Polypeptide ligase mutant and method for preparing polypeptide

Abstract

Provided are a polypeptide ligase mutant and a method for preparing a polypeptide. The polypeptide ligase mutant is a protein obtained by mutation on the basis of a protease derived from Bacillus glycinifermentans and as shown in SEQ ID NO: 1, the mutation comprising a mutation occurring at the S297 site. The present invention can solve the problem in the prior art of poor linking activity of a polypeptide ligase, and is suitable for 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 No. 202411531500.8 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] Polypeptides are compounds composed of multiple amino acids linked by peptide bonds, possessing certain biological activities. They typically consist of 10 to 100 amino acid molecules, linked in the same way as proteins, with a relative molecular mass below 10,000. Compared to small-molecule chemical drugs and protein drugs, peptide drugs exhibit higher activity and selectivity, fewer side effects on the human body, higher stability, and lower immunogenicity. Peptide drugs are widely used in medical fields such as vaccines, anti-tumor drugs, endocrine drugs, and cardiovascular drugs. As of January 2023, approximately 180 peptide drugs were marketed globally, demonstrating broad development prospects.

[0004] Solid-phase synthesis (SPPS) remains the primary and convenient method for obtaining non-natural peptides. However, SPPS suffers from drawbacks such as high elution impurities and difficulties in purification. Furthermore, the yield of SPPS-synthesized peptides decreases exponentially with chain length, requiring two or more HPLC preparations to obtain products with sufficient purity. For medium- to long-chain peptides, the total yield from SPPS is less than 25%.

[0005] To synthesize long peptides, they are typically broken down into several fragments and then ligated. Existing peptide ligation methods fall into two main categories: chemical and enzymatic methods. In chemical ligation, the C-terminus of the fragment is prone to racemization (except for Gly and Pro), and the amino acids in the ligated fragment require full protection, leading to poor solubility and difficult purification. In contrast, enzymatic ligation eliminates the risk of racemization, does not require side chain protection, and is easy to purify. Wells' team engineered a serine protease (Subtilisin) derived from Bacillus amyloliquefaciens, mutating it at two sites (S221C / P225A) to obtain a peptide ligase (Subtiligase) capable of ligating a C-terminal ester-terminated acyl donor fragment and an N-terminal unprotected acyl acceptor fragment in aqueous solution. However, the low ligation efficiency and instability of subtilisase limit its 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.). Subsequently, Enzypep conducted a series of mutation studies and obtained the peptide ligase Omniligase-1, which has further improved stability and catalytic substrate spectrum (Toplak, Ana, et al. "From Thiol-Subtilisin to Omniligase: Design and Structure of a Broadly Applicable Peptide Ligase." Computational and Structural Biotechnology Journal 19.10(2021.).).

[0006] Currently, although peptide ligases have been commercialized, they still face a number of challenges in practical applications. First, their catalytic activity on certain specific peptide drug fragments is limited, thus restricting their application range in drug synthesis. Given these limitations, the development of novel peptide ligases to achieve efficient and stable ligation of various peptide drug fragments is particularly urgent and necessary. Summary of the Invention

[0007] The main objective of this invention is to provide a polypeptide ligase mutant and its application, in order to solve the problem of poor ligation activity of polypeptide ligases 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, 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 S297 site; or (b) a protein having more than 70% homology to the amino acid sequence defined in (a) and having polypeptide ligase activity.

