sequence identity with the peptide ligase shown in SEQ ID NO:3; compared with BPN’ (SEQ ID NO:2), comprising a deletion of the amino acids at positions 75-83; and having a cysteine or selenocysteine, preferably cysteine, at position 221; and having an amino acid that is alanine or asparagine, preferably asparagine, at position 225; and wherein the peptide-stabilizing ligase comprises at least one mutation selected from the group consisting of H17W, S18K, N25G, V30I, S63G, S204N, E251L, N43K and S87D; and preferably one mutation selected from T71V and V72I; wherein the amino acid mutations are defined as mutations relative to the peptide ligase shown in SEQ ID NO:3. Detailed Description

[0038] As used herein, the term "reaction" refers to a reaction catalyzed by a variant of subtilisin BPN’ having ligase activity, in particular a peptidase. Specifically, the term "coupling reaction" or "coupling" refers to the formation of an amide bond (or peptide bond) between an amino group and a carboxyl 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 shorthand form of the phrase "ester or thioester".

[0040] As used herein, the term "enzyme stability" refers to the ability of an enzyme to resist high temperatures, chaotropic agents, and / or organic cosolvents. An enzyme with improved stability refers to an enzyme having a higher melting temperature compared to other enzymes of the same type. It can be determined whether the higher stability results from specific mutations compared to a reference enzyme (i.e., a reference enzyme without these mutations).

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

[0042] As used herein, the term "peptide" refers to any sequence composed of two or more amino acids linearly linked to each other by amide bonds. Peptides are typically formed from α-amino acids, but may also contain other amino acids, such as one or more β-amino acids and / or one or more γ-amino acids. Any peptide is defined by its specific amino acid sequence. Peptides differ from proteins in their shorter length, but the cutoff number of amino acids used to distinguish peptides from proteins is variable in the art. Generally, peptides contain 2 to 500, more typically 2 to 200, or 2 to 100 amino acids. Preferably, peptides contain at least 10 amino acids, more preferably at least 15 amino acids. In addition, the length of the peptide does not exceed 200, preferably does not exceed 100, and most preferably does not exceed 50 amino acids. Thus, most preferably, the length of the peptide is 10 to 50 amino acids.

[0043] Peptides can contain proteinogenic and / or non-proteinogenic amino acids. Proteinogenic amino acids are α-amino acids with an L-configuration 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), β-amino acids, 4-fluorophenylalanine, α-aminoisobutyric acid (Aib), other C-α-alkylated amino acids, and selenocysteine (Sec, U), which is an amino acid with a structure corresponding to cysteine but with a selenium atom replacing the sulfur atom. Peptides can be linear, branched, or cyclic, where branched peptides have at least two interconnected amino acid sequences.

[0044] Peptides can be bioactive peptides. Preferred examples of bioactive peptides include glucagon, GLP-1, GLP-2, and their analogs, such as dasiglucagon, exenatide, liraglutide, semaglutide, lixisenatide, teduglutide, glepaglutide, dulaglutide, elsiglutide, thymosin alpha-1, thymosin alpha-1 analogs, teriparatide, salmon calcitonin, bivalirudin, and peptides containing the sequence of any of these peptides and at least one additional amino acid.

[0045] As used herein, the term "cyclic peptide" refers to a peptide having a cyclic structure, where such peptide is produced by forming an amide bond (also referred to as "cyclization reaction") between the terminal α-amino group and the terminal α-carboxyl group of an amino acid sequence. In particular, such amino acid sequence has at least 12 amino acids.

[0046] As used herein, the term "peptide bond" refers to an amide bond between (i) the amino group of one amino acid and (ii) the carboxyl group of another amino acid. In particular, the peptide bond can be between the α-amino group of one α-amino acid and the α-carboxyl group of another α-amino acid.

[0047] When referring to a protein or an enzyme, the terms "mutated" or "mutation" as used herein mean that in a wild-type or naturally occurring protein or enzyme sequence, at least one amino acid is replaced by a different amino acid, inserted into the sequence, appended to the sequence, or deleted from the sequence by mutagenesis of the nucleic acid encoding these amino acids. Mutagenesis includes, for example, site-directed mutagenesis by PCR or via oligonucleotide-mediated mutagenesis, as described, for example, by Siloto et al. in "(2012) Site saturation mutagenesis: Methods and applications in protein engineering, Biocatalysis and Agricultural Biotechnology, 1, 181-189". When referring to a nucleic acid or a gene, the terms "mutated" or "mutation" as used herein mean that at least one nucleotide in the nucleic acid sequence has been replaced by a different nucleotide, inserted into the sequence, appended to the sequence, or deleted from the sequence, resulting in a protein sequence whose transcriptional function is qualitatively or quantitatively altered, or resulting in a "knockout" of the nucleic acid, which means a nucleic acid that no longer encodes a functional protein that it encoded before the mutation.

[0048] In the present disclosure, a mutation (of an amino acid) is described as the single-letter amino acid code of the amino acid being replaced, followed by a number indicating the position in the amino acid sequence of the protein at which the replacement is made. This number is the amino acid position in the reference amino acid sequence, which is commonly 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 each reference enzyme. Depending on the context, the reference enzyme mentioned herein can be subtilisin BPN’ (SEQ ID NO:2) or a peptide ligase according to any one of SEQ ID NO:3 to SEQ ID NO:6. The relevant reference enzyme has been indicated. Due to one or more other mutations (additions, insertions, deletions, etc.) at lower positions, the actual position in the mutant may not be the same position, but in the embodiments of the present invention, they are exactly the same. A person skilled in the art can use well-known alignment techniques such as NEEDLE to determine the corresponding positions. After the number is the single-letter amino acid code of the amino acid that replaces the reference amino acid. For example, F189W means that phenylalanine (F) at position 189 is replaced by tryptophan (W). X is used to indicate any other native amino acid that may be present at that position in addition to the amino acid being replaced. For example, F189X means that phenylalanine at position 189 is replaced by any other native amino acid.

[0049] As used herein, the term "ligase" refers to an enzyme that has catalytic activity in the coupling of two peptides by catalyzing the formation of a peptide bond between the C-terminus of the first peptide and the N-terminus of another peptide. This activity is also referred to as "ligase activity". When this enzyme has catalytic activity in the formation of an intramolecular peptide bond between the C-terminus and the N-terminus of the same peptide molecule, this activity can also be referred to as "cyclase activity". Thus, the same enzyme can have ligase and / or cyclase activity. When the synthesis / hydrolysis ratio of the enzyme is greater than 1, the enzyme is characterized as having ligase activity. This S / H ratio can be determined by HPLC analysis of the corresponding amounts.

[0050] In the reaction medium used, especially in a reaction medium containing water, more especially an aqueous medium (also referred to as an aqueous solution), the ligase generally has an S / H ratio greater than 1, preferably 2 or greater, especially 5 or greater. The upper limit value of this quotient is not important and can be, for example, 100 or less.

[0051] According to the present invention, a peptide-stabilized ligase is an enzyme having ligase and / or cyclase activity and having at least 80%, preferably at least 85%, more preferably at least 90% sequence identity with each reference enzyme; even more preferably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. According to the present invention, a preferred reference enzyme is the peptide ligase shown in SEQ ID NO: 3. The percent identity is determined according to the NEEDLE EMBOSS method outlined below. Obviously, the percent identity will be less than 100%. The percent identity depends on the number and length of mutations of the peptide (enzyme) being compared to the said homolog.

[0052] For the purposes of the present invention, for determining the percent identity of two amino acid sequences, the complete mature sequences are aligned for optimal comparison such that similar regions are aligned. Any sequence extensions (at the N- or C-terminus), such as commonly used His-tags or other tags for purposes of purification, signal transduction, solubilization and localization, are not considered when determining the percent identity. To optimize the alignment between the two sequences, gaps can be introduced in either of the two sequences being compared. The alignment for determining the sequence identity % value is performed over a length of at least 200 amino acids of the sequences being compared.

