Subtilisin variants and uses thereof

Abstract

The present invention relates to a subtilisin BPN' variant or homologue thereof comprising new mutations compared to subtilisin BPN' as shown in SEQ ID NO: 2 or a homologous sequence thereof. Such mutations can occur at amino acid positions selected from the group consisting of L96, D99, A223 and S224. The present invention also relates to a method for the enzymatic synthesis of a peptide by coupling of peptide fragments, wherein said coupling is catalysed by said subtilisin BPN' variant or homologue thereof.

Description

Technical Field

[0001] This invention relates to an enzyme, which is a variant of the subtilisin BPN' protein or a homolog thereof. The invention also relates to a method for catalyzing peptide fragment coupling with this enzyme and a method for peptide synthesis using this enzyme. Background Technology

[0002] Methods for synthesizing peptides are well known in the art. Relatively short peptides can be chemically synthesized in solution via stepwise methods or through a process known as peptide solid-phase synthesis, which refers to the synthesis of peptides by simultaneously immobilizing one end to a solid phase using highly optimized methods. However, peptides longer than 10–15 amino acids are generally difficult to synthesize due to potential side reactions. As a result, their purification is cumbersome. Therefore, such peptides are typically synthesized by combining a side-chain-protected peptide fragment synthesized in the solid phase with its subsequent chemical condensation in solution. A major drawback of the chemical coupling of side-chain-protected peptide fragments is the racemic reaction that occurs upon activation of the C-terminal amino acid residue of the acyl donor. In contrast, racemic reaction is not observed in the enzymatic coupling of peptide fragments. Another advantage compared to peptide chemical synthesis is the absence of side reactions at the side-chain functional groups. The enzymatic coupling of chemically synthesized peptides is known as peptide chemistry-enzyme synthesis.

[0003] The term peptide chemistry-enzymatic synthesis refers to the enzymatic coupling of multiple peptide fragments, wherein the multiple peptide fragments have been synthesized individually using chemical synthesis (in solution and / or solid phase), fermentation, or a combination of chemical and enzymatic coupling steps.

[0004] Wells et al. (US 5,403,737) discovered that altering the active site of subtilisin BPN', a subtilisin protease derived from Bacillus amyloliquefaciens (SEQ ID NO: 2), significantly improved peptide conjugation in aqueous solution. When two mutations, S221C and P225A, were introduced, a subtilisin BPN' variant, termed subtiligase, was obtained, exhibiting a 500-fold increase in synthesis / hydrolysis ratio (S / H ratio) compared to the wild-type subtilisin BPN'. In further experiments, Wells et al. introduced five additional mutations into the subtiligase—M50F, N76D, N109S, K213R, and N218S—to improve enzyme stability (Proc. Natl. Acad. Sci. USA, 1994, 91, 12544). This new mutant, called a stable ligase, appears to have moderately higher resistance to treatment with sodium dodecyl sulfate and guanidine hydrochloride, but hydrolysis remains the main side reaction.

[0005] WO 2016 / 056913 addresses the problem of undesirable high hydrolytic activity of enzymes such as Bacillus subtilis ligase or stable ligase when used for peptide synthesis in an aqueous environment, and provides a Bacillus subtilis protease BPN' variant exhibiting an improved S / H ratio, characterized by the deletion of amino acids at positions 75–83 and a mutation at amino acid position S221.

[0006] Other BPN' mutants of the subtilisin are provided in WO 2018 / 212658, which have one or more specific mutations in the penultimate pocket near the coupling site, i.e., in the S2' pocket and / or the S2 pocket, thereby broadening the substrate range of the peptide and improving coupling efficiency.

[0007] WO2019170895 and WO2019170918 disclose methods for the enzymatic synthesis of liraglutide and semaglutide by a specific peptide fragment coupling strategy catalyzed by a BPN' mutant of subtilisin.

[0008] However, there is still a need to provide other BPN' variants of subtilisin or their homologs, which can be used for enzymatic synthesis of peptides via fragment coupling or cyclization, thereby improving reaction rates and coupling efficiency while maintaining or enhancing the stability of the BPN' variants.

[0009] Furthermore, there is still a need to find efficient methods for the enzymatic synthesis of novel peptides with pharmacological significance, especially methods suitable for industrial applications.

[0010] The enzymes and methods according to the present invention solve or at least alleviate some or all of the above-mentioned problems. Summary of the Invention

[0011] It has now been found that all or at least some of the problems discussed above can be overcome by providing new enzymes with one or more specific mutations in the acyl donor binding pocket of the BPN' variant of subtilisin or its homolog.

[0012] Therefore, in a first embodiment, the present invention provides a subtilisin BPN' variant or homolog thereof, which has at least 80% sequence identity with subtilisin BPN' SEQ ID NO: 2, comprising a deletion of amino acids 75 to 83 and a mutation at amino acid position S221, said mutation being S221C or S221 selenocysteine, preferably S221C; and the subtilisin BPN' variant or homolog thereof is characterized by having at least one mutation at an amino acid position selected from L96, D99, A223 and S224, wherein said amino acid position is defined according to SEQ ID NO: 2 or its homologous sequence, and wherein said variant or homolog thereof has ligase and / or cyclase activities with improved reaction rate and coupling efficiency.

[0013] The enzyme according to the invention has catalytic activity (coupling activity) in peptide bond formation. This activity is also known as "ligase activity".

[0014] The present invention provides enzymes that preferably exhibit increased reaction rates and enhanced coupling efficiency in aqueous reaction media. The present invention also provides enzymes that exhibit enhanced selectivity for the coupling reaction of (a) a C-terminal (thio)ester of a peptide with (b) a peptide nucleophile having an N-terminal unprotected amine, thereby producing a target peptide.

[0015] Therefore, in a second embodiment, the present invention provides a method for enzymatic synthesis of peptides, the method comprising the step of coupling (a) a C-terminal (thio) ester of a peptide with (b) a peptide nucleophile having an N-terminal unprotected amine, wherein the coupling is preferably carried out in an aqueous solution, and wherein the coupling is catalyzed by a variant of subtilisin BPN' or a homolog thereof, the variant or homolog thereof having at least 80% sequence identity with subtilisin BPN' SEQ ID NO: 2, comprising a deletion of amino acids 75-83 and a mutation at amino acid position S221, the mutation being S221C or S221 selenocysteine, preferably S221C; and the variant or homolog thereof is characterized by having at least one mutation at an amino acid position selected from L96, D99, A223 and S224.

[0016] The positions of the amino acids are defined according to SEQ ID NO: 2 or their homologous sequences. Detailed Implementation

[0017] As used herein, the term "reaction" refers to a reaction that occurs under the catalysis of a BPN' variant of subtilisin or its homolog. Specifically, the terms "coupling reaction" or "coupling" refer 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) or one molecule (intramolecular coupling).

[0018] As used herein, the term "reaction rate" refers to the rate at which the peptidyl donor (thio)ester starting material is consumed by a variant of the subtilisin BPN' or its homolog. When forming an acylase complex, the acyl donor can be coupled to an acyl acceptor (i.e., a peptide nucleophile) (preferred coupling reaction) or can be hydrolyzed by water (an undesirable side reaction). The acyl donor reaction rate, or conversion rate, is the sum of the product formation rate and the hydrolysis rate. Enzymes with increased reaction rates exhibit higher catalytic efficiency compared to wild-type enzymes. Using the same amount of enzyme, the reaction is completed in a shorter time.

[0019] The term “(thio) ester” used in this article is a shortened form of “ester or thioester”.

[0020] As used herein, the term "coupling efficiency" or "coupling reaction efficiency" refers to the amount of peptide-linked product (i.e., target peptide) formed within a given time period catalyzed by a variant of BPN' subtilisin or its homolog. Enzymes with increased coupling efficiency synthesize more product within a given time period compared to wild-type enzymes. Hydrolysis of acyl donor (thio)esters is not considered for coupling efficiency assessment.

[0021] As used herein, the term "synthesis / hydrolysis ratio" (S / H ratio) refers to the amount of peptide-linked product divided by the amount of acyl donor (thio) ester (i.e., acyl donor (thio) acid) that has been hydrolyzed. When the acyl donor (thio) ester is used in excess in the reaction, the S / H ratio can be estimated by dividing the amount of peptide-linked product by the amount of peptide nucleophile.

[0022] As used herein, the term "selectivity" refers to the amount of peptide-linked product divided by the amount of byproducts formed when an acyl donor (thio) ester reacts with the unprotected N-terminus amine of the peptide-linked product. This byproduct formation is highly undesirable, as each byproduct molecule consumes one of the reactants or the target peptide. Therefore, highly selective enzymes are generally preferred.

[0023] As used herein, the term "peptide" refers to any sequence consisting of two or more amino acids linearly linked together 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. Each peptide is defined by its specific amino acid sequence.

[0024] The difference between peptides and proteins lies in their shorter length, but the cutoff number of amino acids used to distinguish between peptides and proteins is variable in the art. Typically, peptides contain 2 to 500 amino acids, more usually 2 to 200, or 2 to 100. Preferably, peptides contain at least 10 amino acids, more preferably at least 15 amino acids. Furthermore, the length of a peptide does not exceed 200, preferably not more than 100, and most preferably not more than 50 amino acids. Therefore, most preferably, the length of a peptide is 10 to 50 amino acids.

[0025] Peptides can contain proteogenic amino acids and / or non-proteogenic amino acids. Proteogenic amino acids are α-amino acids with an L-configuration encoded by the genetic code. Non-proteogenic 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-fluoro-phenylalanine, α-aminoisobutyric acid (Aib), other C-α-alkylated amino acids, and selenocysteine ​​(Sec, U), where selenocysteine ​​is an amino acid whose structure corresponds to cysteine ​​but with a sulfur atom replaced by selenium. Peptides can be linear, branched, or cyclic, wherein branched peptides have at least two interconnected amino acid sequences.

[0026] Peptides can be composed of amino acids or amino acids and protecting groups, wherein the protecting groups can be located at the terminal amino and carboxyl groups or at the amino acid side chain. The term "amino acid side chain" as used herein refers to any proteogenic or non-proteogenic amino acid side chain.

[0027] Peptides can be “coupled peptides,” which are peptide sequences of two or more amino acids attached to another residue, for example: synthetic hydrophilic polymers, such as polyalkylene glycols, preferably polyethylene glycol (PEG); lipophilic moieties or amino acids or combinations thereof; imaging agents; radiotherapy agents; toxins; another non-peptide agent, such as chelating agents or non-peptide bioactive moieties.

[0028] Peptides can be bioactive peptides. Preferred examples of bioactive peptides include glucagon, glp-1, glp-2 and analogues such as dasiglucagon, exenatide, liraglutide, semaglutide, lixisenatide, teduglutide, glepaglutide, dulaglutide, elsiglutide, thymosin-α-1, thymosin-α-1 analogues, teriparatide, salmon calcitonin, bivalirudin, and peptides containing the sequence of any of these peptides and at least one additional amino acid.

[0029] As used herein, the term "cyclic peptide" refers to a peptide with a cyclic structure, wherein such a peptide is produced by the formation of an amide bond (also known as a "cyclization reaction") between the terminal α-amino group and the terminal α-carboxyl group of an amino acid sequence. Specifically, such an amino acid sequence has at least 12 amino acids.

[0030] 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. Specifically, a peptide bond can be between the α-amino group of one α-amino acid and the α-carboxyl group of another α-amino acid.

[0031] When referring to proteins or enzymes, the term “mutated” or “mutated” as used herein means that, in a wild-type or naturally occurring protein or enzyme sequence, at least one amino acid has been substituted for, inserted into, appended to, or deleted from the sequence by mutagenesis of the nucleic acid encoding those amino acids. Mutagenesis includes, for example, site-directed mutagenesis mediated by PCR or oligonucleotides, as described in Siloto et al., “Site saturation mutagenesis: Methods and applications in protein engineering,” Biocatalysis and Agricultural Biotechnology 1, (2012) 181-189. When referring to nucleic acids or genes, the term “mutated” or “mutated” as used herein means a protein sequence in which at least one nucleotide has been substituted for, inserted into, appended to, or deleted from the sequence by mutagenesis, resulting in a qualitative or quantitative alteration of transcriptional function, or a “knockout” of the nucleic acid, which means that the nucleic acid no longer encodes the functional protein previously encoded by the mutation.

[0032] In this disclosure, a mutation is described as a single-letter amino acid code for the substituted amino acid, followed by a number indicating the position in the protein's amino acid sequence where the substitution occurs. This number is the amino acid position in the wild-type amino acid sequence. Therefore, for a mutated amino acid sequence, it is the amino acid position corresponding to the position in the wild-type enzyme with that number. The actual position in the mutant may not be the same due to one or more other mutations (additions, insertions, deletions, etc.) at other positions. Those skilled in the art can determine the corresponding position using well-known alignment techniques such as NEEDLE. Following the number is a single-letter code for the amino acid that replaces the wild-type amino acid. For example, F189W indicates that phenylalanine (F) at position 189 is replaced by tryptophan (W). X is used to indicate any other proteogenic amino acid that may be present at that position besides the amino acid being replaced. For example, F189X indicates that phenylalanine at position 189 is replaced by any other proteogenic amino acid.

[0033] As used herein, the term "ligase" refers to an enzyme that exhibits catalytic activity in the coupling of two peptides by catalyzing the formation of a peptide bond between the C-terminus of one peptide and the N-terminus of another peptide. This activity is also referred to as "ligase activity." When such an enzyme exhibits catalytic activity in forming an intramolecular peptide bond between the C-terminus and N-terminus of the same peptide molecule, this activity can also be referred to as "cyclase activity." Therefore, the same enzyme can possess both ligase and / or cyclase activities. An enzyme can be characterized as having ligase activity when its S / H ratio is greater than 1. This S / H ratio can be determined by HPLC analysis of the corresponding amount.

[0034] In the reaction medium used, particularly in reaction media containing water, and more particularly in aqueous media (also known as aqueous solutions), the ligase typically has an S / H ratio greater than 1, preferably 2 or greater, and especially 5 or greater. The upper limit of this quotient is not important; it can be, for example, 100 or less.

[0035] The term "homology" of an enzyme as used herein refers to an enzyme that has the same intended function as the described enzyme, such as being able to catalyze the same reaction. In this disclosure, such a reaction is a coupling reaction, i.e., the formation of a peptide bond. Specifically, the homologs of the enzymes used herein possess ligase and / or cyclase activities.

[0036] The term homolog is further defined by the level of similarity of its sequence to that of the enzyme. Throughout this application, this level of similarity is referred to as “percentage identity” or “sequence identity.” The terms “percentage identity” and “sequence identity” are used interchangeably herein.

[0037] According to the present invention, a "subtilisin BPN' homolog" is an enzyme having ligase and / or cyclase activity, having at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with subtilisin BPN' (i.e., the enzyme to which the homologous peptide or enzyme is compared). Percentage identity is determined according to the NEEDLE EMBOSS method outlined below. Obviously, percentage identity will be less than 100%. Percentage identity depends on the number of mutations and the length of the peptide (enzyme) to which the homolog is compared.

[0038] For the purposes of this invention, this document defines the process of aligning complete, mature sequences for optimal comparison purposes to determine the percentage identity of two amino acid sequences, such that similar regions are aligned. Any sequence elongation (at the N- or C-terminus), such as commonly used His tags or other tags used for purposes such as purification, signal transduction, dissolution, and localization, is not considered in determining percentage identity. To optimize the alignment between the two sequences, vacancies may be introduced in either of the compared sequences. The alignment used to determine the % sequence identity value is performed over a length of at least 200 amino acids of the compared sequences.