[0009] Further, the mutation is selected from the S297C mutation and any one or more of the following mutations: N194 mutation to N194K; L195 mutation to L195M; S206 mutation to S206A; Y219 mutation to Y219A; V223 mutation to V223M; S258 mutation to S258E; S259 mutation to S259N; S165 mutation to S165E; S320 mutation to S320K; S185 mutation to S185A; P205 mutation to P205G; N294 mutation to N294S; Y285 mutation to Y285A; S237 mutation to S237G; I281 mutation to I281N; T179 mutation to T 179S; N163 mutates to N163R, N163G; I183 mutates to I183V or I183N; S238 mutates to S238Q or S238A; S232 mutates to S232N, S232Q or S232E; S280 mutates to S280T, S280L or S280K; G295 mutates to G295C, G295S or G295H; S287 mutates to S287H, S287M or S287K; S287 mutates to S287N, S287H, S287M or S287K; L202 mutates to L202F, L202M, L202A or L202P; R262 mutates to R26 2L, R262E, R262Q, or R262Y; G84 mutation to G84A, G84S, G84Q, G84D, G84S, or G84P; K246 mutation to K246A, K246D, K246I, K246L, or K246Y; H302 mutation to H302F, H302G, H302T, H302A, or H302G; G137 mutation to G137D, G137I, G137C, G137A, or G137Y; S221 mutation to S221A, S221R, S221W, S221Y, or S221T; T209 mutation to T209A, T209R, T209M, or T209Y. 9S or T209W; T289 mutation to T289R, T289S, T289M, T289K or T289A; I183 mutation to I183V, I183A, I183Q, I183M or I183V; G137 mutation to G137I, G137C, G137D, G137A or G137Y; S177 mutation to S177Q, S177H, S177R, S177K or S177G; K92 mutation to K92A, K92G, K92D, K92E, K92S or K92N; T80 mutation to T80A, T80P, T80S, T80D, T80F, T80G or T80R;Y282 mutations into Y282L, Y28G, Y282H, Y282M, Y282C, or Y282AR; R325 mutations into R325A, R325F, R325G, R325Y, R325E, or R325M; S314 mutations into S314N, S314K, S314C, S314R, S314Y, or S314N; G242 mutations into G242T, G242A, G242Q, G242L, G242S, G242M, or G24 2R; A113 mutation to A113D, A113P, A113M, A113S, A113E, A113R, or A113T; R325 mutation to R325N, R325A, R325G, R325Y, R325E, R325M, or R325D; N134 mutation to N134F, N134D, N134G, N134T, N134R, N134P, N134E, N134L, N134S, N134I, or N134A. The letter before the number represents the original amino acid, and the letter after the number represents the mutated amino acid.

