Polypeptide ligase mutant and method for preparing polypeptide

Abstract

The present invention provides a polypeptide ligase mutant and a method for preparing a polypeptide. The polypeptide ligase mutant comprises: (a) a protein mutated from the amino acid sequence shown in SEQ ID NO: 1, the mutation comprising a mutation occurring at the S307 site; or (b) a protein having 70% or more identity with the amino acid sequence defined in (a) and having polypeptide ligase activity. The present invention can solve the problem in the prior art of poor activity of polypeptide ligases, and is applicable to the field of polypeptide synthesis.

Description

Peptide ligase mutants and methods for preparing peptides

[0001] This application is based on and claims priority to Chinese application CN application number 202411531501.2 filed on October 30, 2024, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates to the field of polypeptide synthesis, and more specifically, to a polypeptide ligase mutant and a method for preparing polypeptides. Background Technology

[0003] Peptides are bioactive compounds formed by multiple amino acids linked by peptide bonds, typically containing 10 to 100 amino acid residues with a molecular weight of less than 10,000. Compared to small molecule drugs and protein drugs, peptide drugs exhibit higher activity and selectivity, fewer side effects in the human body, greater stability, and lower immunogenicity. Peptide drugs have been widely used in the treatment of diseases such as vaccines, cancer, endocrine disorders, and cardiovascular diseases. As of January 2023, approximately 180 peptide drugs had been launched globally, demonstrating enormous development potential.

[0004] Solid-phase synthesis (SPPS) is a major technique for synthesizing non-natural peptides, but it can encounter problems such as numerous elution impurities and purification difficulties during the synthesis process. Furthermore, as the peptide chain length increases, the SPPS yield decreases exponentially, often requiring multiple HPLC purifications to obtain a high-purity product. For medium-length peptides, the total SPPS yield is often less than 25%.

[0005] To synthesize longer peptides, researchers often break down peptides into several smaller fragments and then ligate them. Existing ligation methods are mainly divided into chemical and enzymatic methods. In chemical ligation, the C-terminus of the fragment is prone to racemization, and complete protection of the linked amino acids is required, leading to poor fragment solubility and difficulty in purification. In contrast, enzymatic ligation eliminates the risk of racemization, requires no protection of the side chains, and is easier to purify. Wells' team engineered a serine protease (Subtilisin) from *Bacillus amyloliquefaciens*, creating a subtiligase through mutations at two sites (S221C / P225A) that can ligate a C-terminal ester donor fragment and an acyl acceptor fragment that does not require N-terminal protection in aqueous solution. However, the low ligation efficiency and instability of subtiligases limit their industrial applications (Abrahmsen, Lars, et al. "Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution." Biochemistry 30.17(2012):4151-4159.). Enzypep has developed Omniligase-1, a peptide ligase with greater stability and a broader catalytic substrate spectrum, through a series of mutation studies.

[0006] Although peptide ligases are already commercially available, challenges remain in practical applications. These ligases exhibit limited selectivity for specific amino acid residues, particularly non-natural amino acids and proline, resulting in limited catalytic activity for certain peptide drug fragments and insufficient stability under certain conditions. Therefore, developing novel peptide ligases to achieve efficient and stable ligation of various peptide drug fragments has become an urgent and necessary task. Summary of the Invention

[0007] The main objective of this invention is to provide a polypeptide ligase mutant and a method for preparing polypeptides, so as to solve the problem of poor polypeptide ligase activity in the prior art.

[0008] To achieve the above objectives, according to a first aspect of the present invention, a polypeptide ligase mutant is provided, the polypeptide ligase mutant comprising:

[0009] (a) A protein based on the amino acid sequence shown in SEQ ID NO: 1, the mutation including a mutation at the S307 site; or (b) A protein having more than 70% homology with the amino acid sequence defined in (a) and having polypeptide ligase activity.

[0010] Further, the mutation is selected from the S307C mutation and any one or more of the following mutations: S107 mutation to S107T; S154 mutation to S154A; S186 mutation to S186Y; I192 mutation to I192V; S241 mutation to S241N; K272 mutation to K272L; F275 mutation to F275W; S290 mutation to S290G; L303 mutation to L303H; M308 mutation to M308P; T88 mutation to T88S or T88A; S337 mutation to S337I, S337Y, S337R or S337M; P311 mutation to P311A, P311N, P311Y. P311Q or P311G; H346 mutation to H346A, H346G, H346Y or H346P; S214 mutation to S214P, S214Y, S214A, S214F or S214V; N161 mutation to N161H, N162T, N161C, N161D or N161K; P140 mutation to P14 0A, P140G, P140V, P140R, P140K, or P140D; G92 mutation to G92A, G92S, G92C, G92R, G92H, G92T, G92F, or G92M; N162 mutation to N162T, N162V, N162R, N162E, N162Y, N162S, or N162C.

[0011] Further, the mutation includes any one or more of the following amino acid mutations: S307C, S307C+S107T, S307C+S337I, S307C+G92A, S307C+P140D, S307C+S154A, S307C+N161D, S307C+N162T, S307C+S186Y, S307C+I192V, S307C+S214P, S307C+S241N, S307C+K272L, S307C+F275W, S307C+S290G, S307C+L303H, S307C+M308P, S307C+P311A, S307C+P311G , S307C+P311Y, S307C+H346Y, S307C+T88S, S307C+T88A, S307C+S107T+S3 37I, S307C+S107T+S337Y, S307C+S107T+S337R, S307C+S107T+S337M, S30 7C+S107T+G92A, S307C+S107T+G92S, S307C+S107T+G92C, S307C+S107T+N 162T, S307C+S107T+N162V, S307C+S107T+N162R, S307C+S107T+N162E, S3 07C+S107T+P311A, S307C+S107T+P311N, S307C+S107T+P311Q, S307C+S10 7T+P311G, S307C+S107T+S337I+H346A, S307C+S107T+S337I+H346G, S307 C+S107T+S337I+H346Y, S307C+S107T+S337I+H346P, S307C+S107T+S337I +P311A, S307C+S107T+S337I+P311N, S307C+S107T+S337I+G92A, S307C+S 107T+S337I+G92R, S307C+S107T+S337I+G92H, S307C+S107T+S337I+P311 A+G92T, S307C+S107T+S337I+P311A+G92A, S307C+S107T+S337I+P311A+G 92F、S307C+S107T+S337I+P311A+G92M、S307C+S107T+S337I+P311A+N162 T、S307C+S107T+S337I+P311A+N162Y、S307C+S107T+S337I+P311A+N162S、S307C+S107T+S337I+P311A+N162C, S307C+S107T+S337I+P311A+N161H, S3 07C+S107T+S337I+P311A+N161C, S307C+S107T+S337I+P311A+N161D, S307C +S107T+S337I+P311A+N161K, S307C+S107T+S337I+P311A+G92A+P140A, S3 07C+S107T+S337I+P311A+G92A+P140G, S307C+S107T+S337I+P311A+G92A+P 140V, S307C+S107T+S337I+P311A+G92A+P140R, S307C+S107T+S337I+P311 A+G92A+P140K, S307C+S107T+S337I+P311A+G92A+S214P, S307C+S107T+S33 7I+P311A+G92A+S214Y, S307C+S107T+S337I+P311A+G92A+S214A, S307C+S107T+S337I+P311A+G92A+S214F or S307C+S107T+S337I+P311A+G92A+S214V.