[0053] The sequence comparison and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, such as the Needleman-Wunsch algorithm (Needleman, S.B. and Wunsch, C.D. (1970) J. Mol. Biol. 48(3), pp443-453), which is implemented in the computer program NEEDLE.

[0054] The NEEDLE program from the EMBOSS software package (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) is used to calculate the percent identity. For protein sequences, EBLOSUM62 should be used for the substitution matrix. The parameters for amino acid sequence alignment must be set to a gap opening 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 that show the same amino acid in both sequences divided by the total length of the alignment (the length after subtracting the total number of gaps in the alignment).

[0055] The percent identity as defined herein can be obtained from NEEDLE and is labeled as "longest identity" in the output of said program.

[0056] In particular, the present invention provides an isolated enzyme. As used herein, the term "isolated" means that the enzyme is separated from the organism in which it is expressed, typically a recombinant organism (if it has been produced in an organism), or from the reaction medium in which it is synthesized.

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

[0058] The enzyme according to the present invention can be provided in at least substantially pure form, where the term "substantially pure enzyme" means an enzyme with a purity of at least 75 wt.%, preferably higher than 80 wt.%. The enzyme can also be provided in the form of a mixture with one or more other components, for example in the form of a storage solution, preferably in an aqueous buffer.

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

[0060] The degree of resistance to melting upon increasing temperature is considered an indicator of enzyme stability. By determining the melting temperature T m app of the enzyme, the stability or robustness of the enzyme can be determined. This can be analyzed using an assay, also known as a thermal fluorescence assay, which utilizes a fluorescent dye that binds to the hydrophobic regions of the enzyme and these hydrophobic regions are exposed during thermal denaturation. By slowly increasing the temperature while measuring the fluorescence signal, the T m app of the variant can be determined. The higher the detected T m app , the more stable the enzyme. The assay method used in the experimental section is as described by Lavinder et al. (2009) J. AM. CHEM. SOC. 2009, 131, 3794–3795, where the method is referred to as the high-throughput thermal screening (HTTS) method.

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

[0062] The amino acid sequence of a typical peptide ligase is shown in SEQ ID NO:3, which is a suitable reference enzyme. It is shown as a variant of subtilisin BPN', having an amino acid deletion corresponding to positions 75-83 (the so-called Ca 2+ binding loop), having an S221 mutation of S221C and a P225 mutation of P225N, and another 14 mutations of Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, G169A, S188P, Q206C, N212G, T254A and Q271E, wherein the amino acid mutations are defined as mutations relative to BPN' shown in SEQ ID NO:2.

[0063] Surprisingly, it has been found that for peptide ligases such as those shown in SEQ ID NO:3 or those known in WO2016 / 06913, any single additional mutation selected from the group consisting of H17W, S18K, N25G, V30I, S63G, T71V, V72I, S204N, E251L, N43K and S87D can significantly improve the stability of the enzyme. Combining some of these mutations even further improves the stability of the enzyme.

[0064] Accordingly, the present invention provides a peptide-stabilized ligase having at least 80% sequence identity with the peptide ligase shown in SEQ ID NO:3, characterized by

[0065] compared with BPN', comprising a deletion of the amino acid at positions 75-83; and

[0066] having cysteine or selenocysteine at position 221, preferably cysteine; and

[0067] having an amino acid of alanine or asparagine at position 225, preferably asparagine; and

[0068] 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 as mutations relative to the peptide ligase shown in SEQ ID NO:3.

[0069] Preferably, the peptide-stabilized ligase has at least 90% sequence identity with the peptide ligase shown in SEQ ID NO:3, more preferably 93% sequence identity.

[0070] In a preferred embodiment, the peptide-stabilized ligase further comprises at least one additional mutation selected from T71V and V72I.

[0071] In a further preferred embodiment, the peptide-stabilizing ligase further comprises two mutations, N43K and S87D.

[0072] Thus, the amino acid sequence of the peptide-stabilizing ligase according to the invention differs from SEQ ID NO:3 in at least one stability mutation selected from the group consisting of H17W, S18K, N25G, V30I, S63G, S204N, E251L, N43K and S87D, relative to SEQ ID NO:3.

[0073] Surprisingly, it has also been found that a cumulative effect is observed when two or three or four or five or six mutations among H17W, S18K, N25G, V30I, S63G, T71V, V72I, S204N, E251L, N43K or S87D are combined.

[0074] Thus, in a preferred embodiment, the peptide-stabilizing ligase according to the invention comprises one of the following:

[0075] - one mutation selected from the group consisting of H17W, S18K, N25G, V30I, S63G, T71V, V72I, S204N, E251L, N43K, S87D; or preferably

[0076] - 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 two, three or four mutations selected from the group consisting of H17W, S18K, N25G, V30I, S63G, V72I, S204N, E251L and the combination of N43K and S87D; or more preferably

[0077] - five mutations selected from the group consisting of S18K, N25G, S63G, T71V, S204N, E251L and the combination of N43K and S87D; or five mutations selected from the group consisting of S18K, N25G, S63G, V72I, S204N, E251L and the combination of N43K and S87D; or more preferably

[0078] - six mutations, four of which are selected from the group consisting of S18K, N25G, S63G, T71V and S204N; or selected from the group consisting of S18K, N25G, S63G, V72I, S204N and E251L; and the other two mutations are N43K and S87D.

[0079] The peptide-stabilized ligase according to the present invention preferably has the same amino acids as the peptide ligase of SEQ ID NO:3 at the following positions: a group consisting of K2, C3, S5, A9, L31, F50, L73, A169, P188, C206, G212, A254, and E271; more preferably, one position in a group consisting of K2, S5, A9, L31, F50, L73, A169, P188, G212, A254, and E271; preferably at two or three positions; more preferably at four, five, or six positions; even more preferably at seven, eight, or nine positions; but most preferably at 10 or 11 positions.

[0080] In a preferred embodiment, the peptide-stabilized ligase maintains at least 11 of these positions unchanged compared to the sequence of SEQ ID NO:3. Compared to the wild-type subtilisin BPN’ of SEQ ID NO:2, these amino acids are referred to as mutations Q2K, S3C, P5S, S9A, I31L, M50F, A73L, G169A, S188P, Q206C, N212G, T254A, and Q271E.

[0081] In a preferred embodiment, the peptide-stabilized ligase according to the present 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, and such mutations are 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.

[0082] In another preferred embodiment, the peptide-stabilized ligase according to the present invention further comprises at least one mutation or paired mutations at the amino acid positions of M222 and Y217, wherein the single mutations are preferably selected from the group consisting of M222P, M222G, M222H, Y217H, Y217G, Y217F, Y217L, and Y217R, and wherein the paired 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 increase the stability of the preferred enzyme but can contribute to the coupling specificity and improved yield of the enzyme.

[0083] In one embodiment, the present invention provides a peptide-stabilized ligase that has an increased melting temperature (Tm app )。 The T m app is increased by at least 0.5 °C, more preferably 1 °C, even more preferably 2 °C, and even more preferably 3 °C.

[0084] The peptide-stabilizing ligase according to the invention is preferably immobilized on a solid support. There are several methods of aqueous reaction based on entrapment, microencapsulation, covalent binding, and cross-linking. The technique chosen depends on the peptide-stabilizing ligase chosen and the substrates used in the reaction.

[0085] The enzyme of the present invention is generally produced by recombinant methods, preferably by expressing a mutated subtilisin BPN' DNA such that after expression, a subtilisin BPN' variant is produced, called the peptide-stabilizing ligase according to the invention, which has enzymatic activity.