[0010] Further, the mutation includes any one or more of the following amino acid mutations: S297C, S297C+K92N, S297C+T80S, S297C+G84A, S297C+L202F, S297C+K246A, S297C+R262L, S297C+H302F, S297C+N134D, S297C+N134F, S297C+G137D, S297C+N194K, S297C+L195M, S297C+S206A, S297C+Y219A, S297C+S221A, S297C+V223M, S297C+S238Q, S297C+G242Q, S297C+ K246A, S297C+S258E, S297C+S259N, S297C+Y282L, S297C+S287N, S297C+T2 89R, S297C+T289S, S297C+T289M, S297C+T289K, S297C+T289A, S297C+A113D , S297C+A113P, S297C+A113M, S297C+A113S, S297C+S314N, S297C+S314K, S 297C+S314C, S297C+S314R, S297C+S314I, S297C+S314Y, S297C+R325A, S297 C+R325F, S297C+R325G, S297C+R325Y, S297C+R325E, S297C+R325M, S297C+ A113D+S165E, S297C+A113D+S287N, S297C+A113D+G242L, S297C+A113D+S3 20K, S297C+A113D+N134A, S297C+A113D+N134T, S297C+A113D+N134S, S297 C+A113D+N134I, S297C+A113D+S185A, S297C+A113D+T209A, S297C+S314N+I 183V, S297C+S314N+I183N, S297C+S314N+S185A, S297C+S314N+K92A, S297 C+S314N+G242Q, S297C+T289R+S165E, S297C+T289R+G242S, S297C+T289R+G 242C, S297C+T289R+G137D, S297C+T289R+P205G, S297C+T289R+S238Q, S29 7C+T289R+N294S, S297C+T289R+S165E+S314Y, S297C+T289R+S165E+I183V,S297C+T289R+S165E+S314N、S297C+T289R+S165E+Y285A、S297C+T289R+ S165E+S287H、S297C+T289R+S165E+S287M、S297C+T289R+S165E+S287K、S 297C+T289R+S165E+R325A, S297C+T289R+S165E+S237G, S297C+T289R+S165E+S238A, S297C+T289R+S165E+N134F, S297C+T289R+S165E+N134E, S29 7C+T289R+S165E+N134L、S297C+T289R+S165E+N294S、S297C+T289R+S165 E+I281N、S297C+T289R+S165E+S280T、S297C+T289R+S165E+S280L、S297C +T289R+S165E+S280K、S297C+T289R+S165E+S232N、S297C+T289R+S165E+ S232Q、S297C+T289R+S165E+S232E、S297C+T289R+S165E+G242T、S297C+T 289R+S165E+G242T, S297C+T289R+S165E+G137I, S297C+T289R+S165E+G137C, S297C+T289R+S165E+S177Q, S297C+T289R+S165E+S177H, S297C+T2 89R+S165E+S177R, S297C+T289R+S165E+S177K, S297C+T289R+S165E+S177G, S297C+T289R+S165E+T179S, S297C+T289R+S165E+N163R, S297C+T289 R+S165E+N163G、S297C+T289R+S165E+S287K、S297C+T289R+S165E+S287K +G84A、S297C+T289R+S165E+S287K+G84D、S297C+T289R+S165E+S287K+G8 4S、S297C+T289R+S165E+S287K+G84P、S297C+T289R+S165E+S287K+L202F 、S297C+T289R+S165E+S287K+L202M、S297C+T289R+S165E+S287K+L202A、S297C+T289R+S165E+S287K+L202P, S297C+T289R+S165E+S287K+K246A, S297C+T289R+S165E+S287K+K246D, S297C+T289R+S165E+S287K+K246I, S2 97C+T289R+S165E+S287K+K246L, S297C+T289R+S165E+S287K+K246Y, S297C+T289R+S165E+S287K+R262L, S297C+T289R+S165E+S287K+R262E, S297C +T289R+S165E+S287K+R262Q、S297C+T289R+S165E+S287K+R262Y、S297C+T289R+S165E+S287K+H302F、S297C+T289R+S165E+S287K+H302G、S297C+T2 89R+S165E+S287K+H302T, S297C+T289R+S165E+S287K+H140R, S297C+T289R+S165E+S287K+H140E, S297C+T289R+S165E+S287K+H140V, S297C+T289R +S165E+S287K+H140K, S297C+T289R+S165E+S287K+H140A, S297C+T289R+S165E+S287K+H143E, S297C+T289R+S165E+S287K+H143M, S297C+T289R+S1 65E+S287K+H143V, S297C+T289R+S165E+S287K+H143E, S297C+T289R+S165E+S287K+H143A, S297C+T289R+S165E+S287K+L202F+L293A, S297C+T289R +S165E+S287K+L202F+L293E、S297C+T289R+S165E+S287K+L202F+L293Q、S297C+T289R+S165E+S287K+L202F+L293Y、S297C+T289R+S165E+S287K+L2 02F+G295C, S297C+T289R+S165E+S287K+L202F+G295S, S297C+T289R+S165E+S287K+L202F+G295H, S297C+T289R+S165E+S287K+L202F+G295H+T80SS297C+T289R+S165E+S287K+L202F+G295H+T80A、S297C+T289R+S165E+S2 87K+L202F+G295H+T80P、S297C+T289R+S165E+S287K+L202F+G295H+N134D S297C+T289R+S165E+S287K+L202F+G295H+N134F, S297C+T289R+S165E+S287K+L202F+G295H+N134P, S297C+T289R+S165E+S287K+L202F+G295H+G13 7D, S297C+T289R+S165E+S287K+L202F+G295H+G137A, S297C+T289R+S165E+S287K+L202F+G295H+G137Y, S297C+T289R+S165E+S287K+L202F+G295H+ G137C, S297C+T289R+S165E+S287K+L202F+G295H+G137A+G242Q, S297C+T289R+S165E+S287K+L202F+G295H+G137A+G242A, S297C+T289R+S165E+S287 K+L202F+G295H+G137A+G242K、S297C+T289R+S165E+S287K+L202F+G295H +G137A+K246A、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246M、S 297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+S221A、S297C+T289R+S165E+S287K +L202F+G295H+G137A+K246A+S221R, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+S221W, S297C+T289R+S165E+S287K+L202F+G295H+G1 37A+K246A+S221Y、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+S221T、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282L、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282H, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282G, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282M, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282C, S297C+T289R+S165E+S287 K+L202F+G295H+G137A+K246A+Y282A、S297C+T289R+S165E+S287K+L20 2F+G295H+G137A+K246A+N134F、S297C+T289R+S165E+S287K+L202F+G29 5H+G137A+K246A+N134D, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134F, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K24 6A+N134G, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134T, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R, S2 97C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80D、S29 7C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80F、S29 7C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80G、S297 C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80R、S297C +T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113D、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E 0S+A113R, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113M, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A +N134R+T80S+A113T、S297C+T289R+S165E+S287K+L202F+G295H+G137A+ K246A+N134R+T80S+A113E+R325N、S297C+T289R+S165E+S287K+L202F+G2 95H+G137A+K246A+N134R+T80S+A113E+S314A, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+S314G, S297C+T289R+ S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+S314D、S 297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A11 3E+S314Y、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N13 4R+T80S+A113E+G242A、S297C+T289R+S165E+S287K+L202F+G295H+G137A +K246A+N134R+T80S+A113E+G242M, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G242R, S297C+T289R+S165E+S2 87K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+I183A, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+I183QS297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+I183M R+T80S+A113E+T209R、S297C+T289R+S165E+S287K+L202F+G295H+G137A+ K246A+N134R+T80S+A113E+T209A、S297C+T289R+S165E+S287K+L202F+G29 5H+G137A+K246A+N134R+T80S+A113E+T209M, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209S, S297C+T289R+S1 65E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209W, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K9 2A, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92G, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N1 34R+T80S+A113E+K92D、S297C+T289R+S165E+S287K+L202F+G295H+G137A +K246A+N134R+T80S+A113E+K92E、S297C+T289R+S165E+S287K+L202F+G29 5H+G137A+K246A+N134R+T80S+A113E+K92S, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84A, S297C+T289R+S165 E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84S、S297C+T2 89R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84Q、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+H302F, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+H302A or S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+H302G.