[0012] Further, the polypeptide ligase mutant includes a protein having 75% or more, 80% or more, 85% or more, more preferably 95% or more, and even more preferably 99% or more homology with the amino acid sequence defined in (a) and having polypeptide ligase activity.

[0013] To achieve the above objectives, according to a second aspect of the present invention, a DNA molecule is provided that encodes the aforementioned polypeptide ligase mutant.

[0014] To achieve the above objectives, according to a third aspect of the present invention, a recombinant plasmid is provided, which is linked to the aforementioned DNA molecule.

[0015] To achieve the above objectives, according to a fourth aspect of the present invention, a host cell is provided, which contains the aforementioned DNA molecule or the aforementioned recombinant plasmid; the host cell is not an animal or plant species.

[0016] Further, the host cell includes eukaryotic cells or prokaryotic cells; preferably, the eukaryotic cell includes yeast cells; preferably, the yeast cell includes Pichia pastoris; preferably, the prokaryotic cell includes Escherichia coli or Bacillus subtilis; preferably, Escherichia coli includes BL21(DE3); preferably, Bacillus subtilis includes WB600.

[0017] To achieve the above objectives, according to a fifth aspect of the present invention, a method for preparing a polypeptide is provided, the method comprising: using the above-mentioned polypeptide ligase mutant to catalyze the binding of a substrate peptide chain to prepare a polypeptide.

[0018] Further, the substrate peptide chain comprises 2 to 3 chains. Preferably, the substrate peptide chain comprises 2 chains; preferably, the substrate peptide chain contains 5 to 30 amino acids. Preferably, the amino acids in the substrate peptide chain contain non-natural amino acids.

[0019] Further, when the number of substrate peptide chains is two, the substrate peptide chains include a first substrate peptide chain and a second substrate peptide chain; preferably, the combination of the first substrate peptide chain and the second substrate peptide chain is selected from any one or more of the following groups: 1) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NOs: 2-3 or SEQ ID NOs: 5-23, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: 4; 2) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NO: 24, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: 28; 3) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NO: 25, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: 29; 4) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NO: 26, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: 30; 5) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NO: 27, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: 30. NO: 31; 6) The amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 32, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO: 33; 7) The amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 34, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO: 35; Preferably, the C-terminus of the first substrate peptide chain contains an ester acyl group.

[0020] By applying the technical solution of the present invention, the above-mentioned polypeptide ligase mutant has higher activity than the polypeptide ligase in the prior art, broadens the substrate spectrum of polypeptide ligase, and can achieve the ligation of different polypeptide fragments. Attached Figure Description

[0021] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0022] Figure 1 shows a schematic diagram of the connection of the 5-peptide substrate according to Embodiment 4 of the present invention.

[0023] Figure 2 shows a schematic diagram of the catalytic results of the polypeptide ligase mutant according to Example 7 of the present invention on substrates with different amino acids at the P1 position. Detailed Implementation

[0024] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0025] As mentioned in the background section, existing peptide ligases have low activity, resulting in poor selectivity for specific amino acid residues, a narrow substrate spectrum, limited catalytic activity for certain peptide drug fragments, and insufficient stability. Therefore, in this application, the inventors attempted to develop a novel peptide ligase mutant, and based on this, proposed a series of protection schemes for this application.

[0026] In a first typical embodiment of this application, a polypeptide ligase mutant is provided, comprising: (a) a protein mutated based on the amino acid sequence shown in SEQ ID NO: 1, the mutation including a mutation occurring at the S307 site; or (b) a protein having more than 70% homology to the amino acid sequence defined in (a) and having polypeptide ligase activity.

[0027] In the initial stage of this application, an enzyme library containing 200 proteases was constructed. Activity tests were conducted on these proteases using different polypeptide substrates, revealing that these enzymes only possessed hydrolytic activity and could not be used for peptide bond synthesis. Subsequently, the inventors obtained the three-dimensional structures of these proteases using homology modeling and AI prediction methods. The serine residue near the active site was mutated to cysteine. The linker activity of the enzymes after single-point mutation was tested using pentapeptide substrates (such as Ac-Ala-Asp-Ser-Lys-Leu-O-Cam-Leu-OH (SEQ ID NO: 36) and H-Ala-Leu-Arg-His-Glu-NH2 (SEQ ID NO: 37)), and analyzed by HPLC after the reaction. The results showed that in approximately 10% of the mutants, in addition to the hydrolysis products, a decapeptide was also detected. This indicates that some enzymes, after single-point mutation, retained some hydrolytic activity while also exhibiting linker activity. However, in the remaining approximately 90% of the enzymes, in addition to the absence of observed linker activity, the hydrolytic activity was also significantly reduced. This demonstrates that the serine residue, a key catalytic site near the active site, is crucial for protease catalysis. However, simply mutating it to cysteine ​​to obtain linkage activity is not applicable to most proteases.

[0028] Through the above tests, the inventors of this application screened out a single-point mutant of the protease derived from Bacillus gobiensis (SEQ ID NO: 1), which exhibited high activity during peptide ligation. The active pocket was further modified to improve its activity and substrate selectivity, resulting in a peptide ligase with improved amino acid residue selectivity containing at least an amino acid mutation at the S307 site.