[0039] Sequence comparison and determination of percentage identity between two sequences can be accomplished using mathematical algorithms, such as the Needleman-Wunsch algorithm (Needleman, SB and Wunsch, CD, (1970) J.Mol.Biol.48(3), pp443-453), which is executed in the computer program NEEDLE.

[0040] To calculate percentage identity, the NEEDLE program from the EMBOSS software package (version 2.8.0 or later, EMBOSS: The European Molecular Biology Open Software Suite (2000), Rice, P. Longden, I. and Bleasby, A., Trends in Genetics 16(6) pp 276-277) was used. For protein sequences, EBLOSUM62 should be used for the substitution matrix. The parameters used for amino acid sequence alignment must be set to a vacancy opening penalty of 10 and a vacancy elongation penalty of 0.5. Homology or percentage identity between 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 (after subtracting the total number of vacancy positions in the alignment).

[0041] The percentage identity defined in this article can be obtained from NEEDLE and labeled as "identity" in the output of the program.

[0042] The term "analyte" of a peptide is specifically used herein to refer to a peptide that is a structural and / or functional analogue of the peptide. Functional analogues have the same in vivo target (e.g., the same target receptor on the cell membrane); structural analogues have high similarity in terms of amino acid sequence. A functional analogue of a peptide may have relatively low amino acid sequence identity with the peptide (these analogues are analogues of the peptide) over its entire amino acid sequence, for example, about 50% or less, but high sequence identity (and therefore high structural similarity) in a certain segment of the amino acid sequence, such as a segment near the N-terminus or near the C-terminus. Specifically, a structural analogue comprises an amino acid sequence that has at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 95% sequence identity with the amino acid sequence of the peptide (these structural analogues are analogues of the peptide). The term "analyte" is used herein when referring to the target peptide of a coupling reaction.

[0043] The terms “variant” or “mutant” of an enzyme are used herein to refer to an enzyme that has at least one mutation or a mutated amino acid relative to the enzyme. Specifically, the subtilisin BPN' variant is an enzyme that has at least one mutation relative to the sequence of SEQ ID NO: 2.

[0044] Surprisingly, it has been found that the mutation at position L96 significantly improves the reaction rate and coupling efficiency in the reaction according to the invention compared to the BPN' variant of subtilisin or its homologs known from WO2016 / 06913. Therefore, L96 is a preferred mutation position according to the invention. Similar improvements can be obtained by mutations at positions D99, A223, or S224. Therefore, D99, A223, and S224 are each preferred mutation positions.

[0045] Therefore, the present invention provides a variant of BPN' subtilisin or a homolog thereof, which has at least 80% sequence identity with SEQ ID NO: 2 representing the sequence of BPN' subtilisin, comprising a deletion of amino acids 75-83 and a mutation at amino acid position S221, wherein the mutation is S221C or S221 selenocysteine, preferably S221C; characterized in that

[0046] At least one mutation at the amino acid position selected from L96, D99, A223, and S224.

[0047] The amino acid positions are defined based on the sequence of BPN' (subtilisin BPN) shown in SEQ ID NO: 2 or its homologous sequences.

[0048] The amino acid sequence of subtilisin BPN' is provided in SEQ ID NO: 2 (mature form). The nucleic acid sequence encoding amino acids -107 to 275 of subtilisin BPN' is provided in SEQ ID NO: 1.

[0049] SEQ ID NO: 3 shows a variant of the BPN' protease according to the present invention, which has amino acids corresponding to positions 75-83 (so-called Ca). 2+ The deletion of the binding loop, having the S221 mutation (denoted as S221C), wherein the amino acids marked as X at positions L96, D99, A223, and S224 represent any proteogenic amino acid at that position, thereby including amino acids according to SEQ ID NO: 2 (if said positions are not mutated), provided that at least one X at one of these positions is an amino acid not present at said positions in SEQ ID NO: 2, i.e., at least one X is mutated. Other preferred enzymes may contain one or more additional mutations, particularly one or more other mutations identified elsewhere below.

[0050] Specifically, the present invention provides an isolated enzyme. In this document, the term "isolated" means that it is isolated from the organism in which it is expressed, typically from a recombinant organism (if it has already been produced in the organism), or from the reaction medium in which it is synthesized.

[0051] Specifically, the enzymes according to the invention are isolated or substantially purified in crude form by any suitable technique, such as the single-step purification method disclosed in Smith and Johnson, Gene 67:31-40 (1988).

[0052] The enzymes according to the invention can be provided in a form that is at least substantially pure, wherein the term "substantially pure enzyme" means an enzyme with a purity of at least 75 wt.%, preferably more than 80 wt.%. The enzymes can also be provided as a mixture with one or more other components, for example, as a stock solution, preferably in an aqueous buffer.

[0053] The enzyme according to the invention may contain a terminal His tag, preferably a 6-His tag.

[0054] Surprisingly, a cumulative effect has been observed when two, three, or all four mutations at the aforementioned positions L96, D99, A223, and S224 are combined.

[0055] Therefore, in a preferred embodiment, the BPN' variant or homolog of the subtilisin according to the present invention comprises one of the following:

[0056] - A mutation at the amino acid position selected from L96, D99, A223, and S224; or

[0057] - Two mutations at amino acid positions selected from L96 and D99, L96 and A223, L96 and S224, D99 and A223, D99 and S224, and A223 and S224; or

[0058] - Three mutations at amino acid positions selected from L96, D99 and A223, L96, D99 and S224, L96, A223 and S224, and D99, A223 and S224; or

[0059] - Four mutations at amino acid positions L96, D99, A223, and S224.

[0060] Of the several possible mutations at position L96, eight specific mutations—L96I, L96V, L96M, L96T, L96C, L96Q, L96A, and L96S—performed particularly well. These are highly preferred mutations, with L96I and L96V being even more preferred.

[0061] In another embodiment, the BPN' variant of the subtilisin according to the present invention or its homolog contains a mutation at the L96 position, which is preferably selected from L96I, L96V, L96M, L96T, L96C, L96Q, L96A and L96S, and more preferably selected from L96I and L96V.

[0062] Of the several possible mutations at position D99, 15 mutations—D99R, D99K, D99G, D99F, D99T, D99S, D99N, D99Q, D99Y, D99M, D99I, D99H, D99E, D99L, and D99W—performed particularly well and are therefore preferred. The mutations D99R, D99K, and D99G yielded particularly good results, and are therefore even more preferred.

[0063] In yet another embodiment, the BPN' variant of the subtilisin according to the invention or its homolog contains a mutation at the D99 position, preferably selected from D99R, D99K, D99G, D99F, D99T, D99S, D99N, D99Q, D99Y, D99M, D99I, D99H, D99E, D99L and D99W, and more preferably selected from D99R, D99K and D99G.

[0064] Among the several possible mutations at position A223, mutations A223S and A223G performed particularly well and were therefore preferred.

[0065] In another embodiment, the BPN' variant of the subtilisin according to the invention or its homolog contains a mutation at the A223 position, preferably selected from A223S and A223G.

[0066] Of the several possible mutations at position S224, mutations S224M, S224Q, S224E, S224H, S224L, S224V, and S224I are particularly preferred because they show particularly good results.

[0067] Therefore, in another embodiment, the BPN' variant of the subtilisin according to the present invention or its homolog contains a mutation at position S224, preferably selected from S224M, S224Q, S224E, S224H, S224L, S224V and S224I.

[0068] In a preferred embodiment, the BPN' variant of the subtilisin according to the present invention or its homolog comprises at least one, preferably two, three, or four mutations selected from L96I, L96V, L96M, L96T, L96C, L96Q, L96A, L96S, D99R, D99K, D99G, D99F, D99T, D99S, D99N, D99Q, D99Y, D99M, D99I, D99H, D99E, D99L, D99W, A223S, A223G, S224M, S224Q, S224E, S224H, S224H, S224L, S224V, and S224I; more preferably, the group of mutations The combination is selected from L96V and D99R; L96I and D99R; L96I and D99K; L96I and A223S; L96I and S224V; L96V, D99R and A223S; L96V, D99R and S224V; L96I, D99R and A223S; L96I, D99R and S224V; L96I, D99K and A223S; L96I, D99K and S224V; L96V, D99R, A223S and S224V; L96I, D99R, A223S and S224V; L96V, D99K, A223S and S224V; and L96I, D99K, A223S and S224V.

[0069] The preferred combination of two mutations is selected from L96V and D99R; L96I and D99R; L96I and D99K; L96I and A223S; and L96I and S224V.

[0070] The preferred combination of the three mutations is selected from L96V, D99R and A223S; L96V, D99R and S224V; L96I, D99R and A223S; L96I, D99R and S224V; L96I, D99K and A223S; L96I, A223S and S224V; and L96I, D99K and S224V.

[0071] The preferred combination of the four mutations is selected from L96V, D99R, A223S and S224V; L96I, D99R, A223S and S224V; L96V, D99K, A223S and S224V; and L96I, D99K, A223S and S224V.

[0072] The enzyme according to the invention may have additional mutations compared to BPN', provided that it has ligase and / or cyclase activities as defined above, and preferably one or more additional mutations as described elsewhere herein.

[0073] The enzyme according to the invention preferably also contains a mutation at the amino acid position corresponding to P225, which may be advantageous for improving the S / H ratio in coupling or cyclization reactions of interest.

[0074] Therefore, the present invention provides a BPN' variant of subtilisin or a homolog thereof, which further includes a mutation at amino acid position P225, preferably selected from P225N, P225D, P225S, P225C, P225G, P225A, P225T, P225V, P225I, P225L, P225H and P225Q, more preferably selected from P225N, P225D, P225S, P225C, P225G, P225A and P225T, and even more preferably corresponding to P225N or P225D.

[0075] To obtain good enzyme stability, the BPN' variant of the subtilisin according to the present invention or its homologs preferably further include one or more mutations at amino acid positions selected from Q2, S3, P5, S9, I31, K43, M50, A73, G169, S188, Q206, N212, T254, and Q271. Preferably, the one or more additional mutations are selected from Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, G169A, S188P, Q206C, N212G, T254A, and Q271E.

[0076] Therefore, the present invention provides a BPN' variant of subtilisin or a homolog thereof, which further comprises at least 6, preferably at least 8, more preferably at least 10, and even more preferably at least 12 mutations at amino acid positions selected from Q2, S3, P5, S9, I31, K43, M50, A73, G169, S188, Q206, N212, T254, and Q271, wherein such mutations are preferably selected from Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, G169A, S188P, Q206C, N212G, T254A, and Q271E.

[0077] Furthermore, preferably, the BPN' variant of the subtilisin according to the invention or its homologs further comprises one or more mutations at amino acid positions selected from S33, N62, E156, G166, Y217, N218, and F189, such mutations preferably selected from S33T, N62A, N62R, N62K, E156S, E156N, E156K, E156R, G166S, G166E, G166D, Y217L, Y217H, Y217R, N218S, N218D, and F189W. More preferred combinations of mutations are E156K and G166E; and E156K and G166D.

[0078] In another preferred embodiment, the BPN' variant of the subtilisin according to the invention or its homolog further comprises at least one mutation or paired mutation at amino acid positions M222 and Y217, wherein the single mutation is preferably selected from M222P, M222G, M222H, Y217H, Y217G, Y217F, Y217L and Y217R, and wherein the paired mutation is preferably selected from M222P and Y217H; M222P and Y217G; M222G and Y217F; M222G and Y217G; M222G and Y217L; and M222H and Y217R.

[0079] In other preferred embodiments, the BPN' variant of the subtilisin according to the present invention or its homologs further comprises mutants Q2K, S3C, P5S, S9A, I31L, K43N, M50F, N62A, A73L, E156N, G166E, G169A, S188P, F189W, Q206C, N212G, Y217H, N218D, M222P, P225N, T254A, and Q271E.

[0080] In yet another preferred embodiment, the BPN' variant of the subtilisin according to the invention or its homolog further comprises the mutants Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, I107V, E156S, G166S, G169A, S188P, F189W, Q206C, N212G, Y217H, N218S, M222P, P225N, T254A, and Q271E.

[0081] In a more preferred embodiment, the BPN' variant of the subtilisin according to the present invention or its homolog comprises mutants Q2K, S3C, P5S, S9A, I31L, K43N, M50F, N62A, A73L, E156K, G166D, G169A, S188P, F189W, Q206C, N212G, Y217R, N218D, M222H, P225N, T254A, Q271E, and selected from... The following mutation combinations are: L96I and D99R; L96I and D99K; L96I and A223S; L96I and S224V; L96I, A223S and S224V; L96I, D99R and S224V; L96I, D99R, A223S and S224V; L96I, D99K and A223S; L96I, D99K and S224V; and L96I, D99K, A223S and S224V.

[0082] Other subtilisinases, particularly subtilisinase BPN' homologs, can be used instead of subtilisinase BPN' as template enzymes from which enzymes according to the invention can be derived by mutagenesis. Such variants according to the invention have at least the deletion of amino acids corresponding to L75 up to and including G83 of subtilisinase BPN', a cysteine ​​or selenocysteine ​​at position 221 corresponding to subtilisinase BPN', and at least one of the other mutations taking all possible combinations as defined above at amino acid positions selected from L96, D99, A223, and S224.

[0083] Therefore, in one embodiment, the present invention provides a variant of BPN' subtilisin or a homolog thereof, having at least 80% sequence identity with SEQ ID NO: 2, preferably at least 85%, more preferably at least 90%, or even more preferably at least 95% identity; comprising the deletion of amino acids 75 to 83 and a mutation at amino acid position S221, said mutation being S221C or S221 selenocysteine, preferably S221C; and the variant or homolog thereof is characterized by at least one mutation at an amino acid position selected from L96, D99, A223, and S224, wherein said amino acid position is defined according to the sequence of BPN' subtilisin or a homolog thereof shown in SEQ ID NO: 2, and wherein such variant or homolog thereof has ligase and / or cyclase activities with improved reaction rate and coupling efficiency.

[0084] The enzymes of the present invention are typically produced by recombinant methods, preferably by expressing a mutated subtilisin BPN' DNA, resulting in an enzymatic variant of the present invention, the subtilisin BPN', after expression.

[0085] Therefore, the present invention also provides a method for preparing a recombinant enzyme according to the present invention, the method comprising the following steps:

[0086] a) Provide a recombinant host cell, such as a bacterial cell, like Escherichia coli or Bacillus, that functionally expresses the gene encoding the enzyme;

[0087] b) The host cells are cultured under conditions that provide expression of an enzyme with enzymatic activity; and

[0088] c) Recover the expressed enzymes from the microbial host.

[0089] The present invention also provides a recombinant polynucleotide comprising a sequence encoding an enzyme according to the present invention.

[0090] The present invention also provides a host cell comprising a polynucleotide according to the invention, the polynucleotide being capable of expressing the enzyme.

[0091] According to the present invention, a variant of the subtilisin BPN' or a homolog thereof catalyzes the formation of an amide bond. Therefore, the present invention provides a method for enzymatic synthesis of peptides, the method comprising the step of coupling (a) a C-terminal (thio) ester of a peptide to (b) a peptide nucleophile having an N-terminal unprotected amine, wherein the coupling is preferably carried out in an aqueous solution, and wherein the coupling is catalyzed by a variant of the subtilisin BPN' or a homolog thereof, the variant or homolog having at least 80% sequence identity with SEQ ID NO: 2, comprising the deletion of amino acids 75-83 and a mutation at amino acid position S221, the mutation being S221C or S221 selenocysteine, preferably S221C; and

[0092] The variant or its homolog is characterized by having at least one mutation at an amino acid position selected from L96, D99, A223, and S224.