[0011] 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.

[0012] According to a second aspect of the invention, a DNA molecule is provided that encodes the aforementioned polypeptide ligase mutant.

[0013] According to a third aspect of the invention, a recombinant plasmid is provided, which is linked to the aforementioned DNA molecule.

[0014] According to a fourth aspect of the invention, a host cell is provided containing the aforementioned DNA molecule or the aforementioned recombinant plasmid; the host cell is neither an animal nor a plant species.

[0015] 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.

[0016] According to a fifth aspect of the present invention, a method for preparing a polypeptide is provided, the method comprising using the above-described polypeptide ligase mutant to catalyze the binding of a substrate peptide chain to obtain a polypeptide.

[0017] Further, the substrate peptide chain comprises 2 to 3 chains; preferably, it 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.

[0018] 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:

[0019] 1) The amino acid sequence of the first substrate peptide chain is 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 shown in SEQ ID NO: 4; 2) The amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 24, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO: 28; 3) The amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 25, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO: 29; 4) The amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 26, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO: 30; 5) The amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 27, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID 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: 4. As shown in NO:34, the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO:35.

[0020] By applying the technical solution of the present invention, the above-mentioned polypeptide ligase mutant has higher activity and a broader substrate spectrum than the polypeptide ligase in the prior art, and can achieve the ligation of diverse 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 10 of the present invention on substrates with different amino acids at the P4 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 limited catalytic activity for certain specific peptide drug fragments, a narrow substrate spectrum, 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 S297 site; or (b) a protein having more than 70% homology to the amino acid sequence defined in (a) and having polypeptide ligase activity.