[0029] In a preferred embodiment, the mutation is selected from the S307C mutation and any one or more of the following mutations: S107 mutation to S107T; S154 mutation to S154A; S186 mutation to S186Y; I192 mutation to I192V; S241 mutation to S241N; K272 mutation to K272L; F275 mutation to F275W; S290 mutation to S290G; L303 mutation to L303H; M308 mutation to M308P; T88 mutation to T88S or T88A; S337 mutation to S337I, S337Y, S337R, or S337M; P311 mutation to P311A, P311N, or P3 11Y, P311Q, or P311G; H346 mutation to H346A, H346G, H346Y, or H346P; S214 mutation to S214P, S214Y, S214A, S214F, or S214V; N161 mutation to N161H, N162T, N161C, N161D, or N161K; P140 mutation to P 140A, P140G, P140V, P140R, P140K, or P140D; G92 mutation to G92A, G92S, G92C, G92R, G92H, G92T, G92F, or G92M; N162 mutation to N162T, N162V, N162R, N162E, N162Y, N162S, or N162C.

[0030] In a preferred embodiment, the mutation includes any one or more of the following amino acid mutations: S307C, S307C+S107T, S307C+S337I, S307C+G92A, S307C+P140D, S307C+S154A, S307C+N161D, S307C+N162T, S307C+S186Y, S307C+I192V, S307C+S214P, S307C+S241N, S307C+K272L, S307C+F275W, S307C+S290G, S307C+L303H, S307C+M308P, S307C+P311A, S307C+ P311G, S307C+P311Y, S307C+H346Y, S307C+T88S, S307C+T88A, S307C+S10 7T+S337I, S307C+S107T+S337Y, S307C+S107T+S337R, S307C+S107T+S337M , S307C+S107T+G92A, S307C+S107T+G92S, S307C+S107T+G92C, S307C+S10 7T+N162T, S307C+S107T+N162V, S307C+S107T+N162R, S307C+S107T+N162E , S307C+S107T+P311A, S307C+S107T+P311N, S307C+S107T+P311Q, S307C+ S107T+P311G, S307C+S107T+S337I+H346A, S307C+S107T+S337I+H346G, S3 07C+S107T+S337I+H346Y, S307C+S107T+S337I+H346P, S307C+S107T+S33 7I+P311A, S307C+S107T+S337I+P311N, S307C+S107T+S337I+G92A, S307C+ S107T+S337I+G92R, S307C+S107T+S337I+G92H, S307C+S107T+S337I+P31 1A+G92T, S307C+S107T+S337I+P311A+G92A, S307C+S107T+S337I+P311A+G 92F、S307C+S107T+S337I+P311A+G92M、S307C+S107T+S337I+P311A+N162 T、S307C+S107T+S337I+P311A+N162Y、S307C+S107T+S337I+P311A+N162S、S307C+S107T+S337I+P311A+N162C, S307C+S107T+S337I+P311A+N161H, S3 07C+S107T+S337I+P311A+N161C, S307C+S107T+S337I+P311A+N161D, S307C +S107T+S337I+P311A+N161K, S307C+S107T+S337I+P311A+G92A+P140A, S3 07C+S107T+S337I+P311A+G92A+P140G, S307C+S107T+S337I+P311A+G92A+P 140V, S307C+S107T+S337I+P311A+G92A+P140R, S307C+S107T+S337I+P311 A+G92A+P140K, S307C+S107T+S337I+P311A+G92A+S214P, S307C+S107T+S33 7I+P311A+G92A+S214Y, S307C+S107T+S337I+P311A+G92A+S214A, S307C+S107T+S337I+P311A+G92A+S214F or S307C+S107T+S337I+P311A+G92A+S214V.

[0031] The ligation efficiency of peptide ligases is related to the six substrate recognition pockets (S4-S1, S1', and S2') at the enzyme's active site. S4-S1 corresponds to the amino acid recognition of the acyl donor P4-P3-P2-P1, while S1' and S2' correspond to the amino acid recognition of the acyl acceptor P1'-P2'. In the prior art, the peptide ligase Omnilgase-1 exhibits reduced catalytic activity when amino acids affecting catalytic efficiency (such as histidine, glutamic acid, lysine, aspartic acid, and some non-natural amino acids) are present at the P4 and P1 positions of the peptide substrate. This limits its application in the catalytic synthesis of peptide drugs. Therefore, Omnilgase-1 has a narrow substrate spectrum (Li, Ruifeng, et al. "Traceless enzymatic protein synthesis without ligation sites constraint." National Science Review 5(2022):103-113.), which further restricts its application in the catalytic synthesis of peptide drugs.

[0032] This application modifies a protease derived from Bacillus gobiensis (SEQ ID NO: 1) to obtain a mutant with the aforementioned mutant combination. This mutant exhibits better acceptance of amino acids and non-natural amino acids at the P1 position of the peptide substrate and demonstrates higher catalytic efficiency compared to Omnilgase-1. Furthermore, when the peptide ligase mutant obtained in this application is used in the ligation reaction of smegglutide and liraglutide, the product purity and yield remain at a high level. This indicates that the peptide ligase mutant of this application has high activity, a broad substrate spectrum, and high selectivity for substrate amino acids. Moreover, the peptide ligase of this application is more stable than Omnilgase-1 and can be applied to the synthesis of peptide drugs.

[0033] In a preferred embodiment, the polypeptide ligase mutant comprises a protein having 75% or more, 80% or more, 85% or more, more preferably 95% or more, and even more preferably 99% or more homology with the amino acid sequence defined in (a) and having polypeptide ligase activity.

[0034] All the aforementioned amino acid mutations were experimentally investigated in the embodiments of this application. Compared to the parent protein with the amino acid sequence shown in SEQ ID NO: 1, all mutations exhibited catalytic activity in binding the substrate peptide chain to obtain the target polypeptide. All the above mutation sites were located around the active amino acid site, which improves the binding ability and / or catalytic activity of the mutant to the substrate. Mutations located far from the active site have less impact on the enzyme's catalytic activity. Therefore, proteins with 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% or higher homology to the aforementioned amino acid sequence and possessing the same catalytic activity can be obtained.

[0035] In this specification, homology refers to the "homology" between amino acid sequences, that is, the total ratio of the same type of amino acid residues in the amino acid sequence. The homology of amino acid sequences can be determined using alignment programs such as BLAST (Basic Local Alignment Search Tool) and FASTA.