[0086] Accordingly, the present invention further provides a recombinant method for preparing the enzyme according to the invention, the method comprising the steps of:

[0087] a) providing a recombinant host cell that functionally expresses a gene encoding the enzyme, such as a bacterial cell, such as Escherichia coli (E. coli) or Bacillus;

[0088] b) culturing the host cell under conditions that provide for the expression of an enzymatically active enzyme; and

[0089] c) recovering the expressed enzyme from the microbial host.

[0090] The present invention also provides a recombinant polynucleotide comprising a sequence encoding the enzyme according to the invention.

[0091] The present invention also provides a host cell comprising the polynucleotide according to the invention, the polynucleotide being capable of expressing the enzyme.

[0092] Compared with other known peptide ligases, the enzyme according to the invention shows increased stability towards chaotropic agents (such as, for example, guanidine hydrochloride). Compared with enzyme variants that do not have specific mutations, the enzyme variants according to the invention show higher retention of ligation activity in the presence of different concentrations of guanidine hydrochloride. The enzyme according to the invention retains activity in at least 0.5 M or more of GnCl, more preferably 1 M or more of GnCl, and even more preferably 2 M or more of GnCl, while the activity of the reference enzyme decreases significantly.

[0093] Accordingly, in another embodiment of the present invention, the present invention provides a method for the enzymatic synthesis of a peptide, the method comprising the step of coupling (a) a peptide C-terminal ester or thioester with (b) a peptide nucleophile having an unprotected N-terminal amine,

[0094] The coupling is preferably carried out in an aqueous solution and is catalyzed by a peptide-stabilizing ligase according to the invention; the peptide-stabilizing ligase has at least 80% sequence identity with the peptide ligase shown in SEQ ID NO:3 and contains a deletion of the disposing amino acids at positions 75 - 83 compared to BPN’ (SEQ ID NO:2); and

[0095] has cysteine or selenocysteine, preferably cysteine, at position 221; and

[0096] has an amino acid that is alanine or asparagine, preferably asparagine, at position 225; and

[0097] the peptide-stabilizing ligase contains 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; wherein the amino acid mutations are defined as mutations relative to the peptide ligase shown in SEQ ID NO:3.

[0098] Preferably, a peptide-stabilizing ligase is used that has the amino acids of the peptide ligase of SEQ ID NO:3 at one or more, preferably 11 positions selected from: K2, C3, S5, A9, L31, F50, L73, A169, P188, C206, G212, A254, and E271.

[0099] Preferably, the peptide-stabilizing ligase used in the method has at least 90% sequence identity with the peptide ligase shown in SEQ ID NO:3, and even more preferably 93% sequence identity.

[0100] The use of the enzyme according to the invention extends beyond catalyzing the peptide cyclization reaction and / or the coupling of a peptide C-terminal (thio)ester with a peptide nucleophile as described above. The peptide-stabilizing ligase can be used for the formation of amide bonds other than peptide bonds, but its use in relation to peptide bonds is particularly preferred.

[0101] According to the method of the invention, the reaction is generally carried out in an aqueous solution (preferably containing a buffer). The aqueous solution may also contain an organic solvent or a chaotropic agent. Suitable solvents are co-solvents that are miscible with water.

[0102] It may be advantageous to add an additive to the aqueous solution to increase the solubility of the peptide fragments or to increase the reaction yield. Such an additive, also called a chaotropic agent, can be a salt or an organic molecule. Common chaotropic agents include guanidine hydrochloride, lithium perchlorate, lithium acetate, magnesium chloride, sodium dodecyl sulfate, thiourea, polysorbate, and urea.

[0103] Thus, according to the method of the present invention, preferably, the aqueous solution contains a chaotropic agent. Preferably, the chaotropic agent is selected from the group consisting of guanidine hydrochloride, lithium perchlorate, lithium acetate, magnesium chloride, sodium dodecyl sulfate, thiourea, polysorbate (also known as Tween, ) and urea, and most preferably, the chaotropic agent is guanidine hydrochloride. The suitable concentration range of guanidine hydrochloride in the aqueous solution is 1 to 5 M. This enables the coupling reaction of originally insoluble fragments. Thus, in a preferred embodiment, the aqueous solution contains guanidine hydrochloride in a concentration range of 1 - 5 M, preferably 2 - 4 M.

[0104] Compared with peptide ligases, the peptide stable ligase according to the invention also shows improved stability to the presence of organic co - solvents (such as DMF, DMSO or acetonitrile) in the reaction medium. Thus, in the method according to the present invention, the aqueous solution can contain an organic solvent. Preferably, one or more such organic solvents are selected from the list consisting of N,N - dimethylformamide (DMF), N - methylpyrrolidone (NMP), N - ethylpyrrolidone (NEP), N,N - dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), acetonitrile (ACN), ethers (such as tetrahydrofuran (THF), 2 - methyl - tetrahydrofuran (Me - THF), 1,2 - dimethoxyethane), alcohols (such as methanol, ethanol, isopropanol, tert - butanol, 2,2,2 - trifluoroethanol (TFE) and 1,1,1,3,3,3 - hexafluoroisopropanol).

[0105] Adjust the amount of the co - solvent according to the stability of the peptide ligase and the solubility of the peptide substrate. If the solubility of the peptide fragment or the coupled peptide is low, up to 50% of the aqueous solution can be an organic solvent. Many peptide ligases known in the prior art have reduced activity or are inactivated at such concentrations, but the enzyme according to the present invention is surprisingly tolerant to the presence of such a high content of organic solvents. As shown in the examples, compared with the peptide ligase exemplified in SEQ ID NO:3, the enzyme variant according to the present invention shows a higher activity retention rate with an increase in the concentration of these organic co - solvents. Compared with the reference enzyme, it still maintains activity in at least more than 10% co - solvent, more preferably more than 15% co - solvent, and even more preferably more than 20% co - solvent.

[0106] Thus, according to the method of the present invention, preferably, the concentration range of the organic solvent contained in the aqueous solution is 20 - 50%, preferably 30 - 50%, and most preferably 40 - 50%.

[0107] In principle, the temperature during the peptide ligase coupling or cyclization reaction is not critical, as long as a temperature is selected that allows the enzyme used to exhibit sufficient activity and stability. This temperature can be determined routinely. Usually, the temperature during the coupling or cyclization reaction ranges from 20 to 50 °C. However, ordinary peptide ligases are prone to denaturation at higher temperatures, while the enzymes according to the invention can tolerate higher temperatures. Therefore, according to the method of the invention, preferably, it is carried out at a temperature of 20 °C to 70 °C, more preferably at 30 °C to 70 °C, and most preferably at 35 °C to 70 °C.

[0108] The commonly used peptide C-terminal esters or thioesters (commonly referred to herein as (thio)esters) are activated (thio)esters, i.e., they contain a carboxyl ester or carboxyl thioester group that can participate 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 that can participate in the reaction are methyl-, ethyl-, propyl-, isopropyl-, phenyl-, benzyl- (e.g., p-carboxy-benzyl-), 2,2,2-trichloroethyl-, 2,2,2-trifluoroethyl-, cyanomethyl- and carboxamidomethyl-(thio)esters (OCam(thio)esters).

[0109] For example, esters with particularly good effects, as well as how to synthesize and / or protect these esters, are described in detail in WO2022171667.

[0110] In a preferred embodiment, the peptides synthesized by the method according to the invention are selected from the group consisting of liraglutide, semaglutide, glucagon, dapagliflozin, glepaglutide, exenatide, dulaglutide, thymosin, thymosin-α-1, thymosin-α-1 analogs, calcitonin, lixisenatide, teriparatide, exenatide, bivalirudin or tildrakizumab.