[0093] The amino acid positions are defined according to the sequence of BPN' subtilisin as shown in SEQ ID NO: 2 or its homologous sequence, and the variants or homologs thereof have ligase and / or cyclase activities with improved reaction rate and coupling efficiency.

[0094] The use of the enzyme according to the invention extends beyond catalyzing the cyclization of peptides as described above and / or the coupling of C-terminal (thio) esters of peptides with peptide nucleophiles. The BPN' variant of the subtilisin or its homologs can be used for the formation of amide bonds other than peptide bonds, but its use in relation to peptide bonds is particularly preferred.

[0095] In the method according to the invention, the reaction is typically carried out in an aqueous medium (preferably containing a buffer).

[0096] The water content of the solvent in the aqueous medium is in the range of 10–100 vol%. Particularly good results were obtained in aqueous media in which the solvent contains 70–100 vol%, more particularly 90–100 vol%, 95–100 vol%, or 98–100 vol%. water. More preferably, the solvent is water only.

[0097] Other suitable solvents are water-miscible cosolvents, such as N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), N,N-dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), acetonitrile (ACN), or ethers such as tetrahydrofuran (THF), 2-methyltetrahydrofuran (Me-THF), or 1,2-dimethoxyethane, or (halogenated) alcohols such as methanol, ethanol, isopropanol, tert-butanol, 2,2,2-trifluoroethanol (TFE), 1,1,1,3,3,3-hexafluoroisopropanol, or mixtures thereof. The amount of cosolvent is preferably less than 30 vol%, depending on the stability of the BPN' variant of the subtilisin and the solubility of the peptide substrate.

[0098] The pH of the aqueous buffer medium used for coupling or cyclization reactions can be at least 5, particularly at least 6, and preferably at least 7. The desired pH is typically below 11, particularly below 10, and even more preferably below 9. More preferably, the pH used for coupling or cyclization reactions is between about 7 and 9.

[0099] In principle, any buffer that maintains the pH in the range of 5 to 11 is suitable. Suitable buffers are known to those skilled in the art and are also described, for example, in Good, NE et al., (1966), “Hydrogen Ion Buffers for Biological Research”, Biochemistry 5(2), 467-477. For example, particularly good results have been obtained using Good's buffers such as tricine. The concentration of the buffer can be selected within a wide range, for example, in the range of 10 to 1000 mM, particularly in the range of 25 to 500 mM, and even more particularly in the range of 50 to 250 mM.

[0100] Adding additives to aqueous solutions to improve the solubility of peptide fragments or increase reaction yield can be advantageous. Such additives can be salts or organic molecules, such as guanidine hydrochloride, urea, sodium dodecyl sulfate, or Tween.

[0101] In principle, the temperature during the coupling or cyclization reaction is not critical, as long as the temperature at which the enzyme to be used exhibits sufficient activity and stability is chosen. This temperature can be conventionally determined. Typically, the temperature can be at least 10°C or higher, or at least 20°C or higher, or 70°C or lower, particularly 50°C or lower. Preferably, the temperature during the coupling or cyclization reaction according to the invention is in the range of 20–50°C.

[0102] The C-terminal esters or thioesters of peptides commonly used are activated (thio)esters, meaning they contain a carboxyl ester or carboxythioester group that can participate in the reaction. In principle, any substituted or unsubstituted alkyl group, 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.

[0103] Particularly good results have been obtained using esters (Cam-esters) of the carboxamide methyl type represented by the formula peptide-(C=O)O-CX1X2-C(=O)N-R1R2, wherein each X1 and X2 independently represents a hydrogen atom or an alkyl group, preferably both of which are hydrogen atoms; and wherein each R1 and R2 independently represents a hydrogen atom, or an alkyl group, or an amino acid or peptide residue having a C-terminal carboxamide or carboxylic acid functional group and optionally protected on its side chain. Each alkyl group may independently represent a substituted or unsubstituted C1-C7 alkyl group, preferably a substituted or unsubstituted straight-chain C1-C6 alkyl group, more preferably a substituted or unsubstituted straight-chain C1-C3 alkyl group, and most preferably a methyl group. Preferably, both R1 and R2 represent hydrogen atoms, or R1 represents a hydrogen atom and R2 represents an amino acid or peptide residue having a C-terminal carboxamide or carboxylic acid functional group and optionally protected on its side chain. The use of Cam-AA1-AA2-ester is particularly preferred, wherein R2 is a dipeptide, and AA1 is the first amino acid and AA2 is the second amino acid. Here, AA1 is a hydrophobic amino acid such as alanine, valine, leucine, isoleucine, phenylalanine, methionine, or tryptophan. AA2 is a basic amino acid such as arginine or lysine. AA1 and AA2 typically have free side chains, i.e., they do not contain protecting groups or another residue. A particularly preferred form is the Cam ester peptide -(C=O)O-CH2-C(=O)N-Phe-Arg-NH2 / N-Phe-Lys-NH2.

[0104] Particularly good results were obtained using carboxyl-substituted benzyl esters, particularly those with p- or meta-carboxyl-substituted groups as shown in the formula peptide-(C=O)-O-CH2-C6H4-(C=O)E, where E represents a hydroxyl group, a carboxylate such as an ammonium salt, or an amino acid or peptide residue having a C-terminal carboxamide or carboxylic acid functional group and optionally protected on the amino acid / side chain. Good results were also obtained using p- or meta-carboxyl-substituted benzyl esters as shown in the formula peptide-(C=O)-O-CH2-C6H4-(C=O)E, where E is as defined above, and one or more hydrogen atoms in the phenyl ring (C6H4 in the above formula) are substituted with substituents such as hydroxyl, alkoxy, aryloxy, or halogen.

[0105] The C-terminal (thio) ester of the peptide may be N-terminally unprotected or N-terminally protected. The term "N-terminal protected" is used herein to indicate that the N-terminal amino group of the peptide is provided with a protecting group, typically at least substantially protecting the N-terminal amino group from coupling to the C-terminal carboxyl group of another peptide or the same peptide molecule. Preferably, the C-terminal (thio) ester of the peptide is not N-terminally protected.

[0106] The C-terminal (thio)esters of peptides used in this invention can be synthesized using solid-phase synthesis with high yield and purity, and without racemization. An additional advantage of using carboxamidomethyl type (thio)esters (where R1 represents a hydrogen atom and R2 represents an amino acid or peptide residue having a C-terminal carboxylic acid functional group, optionally protected on a side chain functional group of an amino acid or on one or more side chain functional groups of an amino acid) is that their activated C-terminal (thio)ester groups can be synthesized using commonly used solid-phase supports, such as 2-chlorotriphenylmethyl chloride resin (CTC resin), Rink resin, and Wang resin, via solid-phase synthesis, as also described in embodiments 1-2 of this disclosure.

[0107] The C-terminal (thio)ester of the peptide can also be synthesized via solution-phase synthesis or using microbial fermentation. A reliable method for obtaining peptide (thio)esters using fermentation is through so-called inteptide expression (see, for example, EKLee, Journal of Chemical Technology and Biotechnology, 2010, 9, 11-18). Different inteptide expression system kits are commercially available (e.g., IMPACT). TM (Reagent kit). Other methods for the fermentation production of peptide (sulfur) esters are known in the art.

[0108] The amino acids in C-terminal (thio) esters of peptides (i.e., acyl donor thioesters) can, in principle, be selected from any proteogenic or non-proteogenic amino acids. Optionally, N-terminal protected C-terminal (thio) esters of peptides can be represented by compounds of Formula I:

[0109]

[0110] Where Q represents the OR or SR moiety, and R can represent a substituted or unsubstituted alkyl or substituted or unsubstituted aryl group; and P1 represents a hydrogen or N-terminal protecting group. Suitable N-terminal protecting groups are groups that can be used in peptide synthesis, including urethane or acyl-type protecting groups, such as Cbz (phenylmethoxycarbonyl), Boc (tert-butoxycarbonyl), For (formyl), Fmoc (9-fluorenylmethoxycarbonyl), Smoc (2,7-disulfon-9-fluorenylmethoxycarbonyl), PhAc (phenylacetyl), and Ac (acetyl). For, PhAc, and Ac groups can be introduced, and enzymatic cleavage can be performed using peptide deformylase, PenG acylase, or acyltransferase, respectively. Chemical cleavage methods for all mentioned protecting groups are known in the art.

[0111] Here, n is an integer with a value of at least 2, and depends on the length of the C-terminal (thio) ester of the peptide.

[0112] Here, depending on the amino acid involved, each RA and each RB independently represents a hydrogen atom or an amino acid side chain. Optionally, one or more side chains may contain protecting groups.

[0113] The amino acids of the peptide nucleophile (i.e., acyl acceptor) can, in principle, be selected from any proteogenous or non-proteogenous amino acid. Specifically, the peptide nucleophile can be represented by a compound of formula II:

[0114]

[0115] Where n, RA, and RB are defined as in Equation I above, with necessary modifications.

[0116] The peptide nucleophile can be C-terminally unprotected or C-terminally protected. The term "C-terminally protected" is used herein to indicate that the C-terminal carboxyl group of a peptide, particularly a peptide nucleophile, is provided with a protecting group, generally protecting the carboxyl group from coupling with the N-terminal amino group of another peptide or the same peptide molecule. Here, P2 represents an amine moiety or an OR moiety. In the case where P2 represents an amine moiety, the amine moiety can be represented by the formula NR3R4, where R3 and R4 can each independently represent any substituted or unsubstituted alkyl or any substituted or unsubstituted aryl group. Preferably, one of R3 and R4 is a hydrogen atom, and the other is a substituted or unsubstituted alkyl group; more preferably, both R3 and R4 are hydrogen atoms. In the case where P2 represents an OR moiety, R can represent a carboxylic acid protecting group or a cation, such as a monovalent cation like a trisubstituted or tetrasubstituted ammonium ion or an alkali metal cation or H. When R is a carboxylic acid protecting group, OR can specifically be an ester, wherein R is preferably a tertiary alkyl group such as tert-butyl, 2-methyl-2-butyl and 2,3-dimethyl-2-butyl.

[0117] The peptide nucleophile can be synthesized using methods known in the art, such as solid-phase synthesis, solution-phase synthesis, or microbial fermentation.

[0118] In one embodiment, one or more side chains of the C-terminal (thio) ester of the peptide and / or the peptide nucleophile may contain protecting groups selected from suitable protecting groups known in the art. Carboxylic acid groups may be protected, for example, with cyclohexyl, benzyl, or allyl; amine functional groups may be protected, for example, with alkoxycarbonyl or trifluoroacetyl. In a preferred embodiment, the C-terminal (thio) ester of the peptide and the peptide nucleophile do not contain side-chain protecting groups.

[0119] Because a high S / H ratio can be obtained using the method according to the invention, high yields in coupling reactions are generally not required in large excesses of the peptide C-terminal (thio)ester or the peptide nucleophile. A suitable ratio of (a) the peptide C-terminal (thio)ester to (b) the peptide nucleophile is from 1:5 to 5:1, preferably from 1:3 to 3:1, more preferably from 1.0:2.5 to 2.5:1.0, particularly from 1:2 to 2:1, and even more particularly from 1:1.5 to 1.5:1. A ratio of approximately stoichiometric amounts has been found to be particularly preferred.

[0120] Specifically, this invention provides an enzyme that allows for the efficient preparation of bioactive peptides via the coupling of peptide fragments. These peptides include glucagon, glp-1, glp-2, and analogs such as dasiglucagon, exenatide, liraglutide, semaglutide, lixilamide, teduglutide, glepaglutide, dulaglutide, elsiglutide, etc., thymosin-α-1, thymosin-α-1 analogs, teriparatide, salmon calcitonin, bivalirudin, and peptides containing the sequence of any of these peptides and at least one additional amino acid. Preferably, the glp-1 analogs are liraglutide and semaglutide; the glp-2 analogs are preferably glepaglutide and elsiglutide.

[0121] Therefore, the present invention provides an enzymatic method for synthesizing peptides selected from liraglutide, semaglutide, dasiglucagon, teduglutide, glepaglutide, elsiglutide, teriparatide, salmon calcitonin, bivalirudin, and their analogues.

[0122] The present invention also provides the use of the enzyme according to the invention as a catalyst in a method for the enzymatic synthesis of pharmacologically significant peptides, said peptides including liraglutide, semaglutide, dasiglucagon, tiduglutide, glepaglutide, elsiglutide, teriparatide, salmon calcitonin, bivalirudin, and analogues thereof, wherein dasiglucagon, glepaglutide, elsiglutide, teriparatide, salmon calcitonin, and bivalirudin are particularly preferred. In the method according to the invention, the analogues are preferably structural analogues.

[0123] Liraglutide (SEQ ID NO: 6) is a GLP-1 analog with a palmitic acid substituted at position 20, separated from the ε-amino group of lysine by Glu. Therefore, liraglutide has the formula H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Pal-γ-Glu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH. In Lys(Pal-γ-Glu), the ε-amino group of the Lys residue is linked to the carboxyl side chain of γ-Glu, and the Glu is N-palmitoylated.

[0124] Semaglutide (SEQ ID NO: 7) is also a GLP-1 analog with a substituted ε-amino group of lysine at position 20, and has the formula H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH, wherein AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl is N-(17-carboxy-1-ketoheptadecanoyl)-L-γ-glutamyl-2-[2-(2-aminoethoxy)ethoxy]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl.

[0125] Dasiglucagon (SEQ ID NO: 8) is a novel peptide analog of human glucagon, with the formula H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Aib-Ala-Arg-Ala-Glu-Glu-Phe-Val-Lys-Trp-Leu-Glu-Ser-Thr-OH.

[0126] Glepaglutide (SEQ ID NO: 9) is a GLP-2 analog with the formula H-His-Gly-Glu-Gly-Thr-Phe-Ser-Ser-Glu-Leu-Ala-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-AIa-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys-Lys-NH2.

[0127] Elsiglutide (SEQ ID NO: 10) is a GLP-2 analog with the formula H-His-Gly-Glu-Gly-Ser-Phe-Ser-Ser-Glu-Leu-Ser-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys-Lys-NH2.

[0128] Teriparatide (SEQ ID NO: 11) is a recombinant human parathyroid hormone (rhPTH) with the formula H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH.

[0129] Calcitonin is a peptide hormone. Specifically, salmon calcitonin (SEQ ID NO: 12) has the formula H-Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro-NH2, and also contains a disulfide bond between the cysteine ​​residues at positions 1 and 7.

[0130] Bivalirudin (SEQ ID NO: 13) is a thrombin inhibitor with the formula HD-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Le u-OH.

[0131] Methods for the enzymatic synthesis of dasiglucagon, glepaglutide, elsiglutide, teriparatide, salmon calcitonin, and bivalirudin are unknown in the art.

[0132] In a preferred embodiment, the Bacillus subtilis protease variant of the present invention is highly efficient in catalyzing the preparation of dasiglucagon, glepaglutide, elsiglutide, teriparatide, salmon calcitonin, bivalirudin, liraglutide, and semaglutide involving specific peptide fragment coupling reactions, and is therefore preferred.