[0036] Proteins with 70%, 75%, 80%, 85%, 90%, 95%, and 99% or more (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or even 99.9%) homology and the same function have an active site, active pocket, active mechanism, and protein structure that are highly likely to be the same as the protein provided by sequence (a), and are homologous proteins obtained through amino acid mutation.

[0037] As used herein, the amino acid residue abbreviations are as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0038] Substitution and replacement rules generally apply to amino acids with similar properties; the effects of substituting one another are similar. For example, conserved amino acid substitutions can occur in the aforementioned homologous proteins. "Conserved amino acid substitutions" include, but are not limited to:

[0039] Hydrophobic amino acids (Ala, Cys, Gly, Pro, Met, Val, Ile, Leu) are replaced by other hydrophobic amino acids;

[0040] Hydrophobic amino acids with large side chains (Phe, Tyr, Trp) are replaced by other hydrophobic amino acids with large side chains;

[0041] Amino acids with positively charged side chains (Arg, His, Lys) are replaced by other amino acids with positively charged side chains;

[0042] Amino acids with polar, uncharged side chains (Ser, Thr, Asn, Gln) are replaced by other amino acids with polar, uncharged side chains.

[0043] Those skilled in the art can also perform conservative substitutions of amino acids based on amino acid substitution rules well known to them, such as the "blosum62 score matrix" in the prior art.

[0044] The "AlphaFold2-Multimer" used in this application is a publicly available artificial intelligence model capable of predicting the conformation of protein complexes. Its predictions of protein three-dimensional structures are very close to those observed in real-world experiments using equipment such as cryo-electron microscopy. This allows for the acquisition of relatively realistic protein structures, thereby guiding the investigation of protein structure and activity.

[0045] In a second typical embodiment of this application, a DNA molecule is provided that encodes the aforementioned polypeptide ligase mutant.

[0046] The aforementioned DNA can encode the aforementioned polypeptide ligase mutant and can be ligated onto a recombinant vector to form circular DNA. Both the aforementioned DNA and the recombinant vector can be transcribed and translated under the action of RNA polymerase, ribosomes, tRNA, etc., to obtain the aforementioned polypeptide ligase mutant.

[0047] In a third typical embodiment of this application, a recombinant plasmid is provided that is linked to the aforementioned DNA molecule.

[0048] In a fourth typical embodiment of this application, a host cell containing the aforementioned DNA molecule or the aforementioned recombinant plasmid is provided; the host cell is neither an animal nor a plant species.

[0049] In a preferred embodiment, the host cell includes a eukaryotic cell or a prokaryotic cell; preferably, the eukaryotic cell includes a yeast cell; preferably, the yeast cell includes Pichia pastoris; preferably, Pichia pastoris includes X33; preferably, the prokaryotic cell includes Escherichia coli or Bacillus subtilis; preferably, Escherichia coli includes BL21(DE3); preferably, Bacillus subtilis includes WB600.

[0050] Using the aforementioned host cells, recombinant vectors can be replicated within the host cells, and the DNA molecules carried on the recombinant vectors can be transcribed and translated to obtain a large number of polypeptide ligase mutants. Using existing techniques, polypeptide ligase mutants can be obtained by cleaving and purifying the host cells, followed by crude enzyme catalysis, or other methods, and then catalyzed for subsequent substrate nucleosides. The host cells are not of plant or animal origin. The embodiments of this application verify that the polypeptide ligase mutants of this application can be expressed and high-purity enzyme solutions can be obtained using any of the aforementioned host cells.

[0051] In a fifth typical embodiment of this application, a method for preparing a polypeptide is provided, which utilizes the aforementioned polypeptide ligase mutant to catalyze the binding of substrate peptide chains to prepare a polypeptide.

[0052] In a preferred embodiment, the substrate peptide chain comprises 2 to 3 chains. Preferably, the substrate peptide chain contains 5 to 30 amino acids. More preferably, the amino acids in the substrate peptide chain include non-natural amino acids.

[0053] In a preferred embodiment, when the number of substrate peptide chains is two, the substrate peptide chains include a first substrate peptide chain and a second substrate peptide chain; preferably, the combination of the first substrate peptide chain and the second substrate peptide chain is selected from any one or more of the following groups: 1) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NOs: 2-3 or SEQ ID NOs: 5-23, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: 4; 2) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NO: 24, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: 28; 3) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NO: 25, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: 29; 4) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NO: 26, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: 30; 5) the amino acid sequence of the first substrate peptide chain is as shown in SEQ ID NO: 27, and the amino acid sequence of the second substrate peptide chain is as shown in SEQ ID NO: 30. NO: 31; 6) The amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 32, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO: 33; 7) The amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 34, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO: 35; Preferably, the C-terminus of the first substrate peptide chain contains an ester acyl group.

[0054] The first substrate peptide chain refers to the upper half of the substrate in a polypeptide linkage reaction, while the second substrate peptide chain refers to the lower half. In the linkage reaction, because the C-terminus of the first substrate peptide chain contains a protective ester acyl group, it can also be called an acyl donor, and the second substrate peptide chain can be called an acyl acceptor.

[0055] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0056] Unless otherwise specified, all reagents used in the embodiments of this application are commercially available products.

[0057] Example 1

[0058] Expression and purification of polypeptide ligase mutants in Bacillus subtilis:

[0059] The S307C gene (N-terminal or C-terminal His tag) of the mutant Bacillus gobiensis (SEQ ID NO: 1) was cloned into the restriction enzyme sites MLU I and BamHI of the Escherichia coli-Bacillus subtilis shuttle expression vector pBE-S. After obtaining the recombinant expression plasmid, it was transformed into Bacillus subtilis strain WB600 by the Spizizen method (Spizizen J. Transformation of biochemically deficient strain of B. subtilis by deoxyribonucleate. Proceeding of National Academy of Science USA, 1958, 44:1072-1078.). Transformants were screened on LB plates containing 50 μg / mL kanamycin sulfate.

[0060] Positive transformants were picked and cultured in 5 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate at 37°C and 200 rpm for 16 h. Then, they were transferred at a 1% (v / v) inoculation rate to 500 mL of TB medium (Terrific broth medium, 12 g / L tryptone, 24 g / L yeast extract, 0.4% glycerol) and cultured at 37°C and 200 rpm for 48 h. After fermentation, the supernatant was collected by centrifugation at 4°C and 8000 rpm for 10 min, and the precipitate was discarded.