[0111] In one embodiment, the invention provides a method for the enzymatic synthesis of tildrakizumab, comprising the step of coupling: (a) a peptide C-terminal ester, His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-ester (SEQ ID NO 9) with (b) a peptide nucleophile Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp (SEQ ID NO10) having an unprotected amine at the N-terminus, wherein the coupling is carried out in an aqueous solution, and wherein the coupling is catalyzed by a peptide stable ligase according to the invention.

[0112] In a preferred embodiment, the aqueous solution comprises guanidine hydrochloride at a concentration of 4 M. Preferably, the peptide-stabilizing ligase in this embodiment comprises the following mutations: S18K, N25G, N43K, S87D, S63G, T71V, V72I, and S204N, wherein the amino acid mutations are defined relative to the peptide ligase shown in SEQ ID NO:3.

[0113] Preferably, the peptide-stabilizing ligases according to the present invention are used for the production of therapeutic peptides by enzymatic coupling as described herein in an industrial environment where a high peptide yield is required and the production process needs to be efficient. These peptide-stabilizing ligases have surprising robustness, and after the reaction is completed, these enzymes have a high recovery rate, while also providing a high coupling reaction rate and a high coupling reaction efficiency.

[0114] The present invention will now be illustrated by the following examples.

[0115] Abbreviations

[0116] SPPS Solid-phase peptide synthesis

[0117] CTC 2-Chloro-trityl chloride

[0118] AEEA 2-[2-(2-Aminoethoxy)ethoxy]acetyl

[0119] Cbz Benzyloxycarbonyl

[0120] For Formyl

[0121] Fmoc 9-Fluorenylmethoxycarbonyl

[0122] Boc tert-Butyloxycarbonyl

[0123] Smoc 2,7-Disulfo-9-fluorenylmethoxycarbonyl

[0124] AC Acetyl

[0125] PhAc Phenylacetyl

[0126] Trt Triphenylmethyl (trityl)

[0127] tBu tert-Butyl

[0128] Pbf 2,2,4,6,7-Pentamethyl-dihydrobenzofuran-5-sulfonyl

[0129] Eq Equivalent

[0130] h Hour / second

[0131] min Minute / second

[0132] HPLC High Performance Liquid Chromatography

[0133] DIPEA N,N-Diisopropylethylamine

[0134] TFA Trifluoroacetic Acid

[0135] TIS Triisopropylsilyl

[0136] Ac2O Acetic Anhydride

[0137] DMF N,N-Dimethylformamide

[0138] DMA N,N-Dimethylacetamide

[0139] DCM Dichloromethane

[0140] THF Tetrahydrofuran

[0141] NMP N-Methyl-2-pyrrolidone

[0142] MTBE Methyl tert-butyl ether

[0143] MeOH Methanol

[0144] DCC N,N'-Dicyclohexylcarbodiimide

[0145] EDC N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide

[0146] HOBt 1-Hydroxybenzotriazole

[0147] HOAt 1-Hydroxy-7-azabenzotriazole

[0148] TCEP Tris(2-carboxyethyl)phosphine

[0149] Tricine N-(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine

[0150] OCam ester Carboxamidomethyl ester

[0151] Examples

[0152] Production of an enzyme (or enzyme variant) according to the invention (for use)

[0153] Mutation, Cloning and Expression

[0154] The reference enzyme labeled as SEQ ID NO 4: Ptl-10, is a subtilisin BPN' variant, lacking the amino acids corresponding to positions 75 - 83, and having mutations of Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, Δ75 - 83, E156N, G166D, G169A, S188P, F189W, Q206C, N212G, Y217R, N218S, S221C, M222G, P225N, T254A and Q271E, as well as a 6-His tag. The gene encoding His-tagged Ptl-10 was cloned into an Escherichia coli - Bacillus subtilis shuttle vector (i.e., pBS42 or pBES) based on pUB-110.

[0155] The corresponding amino acid sequence is numbered according to the subtilisin BPN' numbering scheme. Amino acids at positions -107 to -1 contain the signal sequence, pre-sequence, and pro-sequence, which are excised after full maturation. Amino acids at positions 1 - 275 contain the mature enzyme exhibiting full catalytic activity. For rapid and efficient purification, a C-terminal His-tag was attached after amino acid position 275. Due to the removal of the calcium-binding site, Ptl-10 contains a 9-amino acid deletion compared to subtilisin BPN', including 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 the enzymes of the present invention), the numbering jumps from 74 to 84. In the shuttle vector, the expression of the gene is controlled by the aprE promoter. The resulting plasmid pBES-Ptl-10 HIS was propagated in Escherichia coli TOP10 and transformed into Bacillus subtilis BH13, and the gene deletion is shown in the following table. Halmschlag (2020) discloses in detail how to generate such a Bacillus subtilis BH13 strain. Tailored poly-γ-glutamic acid production with Bacillus subtilis 168. Doctoral thesis. Rheinisch- Technische Hochschule Aachen, Germany.

[0156]

[0157]

[0158] Using pBES-Ptl-10 HIS as a template, mutagenesis was carried out by the MISO method (L.A. Mitchell et al., ACS Synth Biol 2013, :473-7). Optionally, other methods of site-directed mutagenesis known in the art can also be used. Optionally, the plasmid was synthesized by Ranomics Inc., CAN.

[0159] Production and purification of a synthetic subtilisin BPN’ variant with a His tag:

[0160] A single microbial colony of Bacillus subtilis containing a plasmid with the gene of the subtilisin variant of interest was inoculated into 5 mL of LB containing kanamycin (10 μg / mL) and cultured in an orbital shaker at 37 °C. 0.6 mL of the overnight culture was added to 30 mL of Terrific broth supplemented with antibiotics (kanamycin 10 μg / mL) and amino acids (100 mg / L Trp, 100 mg / L Met, and 100 mg / L Lys). The cells were grown in an orbital shaker (200 rpm) at 37 °C for 48 h. The cells were harvested by centrifugation (30 min, 4,000 rpm, 4 °C). The medium (30 mL) was poured out, 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 of demineralized water and then equilibrated with 20 mL of buffer A. The crude lysate was loaded onto the column and incubated overnight at 4 °C on an orbital shaker. After incubation, the resin was washed with 100 mL of buffer A. The enzyme was eluted with 15 mL of buffer B (25 mM Tricine, pH 7.5, 0.5 M NaCl, 0.5 M imidazole). The eluate was further incubated with 6 mM TCEP (tris(2-carboxyethyl)phosphine) for 30 min and concentrated by centrifugation (30 min, 4000 rpm, 4 °C) on a device (20 ml, MW cut-off 10 kDa), and the buffer was exchanged into 125 mM Tricine, 0.5 M NaCl, pH 7.5 in three wash / concentration steps (15 ml buffer, 10 min, 4,000 rpm, 4 °C). Optionally, potassium dihydrogen phosphate buffer can be used.

[0161] Purity was determined by SDS-PAGE and densitometry analysis (BioRad GS-900). Enzyme concentration was determined by measuring the absorbance at 280 nm using a Nanodrop (Thermo Scientific), where 1 Abs = 1 mg / ml. The resulting aqueous solution (125 mM Tricine, 0.5 M NaCl, pH 7.5) contained approximately 0.1 - 2 mg / ml of the resulting enzyme. The solution with the enzyme concentration corrected (mg / ml * purity) was used for, e.g., coupling and cyclization reactions.

[0162] For a detailed description of the production and purification of the synthetic subtilisin BPN’ variant reference, see WO2016 / 056913 and WO 2018 / 212658.