[0133] Therefore, in one embodiment, the present invention provides an enzymatic synthesis of dasiglucagon (SEQ ID NO: 8), the method comprising the step of coupling (a) a C-terminal (thio) ester of a peptide to (b) a peptide nucleophile having an N-terminal unprotected amine, wherein said coupling is catalyzed by a variant of the subtilisin BPN' or a homolog thereof according to the present invention as defined above and in claim 1 and its dependent claims.

[0134] The present invention also provides a method for synthesizing a peptide comprising the sequence of dasiglucagon (SEQ ID NO: 8), namely His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Aib-Ala-Arg-Ala-Glu-Glu-Phe-Val-Lys-Trp-Leu-Glu-Ser-Thr, the method comprising the step of conjugating the following substances:

[0135] (a) A C-terminal (thio) ester of a peptide comprising a first peptide fragment having the sequence His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-(thio) ester (SEQ ID NO: 15), and

[0136] (b) A peptide nucleophilic fragment having an N-terminal unprotected amine, comprising a second peptide fragment having the sequence H-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Aib-Ala-Arg-Ala-Glu-Glu-Phe-Val-Lys-Trp-Leu-Glu-Ser-Thr (SEQ ID NO: 16);

[0137] or

[0138] (c) A C-terminal (thio) ester of peptide, comprising a first peptide fragment having the sequence His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp (thio) ester (SEQ ID NO: 17), and

[0139] (d) A peptide nucleophilic fragment having an N-terminal unprotected amine, comprising a second peptide fragment having the sequence H-Tyr-Ser-Lys-Tyr-Leu-Asp-Aib-Ala-Arg-Ala-Glu-Glu-Phe-Val-Lys-Trp-Leu-Glu-Ser-Thr (SEQ ID NO: 18);

[0140] or

[0141] (e) A C-terminal (thio) ester of peptide, comprising a first peptide fragment having the sequence His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr (thio) ester (SEQ ID NO: 19), and

[0142] (f) A peptide nucleophilic fragment having an N-terminal unprotected amine, comprising a second peptide fragment having the sequence H-Ser-Lys-Tyr-Leu-Asp-Aib-Ala-Arg-Ala-Glu-Glu-Phe-Val-Lys-Trp-Leu-Glu-Ser-Thr (SEQ ID NO: 20);

[0143] The coupling is catalyzed by the subtilis protease variant of the present invention.

[0144] In another embodiment, the present invention provides an enzymatic synthesis of glepaglutide (SEQ ID NO: 9), the method comprising the step of coupling (a) a C-terminal (thio) ester of a peptide to (b) a peptide nucleophile having an N-terminal unprotected amine, wherein said coupling is catalyzed by a variant of the subtilisin BPN' or a homolog thereof as defined in the present invention and as defined in claim 1 and its dependent claims.

[0145] The present invention also provides a method for synthesizing a peptide comprising the sequence of glepaglutide (SEQ ID NO: 9), namely His-Gly-Glu-Gly-Thr-Phe-Ser-Ser-Glu-Leu-Ala-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-AIa-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys-Lys, the method comprising the step of conjugating the following substances:

[0146] (a) A C-terminal (thio) ester of peptide comprising a first peptide fragment having the sequence His-Gly-Glu-Gly-Thr-Phe-Ser-Ser-Glu-Leu-Ala-(thio) ester (SEQ ID NO: 21), and

[0147] (b) A peptide nucleophilic fragment having an N-terminal unprotected amine, comprising a second peptide fragment having the sequence H-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-AIa-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys-Lys (SEQ ID NO: 22);

[0148] or

[0149] (c) A C-terminal (thio) ester of peptide, comprising a first peptide fragment having the sequence His-Gly-Glu-Gly-Thr-Phe-Ser-Ser-Glu-Leu-Ala-Thr-Ile-Leu-Asp-Ala-(thio) ester (SEQ ID NO: 23), and

[0150] (d) A peptide nucleophilic fragment having an N-terminal unprotected amine, comprising a second peptide fragment having the sequence H-Leu-Ala-Ala-Arg-Asp-Phe-Ile-AIa-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys (SEQ ID NO: 24);

[0151] The coupling is catalyzed by the subtilis protease variant of the present invention.

[0152] In another embodiment, the present invention provides an enzymatic synthesis of elsiglutide (SEQ ID NO: 10), the method comprising the step of coupling (a) a C-terminal (thio) ester of a peptide to (b) a peptide nucleophile having an N-terminal unprotected amine, wherein said coupling is catalyzed by a variant of the subtilisin BPN' or a homolog thereof as defined in the present invention and as defined in claim 1 and its dependent claims.

[0153] The present invention also provides a method for synthesizing a peptide comprising the sequence of elsiglutide (SEQ ID NO: 10), namely His-Gly-Glu-Gly-Ser-Phe-Ser-Ser-Glu-Leu-Ser-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-AIa-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys-Lys, the method comprising the step of conjugating the following substances:

[0154] (a) A C-terminal (thio) ester of a peptide comprising a first peptide fragment having the sequence His-Gly-Glu-Gly-Ser-Phe-Ser-Ser-Glu-Leu-Ser-(thio) ester (SEQ ID NO: 25), and

[0155] (b) A peptide nucleophilic fragment having an N-terminal unprotected amine, comprising a second peptide fragment having the sequence H-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-AIa-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys-Lys (SEQ ID NO: 26);

[0156] or

[0157] (c) A C-terminal (thio) ester of peptide, comprising a first peptide fragment having the sequence His-Gly-Glu-Gly-Ser-Phe-Ser-Ser-Glu-Leu-Ser-Thr-Ile-Leu-Asp-Ala-(thio) ester (SEQ ID NO: 27), and

[0158] (d) A peptide nucleophilic fragment containing an N-terminal unprotected amine, comprising a second peptide fragment having the sequence H-Leu-Ala-Ala-Arg-Asp-Phe-Ile-AIa-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys (SEQ ID NO: 28).

[0159] The coupling is catalyzed by the subtilis protease variant of the present invention.

[0160] In yet another embodiment, the present invention provides an enzymatic method for synthesizing teriparatide (SEQ ID NO: 11), the method comprising the step of coupling (a) a C-terminal (thio) ester of a peptide to (b) a peptide nucleophile having an N-terminal unprotected amine, wherein said coupling is catalyzed by a variant of the subtilisin BPN' or a homolog thereof as defined in the present invention and as defined in claim 1 and its dependent claims.

[0161] The present invention also provides a method for synthesizing a peptide, wherein the peptide comprises the sequence of teriparatide (SEQ ID NO: 11), i.e.

[0162] Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe, the method includes the step of coupling the following substances:

[0163] (a) A C-terminal (thio) ester of peptide, comprising a first peptide fragment having the sequence Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-(thio) ester (SEQ ID NO: 29), and

[0164] (b) A peptide nucleophilic fragment having an N-terminal unprotected amine, comprising a second peptide fragment having the sequence H-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe (SEQ ID NO: 30);

[0165] The coupling is catalyzed by the subtilis protease variant of the present invention.

[0166] In a preferred embodiment, the enzymatic synthesis of teriparatide comprises the step of coupling (a) a C-terminal (thio) ester of a peptide with (b) a peptide nucleophile having an N-terminal unprotected amine, wherein the coupling is catalyzed by a variant of the BPN' protease according to the invention or a homolog thereof, and wherein the C-terminal (thio) ester of the peptide is N-terminally protected.

[0167] In another embodiment, the present invention provides an enzymatic method for synthesizing salmon calcitonin (SEQ ID NO: 12), the method comprising the step of coupling (a) a C-terminal (thio) ester of a peptide to (b) a peptide nucleophile having an N-terminal unprotected amine, wherein said coupling is catalyzed by a variant of the subtilisin BPN' or a homolog thereof as defined in the present invention and as defined in claim 1 and its dependent claims.

[0168] The present invention also provides a method for synthesizing a peptide comprising the sequence of salmon calcitonin (SEQ ID NO: 12), namely Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro, the method comprising the step of conjugating the following substances:

[0169] (a) A C-terminal (thio) ester of peptide, comprising a first peptide fragment having the sequence Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-(thio) ester (SEQ ID NO: 31), and

[0170] (b) A peptide nucleophilic fragment having an N-terminal unprotected amine, comprising a second peptide fragment having the sequence H-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro (SEQ ID NO: 32);

[0171] or

[0172] (c) A C-terminal (thio) ester of peptide, comprising a first peptide fragment having the sequence Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-(thio) ester (SEQ ID NO: 33), and

[0173] (d) A peptide nucleophilic fragment containing an N-terminal unprotected amine, comprising a second peptide fragment having the sequence H-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro (SEQ ID NO: 34).

[0174] The coupling is catalyzed by the subtilis protease variant of the present invention.

[0175] In another embodiment, the present invention provides an enzymatic synthesis of bivalirudin (SEQ ID NO: 13), the method comprising the step of coupling (a) a C-terminal (thio) ester of a peptide to (b) a peptide nucleophile having an N-terminal unprotected amine, wherein said coupling is catalyzed by a variant of the subtilisin BPN' or a homolog thereof as defined in the present invention and as defined in claim 1 and its dependent claims.

[0176] The present invention also provides a method for synthesizing a peptide, wherein the peptide comprises the sequence of bivalirudin (SEQ ID NO: 13), i.e.

[0177] D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu, the method includes the step of coupling the following substances:

[0178] (a) The C-terminal (thio) ester of the peptide comprises a first peptide fragment having the sequence D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-(thio) ester (SEQ ID NO: 35), and

[0179] (b) A peptide nucleophilic fragment having an N-terminal unprotected amine, comprising a second peptide fragment having the sequence H-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu (SEQ ID NO: 36);

[0180] The coupling is catalyzed by the subtilis protease variant of the present invention.

[0181] In a preferred embodiment, the enzymatic synthesis of bivalirudin (SEQ ID NO: 13) comprises the step of coupling (a) a C-terminal thioester of a peptide with (b) a peptide nucleophile having an N-terminal unprotected amine, wherein the coupling is catalyzed by a variant of the subtilisin BPN' or a homolog thereof as defined in the present invention and as defined in claim 1 and its dependent claims.

[0182] In another embodiment, the present invention provides an enzymatic method for the synthesis of liraglutide (SEQ ID NO: 6), the method comprising the step of coupling (a) a C-terminal (thio) ester of a peptide to (b) a peptide nucleophile having an N-terminal unprotected amine, wherein said coupling is catalyzed by a variant of the subtilisin BPN' or a homolog thereof as defined in the present invention and as defined in claim 1 and its dependent claims.

[0183] In yet another embodiment, the present invention provides an enzymatic method for the synthesis of semaglutide (SEQ ID NO: 7), the method comprising the step of coupling (a) a C-terminal (thio) ester of a peptide to (b) a peptide nucleophile having an N-terminal unprotected amine, wherein said coupling is catalyzed by a variant of the subtilisin BPN' or a homolog thereof as defined in the present invention and as defined in claim 1 and its dependent claims.

[0184] This invention provides a method for synthesizing peptides, said peptides comprising the sequence His-W-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Z-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly, wherein

[0185] For the synthesis of liraglutide, W is Ala and Z is selected from Lys, Lys(PG), Lys(γ-Glu), and Lys(Pal-γ-Glu); or wherein...

[0186] For the synthesis of semaglutide, W is Aib, and Z is selected from Lys, Lys(PG), and Lys(AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl).

[0187] The method includes the step of coupling the following substances:

[0188] (a) A C-terminal (thio) ester of a peptide comprising a first peptide fragment having the sequence His-W-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio) ester (SEQ ID NO: 37), and

[0189] (b) A peptide nucleophilic fragment having an N-terminal unprotected amine, comprising a second peptide fragment having the sequence H-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Z-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (SEQ ID NO: 38);

[0190] The coupling is catalyzed by the Bacillus subtilis protease variant of the present invention, and wherein PG is a protecting group of the Lys side chain amino group.

[0191] In another preferred embodiment, the present invention provides a method for enzymatically synthesizing a peptide comprising the sequence of liraglutide (SEQ ID NO: 6), namely His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Z-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly.

[0192] Wherein Z is selected from Lys, Lys(PG), Lys(γ-Glu), and Lys(Pal-γ-Glu); the method includes the step of coupling the following substances:

[0193] a) A C-terminal (thio) ester of a peptide comprising a first peptide fragment having the sequence His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio) ester (SEQ ID NO: 37), and

[0194] (b) A peptide nucleophilic fragment having an N-terminal unprotected amine, comprising a second peptide fragment having the sequence H-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Z-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH (SEQ ID NO: 38).

[0195] The coupling is preferably carried out in an aqueous solution, and the coupling is catalyzed by a variant of BPN' subtilisin, which has at least 80% sequence identity with BPN' SEQ ID NO: 2, comprising a deletion of amino acids 75-83 and a mutation at amino acid position S221, wherein the mutation is S221C or S221 selenocysteine, preferably S221C; and

[0196] The variant is characterized by having at least one mutation at an amino acid position selected from L96, D99, A223, and S224.

[0197] The amino acid positions are defined according to SEQ ID NO: 2.

[0198] Furthermore, these variants or their homologs exhibit selectively enhanced ligase activity.

[0199] In a more preferred embodiment, the BPN' variant of the subtilisin in the above method is further characterized as having a combination of mutations selected from the following:

[0200] L96V and D99R; L96I and D99R; L96I and D99K; L96I and A223S; L96I and S224V;

[0201] L96V, D99R and A223S; L96V, D99R and S224V; L96I, A223S and S224V; L96I, D99R and A223S; L96I, D99R and S224V; L96I, D99K and S224V;

[0202] L96V, D99R, A223S and S224V; L96I, D99R, A223S and S224V; and L96I, D99K, A223S and S224V.

[0203] Even more preferably, the BPN' variant of subtilisin in the above method has one or more combinations of mutations selected from the following:

[0204] E156K and G166E; E156K and G166D; and M222H and Y217R.

[0205] For example, preferred mutation combinations are: E156K, G166E, L96I, and D99R;

[0206] E156K, G166E, L96I, and A223S;

[0207] E156K, G166E, L96I, and S224V;

[0208] E156K, G166E, L96I, A223S and S224V;

[0209] E156K, G166E, L96I, D99R, and A223S;

[0210] E156K, G166E, L96I, D99R, and S224V;

[0211] E156K, G166E, L96I, D99R, A223S and S224V;

[0212] Y217R, M222H, L96I, E156K, and G166D;

[0213] Y217R, M222H, L96I, E156K, G166D and D99R;

[0214] Y217R, M222H, L96I, E156K, G166D and D99K;

[0215] Y217R, M222H, L96I, E156K, G166D and A223S;

[0216] Y217R, M222H, L96I, E156K, G166D and S224V;

[0217] Y217R, M222H, L96I, E156K, G166D, A223S and S224V;

[0218] Y217R, M222H, L96I, E156K, G166D, D99R, and S224V;

[0219] Y217R, M222H, L96I, E156K, G166D, D99R, A223S and S224V;

[0220] Y217R, M222H, L96I, E156K, G166D, D99K, and S224V; and

[0221] Y217R, M222H, L96I, E156K, G166D, D99K, A223S, and S224V.