[0027] Existing peptide ligases exhibit low activity, resulting in limited catalytic activity and insufficient stability for certain specific peptide drug fragments. In our previous work, we constructed an enzyme library containing 200 proteases. Activity tests on these proteases using different peptide substrates revealed that they 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. We mutated a serine residue near the active site to cysteine, and tested the ligation activity of the enzymes after a single point mutation 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)). The results were 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 of these hydrolases, after single-point mutation, retained some hydrolytic activity while also exhibiting linkage activity. However, the remaining approximately 90% of enzymes, after single-point mutation, showed a significant reduction in hydrolytic activity in addition to the absence of linkage activity. This suggests that the key catalytic site of serine 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 glycinifermentans (SEQ ID NO: 1), which exhibits high activity during peptide ligation. The active pocket of the protease 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 S297 site.

[0029] In a preferred embodiment, the mutation is selected from the S297C mutation and any one or more of the following mutations: N194 mutation to N194K; L195 mutation to L195M; S206 mutation to S206A; Y219 mutation to Y219A; V223 mutation to V223M; S258 mutation to S258E; S259 mutation to S259N; S165 mutation to S165E; S320 mutation to S320K; S185 mutation to S185A; P205 mutation to P205G; N294 mutation to N294S; Y285 mutation to Y285A; S237 mutation to S237G; I281 mutation to I281N; T179 Mutations include: T179S; N163 becoming N163R or N163G; I183 becoming I183V or I183N; S238 becoming S238Q or S238A; S232 becoming S232N, S232Q, or S232E; S280 becoming S280T, S280L, or S280K; G295 becoming G295C, G295S, or G295H; S287 becoming S287H, S287M, or S287K; S287 becoming S287N, S287H, S287M, or S287K; L202 becoming L202F, L202M, L202A, or L202P; and R262 becoming... R262L, R262E, R262Q, or R262Y; G84 mutation to G84A, G84S, G84Q, G84D, G84S, or G84P; K246 mutation to K246A, K246D, K246I, K246L, or K246Y; H302 mutation to H302F, H302G, H302T, H302A, or H302G; G137 mutation to G137D, G137I, G137C, G137A, or G137Y; S221 mutation to S221A, S221R, S221W, S221Y, or S221T; T209 mutation to T209A, T209R, T209M, or T209Y. 09S or T209W; T289 mutation to T289R, T289S, T289M, T289K or T289A; I183 mutation to I183V, I183A, I183Q, I183M or I183V; G137 mutation to G137I, G137C, G137D, G137A or G137Y; S177 mutation to S177Q, S177H, S177R, S177K or S177G; K92 mutation to K92A, K92G, K92D, K92E, K92S or K92N; T80 mutation to T80A, T80P, T80S, T80D, T80F, T80G or T80R;Y282 mutations into Y282L, Y28G, Y282H, Y282M, Y282C, or Y282AR; R325 mutations into R325A, R325F, R325G, R325Y, R325E, or R325M; S314 mutations into S314N, S314K, S314C, S314R, S314Y, or S314N; G242 mutations into G242T, G242A, G242Q, G242L, G242S, G242M, or G24 2R; A113 mutation to A113D, A113P, A113M, A113S, A113E, A113R, or A113T; R325 mutation to R325N, R325A, R325G, R325Y, R325E, R325M, or R325D; N134 mutation to N134F, N134D, N134G, N134T, N134R, N134P, N134E, N134L, N134S, N134I, or N134A. The letter before the number represents the original amino acid, and the letter after the number represents the mutated amino acid.