[0061] The fermentation broth was purified after being treated with a 10 kDa membrane. The specific procedure was as follows: the sample was loaded at a flow rate of 2 mL / min, then washed with buffer A (25 mM Tricine, pH 7.5, 0.5 M NaCl, 20 mM imidazole) until all unbound proteins were eluted. Next, five column volumes of contaminating proteins were eluted with a linear gradient of imidazole (imidazole concentration increased from 20 mM to 50 mM), followed by elution of the target protein at 200 mM. The affinity-purified protein was further centrifuged using ultrafiltration tubes to remove imidazole and salts, and stored at -20°C. Protein concentration was determined using the Bradford method, and purity was analyzed by 12% separating gel SDS-PAGE. Finally, an aqueous solution (50 mM Tricine, 0.1 M NaCl, pH 8.0) containing approximately 2 mg / mL (purity >90%) of the obtained enzyme was used for peptide fragment ligation.

[0062] Example 2

[0063] Expression and purification of polypeptide ligases in Escherichia coli:

[0064] The mutant S307C gene (N-terminal or C-terminal His tag) from Bacillus gobiensis (SEQ ID NO: 1) was cloned into the restriction enzyme sites Nco I-BamH I of the expression vector pET28a(+) and transformed into competent Escherichia coli BL21(DE3). The strain was cultured on LB agar plates containing 50 μg / mL kanamycin sulfate at 37°C for 16 hours. The culture was then picked and inoculated into LB liquid medium containing 50 μg / mL kanamycin sulfate. The culture was incubated at 37°C until OD200 reached. 600 When the concentration reaches approximately 0.8, add 0.1 mM IPTG and continue incubating at 18°C ​​for 17 hours before collecting the mycelium.

[0065] Weigh the bacterial sludge and add lysis buffer (50 mM Tris-HCl, 500 mM NaCl, 0.1% Triton, pH 8.0). Lyse the bacterial cells using an ultrasonic homogenizer. Centrifuge the lysate (12000 rpm for 20 minutes) and collect the supernatant for purification. The specific procedure is as follows: Load the sample at a flow rate of 2 mL / min, then wash with buffer A (25 mM Tricine, pH 7.5, 0.5 M NaCl, 20 mM imidazole) until all unbound proteins are eluted. Next, elute contaminating proteins with a linear gradient of imidazole for 5 column volumes (imidazole concentration increased from 20 mM to 50 mM), and then elute the target protein at 200 mM. The affinity-purified protein is further purified by ultrafiltration, centrifugation, and medium exchange to remove imidazole and salts, and stored at -20°C for later use. Protein concentration is determined using the Bradford method, and purity is analyzed by 12% separating gel SDS-PAGE. The final product was an aqueous solution (50 mM Tricine, 0.1 M NaCl, pH 8.0) containing approximately 5 mg / mL (purity greater than 90%) of the enzyme, which was used for peptide fragment ligation.

[0066] Example 3

[0067] Expression and purification of polypeptide ligase mutants in yeast:

[0068] The S307C gene (N-terminal or C-terminal His tag) from the mutant Bacillus gobiensis (SEQ ID NO: 1) was cloned into the restriction enzyme sites EcoR I and Not I of the Pichia pastoris expression vector pPICZ A to obtain the recombinant expression plasmid. The plasmid was then linearized by Sac I restriction endonuclease digestion at 37°C. After confirming complete digestion by agarose gel electrophoresis, the linearized plasmid was recovered. The linearized plasmid was transformed into Pichia pastoris strain X33 via electroporation and plated on low-salt YPD plates containing 100 mg / mL bleomycin. The plates were incubated at 30°C for 3 days. Transformants were picked and transferred to 10 mL of BMGY liquid medium (10 g / L yeast extract, 20 g / L tryptone, 100 mM potassium phosphate, pH 6.0, 13.4 g / L YNB, 4 × 10⁻⁶ oz.). -4 After culturing in a solution of 1 g / L biotin and 10 g / L glycerol at 30°C and 200 rpm for 18 h, the culture was transferred to 100 mL of BMGY liquid medium at a 1% (v / v) inoculation rate and cultured for an additional 36 h at 30°C and 200 rpm.

[0069] Collect bacterial cells by centrifugation at 1500-3000g at room temperature, and culture in BMMY liquid medium (10g / L yeast extract, 20g / L tryptone, 100mM potassium phosphate, pH 6.0, 13.4g / L YNB, 4×10⁻⁶ ppm). -4 Resuspend bacterial cells at OD500 (g / L biotin, 5g / L methanol) 600 ≈1.0, cultured at 30℃ and 200rpm, with methanol added every 24 hours to induce expression at a final concentration of 5g / L.

[0070] After 5 days of fermentation, the supernatant of the fermentation broth was collected by centrifugation at 8000 rpm for 10 min at 4℃. The precipitate was discarded, and the supernatant was used for subsequent purification. The specific procedure was as follows: the sample was loaded at a flow rate of 2 mL / min, and then washed with buffer A (20 mM KPB, pH 7.5, 0.5 M NaCl, 20 mM imidazole) until all unbound proteins were eluted. Then, five column volumes of contaminating proteins were eluted with a linear gradient imidazole (imidazole concentration increased from 20 mM to 100 mM), and then the target protein was eluted with 200 mM. The affinity-purified protein was further centrifuged using an ultrafiltration tube to remove imidazole and salt, and stored at -20℃ for later use.

[0071] Protein concentration was determined using the Bradford method, and purity was analyzed by 12% separating gel SDS-PAGE. The final product, an aqueous solution (50 mM Tricine, 0.1 M NaCl, pH 8.0) containing 2 mg / mL (purity >90%) of the enzyme, was used for peptide fragment ligation.

[0072] Other polypeptide ligase mutants of this application can be obtained by using any of the expression and purification methods in Examples 1 to 3 to obtain the enzyme solution of the mutant, which can be used to catalyze the ligation of polypeptide substrates.