[0222] Therefore, another embodiment of the present invention is a variant of BPN' subtilisin, which has at least 80% sequence identity with BPN' SEQ ID NO: 2, comprising the deletion of amino acids 75-83 and a mutation at amino acid position S221, said mutation being S221C or S221 selenocysteine, preferably S221C; and said variant is characterized by the mutations and combinations of mutations listed above, and said variant has ligase activity with improved reaction rate, coupling efficiency and selectivity for the enzymatic synthesis of liraglutide.

[0223] Using the subtilisin BPN' variant according to the invention described in Example 9, remarkably good results were obtained in the reaction for liraglutide in terms of coupling efficiency and selectivity, wherein the mutation referred to as the Ptl-84 (SEQ ID NO: 4) ligase sequence has a mutation relative to the wild-type subtilisin BPN' (SEQ ID NO: 2). For example, at position 156, SEQ ID NO: 2 has an E, while SEQ ID NO: 4 has an N. Thus, for example, in Table 9 of Example 9, the subtilisin BPN' variant according to the invention indicated as Ptl-84+N156K+E166E+L96I has a mutation at position 156 indicated as N156K, instead of the E156K mutation in the case referred to as SEQ ID NO: 2.

[0224] The present invention will now be described through the following embodiments.

[0225] Abbreviations

[0226] SPPS Peptide Solid Phase Synthesis

[0227] CTC 2-Chlorotriphenylmethyl chloride

[0228] AEEA 2-[2-(2-aminoethoxy)ethoxy]acetyl

[0229] Cbz benzyloxycarbonyl

[0230] For formyl group

[0231] Fmoc 9-fluorenylmethoxycarbonyl

[0232] Boc tert-butoxycarbonyl

[0233] Smoc 2,7-disulfonyl-9-fluorenylmethoxycarbonyl

[0234] Acetyl group

[0235] PhAc phenylacetyl

[0236] Trt Triphenylmethyl (Triphenylmethyl)

[0237] tBu tert-butyl

[0238] Pbf 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl

[0239] eq equivalent

[0240] h hours

[0241] min minutes

[0242] HPLC (High Performance Liquid Chromatography)

[0243] DIPEA N,N-Diisopropylethylamine

[0244] TFA (trifluoroacetic acid)

[0245] TIS Triisopropylsilane

[0246] Ac2O Acetic anhydride

[0247] DMF N,N-dimethylformamide

[0248] DMA N,N-dimethylacetamide

[0249] DCM dichloromethane

[0250] THF Tetrahydrofuran

[0251] NMP (N-methyl-2-pyrrolidone)

[0252] MTBE (methyl tert-butyl ether)

[0253] MeOH (methanol)

[0254] DCC N,N'-Dicyclohexylcarbodiimide

[0255] EDC N-(3-Dimethylaminopropyl)-N′-Ethylcarbodiimide

[0256] HOBt 1-hydroxybenzotriazole

[0257] HOAt 1-hydroxy-7-azabenzotriazole

[0258] TCEP tris(2-carboxyethyl)phosphine

[0259] Tricine N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine

[0260] Examples

[0261] Production of the enzyme (or enzyme variant) according to the invention (for use)

[0262] Mutagenesis, cloning and expression

[0263] The reference enzyme, designated Ptl-84 (SEQ ID NO: 4), corresponds to SEQ ID NO: 2, wherein the amino acids corresponding to positions 75–83 are missing (Δ75–83, Ca). 2+The gene encoding Ptl-84 with a His tag was cloned into an Escherichia coli-Bacillus subtilis shuttle vector (i.e., pBS42 or pBES) based on pUB-110. Ptl-84 is previously disclosed in WO2019170918 (wherein it corresponds to SEQ ID NO: 3).

[0264] The corresponding amino acid sequences are numbered according to the subtilisin BPN' numbering scheme. Amino acids -107 to -1 contain the signal sequence, pre-sequence, and pro-sequence, which are cleaved after full maturation. Amino acids 1 to 275 contain the mature enzyme exhibiting full catalytic activity. To enable rapid and efficient purification, a C-terminal His tag is attached after amino acid 275. Due to the removal of the calcium-binding site, Ptl-84 contains a 9-amino acid deletion compared to subtilisin BPN', including 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-84 (and the enzyme of this invention), the numbering jumps from 74 to 84. In the shuttle vector, gene expression is controlled by the aprE promoter. The obtained plasmid pBES-Ptl-84 HIS was propagated in Escherichia coli TOP10 and transformed into Bacillus subtilis GX4935 (trpC2metB10 lys-3ΔnprEΔaprE). Using pBES-Ptl-84 HIS as a template, mutations were performed using the Quikchange method (Agilent). Alternatively, other methods known in the art for site-directed mutagenesis can be used. Alternatively, the DNA was synthesized by GenScript, USA, and integrated into a suitable shuttle vector.

[0265] Production and purification of synthetic subtilisin BPN' variant with His tag:

[0266] Single colonies of Bacillus subtilis containing plasmids with the gene of the subtilis protease variant of interest were inoculated into 5 mL of LB broth containing kanamycin (10 μg / mL) and cultured in a shaking incubator 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). Cells were grown in a shaking incubator (200 rpm) at 37 °C for 48 h. Cells were harvested by centrifugation (30 min, 4,000 rpm, 4 °C). 30 mL of culture medium was decanted and concentrated using an Amicon centrifuge (15 mL, 10 kDa cutoff MW) via two centrifugation steps (15 min, 4,000 rpm, 4 °C). The concentrated culture medium (0.5 mL) was then exchanged with buffer A (25 mM Tricine, pH 7.5, 0.5 M NaCl) in three wash / concentration steps (14 mL buffer A, 10 min, 4,000 rpm, 4 °C). For His-tag purification, Talon resin (2.5 mL, Clonetech) was added to a plastic column sleeve. The resin was washed with 20 mL of demineralized water and equilibrated with 20 mL buffer A. The crude enzyme was loaded onto the column and incubated overnight on a track-setting 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 NaCl, 0.5 M imidazole). The eluent 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 an Amicon centrifuge (15 ml, 10 kDa cutoff MW). The buffer was then exchanged for 25 mM Tricine at pH 7.5 in three wash / concentration steps (15 ml buffer, 10 min, 4000 rpm, 4 °C).

[0267] Purity was determined by SDS-PAGE and density measurement analysis (BioRad GS-900). Enzyme concentration was determined by measuring absorbance at 280 nm using a Nanodrop (Thermo Scientific), with 1 Abs = 1 mg / ml. The resulting aqueous solution (25 mM Tricine, pH 7.5) contained approximately 0.1–2 mg / ml of the obtained enzyme. Solutions with enzyme concentrations corrected (mg / ml * purity) were used for coupling and cyclization reactions.

[0268] For a detailed description of the production and purification of the synthetic BPN' variant reference, see WO2016 / 056913 and WO 2018 / 212658.

[0269] Examples of fragment enzyme method coupling

[0270] Materials and methods

[0271] Unless otherwise stated, the chemicals were obtained from commercial sources and can be 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, a linear gradient uniformity of 5% to 98% eluent B; 25.1–30 min, 5% eluent B (eluent A: 0.5 mL / L methanesulfonic acid (MSA) in H₂O solution, eluent B: 0.5 mL / L MSA in acetonitrile solution). The flow rate was 1 mL / min from 0–25.1 min, 2 mL / min from 25.2–29.8 min, then back to 1 mL / min until stopping 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 programs were as described for analytical HPLC. Molecular weights were determined using an Agilent 6130 quadrupole LC / MS system.

[0272] Option 1: Preparation of peptide-OCam-Leu-OH ester

[0273] 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 then deprotected with 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 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 Fmoc-protected amino acids using 4 eq. Fmoc-Xxx-OH and 10 eq. DIPEA in DMF / THF (1 / 1, v / v, 10 mL) at 50 °C for 20 h. Herein and elsewhere in this disclosure, “Xxx” represents an amino acid (which may vary depending on the target peptide, as shown in the examples below).

[0274] 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 extended according to the standard SPPS protocol. Deprotection of the cleavage and side chains from the resin was performed for 120 min using a mixture of TFA, TIS, and water (95 / 2.5 / 2.5, v / v / v, 15 mL). 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), then lyophilized in acetonitrile / water (1 / 1, v / v, 50 mL).

[0275] Option 2: Preparation of peptide-OCam-Phe-Lys-NH2 ester

[0276] 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), followed by Fmoc deprotection with piperidine / DMF (1 / 4, v / v, 2 × 8 min, 10 mL). Fmoc-Lys(Boc)-OH was coupled according to the standard SPPS protocol, followed by Fmoc-Phe-OH. After Fmoc deprotection with 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). 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 (2×2 min, 10 mL), DCM (2×2 min, 10 mL), and THF (2×2 min, 10 mL), the resin was loaded with Fmoc-protected amino acids at 50 °C for 20 h using Fmoc-Xxx-OH (4 eq) and DIPEA (10 eq) in DMF / THF (1 / 1, v / v, 10 mL).

[0277] 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 extended according to the standard SPPS protocol. Deprotection of the resin cleavage and side chains was performed for 120 min using a mixture of TFA, TIS, and water (95 / 2.5 / 2.5, v / v / v, 15 mL). 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 vacuum dried to provide the title ester (also labeled peptide-OCam-FK-NH2 ester). The crude peptide was purified by preparative HPLC prior to enzymatic ligation, and the pure fraction was then lyophilized.

[0278] Similarly, peptide-OCam-FR-NH2 ester was prepared.

[0279] Scheme 3a: Preparation of C-terminal amide peptide nucleophile

[0280] 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), followed by Fmoc deprotection with piperidine / DMF (1 / 4, v / v, 2 × 8 min, 10 mL). The peptide was extended according to the standard SPPS protocol. Deprotection of the resin cleavage and side chains was performed for 120 min using a mixture of TFA / TIS / water (95 / 2.5 / 2.5, v / v / v, 15 mL). The crude peptide was precipitated with 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 vacuum dried. The crude peptide was purified by preparative HPLC prior to enzymatic ligation, and the pure fraction was then lyophilized.

[0281] Scheme 3b: Preparation of nucleophilic reagent for C-terminal carboxylic acid peptides

[0282] One gram of pre-loaded Fmoc-Xxx-Wang resin (loading 0.30 mmol / g) was washed with DCM (2 × 2 min, 10 mL) and DMF (2 × 2 min, 10 mL), and then deprotected with piperidine / DMF (1 / 5, v / v, 2 × 8 min, 10 mL). The peptide was extended according to the standard SPPS protocol. Deprotection of the side chains from the resin was performed for 120 min using a mixture of TFA / TIS / water (95 / 2.5 / 2.5, v / v / v, 15 mL). The crude peptide was precipitated with 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 under vacuum. The crude peptide was purified by preparative HPLC before enzymatic ligation, and the pure fraction was then lyophilized.

[0283] Option 4: Preparation of N-acetyl-protected peptide-activated esters

[0284] After SPPSing the desired sequence according to either Scheme 1 or 2, the resin-bound peptide was deprotected using piperidine / DMF (1 / 4, v / v, 2 × 8 min, 10 mL) with Fmoc. 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 N-terminal amine functional group of the peptide was acetylated with a mixture of Ac2O (10 vol%), DIPEA (5 vol%), and 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). Deprotection of the cleavage and side chains from the resin was performed for 120 min using a mixture of TFA / TIS / water (95 / 2.5 / 2.5, v / v / v, 15 mL). 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 under vacuum. The crude peptide was purified by preparative HPLC prior to enzymatic ligation, and the purified fraction was then lyophilized.

[0285] Similarly, by replacing Ac2O with PhAc2O, N-phenylacetyl-protected peptide-activated esters were also prepared.

[0286] Option 5: Screening for novel enzyme variants from the synthesis of liraglutide (SEQ ID NO: 6) from two fragments.

[0287] Prepare 5 mg / mL H-Lira(1-11)-OCam-FK-NH2 (i.e., H- prepared according to Scheme 2) 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser-OCam-Phe-Lys-NH2·3TFA (SEQ ID NO: 39)) and 5 mg / mL H-Lira(12-31)-OH (prepared according to scheme 3b) 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala-19 Ala- 20 Lys(Pal-γ-Glu)- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 A stock solution of Gly-OH·3TFA (SEQ ID NO: 40), also see WO2019170918, was dissolved in a buffer solution containing 50 mM Tricine, 220 mM potassium trifluoroacetate, and 2 mg / mL TCEP, and the pH was set to 7.9 using 1N KOH aqueous solution. This stock solution could be aliquoted and stored at -20°C for further use. 50 μL of the above peptide stock solution was added to a glass vial, and the coupling reaction was initiated by mixing with 25 μL of the enzyme variant solution (0.2 mg / mL, in a buffer solution containing 25 mM Tricine at pH 7.5). At different time points (typically 0, 15, 30, 60, and 120 min), 10 μL of the reaction mixture was quenched in 250 μL of demineralized aqueous solution of methanesulfonic acid (5 mL / L) to terminate any enzyme activity. The quenched samples were analyzed using HPLC / MS. The HPLC peak integrals of the indicated conjugate liraglutide (H-Lira(1-31)-OH) are presented as HPLC area in the table for each example.

[0288] Note: The reaction is generally not complete after 60 min, so this time point is used to highlight the differences between variants (slower enzymes may still give the same product yield at later time points). The 60 min time point was used in Examples 1, 2, 3, 5, 6, 8 and Examples 4, 10, 11, 12 and 13 related to liraglutide.

[0289] Option 6: Screening for novel enzyme variants in the synthesis of the 8-mer model peptide (SEQ ID NO: 14)

[0290] Stock solutions of 9.4 mg / mL ester (Ac-Asp-Phe-Ser-Lys-Leu-OCam-Leu-OH·1TFA (SEQ ID NO: 41) prepared according to schemes 1 and 4) and 8.8 mg / mL amine (H-Ala-Leu-Arg-NH2·2TFA prepared according to scheme 3a) were prepared by dissolving them in a buffer solution containing 50 mM Tricine, 220 mM potassium trifluoroacetate, and 2 mg / mL TCEP, and the pH was set to 7.9 using 1 NKOH aqueous solution. This stock solution could be aliquoted and stored at -20°C for further use. Add 25 μL of the above-described peptide stock solution and 65 μL of buffer containing 50 mM Tricine and 220 mM potassium trifluoroacetate to a glass vial, and initiate the reaction by mixing with 12.5 μL of enzyme variant solution (0.2 mg / mL in buffer containing 25 mM Tricine at pH 7.5, thus totaling 2.5 μg of enzyme). At different time points (typically 0, 15, 30, 60, and 120 min), quench 10 μL of the reaction mixture in 190 μL of demineralized aqueous solution of methanesulfonic acid (5 mL / L) to terminate any enzyme activity. Analyze the quenched sample using HPLC / MS. For each enzyme variant, the HPLC peak integral of the conjugate liraglutide (Ac-Asp-Phe-Ser-Lys-Leu-Ala-Leu-Arg-NH2, SEQ ID NO: 14) will be indicated and expressed as HPLC area % in the table of the corresponding examples.

[0291] Option 7: Selectivity screening for novel enzyme variants in the synthesis of liraglutide (SEQ ID NO: 6) from two fragments.