[0030] In a preferred embodiment, the mutation includes any one or more of the following amino acid mutations: S297C, S297C+K92N, S297C+T80S, S297C+G84A, S297C+L202F, S297C+K246A, S297C+R262L, S297C+H302F, S297C+N134D, S297C+N134F, S297C+G137D, S297C+N194K, S297C+L195M, S297C+S206A, S297C+Y219A, S297C+S221A, S297C+V223M, S297C+S238Q, S297C+G2 42Q, S297C+K246A, S297C+S258E, S297C+S259N, S297C+Y282L, S297C+S28 7N, S297C+T289R, S297C+T289S, S297C+T289M, S297C+T289K, S297C+T289 A. S297C+A113D, S297C+A113P, S297C+A113M, S297C+A113S, S297C+S314N , S297C+S314K, S297C+S314C, S297C+S314R, S297C+S314I, S297C+S314Y, S 297C+R325A, S297C+R325F, S297C+R325G, S297C+R325Y, S297C+R325E, S2 97C+R325M, S297C+A113D+S165E, S297C+A113D+S287N, S297C+A113D+G24 2L, S297C+A113D+S320K, S297C+A113D+N134A, S297C+A113D+N134T, S297 C+A113D+N134S, S297C+A113D+N134I, S297C+A113D+S185A, S297C+A113D+ T209A, S297C+S314N+I183V, S297C+S314N+I183N, S297C+S314N+S185A, S 297C+S314N+K92A, S297C+S314N+G242Q, S297C+T289R+S165E, S297C+T28 9R+G242S, S297C+T289R+G242C, S297C+T289R+G137D, S297C+T289R+P205 G. S297C+T289R+S238Q, S297C+T289R+N294S, S297C+T289R+S165E+S314Y,S297C+T289R+S165E+I183V, S297C+T289R+S165E+S314N, S297C+T289R+S165E+Y285A, S297C+T289R+S165E+S287H, S297C+T289R+S165E+S287M, S297 C+T289R+S165E+S287K, S297C+T289R+S165E+R325A, S297C+T289R+S165E+S237G, S297C+T289R+S165E+S238A, S297C+T289R+S165E+N134F, S297C+T28 9R+S165E+N134E, S297C+T289R+S165E+N134L, S297C+T289R+S165E+N294S, S297C+T289R+S165E+I281N, S297C+T289R+S165E+S280T, S297C+T289R+S1 65E+S280L、S297C+T289R+S165E+S280K、S297C+T289R+S165E+S232N、S297 C+T289R+S165E+S232Q、S297C+T289R+S165E+S232E、S297C+T289R+S165E+G 242T, S297C+T289R+S165E+G242T, S297C+T289R+S165E+G137I, S297C+T289R+S165E+G137C, S297C+T289R+S165E+S177Q, S297C+T289R+S165E+S177H S297C+T289R+S165E+S177R, S297C+T289R+S165E+S177K, S297C+T289R+S165E+S177G, S297C+T289R+S165E+T179S, S297C+T289R+S165E+N163R, S297 C+T289R+S165E+N163G, S297C+T289R+S165E+S287K, S297C+T289R+S165E+S287K+G84A, S297C+T289R+S165E+S287K+G84D, S297C+T289R+S165E+S287K +G84S、S297C+T289R+S165E+S287K+G84P、S297C+T289R+S165E+S287K+L20 2F、S297C+T289R+S165E+S287K+L202M、S297C+T289R+S165E+S287K+L202A、S297C+T289R+S165E+S287K+L202P, S297C+T289R+S165E+S287K+K246A, S297C+T289R+S165E+S287K+K246D, S297C+T289R+S165E+S287K+K246I, S2 97C+T289R+S165E+S287K+K246L, S297C+T289R+S165E+S287K+K246Y, S297C+T289R+S165E+S287K+R262L, S297C+T289R+S165E+S287K+R262E, S297C +T289R+S165E+S287K+R262Q、S297C+T289R+S165E+S287K+R262Y、S297C+T289R+S165E+S287K+H302F、S297C+T289R+S165E+S287K+H302G、S297C+T2 89R+S165E+S287K+H302T, S297C+T289R+S165E+S287K+H140R, S297C+T289R+S165E+S287K+H140E, S297C+T289R+S165E+S287K+H140V, S297C+T289R +S165E+S287K+H140K, S297C+T289R+S165E+S287K+H140A, S297C+T289R+S165E+S287K+H143E, S297C+T289R+S165E+S287K+H143M, S297C+T289R+S1 65E+S287K+H143V, S297C+T289R+S165E+S287K+H143E, S297C+T289R+S165E+S287K+H143A, S297C+T289R+S165E+S287K+L202F+L293A, S297C+T289R +S165E+S287K+L202F+L293E、S297C+T289R+S165E+S287K+L202F+L293Q、S297C+T289R+S165E+S287K+L202F+L293Y、S297C+T289R+S165E+S287K+L2 02F+G295C, S297C+T289R+S165E+S287K+L202F+G295S, S297C+T289R+S165E+S287K+L202F+G295H, S297C+T289R+S165E+S287K+L202F+G295H+T80SS297C+T289R+S165E+S287K+L202F+G295H+T80A、S297C+T289R+S165E+S2 87K+L202F+G295H+T80P、S297C+T289R+S165E+S287K+L202F+G295H+N134D S297C+T289R+S165E+S287K+L202F+G295H+N134F, S297C+T289R+S165E+S287K+L202F+G295H+N134P, S297C+T289R+S165E+S287K+L202F+G295H+G13 7D, S297C+T289R+S165E+S287K+L202F+G295H+G137A, S297C+T289R+S165E+S287K+L202F+G295H+G137Y, S297C+T289R+S165E+S287K+L202F+G295H+ G137C, S297C+T289R+S165E+S287K+L202F+G295H+G137A+G242Q, S297C+T289R+S165E+S287K+L202F+G295H+G137A+G242A, S297C+T289R+S165E+S287 K+L202F+G295H+G137A+G242K、S297C+T289R+S165E+S287K+L202F+G295H +G137A+K246A、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246M、S 297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+S221A、S297C+T289R+S165E+S287K +L202F+G295H+G137A+K246A+S221R, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+S221W, S297C+T289R+S165E+S287K+L202F+G295H+G1 37A+K246A+S221Y、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+S221T、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282L、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282H, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282G, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282M, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282C, S297C+T289R+S165E+S287 K+L202F+G295H+G137A+K246A+Y282A、S297C+T289R+S165E+S287K+L20 2F+G295H+G137A+K246A+N134F、S297C+T289R+S165E+S287K+L202F+G29 5H+G137A+K246A+N134D, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134F, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K24 6A+N134G, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134T, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R, S2 97C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80D、S29 7C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80F、S29 7C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80G、S297 C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80R、S297C +T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113D、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E 0S+A113R, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113M, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A +N134R+T80S+A113T、S297C+T289R+S165E+S287K+L202F+G295H+G137A+ K246A+N134R+T80S+A113E+R325N、S297C+T289R+S165E+S287K+L202F+G2 95H+G137A+K246A+N134R+T80S+A113E+S314A, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+S314G, S297C+T289R+ S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+S314D、S 297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A11 3E+S314Y、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N13 4R+T80S+A113E+G242A、S297C+T289R+S165E+S287K+L202F+G295H+G137A +K246A+N134R+T80S+A113E+G242M, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G242R, S297C+T289R+S165E+S2 87K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+I183A, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+I183QS297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+I183M R+T80S+A113E+T209R、S297C+T289R+S165E+S287K+L202F+G295H+G137A+ K246A+N134R+T80S+A113E+T209A、S297C+T289R+S165E+S287K+L202F+G29 5H+G137A+K246A+N134R+T80S+A113E+T209M, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209S, S297C+T289R+S1 65E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209W, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K9 2A, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92G, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N1 34R+T80S+A113E+K92D、S297C+T289R+S165E+S287K+L202F+G295H+G137A +K246A+N134R+T80S+A113E+K92E、S297C+T289R+S165E+S287K+L202F+G29 5H+G137A+K246A+N134R+T80S+A113E+K92S, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84A, S297C+T289R+S165 E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84S、S297C+T2 89R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84Q、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+H302F, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+H302A or S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+H302G.