[0073] Example 4

[0074] This embodiment modifies a protease derived from Bacillus gobiensis (SEQ ID NO: 1) by mutating a serine residue near the active site to cysteine. Using a pentapeptide (acyl donor P1 position with the non-natural amino acid Aib, α-aminoisobutyric acid) as a substrate, a ligation reaction was performed to verify the activity of the mutant S307C and Omnilgase-1. The reaction system is as follows:

[0075] The 1 mL reaction system contained: 2.5 mM acyl donor Ac-Asp-Aib-Tyr-Ser-Leu-O-Cam-Leu-OH (SEQ ID NO: 2) or Ac-Asp-Phe-Tyr-Ser-Leu-O-Cam-Leu-OH (SEQ ID NO: 3), 3.75 mM acyl acceptor H-Met-Leu-Val-Lys-Ala-NH2 (SEQ ID NO: 4), enzyme 0.5 mg / mL, and reaction buffer (0.1 M Tricine, pH 8.0, 0.8 mg / mL TCEP). The reaction was carried out at 25 °C for 4 h, followed by the addition of one volume of anhydrous ethanol to terminate the reaction. After thorough shaking and mixing, the mixture was centrifuged at 8000 rpm for 1 min to obtain the supernatant aqueous phase, which was then analyzed by HPLC to determine the conversion rate. The specific results are shown in Table 1.

[0076] Table 1 Note: In Table 1, * represents a conversion rate less than 0.1%, ** represents a conversion rate greater than or equal to 0.1% and less than 10% (excluding 10%), **** represents a conversion rate greater than or equal to 30% and less than 50% (excluding 50%), ***** represents a conversion rate greater than or equal to 50% and less than 70% (excluding 70%), and ****** represents a conversion rate greater than or equal to 70% and less than 90% (excluding 90%). Acyl donors refer to the substrates in the first half of the linkage reaction that contain an ester acyl group at their C-terminus; acyl acceptors refer to the substrates in the second half of the linkage reaction.

[0077] The polypeptide ligation reaction in this embodiment is illustrated in Figure 1. In Figure 1, P1-P4 and P1'-P2' refer to the amino acid sites of the acyl donor and acyl acceptor, respectively. In the sequence of the acyl donor, "Ac" represents an acetyl group attached to the N-terminus of the pentapeptide substrate, and "O-Cam" represents a carboxyamide methyl ester group attached to the C-terminus of the pentapeptide substrate. Following "O-Cam" is a "Leu-OH" group, which is a leucine + hydroxyl group. The purpose of attaching the acetyl group at the N-terminus is to prevent the polypeptide ligase from self-ligating. The "O-Cam" group is a protecting group at the C-terminus of the polypeptide substrate, and the "Leu-OH" group is a group attached to the "O-Cam" group. "Leu" following "O-Cam" is an amino acid residue known to those skilled in the art to improve the ligation efficiency in the ligation reaction. The specific structure of "O-Cam-Leu-OH" is shown in Figure 1. The connection methods and purposes of the same groups on the sequences of the remaining substrate peptide chains in this application are described herein.

[0078] In this embodiment, both the peptide ligase mutants S307C and Omniligase-1 catalyze ligation reactions in which the acyl donor does not contain a non-natural amino acid, and exhibit high conversion rates. However, in reactions where the acyl donor has a non-natural amino acid Aib at the P1 position, the catalytic activity of S307C is approximately tens of times that of Omniligase-1. This indicates that mutants derived from Bacillus gobiensis possess excellent catalytic potential for ligation reactions in which the non-natural amino acid Aib is present at the P1 position.

[0079] Example 5

[0080] Enzymatic evolution was continued on the mutant S307C derived from Bacillus gobiensis (SEQ ID NO: 1), using Ac-His-Ala-Gly-Ser-Aib-O-Cam-Leu-OH as the acyl donor and H-Ala-Tyr-Gln-Lys-Ser-NH2 as the acyl acceptor. The resulting mutant was subjected to a ligation reaction to verify its activity. The reaction system is as follows:

[0081] The 1 mL reaction system contained: 5 mM acyl donor Ac-His-Ala-Gly-Ser-Aib-O-Cam-Leu-OH (SEQ ID NO: 2), 3.75 mM acyl acceptor H-Ala-Tyr-Gln-Lys-Ser-NH2 (SEQ ID NO: 4), enzyme concentration reduced to 0.05 mg / mL, and reaction buffer (0.1 M Tricine, pH 8.0, 0.8 mg / mL TCEP). The reaction was carried out at 25 °C for 2 h, followed by the addition of one volume of anhydrous ethanol to terminate the reaction. After thorough shaking and mixing, the mixture was centrifuged at 8000 rpm for 1 min to obtain the supernatant aqueous phase, which was then sent for HPLC analysis to determine the conversion rate.

[0082] After initial and secondary screening, the following mutants were obtained, which showed increased activity. The specific results are shown in Table 2.

[0083] Table 2 Note: In Table 2, * represents a conversion rate less than 0.1% (excluding 0.1%), ** represents a conversion rate greater than or equal to 0.1% and less than 10% (excluding 10%), *** represents a conversion rate greater than or equal to 10% and less than 30% (excluding 30%), **** represents a conversion rate greater than or equal to 30% and less than 50% (excluding 50%), ***** represents a conversion rate greater than or equal to 50% and less than 70% (excluding 70%), and ****** represents a conversion rate greater than or equal to 70% and less than 90%.

[0084] In this embodiment, a single-point mutation S307C was used as the parent mutant for evolution, and substrates with Aib at P1 position were used for screening and activity testing to obtain a series of peptide ligase mutants with enhanced activity.

[0085] Example 6

[0086] Enzyme stability test:

[0087] Stability tests were performed on some of the mutants obtained in Example 5 to screen for mutants with improved stability. The mutants were used to prepare enzyme solutions according to the methods described in Examples 1, 2, or 3. The enzyme solutions were treated at 70°C for one hour, and then the mutants were tested for activity according to the following reaction system.

[0088] The 1 mL reaction system included: 2.5 mM acyl donor Ac-His-Ala-Gly-Ser-Aib-O-Cam-Leu-OH (SEQ ID NO: 2), 3.75 mM acyl acceptor H-Ala-Tyr-Gln-Lys-Ser-NH2 (SEQ ID NO: 4), enzyme concentration of 0.1 mg / mL (treated at 70 °C for one hour), and reaction buffer (0.1 M Tricine, pH 8.0, 0.8 mg / mL TCEP). The reaction was carried out at 25 °C for 2 hours, followed by the addition of one volume of anhydrous ethanol to terminate the reaction. After thorough shaking and mixing, the mixture was centrifuged at 8000 rpm for 1 min to obtain the supernatant aqueous phase, which was then analyzed by HPLC to determine the conversion rate. The stability test results of some mutants are shown in Table 3.