[0163] Examples of enzymatic fragment coupling

[0164] Materials and Methods

[0165] Unless otherwise stated, chemicals were obtained from commercial sources and used directly without further purification. Analytical HPLC was performed on an HP1090 liquid chromatograph using a reversed-phase column (Phenomenex, C18, 5 μm particle size, 150 × 4.6 mm) at 40 °C. UV detection was performed at 220 nm using a UV-VIS204 linear spectrometer. The gradient program was: 0 - 25 min, linear gradient from 5% to 98% of eluent B; 25.1 - 30 min, 5% eluent B (eluent A: H2O solution of 0.5 mL / L methanesulfonic acid (MSA), eluent B: acetonitrile solution of 0.5 mL / L MSA). The flow rate was 1 mL / min from 0 - 25.1 min, 2 mL / min from 25.2 - 29.8 min, and then back to 1 mL / min until stopped at 30 min. The injection volume was 20 μL. Preparative HPLC was performed on a Varian PrepStar system using a stationary-phase column (Pursuit XRs, C18, 10 μm particle size, 500 × 41.4 mm). LC-MS was performed on an Agilent 1200 series liquid chromatograph using a reversed-phase column (Phenomenex, C18, 5 μm particle size, 150 × 4.6 mm) at 40 °C. UV detection and gradient program were as described for analytical HPLC. The molecular weight was determined using an Agilent 6130 quadrupole LC / MS system.

[0166] Scheme 1: Preparation of the peptide - OCam - Leu - OH ester

[0167] 1 g of Fmoc-Leu-Wang resin (loading 0.72 mmol / g) was washed with DCM (2×2 min, 10 mL) and DMF (2×2 min, 10 mL), and Fmoc deprotection was carried out using piperidine / DMF (1 / 4, v / v, 2×8 min, 10 mL). After washing with DMF (2×2 min, 10 mL), DCM (2×2 min, 10 mL) and DMF (2×2 min, 10 mL), iodoacetic acid (4 eq) was coupled to the resin using a solution of DCC (4 eq) and HOAt (4 eq) in DCM (45 min, 10 mL). After washing with DMF (2×2 min, 10 mL), DCM (2×2 min, 10 mL) and THF (2×2 min, 10 mL), the resin was loaded with the Fmoc-protected amino acid (4 eq. Fmoc-XXX-OH and 10 eq. DIPEA in a DMF / THF (1 / 1, v / v, 10 mL) solution) at 50 °C for 20 h. Here and in other parts of the present disclosure "XXX" represents an amino acid (which can vary with the target peptide as shown in the examples below).

[0168] After washing with DMF (2×2 min, 10 mL), DCM (2×2 min, 10 mL) and DMF (2×2 min, 10 mL), the peptide was elongated according to the standard SPPS protocol. Resin cleavage and side-chain deprotection were carried out 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-heptane (1 / 1, v / v, 50 mL). The precipitated peptide was collected by centrifugation and washed twice with MTBE / n-heptane (1 / 1, v / v, 50 mL), and then lyophilized from acetonitrile / water (1 / 1, v / v, 50 mL).

[0169] Protocol 2: Preparation of C-terminal amide peptide nucleophiles

[0170] 1 g of Rink resin (4-((2,4-dimethoxyphenyl)(Fmoc-amino)methyl)phenoxyalkyl linker, loading 0.64 mmol / g) was washed with DCM (2×2 min, 10 mL) and DMF (2×2 min, 10 mL), and then Fmoc deprotection was carried out using piperidine / DMF (1 / 4, v / v, 2×8 min, 10 mL). The peptide was elongated according to the standard SPPS protocol. Resin cleavage and side-chain deprotection were carried out 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-heptane (1 / 1, v / v, 50 mL). The precipitated peptide was collected by centrifugation, washed twice with MTBE / n-heptane (1 / 1, v / v, 50 mL) and dried in vacuo. Before enzymatic ligation, the crude peptide was purified by preparative HPLC and then the pure fractions were lyophilized.

[0171] Protocol 3: Preparation of C-terminal amide peptide nucleophiles

[0172] Pre-loaded Wang resin (Fmoc-Xxx-Wang resin, loading 0.3 mmol / g) was washed with DCM (2×2 min, 10 mL) and DMF (2×2 min, 10 mL), and Fmoc deprotection was carried out using piperidine / DMF (1 / 4, v / v, 2×8 min, 10 mL). The peptide was elongated according to the standard SPPS protocol. Resin cleavage and side-chain deprotection were carried out 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-heptane (1 / 1, v / v, 50 mL). The precipitated peptide was collected by centrifugation, washed twice with MTBE / n-heptane (1 / 1, v / v, 50 mL) and dried in vacuo. Before enzymatic ligation, the crude peptide was purified by preparative HPLC and then the pure fractions were lyophilized.

[0173] Protocol 4: Preparation of N-acetyl protected peptide activated esters

[0174] After SPPS of the desired sequence according to one of Scheme 1 or 2, the resin-bound peptide was subjected to Fmoc deprotection using piperidine / DMF (1 / 4, v / v, 2×8 min, 10 mL). The resin was washed with DMF (2×2 min, 10 mL), DCM (2×2 min, 10 mL) and DMF (2×2 min, 10 mL), and the peptide N-terminal amine functional group was acetylated using a mixture of Ac2O (10 vol%), DIPEA (5 vol%), HOBt (0.2 wt%) in DMF (2×10 min, 10 mL). The resin was washed with DMF (3×2 min, 10 mL) and DCM (3×2 min, 10 mL). Resin cleavage and side-chain deprotection were carried out 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-heptane (1 / 1, v / v, 50 mL). The precipitated peptide was collected by centrifugation, washed twice with MTBE / n-heptane (1 / 1, v / v, 50 mL) and dried in vacuo. Before enzymatic ligation, the crude peptide was purified by preparative HPLC, and then the pure fractions were lyophilized.

[0175] Similarly, N-phenylacetyl-protected peptide activated esters were also prepared by replacing Ac2O with PhAc2O.

[0176] The peptide ligase used as a reference enzyme in the experimental section will be described in detail below:

[0177] The reference enzyme Ptl-10 (thymoligase GMO400) is a subtilisin BPN' variant (not belonging to the present invention), which contains an amino acid deletion at positions 75-83 compared to BPN' (SEQ ID NO: 2); has a cysteine at position 221; has an asparagine at position 225; and also has the following mutations relative to SEQ ID NO: 2: Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, E156N, G166D, G169A, S188P, F189W, Q206C, N212G, Y217R, N218S, M222G, T254A and Q271E, and 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 provides the following mutations different from SEQ ID NO: 3, E156N, G166D, F189W, Y217R, N218S and M222G, which are not related to stability. It is shown by SEQ ID NO: 4.

[0178] The reference enzyme Ptl-02 (omniligase EU GMO 79, a subtilisin BPN' variant not belonging to the present invention) contains an amino acid deletion at positions 75 - 83 compared to BPN' (SEQ ID NO:2); has a cysteine at position 221; has an asparagine at position 225; and also has the following mutations relative to SEQ ID NO:2: Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, I107V, E156S, G166S, G169A, S188P, F189W, Q206C, N212G, Y217H, N218S, M222P, T254A, and Q271E, as well as 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 provides the following mutations different from SEQ ID NO:3, I107V, E156S, G166S, F189W, Y217H, N218S, and M222P, which are not related to stability. It is shown by SEQ ID NO:5.

[0179] The reference enzyme Ptl-84 is a subtilisin BPN' variant (not belonging to the present invention). Compared with BPN' (SEQ ID NO:2), it contains an amino acid deletion at positions 75 - 83; has a cysteine at position 221; has an asparagine at position 225; and also has the following mutations relative to SEQ ID NO:2: Q2K, S3C, P5S, S9A, I31L, K43N, M50F, N62A, A73L, E156N, G166E, G169A, S188P, F189W, Q206C, N212G, Y217H, N218D, M222P, T254A, and Q271E, as well as 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 provides the following mutations different from SEQ ID NO:3, E156N, G166E, F189W, Y217H, N218D, and M222P. It is shown by SEQ ID NO:6.