[0292] Preparation of 7.87 mM H-Lira(1-11)-OCam-FK-NH2 (H- prepared according to Scheme 2) 1 His- 2 Ala- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser-OCam-Phe-Lys-NH2 (SEQ ID NO: 39)) and 4.49 mMMH-Lira(12-31)-OH (H- prepared according to scheme 3b) 12 Ser- 13 Tyr- 14 Leu- 15Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys(Pal-γ-Glu)- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 A stock solution of Gly-OH (SEQ ID NO: 40, see also WO2019170918) was dissolved in a buffer solution containing 50 mM Tricine, 220 mM potassium trifluoroacetate, and 0.25 mg / mL TCEP, and the pH was set to 7.9 using a 3N KOH aqueous solution. This stock solution could be aliquoted and stored at -20°C for further use. 85 μL of the above peptide stock solution was added to a glass vial and the reaction was initiated by mixing with 15 μL of the enzyme variant solution (0.2 mg / mL, in a pH 7.5 buffer solution containing 25 mM Tricine and 125 mM NaCl). At different time points (typically 0, 15, 30, 60, 120, and 360 min), 10 μL of the reaction mixture was quenched in 250 μL of demineralized aqueous solution of methanesulfonic acid (5 mL / L) to terminate any enzyme activity. The quenched samples were analyzed using HPLC / MS. The conversion to the product was calculated by integrating the peaks of the liraglutide (H-Lira(1-31)-OH) product and the unreacted peptide amine (H-Lira(12-31)-OH) peak, and is expressed as HPLC area % in the tables of each example. Selectivity was calculated by dividing the amount (area %) of the liraglutide (H-Lira(1-31)-OH) product by the amount (area %) of the unwanted byproduct H-Lira(1-11-1-31)-OH. The conversion and selectivity values ​​were calculated after complete conversion of the acyl donor ester (between 30 and 360 min).

[0293] Examples of coupling

[0294] Note: As described above, the reference enzyme named Ptl-84 (SEQ ID NO: 4) corresponds to SEQ ID NO: 2 (wild-type Bacillus subtilis protease BPN') which is missing amino acids corresponding to positions 75-83 and includes additional mutations Q2K, S3C, P5S, S9A, I31L, K43N, M50F, N62A, A73L, E156N, G166E, G169A, S188P, F189W, Q206C, N212G, Y217H, N218D, S221C, M222P, P225N, T254A, and Q271E and is tagged with a 6-His.

[0295] Another reference enzyme was named Ptl-79 (SEQ ID NO: 5), which corresponds to SEQ ID NO: 2 (wild-type Bacillus subtilis protease BPN') with the amino acids corresponding to positions 75-83 missing and including additional mutations Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, I107V, E156S, G166S, G169A, S188P, F189W, Q206C, N212G, Y217H, N218S, S221C, M222P, P225N, T254A, and Q271E, and tagged with a 6-His.

[0296] All enzymes used in Examples 1-13 have all the mutations of the reference enzymes (Ptl-84 or Ptl-79) mentioned above, plus the additional mutations mentioned in each example. Ptl-84, Ptl-79, any other reference enzymes, and the enzymes of the present invention are all produced using the techniques described above.

[0297] Example 1: Synthesis of liraglutide (SEQ ID NO: 6) from two fragments using the Ptl-84+L96X variant

[0298] To determine the effect of the L96 mutation on liraglutide synthesis, screening was performed according to scheme 5. The amounts of the conjugated product liraglutide (H-Lira(1-31)-OH) after 60 min of reaction for representative enzyme variants are listed in the table below. The reference enzyme Ptl-84, which does not contain the mutation at the L96 position, is indicated in bold.

[0299] Number Enzyme variant Area % of H-Lira(1-31)-OH 1 Ptl-84 + L96I 51,50 2 Ptl-84 + L96V 46,11 3 Ptl-84 + L96M 43,84 4 Ptl-84 + L96T 41,82 5 Ptl-84 + L96A 41,18 6 Ptl-84 + L96C 40,24 7 Ptl-84 + L96S 39,81 8 Ptl-84 + L96Q 36,51 9 Ptl-84 + L96H 35,28 10 Ptl-84 33,93

[0300] Clearly, the L96 position has a significant impact on reaction rate and coupling efficiency. The nine variants (Ptl-84+L96I / V / M / T / A / C / S / Q / H) scored significantly better than the wild-type enzyme. Compared to the wild-type enzyme, these variants can be used to produce liraglutide in higher yields while requiring less enzyme for the same conversion.

[0301] Example 2: Synthesis of liraglutide (SEQ ID NO: 6) from two fragments using the Ptl-84+D99X variant

[0302] To determine the effect of the D99 mutation on liraglutide synthesis, screening was performed according to scheme 5. The amounts of the conjugate liraglutide (H-Lira(1-31)-OH) after 60 min of reaction for representative enzyme variants are listed in the table below.

[0303]

[0304]

[0305] Clearly, the D99 position has a significant impact on reaction rate and coupling efficiency. Seventeen variants (Ptl-84+D99R / K / G / F / T / S / Q / N / Y / A / M / I / V / H / L / E / W) scored significantly better than the wild-type. These variants can be used to produce liraglutide at higher yields compared to the wild-type, while requiring less enzyme for the same conversion.

[0306] Example 3: Synthesis of liraglutide (SEQ ID NO: 6) from two fragments using the Ptl-84+A223X variant

[0307] To determine the effect of the A223 mutation on liraglutide synthesis, screening was performed according to scheme 5. The amounts of the conjugated product liraglutide (H-Lira(1-31)-OH) after 60 min of reaction for representative enzyme variants are listed in the table below.

[0308] Number Enzyme variant Area % of H-Lira(1-31)-OH 1 Ptl-84 + A223S 51,54 2 Ptl-84 + A223G 50,14 3 Ptl-84 45,13

[0309] Clearly, the A223 position affects the reaction rate and coupling efficiency. Surprisingly, the A223S and A223G mutations have a very positive effect on both reaction rate and efficiency. Compared to the wild-type enzyme, these variants can be used to produce liraglutide in higher yields, while requiring less enzyme for the same conversion.

[0310] Example 4: Synthesis of 8-mer model peptide (SEQ ID NO: 14) using the Ptl-84+S224X variant

[0311] To determine the effect of the S224 mutation on the synthesis of the 8-mer model peptide, screening was performed according to scheme 6. The amounts of the conjugate Ac-Asp-Phe-Ser-Lys-Leu-Ala-Leu-Arg-NH2 (SEQ ID NO: 14) after 60 min of reaction for representative enzyme variants are listed in the table below.

[0312] Number Enzyme variant [Ac-Asp-Phe-Ser-Lys-Leu-Ala-Leu-Arg-NH2] 1 Ptl-84 + S224M 95,64％ 2 Ptl-84 + S224Q 89,20％ 3 Ptl-84 + S224E 76,13％ 4 Ptl-84 + S224H 72,10％ 5 Ptl-84 + S224L 70,16％ 6 Ptl-84 + S224V 68,41％ 7 Ptl-84 + S224I 66,90％ 8 Ptl-84 61,08％

[0313] Clearly, the S224 position has a significant impact on reaction rate and coupling efficiency. The seven variants (Ptl-84+S224I / V / L / H / E / Q / M) scored significantly better than the wild-type enzyme. Compared to the wild-type enzyme, these variants can be used to produce peptides at higher yields, while requiring less enzyme for the same conversion.

[0314] Example 5: Synthesis of liraglutide (SEQ ID NO: 6) from two fragments by adding the L96 mutation to several different enzyme variants.

[0315] To determine the effect of the L96 mutation on liraglutide, screening was performed according to scheme 5. The amounts of the conjugated product liraglutide (H-Lira(1-31)-OH) after 60 min of reaction for representative enzyme variants are listed in the table below. Nine reference enzymes were compared with their corresponding mutants containing the mutation at the L96 position. For each series, reference enzymes without the mutation at the L96 position are indicated in bold. Each reference enzyme (numbered 1, 4, 7, 10, 13, 16, 18, 21, 23) has the Ptl-84 mutation plus additional mutations indicated at positions 156, 166, 33, 62, 217, and 222.

[0316]

[0317]

[0318] Clearly, the L96 mutation has a positive effect on reaction rate and coupling efficiency. This mutation remains beneficial when combined with other mutations at previously described mutation sites (WO 2018 / 212658), such as S33, N62, E156, G166, Y217, and / or M222.

[0319] Example 6: Synthesis of liraglutide (SEQ ID NO: 6) from two fragments using a variant with the D99 mutation

[0320] To determine the effect of the D99 mutation on liraglutide synthesis, screening was performed according to scheme 5, but using only 15 μL of enzyme variant solution (instead of 25 μL). The amount of the conjugated product liraglutide (H-Lira(1-31)-OH) after 60 min of reaction for each representative enzyme variant is listed in the table below. The two reference enzymes were compared with the corresponding mutants with the mutation at the D99 position. For each series, the reference enzyme without the mutation at the D99 position is indicated in bold. Reference enzyme (3) has the Ptl-84 mutation plus an additional indicated mutation.

[0321] Number Enzyme variant Area % of H-Lira(1-31)-OH 1 Ptl-84 30,96 2 Ptl-84 + D99R 39,46 3 Ptl-84 + N156K 41,20 4 Ptl-84 + N156K + D99R 46,04 5 Ptl-84 + N156K + D99K 45,68 6 Ptl-84 + N156K + D99G 48,69

[0322] Clearly, the D99 mutation has a positive impact on reaction rate and coupling efficiency.

[0323] Example 7: Synthesis of 8-mer model peptide (SEQ ID NO: 14) using combinatorial variants

[0324] To determine the effect of the mutant combinations of the present invention on the synthesis of the 8-mer model peptide, screening was performed according to scheme 6. Since the enzyme is an improved variant, a smaller amount of enzyme (5 μL enzyme variant solution, totaling 1 μg enzyme) was used to better distinguish the effects of the mutations. Furthermore, after 15 min, the HPLC peaks of both the target peptide Ac-Asp-Phe-Ser-Lys-Leu-Ala-Leu-Arg-NH2 (SEQ ID NO: 14) and the hydrolyzed peptide ester Ac-Asp-Phe-Ser-Lys-Leu-OH (SEQ ID NO: 41) were integrated, and the S / H ratio was calculated by dividing the area % of the target peptide by the area % of the hydrolyzed peptide ester (last column). The reference enzyme is shown in bold.

[0325]

[0326]

[0327] Clearly, each single mutation has a positive effect on the reaction rate and also leads to an increase in the S / H ratio. Specifically, the combination of L96V and D99R (entry 4) shows a synergistic effect on both reaction rate and S / H ratio compared to the single L96V (entry 2) and D99R (entry 3) variants.

[0328] Example 8: Synthesis of liraglutide (SEQ ID NO: 6) from two fragments using a variant with combined mutations

[0329] To determine the effect of mutant combinations on liraglutide synthesis, screening was performed according to scheme 5, but using only 15 μL of enzyme variant solution (instead of 25 μL). The amount of the conjugated product liraglutide (H-Lira(1-31)-OH) after 60 min of reaction for each representative variant is listed in the table below. The reference enzyme Ptl-84 is indicated in bold.

[0330] Number Enzyme variant Area % of H-Lira(1-31)-OH 1 Ptl-84 28,48 2 Ptl-84 + D99R 39,46 3 Ptl-84 + L96V 51,48 4 Ptl-84 + L96V + D99R 59,38

[0331] Clearly, mutations at the L96 and D99 positions have a positive impact on reaction rate and coupling efficiency, both as single mutations and in combination.

[0332] Example 9: Synthesis of liraglutide (SEQ ID NO: 6) from two fragments to determine the effect on selectivity of the combined variant.

[0333] To determine the effect of mutant combinations on coupling efficiency and selectivity, screening was performed according to scheme 7. To assess the conversion rate to the coupling product, the amounts of the coupling product liraglutide (H-Lira(1-31)-OH, SEQ ID NO: 6), unreacted peptide amine (H-Lira(12-31)-OH, SEQ ID NO: 40), and byproduct H-Lira(1-11-1-31)-OH (SEQ ID NO: 42, formed by the reaction of the acyl donor H-Lira(1-11)-OCam-FK-NH2 (SEQ ID NO: 39) with H-Lira(1-31)-OH (SEQ ID NO: 6)) were measured within 30–360 min after complete conversion of the acyl donor ester fragment (SEQ ID NO: 39). The amount of liraglutide and the corresponding selectivity values ​​for each tested enzyme variant are listed in the table below.

[0334]

[0335]

[0336] Clearly, the mutations of the present invention improve both the reaction rate and coupling efficiency of the enzyme compared to the reference Ptl-84, as well as its selectivity. Single mutations at positions 99, 96, and 224 (entries 2, 3, and 5) improve the selectivity of the Ptl-84 enzyme. Mutations at positions 96 and 99 can be combined with mutations at positions 223 and 224 to show improvements in reaction rate and coupling efficiency while maintaining very high selectivity.

[0337] Example 10: Synthesis of teriparatide (SEQ ID NO: 11) from two fragments using different enzyme variants

[0338] Preparation of 1.3 mg / ml PhAc-Teri(1-11)-OCam-L-OH (PhAc- prepared according to schemes 1 and 4) 1 Ser- 2 Val- 3 Ser- 4 Glu- 5 Ile- 6 Gln- 7 Leu- 8 Met- 9 His- 10 Asn- 11 Leu-OCam-Leu-OH·1TFA (SEQ ID NO: 43)) and 4.98 mg / ml H-Teri(12-34)-OH (H- prepared according to scheme 3b) 12 Gly- 13 Lys-14 His- 15 Leu- 16 Asn- 17 Ser- 18 Met- 19 Glu- 20 Arg- 21 Val- 22 Glu- 23 Trp- 24 Leu- 25 Arg- 26 Lys- 27 Lys- 28 Leu- 29 Gln- 30 Asp- 31 Val- 32 His- 33 Asn- 34 A stock solution of Phe-OH·6TFA (SEQ ID NO: 30) was dissolved in a buffer solution containing 200 mM Tricine and 2 mg / mL TCEP, and the pH was set to 7.9 using 3N KOH aqueous solution. This stock solution could be aliquoted and stored at -20°C for further use. 80 μL of the above peptide stock solution was added to a glass vial and the reaction was initiated by mixing with 20 μL of the enzyme variant solution (0.1 mg / mL, in a buffer solution containing 25 mM Tricine and 125 mM NaCl at pH 7.5). At different time points (typically 0, 15, 30, 60, and 120 min), 10 μL of the reaction mixture was quenched in 250 μL of demineralized aqueous solution of methanesulfonic acid (5 mL / L) to terminate any enzyme activity. The quenched samples were analyzed using HPLC / MS. The HPLC peak integral of the indicator conjugate teriparatide (PhAc-Teri(1-34)-OH) (SEQ ID NO: 11) is expressed as HPLC area % in the table below (60 min sample).

[0339] Number Enzyme variant Area % of PhAc-Teriparatide(1-34)-OH 1 Ptl-79 30,99 2 Ptl-79 + L96V 31,00 3 Ptl-79 + D99R 29,67 4 Ptl-79 + L96V + D99R 34,81 5 Ptl-79 + G100D 35,18

[0340] Although single mutations at the L96 and D99 positions did not individually show an effect on reaction rate and coupling efficiency compared to the Ptl-79 reference enzyme, their combination resulted in a synergistic effect.