[0089] Table 3 Note: The residual activity in Table 3 refers to the conversion rate of the enzyme solution after treatment at 70℃ for 1 hour, divided by the conversion rate of the enzyme solution directly added to the reaction without high-temperature treatment, multiplied by 100%. Wherein, * represents residual activity greater than or equal to 10% and less than 30% (excluding 30%), ** represents residual activity greater than or equal to 30% and less than 50% (excluding 50%), *** represents residual activity greater than or equal to 50% and less than 70% (excluding 70%), and **** represents residual activity greater than or equal to 70% and less than 90%.

[0090] The peptide ligase mutant in this embodiment exhibits significantly improved thermal stability compared to Omniligase-1.

[0091] Example 7

[0092] Substrate spectral analysis:

[0093] Using the peptide ligase mutant S307C+S107T+S337I+P311A+G92A+S214A as a catalyst, the catalytic activity of 5-peptide acyl donors with different amino acids at the P1 position was tested. The acyl donor was Ac-His-Ala-Gly-Ser-Xxx-O-Cam-Leu-OH (Xxx refers to 20 different amino acids, including I, A (SEQ ID NO: 3), W, Y, V, K, Q, F, M, P, T, N, E, H, S, L, R, G, C, and D, corresponding to SEQ ID NO: 5–23, respectively), and the acyl acceptor was H-Ala-Tyr-Gln-Lys-Ser-NH2 (SEQ ID NO: 4). The reaction system is as follows:

[0094] The 1 mL reaction system included: 5 mM acyl donor, 7.5 mM acyl acceptor, enzyme concentration of 0.05 mg / mL, and reaction buffer (0.1 M Tricine, pH 8.0, 0.8 mg / mL TCEP). The reaction was carried out at 25 °C for 2 h, followed by the addition of one volume of anhydrous ethanol to terminate the reaction. After thorough shaking and mixing, the mixture was centrifuged at 8000 rpm for 1 min to obtain the supernatant aqueous phase, which was then sent to HPLC for conversion analysis.

[0095] Figure 2 shows the catalytic results of the above-mentioned polypeptide ligase mutants for different amino acids at the P1 position.

[0096] In this embodiment, enzyme evolution screening was performed using a 5-peptide containing the non-natural amino acid Aib at position P1 as the target substrate. The resulting mutants exhibited a wider range of substrate adaptability at position P1.

[0097] Example 8

[0098] Using the peptide ligase mutant S307C+S107T+S337I+P311A+G92A+S214A as a catalyst, ligation reactions of substrate combinations of different lengths were tested (see Table 7 for details): 8+8, 12+12, 15+15, and 20+18 (the numbers represent the lengths of the acyl donor and acyl acceptor, respectively). The reaction systems are shown below:

[0099] The 1 mL reaction system contained: 2.5 mM acyl donor, 3.75 mM acyl acceptor, enzyme concentration of 0.1 mg / mL, and reaction buffer (0.1 M Tricine, pH 8.0, 0.8 mg / mL TCEP). The reaction was carried out at 25 °C for 2 h, followed by the addition of one volume of anhydrous ethanol to terminate the reaction. After thorough mixing, the mixture was centrifuged at 8000 rpm for 1 min to obtain the supernatant aqueous phase, which was then analyzed by HPLC to determine the conversion rate. The reaction results are shown in Table 4.

[0100] Table 4 Note: In Table 4, S / H refers to the ratio of the amount of enzyme-synthesized product to the amount of enzyme-hydrolyzed substrate.

[0101] Example 9

[0102] Smegglutinin was synthesized enzymatically using a 17-mer + 14-mer (acyl donor + acyl acceptor) fragment:

[0103] Add 5 mM of acyl donor His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Gln-Gly-Gln-O-Cam-Leu-OH (SEQ ID NO: 32) and 7.5 mM of acyl acceptor H-Ala-Ala-Lys(AEEA-AEEA-γ-Glu-17-carboxyheptadecanoyl)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-NH2 (SEQ ID NO: 33) to a 200 mL four-necked flask, then add 0.01 mg / mL of the mutant (S307C+S107T+S337I+P311A+G92A+S214A) mutated from SEQ ID NO: 1, and finally add buffer (0.1 M Tricine, pH 10). Add 8.0, 0.8 mg / mL TCEP) to a total volume of 50 mL, adjust pH to 7.8–8.2, and stir at 25 °C for 2 h. HPLC analysis showed complete reaction of the donor and substrate, with a conversion rate of 93% (no product isomers detected), and an S / H ratio of 44. Then, adjust the system to pH 1–2 to denature the protein. Centrifuge at 8000 rpm for 10 min to obtain the supernatant aqueous phase, which was then purified by preparative HPLC and lyophilized to obtain the product smegglutinin.

[0104] The purity of the product was >98% and the yield was 75%, as determined by HPLC and Q-NMR.

[0105] Example 10

[0106] Liraglutide was synthesized enzymatically using a 17-mer + 14-mer (acyl donor + acyl acceptor) fragment:

[0107] Add 5 mM of the acyl donor His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-O-Cam-Leu-OH (SEQ ID NO: 34) and 7.5 mM of the acyl acceptor H-Ala-Ala-Lys(Pal-γ-Glu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-NH2 (SEQ ID NO: 35) to a 200 mL four-necked flask, then add 0.01 mg / mL of the mutant (S307C+S107T+S337I+P311A+G92A+S214A) mutated from SEQ ID NO: 1, and finally add buffer (0.1 M Tricine, pH 8.0, 0.8 mg / mL). The mixture was prepared by adding TCEP (total volume 50 mL), adjusting the pH to 7.8–8.2, and stirring at 25°C for 3 h. HPLC analysis showed complete reaction of the donor and substrate, with a conversion rate of 91% (no product isomers detected), and an S / H ratio of 43. The system was then acidified to pH 1–2 to denature the protein. The system was centrifuged at 8000 rpm for 10 min to obtain the supernatant aqueous phase, which was then purified by preparative HPLC and lyophilized to obtain the product liraglutide.

[0108] HPLC and Q-NMR analysis showed that the product purity was >98% and the yield was 73%.