[0180] The peptide-stabilizing ligase (i.e., the enzyme according to the present invention) differs from SEQ ID NO:3 in having at least one of the following mutations: H17W, S18K, N25G, V30I, S63G, S204N, E251L, N43K, and S87D, and preferably also having one or two additional mutations: T71V and V72I. Examples thereof are shown in SEQ ID NO:7 and SEQ ID NO:8.

[0181] Example 1: A stabilized variant of Ptl-10, according to sequence SEQ ID NO:4.

[0182] The melting temperatures of the reference enzyme Ptl-10 (referred to as wild-type in the table) and its variants carrying single-point stability mutations were determined by the dye-binding thermal shift screening method, as described in detail in Lavinder et al. (2009) J. AM. CHEM. SOC. 2009, 131, 3794–3795, where the method is referred to as the high-throughput thermal screening (HTTS) method. 19 μL of the enzyme stock solution was mixed with 1 μL of Sypro Orange (1000× stock solution). The following table lists the T m app (hereinafter referred to as Tm). Shown in the table are the averages of repeated measurements in this experiment.

[0183] Sample ID Temperature (°C) Wild type 78.6 Wild type + E251L 80.0 Wild type + H17W 79.5 Wild type + N25G 80.5 Wild type + N43K 79.5 Wild type + S18K 81.5 Wild type + S204N 80.0 Wild type + S87D 79.5 Wild type + S63G 81.5 Wild type + T71V 81.5 Wild type + V30I 80.5 Wild type + V72I 81.5

[0184] Obviously, each of the single mutations H17W, S18K, N25G, V30I, S63G, T71V, V72I, S204N, E251L, N43K, and S87D has a positive effect on the stability of the enzyme, which is manifested as an increase in the melting temperature Tm.

[0185] Example 2: According to sequence SEQ ID NO:4, combining positive mutations on Ptl-10 to form a peptide-stabilizing ligase according to the present invention.

[0186] Subsequently, several positive single mutations from Example 1 were added to the reference enzyme. The melting temperature was measured using the same detection method as above, i.e., 19 μL of the enzyme stock solution was mixed with 1 μL of Sypro Orange (1000× stock solution). The melting temperatures of the variants are shown in the following table. The T m app values ("Tm") shown in the first column were collected from multiple experiments. The T m app values shown in the second column are from a single experiment and are very close, indicating that this is a reproducible effect.

[0187]

[0188] Combining several single mutations can further increase the melting temperature. The subsequent addition of N43K, S87D, S18K, S63G, T71V, V72I, N25G, S204N resulted in a 9 °C increase in the melting temperature of the thermostable enzyme variant.

[0189] Example 3: Based on sequence SEQ ID NO:5 and variant sequence SEQ ID NO:6, 8 of these identified mutations were incorporated onto other enzyme scaffolds.

[0190] The melting temperatures of several reference enzymes (such as Ptl-10; Ptl-02 + A9S + L31I + 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 stable variants according to the present invention were determined using the same detection method as above, namely, 19 μL of the enzyme stock solution was mixed with 1 μL of Sypro Orange (1000× stock solution). The melting temperatures of each variant are shown in the table below.

[0191] Reference enzyme: Ptl-10 (SEQ ID NO:4)

[0192]

[0193]

[0194] Reference enzyme: Ptl-02 + A9S + L31I + S156E + G212N + C3S + C206Q according to SEQ ID NO:5 (mutations relative to SEQ ID NO:5)

[0195] Sample ID Tm (°C) Reference enzyme 64.3 Reference enzyme + N43K + S87D + S18K + S63G + T71V + V72I + N25G + S204N 75.5

[0196] Reference enzyme: Ptl-02 + C3S + C206Q according to SEQ ID NO:5 (mutations relative to SEQ ID NO:5)

[0197] Sample ID Tm (°C) Reference enzyme 68.5 Reference enzyme + N43K + S87D + S18K + S63G + T71V + V72I + N25G + S204N 79.8

[0198] Reference enzyme: Ptl-84 + L96I + N156K + E166D + C3S + C206Q (mutations relative to SEQ ID NO:6)

[0199] Sample ID Tm (°C) Reference enzyme 65 Reference enzyme + N43K + S87D + S18K + S63G + T71V + V72I + N25G + S204N 77

[0200] Reference enzyme: Ptl-84+P222H+H217R+N156K+E166D+L96I+D99R+S224V+C3S+C206Q (mutation relative to SEQ ID NO: 6)

[0201] Sample ID Tm (°C) Reference enzyme 65.8 Reference enzyme + N43K + S87D + S18K + S63G + T71V + V72I + N25G + S204N 69.5

[0202] Obviously, the addition of the thermostable mutations of N43K+S87D+S18K+S63G+T71V+V72I+N25G+S204N increased the melting temperature, thus improving the robustness and stress resistance of different enzyme variants.

[0203] Example 4: In the synthesis of thymosin α1, compared with Ptl-10 (according to SEQ ID NO: 4) (referred to here as the reference enzyme), the improvement in the stability of the thermostable variant of Ptl-10 against chaotropic agents.

[0204] The enzyme activities of the reference enzyme Ptl-10 and the thermostable variant (Ptl-10+C3S+N43K+S87D+S18K+S63G+T71V+V72I+N25G+S204N+C206Q (mutation relative to SEQ ID NO: 4)) in the reaction buffer for different concentrations of guanidine hydrochloride (GnCl) were measured. 1 μL of TCEP (100 mg / mL; pH 8.0), 72 μL of incubation buffer (82 mM Tricine+X M GnCl, pH 8.5, where X = 0, 1.5, 2.9, 4.4, and 5.8) were mixed with 6.5 μL of enzyme stock solution (0.06 mg / mL) for the reaction. The enzyme was incubated at room temperature for 30 minutes in this (chaotropic denaturing) mixture. The reaction was initiated by adding 25 μL of thymosin α1 peptide mixture (0.7 mg Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-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 50 mM Tricine; pH 8.0). After 30 minutes, the amount of product formed was analyzed by HPLC-MS (peak integration of the starting material acyl donor, hydrolyzed ester, and ligation product), and the following table reports the percentage of thymosin α1 product (produced by the ligation of two fragments) in conversion %.

[0205]

[0206] Obviously, when co-incubated with the chaotropic agent guanidine hydrochloride (GnCl), the thermostable variant showed higher retained activity compared to the reference enzyme Ptl-10. At high concentrations, all of the reference enzyme Ptl-10 denatured and no activity was observed, while the stable variant was still able to form the desired product.

[0207] Example 5: Stability of the thermostable variant of Ptl-10 towards various organic cosolvents in thymosin α1 synthesis

[0208] The enzyme activity of the thermostable variant (Ptl-10 + C3S + N43K + S87D + S18K + S63G + T71V + V72I + N25G + S204N + C206Q (mutations relative to SEQ ID NO: 4)) in reaction buffer solutions with different cosolvents (DMSO, DMF, and ACN) and their different concentrations was determined. 1 μL of TCEP (100 mg / mL; pH 8.0), 72 μL of incubation buffer (82 mM Tricine pH 8.5 containing 0, 14.5%, 29.0%, 43.5%, 58.1%, and 72.6% cosolvent) were mixed with 6.5 μL of enzyme stock solution (0.06 mg / mL) for the reaction. The enzyme was incubated in this denaturing organic cosolvent mixture at room temperature for 30 minutes. The reaction was initiated by adding 25 μL of thymosin α1 peptide mixture (0.7 mg Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-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 50 mM Tricine; pH 8.0). After 30 minutes, the amount of product formed was analyzed by HPLC-MS (peak integration for the starting material acyl donor, hydrolyzed ester, and ligation product), and the following table reports the percentage of thymosin α1 product (formed by ligation of two fragments) as % conversion.