[0341] Example 11: Synthesis of semaglutide (SEQ ID NO: 7) from two fragments using several different enzyme variants

[0342] Preparation of 6.06 mM H-Sema(1-11)-OCam-FK-NH2 (H- prepared according to Scheme 2)1 His- 2 Aib- 3 Glu- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser- 9 Asp- 10 Val- 11 Ser-OCam-Phe-Lys-NH2 (SEQ ID NO: 44) and 4.04 mMMH-Sema(12-31)-OH (H- prepared according to scheme 3b) 12 Ser- 13 Tyr- 14 Leu- 15 Glu- 16 Gly- 17 Gln- 18 Ala- 19 Ala- 20 Lys(AEEA-AEEA-γ-Glu-17-Carboxyheptadecanoyl)- 21 Glu- 22 Phe- 23 Ile- 24 Ala- 25 Trp- 26 Leu- 27 Val- 28 Arg- 29 Gly- 30 Arg- 31 A stock solution of Gly-OH (SEQ ID NO: 45) was dissolved in a buffer solution containing 50 mM Tricine, 220 mM potassium trifluoroacetate, and 0.25 mg / mL TCEP, and the pH was set to 7.9 using 3N KOH aqueous solution. This stock solution could be aliquoted and stored at -20°C for further use. 83.5 μL of the above peptide stock solution was added to a glass vial, and the reaction was initiated by mixing with 16.7 μL of the enzyme variant solution (0.15 mg / mL, in a buffer solution at pH 7.5 containing 25 mM Tricine and 125 mM NaCl). At different time points (typically 0, 15, 30, 60, and 120 min), 5 μL of the reaction mixture was quenched in 250 μL of demineralized aqueous solution of methanesulfonic acid (5 mL / L) to terminate any enzyme activity. The quenched samples were analyzed using HPLC / MS. The HPLC peak integral of the indicator semaglutide (H-Sema(1-31)-OH) is expressed as HPLC area % in the table below (60 min sample).

[0343] Number Enzyme variant Area % of H-Sema(1-31)-OH 1 Ptl-84 61,84 2 Ptl-84 + D99R 76,92 3 Ptl-84 + L96V 91,33 4 Ptl-84 + L96V + D99R 87,73

[0344] Clearly, the mutations at the L96 and D99 positions have a positive impact on the reaction rate and coupling efficiency compared to the Ptl-84 reference enzyme.

[0345] Example 12: Synthesis of salmon calcitonin (SEQ ID NO: 12) from two fragments using an enzyme variant

[0346] Preparation of 1.7 mg / ml H-Calci(1-12)-OCam-L-OH (H- prepared according to Scheme 1) 1 Cys- 2 Ser- 3 Asn- 4 Leu- 5 Ser- 6 Thr- 7 Cys- 8 Val- 9 Leu- 10 Gly- 11 Lys- 12 Leu-OCam-Leu-OH·1TFA (SEQ ID NO: 31)) and 1.7 mg / ml H-Calci(13-32)-NH2 (H- prepared according to scheme 3a) 13 Ser- 14 Gln- 15 Glu- 16 Leu- 17 His- 18 Lys- 19 Leu- 20 Gln- 21 Thr- 22 Tyr- 23 Pro- 24 Arg- 25 Thr- 26 Asn- 27 Thr- 28 Gly- 29 Ser- 30 Gly- 31 Thr- 32A stock solution of Pro-NH2·3TFA (SEQ ID NO: 32) was dissolved in a buffer solution containing 2M guanidine hydrochloride, 50mM Tricine, and 2 mg / mL TCEP, and the pH was set to 7.9 using 3N KOH aqueous solution. This stock solution could be aliquoted and stored at -20°C for further use. 70 μL of the above peptide stock solution was added to a glass vial and the reaction was initiated by mixing with 30 μL of the enzyme variant solution (0.1 mg / mL, in a buffer solution containing 25 mM Tricine and 125 mM NaCl at pH 7.5). At different time points (typically 0, 15, 30, 60, and 120 min), 10 μL of the reaction mixture was quenched in 250 μL of demineralized aqueous solution of methanesulfonic acid (5 mL / L) to terminate any enzyme activity. The quenched samples were analyzed using HPLC / MS. The HPLC peak integral of the indicator conjugate, salmon calcitonin (H-Cal(1-32)-NH2), is shown in the table below as HPLC area % (60 min sample).

[0347] Enzyme variant ​ Ptl-79 + D99R 60,25

[0348] This embodiment shows that the enzyme variant containing the variant according to the present invention can be effectively used to synthesize salmon calcitonin.

[0349] Example 13: Synthesis of dasiglucagon (SEQ ID NO: 8) from two fragments using an enzyme variant

[0350] Preparation of 3.7 mg / ml H-Dasi(1-8)-OCam-L-OH (H- prepared according to Scheme 1) 1 His- 2 Ser- 3 Gln- 4 Gly- 5 Thr- 6 Phe- 7 Thr- 8 Ser-OCam-Leu-OH·2TFA (SEQ ID NO: 15)) and 5.9 mg / ml H-Dasi(9-29)-OH (H- prepared according to scheme 3b) 9 Asp- 10 Tyr- 11 Ser- 12 Lys- 13 Tyr- 14 Leu- 15 Asp- 16 Aib- 17 Ala- 18 Arg- 19Ala- 20 Glu- 21 Glu- 22 Phe- 23 Va l- 24 Lys- 25 Trp- 26 Leu- 27 Glu- 28 Ser- 29 A stock solution of Thr-OH·4TFA (SEQ ID NO: 16) was dissolved in a buffer solution containing 50 mM Tricine, 220 mM potassium trifluoroacetate, and 1 mg / mL TCEP, and the pH was set to 7.9 using 3N KOH aqueous solution. This stock solution could be aliquoted and stored at -20°C for further use. 100 μL of the above peptide stock solution was added to a glass vial and the reaction was initiated by mixing with 2.5 μL of the enzyme variant solution (2 mg / mL, in a pH 7.5 buffer solution containing 25 mM Tricine and 125 mM NaCl). At different time points (typically 0, 15, 30, 60, and 120 min), 10 μL of the reaction mixture was quenched in 250 μL of demineralized aqueous solution of methanesulfonic acid (5 mL / L) to terminate any enzyme activity. The quenched samples were analyzed using HPLC / MS. The HPLC peak integral of the indicator coupling product dashiglucagon (H-Dasi(1-29)-OH) is expressed as HPLC area % in the table below (60 min sample).

[0351] Enzyme variant Area % of H-Dasi(1-29)-OH Ptl-84 + N156K + E166E + L96I 80,59

[0352] This embodiment shows that the enzyme variant containing the variant according to the present invention can be effectively used to synthesize dasiglucagon.

[0353] sequence

[0354] SEQ ID NO: 1: Wild-type gene encoding amino acids -107 to 275 of BPN' (subtilisin)

[0355] ENA|K02496|K02496.1 B. Bacillus subtilis protease BPN' Bacillus amyloliquefaciens

[0356]

[0357] SEQ ID NO: 2: Wild-type Bacillus subtilis protease BPN' (mature form)

[0358] >SUBT_BACAM Bacillus subtilis protease BPN' mature Bacillus amylolyticus 1 to 275

[0359] Amino acids at positions 75-83, 96, 99, 223, and 224 are indicated in bold.

[0360] AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETNPFQD 60

[0361] NNSHGTHVAGTVAALNNSIGVLGVAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPS130

[0362] GSAALKAAVDKAVASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFS 190

[0363] SVGPELDVMAPGVSIQSTLPGNKYGAYNGTSMASPHVAGAAALILSKHPNWTNTQVRSSL 250

[0364] ENTTTKLGDSFYYGKGLINVQAAAQ

[0365] SEQ ID NO: 3: The BPN' variant of the subtilisin according to the present invention, having amino acids corresponding to positions 75-83 (Ca). 2+ The deletion of the binding loop, the S221 mutation (S221C), and possible L96, D99, A223, and S224 mutations (all four are marked with bold X) are tagged with 6-His.

[0366] AQSVPYGVSQIKAPALHSQGYTGSNVKVAVIDSGIDSSHPDLKVAGGASMVPSETNPFQD 60

[0367] NNSHGTHVAGTVAA-VAPSASLYAVKVXGAXGSGQYSWIINGIEWAIANNMDVINMSLGGPS 130(-9)

[0368] GSAALKAAVDKAVASGVVVVAAAGNEGTSGSSSTVGYPGKYPSVIAVGAVDSSNQRASFS 190(-9)

[0369] SVGPELDVMAPGVSIQSTLPGNKYGAYNGTCMXXPHVAGAAALILSKHPNWTNTQVRSSL 250(-9)

[0370] ENTTTKLGDSFYYGKGLINVQAAAQHHHHHH

[0371] SEQ ID NO: 4-Reference enzyme Ptl-84

[0372] The BPN' variant of subtilisin contains mutants Q2K, S3C, P5S, S9A, I31L, K43N, M50F, N62A, A73L, Δ75-83, E156N, G166E, G169A, S188P, F189W, Q206C, N212G, Y217H, N218D, S221C, M222P, P225N, T254A, and Q271E, plus a 6-His tag.

[0373] AKCVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQD 60

[0374] NASHGTHVAGTVLA-VAPSASLYAVKVLGADGSGQYSWIINGIEWAIANNMDVINMSLGGPS 130(-9)

[0375] GSAALKAAVDKAVASGVVVVAAAGNNGTSGSSSTVEYPAKYPSVIAVGAVDSSNQRAPWS190(-9)

[0376] SVGPELDVMAPGVSICSTLPGGKYGAHDGTCPASNHVAGAAALILLSKHPNWTNTQVRSSL 250(-9)

[0377] ENTATKLGDSFYYGKGLINVEAAAQHHHHHH

[0378] SEQ ID NO: 5-Reference enzyme Ptl-79

[0379] The BPN' variant of subtilisin contains the mutants Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, Δ75-83, I107V, E156S, G166S, G169A, S188P, F189W, Q206C, N212G, Y217H, N218S, S221C, M222P, P225N, T254A, and Q271E, plus a 6-His tag.

[0380] AKCVSYGVAQIKAPALHSQGYTGSNVKVAVLDSGIDSSHPDLNVAGGASFVPSETNPFQD 60

[0381] NNSHGTHVAGTVLAVAPSASLYAVKVLGADGSGQYSWVINGIEWAIANNMDVINMSLGGPS130(-9)

[0382] GSAALKAAVDKAVASGVVVVAAAGNSGTSGSSSTVSYPAKYPSVIAVGAVDSSNQRAPWS190(-9)

[0383] SVGPELDVMAPGVSICSTLPGGKYGAHSGTCPASNHVAGAAALILLSKHPNWTNTQVRSSL 250(-9)

[0384] ENTATKLGDSFYYGKGLINVEAAAQHHHHHH

[0385] SEQ ID NO: 6-liraglutide

[0386] HAEGTFTSDV SSYLEGQAAK EFIAWLVRGR G

[0387] Where K is Lys(Pal-γ-Glu)

[0388] SEQ ID NO: 7-Semaglutide

[0389] HXEGTFTSDV SSYLEGQAAK EFIAWLVRGR G

[0390] Where X is Aib, and K is Lys(AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl)

[0391] SEQ ID NO: 8–dasiglucagon

[0392] HSQGTFTSDY SKYLDXARAE EFVKWLEST

[0393] Where X is Aib

[0394] SEQ ID NO: 9–glepaglutide

[0395] HGEGTFSSEL ATILDALAAR DFIAWLIATK ITDKKKKKK

[0396] The C-terminus is an amide.

[0397] SEQ ID NO: 10–elsiglutide

[0398] HGEGSFSSEL STILDALAAR DFIAWLIATK ITDKKKKKK

[0399] The C-terminus is an amide.

[0400] SEQ ID NO: 11 – Teriparatide

[0401] SVSEIQLMHN LGKHLNSMER VEWLRKKLQD VHNF

[0402] In the teriparatide precursor, the N-terminus can be phenylacetylated.

[0403] SEQ ID NO: 12-Salmon Calcitonin

[0404] CSNLSTCVLG KLSQELHKLQ TYPRTNTGSG TP

[0405] Cys 1 and Cys 7 It is linked by disulfide bonds and has a C-terminal amide.

[0406] SEQ ID NO: 13-Bivaludin

[0407] XPRPGGGGNG DFEEIPEEYL

[0408] Where X is D-Phe

[0409] SEQ ID NO: 14-8-mer model peptide

[0410] DFSKLALR

[0411] The peptide is acetylated at the N-terminal amino acid and has a C-terminal amide.

[0412] SEQ ID NO: 15–Dasi 1-8

[0413] HSQGTFTS

[0414] The C-terminus is a (thio) ester.

[0415] SEQ ID NO: 16–Dasi 9-29

[0416] DYSKYLDXAR AEEFVKWLES T

[0417] Where X is Aib

[0418] SEQ ID NO: 17–Dasi 1-9

[0419] HSQGTFTSD

[0420] The C-terminus is a (thio) ester.

[0421] SEQ ID NO: 18–Dasi 10-29

[0422] YSKYLDXARA EEFVKWLEST

[0423] Where X is Aib

[0424] SEQ ID NO: 19–Dasi 1-10

[0425] HSQGTFTSDY

[0426] The C-terminus is a (thio) ester.

[0427] SEQ ID NO: 20–Dasi 11-29

[0428] SKYLDXARA EEFVKWLEST

[0429] Where X is Aib

[0430] SEQ ID NO: 21–Glepa 1-11

[0431] HGEGTFSSEL A

[0432] The C-terminus is a (thio) ester.

[0433] SEQ ID NO: 22–Glepa 12-39

[0434] TILDALAARD FIAWLIATKI TDKKKKKK

[0435] SEQ ID NO: 23–Glepa 1-16

[0436] HGEGTFSSEL ATILDA

[0437] The C-terminus is a (thio) ester.

[0438] SEQ ID NO: 24–Glepa 17-39

[0439] LAARD FIAWLIATKI TDKKKKKK

[0440] SEQ ID NO: 25–Elsi 1-11

[0441] HGEGSFSSEL S

[0442] The C-terminus is a (thio) ester.

[0443] SEQ ID NO: 26–Elsi 12-39

[0444] TILDALAARD FIAWLIATKI TDKKKKKK

[0445] SEQ ID NO: 27–Elsi 1-16

[0446] HGEGSFSSEL STILDA

[0447] The C-terminus is a (thio) ester.

[0448] SEQ ID NO: 28–Elsi 17-39

[0449] LAARDFIAWL IATKITDKKK KKK

[0450] SEQ ID NO: 29–Teri 1-11

[0451] SVSEIQLMHN L

[0452] The C-terminus is a (thio) ester.

[0453] SEQ ID NO: 30–Teri 12-34

[0454] GKHLNSMERV EWLRKKLQDV HNF

[0455] SEQ ID NO: 31–Calci 1-12

[0456] CSNLSTCVLG KL

[0457] The C-terminus is a (thio) ester.

[0458] SEQ ID NO: 32–Calci 13-32

[0459] SQELHKLQTY PRTNTGSGTP

[0460] The C-terminus may be an amide.

[0461] SEQ ID NO: 33–Calci 1-20

[0462] CSNLSTCVLG KLSQELHKLQ

[0463] The C-terminus is a (thio) ester.

[0464] SEQ ID NO: 34–Calci 21-32

[0465] TYPRTNTGSG TP

[0466] The C-terminus may be an amide.

[0467] SEQ ID NO: 35–Biva 1-10

[0468] XPRPGGGGNG

[0469] Where X is D-Phe

[0470] SEQ ID NO: 36–Biva 11-20

[0471] DFEEIPEEYL

[0472] SEQ ID NO: 37–Lira-Sema 1-11

[0473] HXEGTFTSDV S

[0474] Where X is Ala or Aib, and the C-terminus is a (thio)ester.