[0109] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: (1) Through enzyme evolution, a ligase with high peptide ligation activity was obtained, which showed good catalytic potential for the reaction in which the non-natural amino acid Aib is at the P1 position. (2) Using the pentapeptide with Aib at the P1 position as a substrate, further enzyme evolution was carried out to obtain mutants with further improved activity. These mutants also showed higher stability and catalytic ability for a wider substrate spectrum. (3) The obtained peptide ligase mutants can efficiently catalyze the synthesis of peptide substrates of different lengths. (4) The peptide ligase mutants obtained in this application can be used for the efficient synthesis of various peptide drug APIs. (5) Compared with traditional chemical methods, enzymatic methods do not require complex processes and steps, are simpler and milder to operate, do not cause racemization during the reaction process, do not generate isomers, generate fewer impurities in the system, are easier to purify, and are more suitable for industrial-scale production.

[0110] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A polypeptide ligase mutant, characterized in that, The polypeptide ligase mutant includes: (a) A protein with mutations based on the amino acid sequence shown in SEQ ID NO: 1, including mutations occurring at the S307 site; or (b) A protein that has more than 70% homology with the amino acid sequence defined in (a) and has polypeptide ligase activity.

2. The polypeptide ligase mutant according to claim 1, characterized in that, The mutation is selected from the S307C mutation and any one or more of the following mutations: S107 mutates to S107T; S154 mutates to S154A; S186 mutates to S186Y; I192 mutates to I192V; S241 mutates to S241N; K272 mutates into K272L; F275 mutates to F275W; S290 mutates to S290G; L303 mutates to L303H; M308 mutates to M308P; T88 mutates into T88S or T88A; S337 mutations can be S337I, S337Y, S337R, or S337M; P311 mutations can be P311A, P311N, P311Y, P311Q, or P311G; H346 mutation can be converted into H346A, H346G, H346Y, or H346P; S214 mutates into S214P, S214Y, S214A, S214F, or S214V; N161 mutations into N161H, N162T, N161C, N161D, or N161K; P140 mutations can be P140A, P140G, P140V, P140R, P140K, or P140D. G92 mutations can result in G92A, G92S, G92C, G92R, G92H, G92T, G92F, or G92M. N162 mutations can be N162T, N162V, N162R, N162E, N162Y, N162S, or N162C.

3. The polypeptide ligase mutant according to claim 1, characterized in that, The mutation includes any one or more of the following amino acid mutations: S307C、S307C+S107T、S307C+S337I、S307C+G92A、S307C+P140D、S307C+S154A、S307C+N161D、S307C+N162T、S307C+S186Y、S307C+I192V、S307C+S214P、S307C+S241N、S307C+K272L、S307C+F275W、S307C+S290G、S307C+L303H、S307C+M308P、S307C+P311A、S307C+P311G、S307C+P311Y、S307C+H346Y、S307C+T88S、S307C+T88A、S307C+S107T+S337I、S307C+S107T+S337Y、S307C+S107T+S337R、S307C+S107T+S337M、S307C+S107T+G92A、S307C+S107T+G92S、S307C+S107T+G92C、S307C+S107T+N162T、S307C+S107T+N162V、S307C+S107T+N162R、S307C+S107T+N162E、S307C+S107T+P311A、S307C+S107T+P311N、S307C+S107T+P311Q、S307C+S107T+P311G、S307C+S107T+S337I+H346A、S307C+S107T+S337I+H346G、S307C+S107T+S337I+H346Y、S307C+S107T+S337I+H346P、S307C+S107T+S337I+P311A、S307C+S107T+S337I+P311N、S307C+S107T+S337I+G92A、S307C+S107T+S337I+G92R、S307C+S107T+S337I+G92H、S307C+S107T+S337I+P311A+G92T、S307C+S107T+S337I+P311A+G92A、S307C+S107T+S337I+P311A+G92F、S307C+S107T+S337I+P311A+G92M、S307C+S107T+S337I+P311A+N162T、S307C+S107T+S337I+P311A+N162Y、S307C+S107T+S337I+P311A+N162S、S307C+S107T+S337I+P311A+N162C、S307C+S107T+S337I+P311A+N161H, S307C+S107T+S337I+P311A+N161 C. S307C+S107T+S337I+P311A+N161D, S307C+S107T+S337I+P311A+N16 1K、S307C+S107T+S337I+P311A+G92A+P140A、S307C+S107T+S337I+P31 1A+G92A+P140G, S307C+S107T+S337I+P311A+G92A+P140V, S307C+S107 T+S337I+P311A+G92A+P140R, S307C+S107T+S337I+P311A+G92A+P140 K. S307C+S107T+S337I+P311A+G92A+S214P, S307C+S107T+S337I+P311 A+G92A+S214Y, S307C+S107T+S337I+P311A+G92A+S214A, S307C+S107T +S337I+P311A+G92A+S214F or S307C+S107T+S337I+P311A+G92A+S214V. , 4. The polypeptide ligase mutant according to any one of claims 1 to 3, characterized in that, The polypeptide ligase mutant includes a protein having 75% or more, 80% or more, 85% or more, more preferably 95% or more, and even more preferably 99% or more homology with the amino acid sequence defined in (a) and having polypeptide ligase activity.

5. A DNA molecule, characterized in that, The DNA molecule encodes the polypeptide ligase mutant according to any one of claims 1 to 4.

6. A recombinant plasmid, characterized in that, The recombinant plasmid is ligated with the DNA molecule of claim 5.

7. A host cell, characterized in that, The host cell contains the DNA molecule of claim 5 or the recombinant plasmid of claim 6; the host cell is not an animal or plant species.

8. The host cell according to claim 7, characterized in that, The host cells include eukaryotic cells or prokaryotic cells; Preferably, the eukaryotic cells include yeast cells; Preferably, the yeast cells include Pichia pastoris; Preferably, the prokaryotic cells include Escherichia coli or Bacillus subtilis; Preferably, the *Escherichia coli* includes BL21(DE3); Preferably, the Bacillus subtilis includes WB600.

9. A method for preparing a polypeptide, characterized in that, The preparation method includes: using the polypeptide ligase mutant according to any one of claims 1 to 4 to catalyze the binding of the substrate peptide chain to prepare the polypeptide.

10. The preparation method according to claim 9, characterized in that, The substrate peptide chain has 2 to 3 strands.

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