[0209] Incubation buffer Reaction buffer Conversion rate % 82 mM Tricine 68.7 mM Tricine 86.9 82 mM Tricine + 14.5% DMSO 68.7 mM Tricine + 10% DMSO 78.7 82 mM Tricine + 29.0% DMSO 68.7 mM Tricine + 20% DMSO 70.7 82 mM Tricine + 43.5% DMSO 68.7 mM Tricine + 30% DMSO 59.9 82 mM Tricine + 58.1% DMSO 68.7 mM Tricine + 40% DMSO 52.4 82 mM Tricine + 72.6% DMSO 68.7 mM Tricine + 50% DMSO 38.9

[0210]

[0211]

[0212] Incubation buffer Reaction buffer Conversion rate % 82 mM Tricine 68.7 mM Tricine 86.9 82 mM Tricine + 14.5% ACN 68.7 mM Tricine + 10% ACN 85.5 82 mM Tricine + 29.0% ACN 68.7 mM Tricine + 20% ACN 77.3 82 mM Tricine + 43.5% ACN 68.7 mM Tricine + 30% ACN 62.0 82 mM Tricine + 58.1% ACN 68.7 mM Tricine + 40% ACN 49.1 82 mM Tricine + 72.6% ACN 68.7 mM Tricine + 50% ACN 55.6

[0213] A thermostable variant with the mutations C3S+N43K+S87D+S18K+S63G+T71V+V72I+N25G+S204N+C206Q shows activity retention even in the presence of different cosolvents at higher concentrations.

[0214] Example 6: Stability of the thermostable variant of Ptl-02 in a ligation reaction (hexapeptide and tripeptide) towards several organic cosolvents and chaotropes

[0215] The enzymatic activity of the thermostable variant (Ptl-02+C3S+N43K+S87D+S18K+S63G+T71V+V72I+N25G+S204N+C206Q+A9S+L31I+S156E+G212N (mutations relative to SEQ ID NO:5)) in reaction buffers containing different cosolvents (DMSO, DMF, ACN) and chaotropes was determined. A incubation mixture was prepared by mixing 2.5 μL of TCEP (100 mg / mL; pH 8.0), 72 μL of incubation buffer (82 mM Tricine pH 8.5 containing different concentrations of GnCl or cosolvent), 3.75 μL of milliQ water, and 1.25 μL of enzyme stock solution (0.1 mg / mL). The enzyme was incubated in this denaturing organic cosolvent or chaotrope mixture at room temperature for 30 minutes. The reaction was initiated by adding 25 μL of peptide mixture (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 (peak integration of starting material acyl donor, hydrolyzed ester, and ligation product) was analyzed using HPLC-MS, and the percentage of the ligation product (Ac-Asp-Phe-Ser-Lys-Leu-Ala-Leu-Arg-NH2) is reported in the table below.

[0216] Incubation buffer Reaction buffer Conversion rate % 82.3 mM Tricine 56.7 mM Tricine 70.0 82.3 mM Tricine + 1.5 M GnCl 56.7 mM Tricine + 1 M GnCl 82.2 82.3 mM Tricine + 2.9 M GnCl 56.7 mM Tricine + 2 M GnCl 61.7 82.3 mM Tricine + 4.4 M GnCl 56.7 mM Tricine + 3 M GnCl 30.8

[0217]

[0218]

[0219] Incubation buffer Reaction buffer Conversion rate % 82.3 mM Tricine 56.7 mM Tricine 70.0 82.3 mM Tricine + 14.5% ACN 56.7 mM Tricine + 10% ACN 76.1 82.3 mM Tricine + 29.0% ACN 56.7 mM Tricine + 20% ACN 76.6 82.3 mM Tricine + 43.5% ACN 56.7 mM Tricine + 30% ACN 57.5 82.3 mM Tricine + 58.0% ACN 56.7 mM Tricine + 40% ACN 30.4 82.3 mM Tricine + 72.6% ACN 56.7 mM Tricine + 50% ACN 12.9

[0220] The above thermostable variant of Ptl-02 shows good activity towards chaotropes and organic cosolvents. The stability mutations according to the present invention can be added to different enzyme scaffolds and still show a stabilizing effect.

[0221] Experiments were carried out using the reference enzyme Ptl-02 (SEQ ID NO:5). When the reaction increased from 1M GnCl to 2M GnCl, the conversion rate % value decreased by 90%. When guanidine hydrochloride was further increased from 2M GnCl to 3M GnCl, the conversion rate % decreased by 90% again. In the above-mentioned stable variants, the conversion rate % values only decreased by 25% and 50% respectively.

[0222] The inventors repeated the experiments on another thermostable variant (Ptl-02+C3S+N43K+S87D+S18K+S63G+T71V+V72I+N25G+S204N+C206Q (mutation relative to SEQ ID NO:5)). Compared with the above variants, this variant did not carry the four mutations A9S, L31I, S156E and G212N. The same trend was observed, and compared with the reference enzyme (BPN' variant without these mutations), the performance was more stable (maintaining a higher conversion rate %).

[0223] Example 7: Synthesis of teduglutide using a chaotropic agent containing a thermostable variant of Ptl-10 compared to the same reaction using the reference enzyme Ptl-10 (SEQ ID NO:4)

[0224] Using the reference enzyme Ptl-10 or the thermostable variant (Ptl-10+C3S+N43K+S87D+S18K+S63G+T71V+V72I+N25G+S204N+C206Q), the synthesis of teduglutide was studied under conditions with / without GnCl. In the reaction, 2mg H-His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-OCam-Leu-OH (SEQ ID NO:9) and 1mg H-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-OH (SEQ ID NO:10) were dissolved / suspended in 66.6 μL of 300 mM Tricine (pH 8.5) and 133.3 μL of milliQ water or 133.3 μL of 6M GnCl (pH 7.7). 2 μL of TCEP (100 mg / mL; pH 8.0) was added to the peptide mixture, and then the pH was adjusted to 8.0 with an aqueous solution of 3M NaOH. After adding 5 μg of the enzyme, the reaction was initiated. After 30 minutes, the amount of the formed product was analyzed by HPLC-MS (peak integration for the starting material acyl donor, hydrolyzed ester and linked product), and the percentage of the teduglutide product was reported as the conversion rate % in the following table.

[0225]

[0226] Since the solubility of the teduglutide fragment in aqueous buffer is very low, no product was formed in the reaction in the absence of guanidine hydrochloride. When the fragment was dissolved with 4 M GnCl, the reference enzyme Ptl-10 was denatured and no product was formed. However, using the stable variant, the ligation reaction proceeded smoothly in 4 M GnCl.

[0227] Sequence

[0228] SEQ ID NO:1: Wild-type gene encoding amino acids -107 to 275 of subtilisin BPN'

[0229] ENA|K02496|K02496.1 B. subtilisin BPN' Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens

[0230] SEQ ID NO:2: Wild-type subtilisin BPN' (mature form)

[0231] >SUBT_BACAM Subtilisin BPN' from Bacillus amyloliquefaciens mature form 1 to 275

[0232]

[0233] SEQ ID NO:3: Shows the sequence of a peptide ligase, namely a variant of subtilisin BPN', which has, compared to SEQ ID NO:2, a deletion corresponding to the amino acids at positions 75 - 83 (the so-called Ca 2+ binding loop), has an S221 mutation (designated S221C) and a P225 mutation (designated P225N) as well as some mutations, resulting in the following amino acids K2, C3, S5, A9, L31, N43, F50, L73, A169, P188, C206, G212, A254 and E271.