[0475] SEQ ID NO: 38-Lira-Sema 12-31

[0476] SYLEGQAAKE FIAWLVRGRG

[0477] Lys is selected from Lys, Lys(PG), Lys(γ-Glu), Lys(Pal-γ-Glu) and Lys(AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl).

[0478] SEQ ID NO: 39–Lira 1-11

[0479] HAEGTFTSDV S

[0480] The C-terminus is an -OCam-Phe-Lys-NH2 ester.

[0481] SEQ ID NO: 40-Lira 12-31

[0482] SYLEGQAAKE FIAWLVRGRG

[0483] Where Lys is Lys(Pal-γ-Glu)

[0484] SEQ ID NO: 41 – Model peptide fragment

[0485] DFSKL

[0486] The N-terminus is acetylated, and the C-terminus is an -OCam-Leu-OH ester or OH.

[0487] SEQ ID NO: 42–lira 1-11-1-31

[0488] HAEGTFTSDV SHAEGTFTSD VSSYLEGQAA KEFIAWLVRG RG

[0489] Where Lys is Lys(Pal-γ-Glu)

[0490] SEQ ID NO: 43–Teri 1-11 ester

[0491] SVSEIQLMHN L

[0492] The N-terminus is phenylacetylated, and the C-terminus is an -OCam-Leu-OH ester.

[0493] SEQ ID NO: 44–Sema 1-11

[0494] HXEGTFTSDV S

[0495] Where X is Aib, and the C-terminus is an -OCam-Phe-Lys-NH2 ester.

[0496] SEQ ID NO: 45-Sema 12-31

[0497] SYLEGQAAKE FIAWLVRGRG

[0498] Lys is Lys(AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl)

Claims

1. A variant of BPN' subtilisin, having an amino acid sequence comprising a deletion of amino acids 75-83 and a mutation at amino acid position S221, said mutation being S221C or S221 selenocysteine, compared to BPN' SEQ ID NO: 2; and Its features are, The BPN' variant of the subtilisin has at least one mutation at an amino acid position selected from L96, D99, A223, and S224, wherein the at least one mutation is selected from L96H, L96I, L96V, L96M, L96T, L96C, L96Q, L96A, L96S, D99A, D99V, D99R, D99K, D99G, D99F, D99T, D99S, D99N, D99Q, D99Y, D99M, D99I, D99H, D99E, D99L, D99W, A223S, A223G, S224M, S224Q, S224E, S224H, S224L, S224V, and S224I. The variants mentioned include additional mutations Q2K, S3C, P5S, S9A, I31L, K43N, M50F, N62A, A73L, E156N, G166E, G169A, S188P, F189W, Q206C, N212G, Y217H, N218D, M222P, P225N, T254A, and Q27. 1E, or additional mutations in Q2K, S3C, P5S, S9A, I31L, K43N, M50F, A73L, I107V, E156S, G166S, G169A, S188P, F189W, Q206C, N212G, Y217H, N218S, M222P, P225N, T254A, and Q271E. The amino acid positions are defined according to SEQ ID NO:

2. Furthermore, these variants exhibit ligase and / or cyclase activities with improved reaction rates and coupling efficiency.

2. The BPN' variant of subtilisin according to claim 1, wherein the mutation at S221 is S221C.

3. The BPN' variant of subtilisin according to claim 1, comprising one of the following: A mutation at the amino acid position selected from L96, D99, A223, and S224; or Two mutations at the amino acid positions selected from L96 and D99, L96 and A223, L96 and S224, D99 and A223, D99 and S224, and A223 and S224; or Three mutations at the amino acid positions selected from L96, D99 and A223, L96, D99 and S224, L96, A223 and S224, and D99, A223 and S224; or Four mutations at amino acid positions L96, D99, A223, and S224.

4. The BPN' variant of subtilisin according to any one of claims 1-3, comprising a mutation at the L96 position.

5. The BPN' variant of subtilisin according to claim 4, wherein the mutation is selected from L96I, L96V, L96M, L96T, L96C, L96Q, L96A and L96S.

6. The BPN' variant of subtilisin according to claim 5, wherein the mutation is selected from L96I and L96V.

7. The BPN' variant of subtilisin according to any one of claims 1-3, comprising a mutation at the D99 position.

8. The BPN' variant of subtilisin according to claim 7, wherein the mutation is selected from D99R, D99K, D99G, D99F, D99T, D99S, D99N, D99Q, D99Y, D99M, D99I, D99H, D99E, D99L and D99W.

9. The BPN' variant of subtilisin according to claim 8, wherein the mutation is selected from D99R, D99K and D99G.

10. The BPN' variant of subtilisin according to any one of claims 1-3, comprising a mutation at position A223.

11. The BPN' variant of subtilisin according to claim 10, wherein the mutation is selected from A223S and A223G.

12. The BPN' variant of subtilisin according to any one of claims 1-3, comprising a mutation at position S224.

13. The BPN' variant of subtilisin according to claim 12, wherein the mutation is selected from S224M, S224Q, S224E, S224H, S224L, S224V and S224I.

14. The BPN' variant of subtilisin according to any one of claims 1-3, wherein at least two, three, or four mutations are selected from L96H, L96I, L96V, L96M, L96T, L96C, L96Q, L96A, L96S, D99A, D99V, D99R, D99K, D99G, D99F, D99T, D99S, D99N, D99Q, D99Y, D99M, D99I, D99H, D99E, D99L, D99W, A223S, A223G, S224M, S224Q, S224E, S224H, S224L, S224V, and S224I.

15. The BPN' variant of subtilisin according to claim 14, wherein the combination of said mutations is selected from L96V and D99R; L96I and D99R; L96I and D99K; L96I and A223S; L96I and S224V; L96V, D99R and A223S; L96V, D99R and S224V; L96I, D99R and A223S; L96I, D99K R and S224V; L96I, D99K and A223S; L96I, A223S and S224V; L96I, D99K and S224V; L96V, D99R, A223S and S224V; L96I, D99R, A223S and S224V; L96V, D99K, A223S and S224V; and L96I, D99K, A223S and S224V.

16. The BPN' variant of subtilisin according to any one of claims 1-3, further comprising at least one mutation at an amino acid position selected from S33 and N62.

17. The BPN' variant of subtilisin according to claim 16, wherein the mutation is selected from S33T, N62A, N62R and N62K.

18. The BPN' variant of subtilisin according to any one of claims 1-3, comprising mutants Q2K, S3C, P5S, S9A, I31L, K43N, M50F, N62A, A73L, E156N, G166E, G169A, S188P, F189W, Q206C, N212G, Y217H, N218D, M222P, P225N, T254A, Q271E, and selected... Mutation combinations from L96I and D99R, L96I and D99K, L96I and A223S, L96I and S224V, L96I, A223S and S224V, L96I, D99R and S224V, L96I, D99R, A223S and S224V, L96I, D99K and A223S, L96I, D99K and S224V, and L96I, D99K, A223S and S224V.

19. A method for enzymatic synthesis of peptides, the method comprising the step of coupling (a) a C-terminal (thio) ester of a peptide with (b) a peptide nucleophile having an N-terminal unprotected amine. The coupling is carried out in an aqueous solution, and the coupling is catalyzed by a subtilisin BPN' variant, which, compared to subtilisin BPN' SEQ ID NO: 2, has an amino acid sequence containing the deletion of amino acids at positions 75-83 and a mutation at amino acid position S221, the mutation being S221C or S221 selenocysteine; and Its features are, The BPN' variant of the subtilisin has at least one mutation at an amino acid position selected from L96, D99, A223, and S224. The amino acid positions are defined according to SEQ ID NO:

2. The BPN' variant of the subtilisin is defined as in any one of claims 1 to 18. Furthermore, these variants exhibit ligase and / or cyclase activities with improved reaction rates and coupling efficiency.

20. The method of claim 19, wherein the mutation at S221 is S221C.

21. The method of claim 19, wherein the synthesized peptide is selected from liraglutide, semaglutide, dassiglitazone, tiduglutide, gliperagglutide, esgglutide, teriparatide, salmon calcitonin, bivalirudin, and analogues thereof.

22. The method of claim 19, wherein the method is used to synthesize a peptide comprising the dasiglitazone sequence as shown in SEQ ID NO: 8, the method comprising the step of conjugating the following substance: (a) A C-terminal (thio) ester of a peptide comprising a first peptide fragment, wherein the first peptide fragment has the sequence His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-(thio) ester as shown in SEQ ID NO: 15, and (b) A peptide nucleophilic fragment comprising a second peptide fragment having an N-terminal unprotected amine, wherein the second peptide fragment has the sequence H-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Aib-Ala-Arg-Ala-Glu-Glu-Phe-Val-Lys-Trp-Leu-Glu-Ser-Thr as shown in SEQ ID NO: 16; or (c) A C-terminal (thio) ester of a peptide comprising a first peptide fragment, wherein the first peptide fragment has the sequence His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp (thio) ester as shown in SEQ ID NO: 17, and (d) A peptide nucleophilic fragment containing a second peptide fragment having an N-terminal unprotected amine, wherein the second peptide fragment has the sequence H-Tyr-Ser-Lys-Tyr-Leu-Asp-Aib-Ala-Arg-Ala-Glu-Glu-Phe-Val-Lys-Trp-Leu-Glu-Ser-Thr as shown in SEQ ID NO: 18; or (e) A C-terminal (thio) ester of a peptide comprising a first peptide fragment, wherein the first peptide fragment has the sequence His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr (thio) ester as shown in SEQ ID NO: 19, and (f) A peptide nucleophile fragment comprising a second peptide fragment having an N-terminal unprotected amine, wherein the second peptide fragment has the sequence H-Ser-Lys-Tyr-Leu-Asp-Aib-Ala-Arg-Ala-Glu-Glu-Phe-Val-Lys-Trp-Leu-Glu-Ser-Thr as shown in SEQ ID NO: 20; Alternatively, the method is used to synthesize a peptide comprising the glipagglutinin sequence shown in SEQ ID NO: 9, the method comprising the step of conjugating the following substance: (a) A C-terminal (thio) ester of a peptide comprising a first peptide fragment, wherein the first peptide fragment has the sequence His-Gly-Glu-Gly-Thr-Phe-Ser-Ser-Glu-Leu-Ala-(thio) ester as shown in SEQ ID NO: 21, and (b) A peptide nucleophilic fragment comprising a second peptide fragment having an N-terminal unprotected amine, wherein the second peptide fragment has the sequence H-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-AIa-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys-Lys; or (c) A C-terminal (thio) ester of a peptide comprising a first peptide fragment, wherein the first peptide fragment has the sequence His-Gly-Glu-Gly-Thr-Phe-Ser-Ser-Glu-Leu-Ala-Thr-Ile-Leu-Asp-Ala-(thio) ester as shown in SEQ ID NO: 23, and (d) A peptide nucleophile fragment comprising a second peptide fragment having an N-terminal unprotected amine, wherein the second peptide fragment has the sequence H-Leu-Ala-Ala-Arg-Asp-Phe-Ile-AIa-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys-Lys as shown in SEQ ID NO: 24; Alternatively, the method is used to synthesize a peptide comprising the esoglutide sequence as shown in SEQ ID NO: 10, the method comprising the step of conjugating the following substances: (a) A C-terminal (thio) ester of a peptide comprising a first peptide fragment, wherein the first peptide fragment has the sequence His-Gly-Glu-Gly-Ser-Phe-Ser-Ser-Glu-Leu-Ser-(thio) ester as shown in SEQ ID NO: 25, and (b) A peptide nucleophilic fragment comprising a second peptide fragment having an N-terminal unprotected amine, wherein the second peptide fragment has the sequence H-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-AIa-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys-Lys; or (c) A C-terminal (thio) ester of a peptide comprising a first peptide fragment, wherein the first peptide fragment has the sequence His-Gly-Glu-Gly-Ser-Phe-Ser-Ser-Glu-Leu-Ser-Thr-Ile-Leu-Asp-Ala-(thio) ester as shown in SEQ ID NO: 27, and (d) A peptide nucleophilic fragment containing a second peptide fragment having an N-terminal unprotected amine, wherein the second peptide fragment has the sequence H-Leu-Ala-Ala-Arg-Asp-Phe-Ile-AIa-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys-Lys as shown in SEQ ID NO: 28; Alternatively, the method is used to synthesize a peptide comprising the teriparatide sequence shown in SEQ ID NO: 11, the method comprising the step of conjugating the following substance: (a) A C-terminal (thio) ester of a peptide comprising a first peptide fragment, wherein the first peptide fragment has the sequence Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-(thio) ester as shown in SEQ ID NO: 29, and (b) A peptide nucleophilic fragment comprising a second peptide fragment having an N-terminal unprotected amine, wherein the second peptide fragment has the sequence H-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe as shown in SEQ ID NO: 30; Alternatively, the method is used to synthesize a peptide comprising the salmon calcitonin sequence shown in SEQ ID NO: 12, the method comprising the step of conjugating the following substances: (a) A C-terminal (thio) ester of a peptide comprising a first peptide fragment, wherein the first peptide fragment has the sequence Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-(thio) ester as shown in SEQ ID NO: 31, and (b) A peptide nucleophilic fragment comprising a second peptide fragment having an N-terminal unprotected amine, wherein the second peptide fragment has the sequence H-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro as shown in SEQ ID NO: 32; or (c) A C-terminal (thio) ester of a peptide comprising a first peptide fragment, wherein the first peptide fragment has the sequence shown in SEQ ID NO: 33: Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-(thio) ester, and (d) A peptide nucleophile fragment containing a second peptide fragment having an N-terminal unprotected amine, wherein the second peptide fragment has the sequence H-Thr-Tyr-Pro-Arg-Thr-Asn-Thr-Gly-Ser-Gly-Thr-Pro as shown in SEQ ID NO: 34; Alternatively, the method is used to synthesize a peptide comprising the bivalirudin sequence as shown in SEQ ID NO: 13, the method comprising the step of conjugating the following substance: (a) A C-terminal (thio) ester of a peptide comprising a first peptide fragment, wherein the first peptide fragment has the sequence D-Phe-Pro-Arg-Pro-Gly-Gly-Gly-Gly-Asn-Gly-(thio) ester as shown in SEQ ID NO: 35, and (b) A peptide nucleophilic fragment comprising a second peptide fragment having an N-terminal unprotected amine, wherein the second peptide fragment has the sequence H-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu as shown in SEQ ID NO: 36; Alternatively, the method is used to synthesize liraglutide or semaglutide comprising the sequence His-W-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Z-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly. in For the synthesis of liraglutide as shown in SEQ ID NO: 6, W is Ala and Z is selected from Lys, Lys(PG), Lys(γ-Glu), and Lys(Pal-γ-Glu), or For the synthesis of semaglutide as shown in SEQ ID NO: 7, W is Aib and Z is selected from Lys, Lys(PG) and Lys(AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl). Furthermore, PG is a protecting group for the amino group on the Lys side chain; The method includes the step of coupling the following substances: (a) A C-terminal (thio) ester of a peptide comprising a first peptide fragment, wherein the first peptide fragment has the sequence His-W-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-(thio) ester as shown in SEQ ID NO: 37, and (b) A peptide nucleophilic fragment containing a second peptide fragment having an N-terminal unprotected amine, wherein the second peptide fragment has the sequence H-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Z-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH as shown in SEQ ID NO: 38.

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