[0234] AKCVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQD 60

[0235] NNSHGTHVAGTVLA-VAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPS 130(-9)

[0236] GSAALKAAVDKAVASGVVVVAAAGNEGTSGSSSTVGYPAKYPSVIAVGAVDSSNQRAPFS 190(-9)

[0237] SVGPELDVMAPGVSICSTLPGGKYGAYNGTCMASNHVAGAAALILSKHPNWTNTQVRSSL 250(-9)ENTATKLGDSFYYGKGLINVEAAAQ

[0238] SEQ ID NO 4: Reference enzyme Ptl-10 thymic ligase GMO400, a subtilisin BPN' variant with a deletion at Δ75-83, mutations S221C and P225N, having amino acids K2, C3, S5, A9, L31, N43, F50, L73, A169, P188, C206, G212, A254, and E271; having mutations E156N, G166D, F189W, Y217R, N218S, M222G, and a 6-His tag.

[0239] AKCVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQD 60

[0240] NNSHGTHVAG TVLAVAPSAS LYAVKVLGAD GSGQYSWIIN GIEWAIANNMDVINMSLGGPS 130(-9)

[0241] GSAALKAAVDKAVASGVVVVAAAGNNGTSGSSSTVDYPAKYPSVIAVGAVDSSNQRAPWS 190(-9)

[0242] SVGPELVMAPGVSICSTLPGKYGARSGTCGASNHVAGAALILKHPNWTNQVRSSL 250(-9)

[0243] ENTATKLGDSFYYGKGLINVEAAAQHHHHHH

[0244] SEQ ID NO 5: Reference enzyme Ptl-02 omniligase EU GMO 79, a subtilisin BPN' variant with a deletion at Δ75-83, mutations S221C and P225N, having amino acids K2, C3, S5, A9, L31, N43, F50, L73, A169, P188, C206, G212, A254, and E271; having mutations I107V, E156S, G166S, F189W, Y217H, N218S, M222P, and a 6-His tag.

[0245] AKCVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQD 60

[0246] NNSHGTHVAGTVLA-VAPSASLYAVKVLGADGSGQYSWVINGIEWAIANNMDVINMSLGGPS 130(-9)

[0247] GSAALKAAVDKAVASGVVVVAAAGNSGTSGSSSTVSYPAKYPSVIAVGAVDSSNQRAPWS 190(-9)

[0248] SVGPELDVMAPGVSICSTLPGGKYGAHSGTCPASNHVAGAAALILSKHPNWTNTQVRSSL 250(-9)

[0249] ENTATKLGDSFYYGKGLINVEAAAQHHHHHH

[0250] SEQ ID NO 6: Reference enzyme Ptl-84, a subtilisin BPN' variant with a deletion at Δ75-83, mutations S221C and P225N, having amino acids K2, C3, S5, A9, L31, N43, F50, L73, A169, P188, C206, G212, A254 and E271; having mutations E156N, G166E, F189W, Y217H, N218D, M222P, and a 6-His tag.

[0251] AKCVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQD 60

[0252] NASHGTHVAGTVLAVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPS 130(-9)

[0253] GSAALKAAVDKAVASGVVVVAAAGNNGTSGSSSTVEYPAKYPSVIAVGAVDSSNQRAPWS 190(-9)

[0254] SVGPELDVMAPGVSICSTLPGGKYGAHDGTCPASNHVAGAAALILSKHPNWTNTQVRSS 250(-9)

[0255] LENTATKLGDSFYYGKGLINVEAAAQHHHHHH

[0256] SEQ ID NO:7 is an example of the peptide-stabilized ligase used in Example 7, in which S18K, N25G, N43K, S63G, T71V, V72I, S87D, and S204N are mutated

[0257]

[0258] SEQ ID NO:8 is another example of the peptide-stabilized ligase, in which S18K, N25G, N43K, S63G, T71V, V72I, S87D, and S204N are mutated

[0259]

[0260] SEQ ID NO:9 provides the amino acid sequence of the first teduglutide fragment in Example 7

[0261] His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-OCam-Leu-OH

[0262] SEQ ID NO:10 provides the amino acid sequence of the second teduglutide fragment in Example 7

[0263] Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp。

Claims

1. A peptide-stabilized ligase having at least 80% sequence identity with the peptide ligase shown in SEQ ID NO:3, characterized in that - compared with BPN’, it contains a deletion of the amino acids at positions 75 - 83; and - has a cysteine or selenocysteine at position 221, preferably cysteine; and - has an amino acid which is alanine or asparagine at position 225, preferably asparagine; and - contains at least one mutation selected from the group consisting of H17W, S18K, N25G, V30I, S63G, S204N, E251L, N43K and S87D; wherein the amino acid mutation is defined as a mutation relative to the peptide ligase shown in SEQ ID NO:

3.

2. The peptide-stabilized ligase according to claim 1, having at least 90% sequence identity with the peptide ligase shown in SEQ ID NO:

3.

3. The peptide-stabilized ligase according to any one of the preceding claims, further comprising at least one additional mutation selected from T71V and V72I.

4. The peptide-stabilized ligase according to any one of the preceding claims, containing two mutations N43K and S87D.

5. The peptide-stabilized ligase according to any one of the preceding claims, containing 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 two, three or four mutations selected from the group consisting of H17W, S18K, N25G, V30I, S63G, V72I, S204N, E251L and the combination of N43K and S87D.

6. The peptide-stabilized ligase according to any one of claims 1 - 4, containing five mutations selected from the group consisting of S18K, N25G, S63G, T71V, S204N, E251L and the combination of N43K and S87D; or five mutations selected from the group consisting of S18K, N25G, S63G, V72I, S204N, E251L and the combination of N43K and S87D.

7. The peptide-stabilized ligase according to any one of claims 1 - 4, containing six mutations, wherein four mutations are selected from the group consisting of S18K, N25G, S63G, T71V and S204N; or selected from the group consisting of S18K, N25G, S63G, V72I, S204N and E251L; and the other two mutations are N43K and S87D.

8. The peptide-stabilized ligase according to any one of claims 1 - 4, containing the mutations S18K, N25G, S63G, T71V, V72I and S204N, and N43K and S87D.

9. The peptide-stabilized ligase according to any one of claims 1-8, characterized in that, The peptide ligase having SEQ ID NO:3 at the amino acid at one or more, preferably 11 positions selected from: K2, C3, S5, A9, L31, F50, L73, A169, P188, C206, G212, A254 and E271.

10. The peptide-stabilizing ligase according to any one of the preceding claims, wherein the peptide-stabilizing ligase is immobilized on a solid support.

11. A method for the enzymatic synthesis of a peptide, the method comprising the step of coupling (a) a peptide C-terminal ester or thioester with (b) a peptide nucleophile having an unprotected amine at the N-terminus, wherein the coupling is carried out in an aqueous solution, and wherein the coupling is catalyzed by a peptide-stabilizing ligase having at least 80% sequence identity, preferably at least 90% sequence identity, with the peptide ligase shown in SEQ ID NO:3, compared to BPN’ (SEQ ID NO:2), contains a deletion of the amino acids at positions 75 - 83; and has a cysteine or selenocysteine, preferably cysteine, at position 221; and has an amino acid which is alanine or asparagine, preferably asparagine, at position 225; and wherein the peptide-stabilizing ligase contains 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 mutation is defined as a mutation relative to the peptide ligase shown in SEQ ID NO:

3.

12. The method according to claim 10, wherein the peptide-stabilizing ligase is further characterized by having the amino acids of the peptide ligase of SEQ ID NO:3 at one or more, preferably 11 positions selected from: 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 contains a chaotropic agent.

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

15. The method according to claim 12, wherein the aqueous solution contains guanidine hydrochloride.

16. The method according to claim 12, wherein the aqueous solution contains guanidine hydrochloride in a concentration range of 1 - 5 M, preferably in a concentration range of 2 - 4 M.

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