[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 existing peptide ligases, 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.

[0032] This application modifies a protease derived from Bacillus glycinifermentans (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 P4 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, which is linked to the aforementioned DNA molecule.

[0048] In a fourth typical embodiment of this application, a host cell is provided, which contains the aforementioned DNA molecule or the aforementioned recombinant plasmid; 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 includes using the above-mentioned 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 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.

[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:

[0054] 1) The amino acid sequence of the first substrate peptide chain is 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 shown in SEQ ID NO: 4; 2) The amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 24, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO: 28; 3) The amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 25, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO: 29; 4) The amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 26, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID NO: 30; 5) The amino acid sequence of the first substrate peptide chain is shown in SEQ ID NO: 27, and the amino acid sequence of the second substrate peptide chain is shown in SEQ ID 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: 4. As shown in NO: 34, 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.

[0055] 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.

[0056] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0057] Unless otherwise specified, all reagents used in the embodiments of this application are commercially available products.

[0058] Example 1

[0059] Expression and purification of polypeptide ligase mutants in Bacillus subtilis:

[0060] The mutant S297C gene (N-terminal or C-terminal His tag) from Bacillus glycinifermentans (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 using 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.

[0061] Positive transformants were picked and cultured in 5 mL of LB liquid medium containing 50 μg / mL kanamycin sulfate for 16 h at 37°C and 200 rpm. 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.

[0062] 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.

[0063] Example 2

[0064] Expression and purification of polypeptide ligase mutants in E. coli:

[0065] The mutant S297C gene (N-terminal or C-terminal His tag) from Bacillus glycinifermentans (SEQ ID NO: 1) was cloned into the restriction enzyme sites Nco I-BamHI 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 OD... 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.

[0066] 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 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.

[0067] Example 3

[0068] Expression and purification of polypeptide ligase mutants in yeast:

[0069] The mutant S297C gene (N-terminal or C-terminal His tag) from Bacillus glycinifermentans (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 ppm). -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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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 this application, which can be used to catalyze the ligation of polypeptide substrates.

[0074] Example 4

[0075] This embodiment modifies a protease derived from Bacillus glycinifermentans (SEQ ID NO: 1) by mutating a serine residue near the active site to cysteine. Using a pentapeptide (acyl donor P4 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 S297C and Omnilgase-1. The reaction system is as follows:

[0076] 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.

[0077] 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.

[0078] 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.

[0079] In this embodiment, both mutant S297C and Omniligase-1 catalyze the ligation reaction in which the acyl donor does not contain a non-natural amino acid, and exhibit high conversion rates. However, in the reaction where the acyl donor has a non-natural amino acid Aib at the P4 position, the catalytic activity of S297C is approximately several hundred times that of Omniligase-1. This indicates that mutants derived from Bacillus glycinifermentans have catalytic potential for ligation reactions in which the non-natural amino acid Aib is present at the P4 position.

[0080] Example 5

[0081] Enzymatic evolution was continued on the mutant S297C derived from Bacillus glycinifermentans. Ac-Asp-Aib-Tyr-Ser-Leu-O-Cam-Leu-OH (SEQ ID NO: 2), with a non-natural amino acid at position P4, was used as the acyl donor, and H-Met-Leu-Val-Lys-Ala-NH2 (SEQ ID NO: 4) as the acyl acceptor. The resulting polypeptide ligase mutants were screened. The reaction system is as follows:

[0082] The 1 mL reaction system included: 2.5 mM acyl donor, 3.75 mM acyl acceptor, enzyme concentration reduced to 0.3 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 sent to HPLC for conversion analysis.

[0083] After initial screening and secondary screening, the following mutants exhibiting enhanced activity were obtained, and the specific results are shown in Table 2.

[0084] Table 2 Note: In Table 2, ** 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%), and **** represents a conversion rate greater than or equal to 30% and less than 50%.

[0085] In this embodiment, a combination of mutants was constructed using the single-point mutation S297C as the parent material, and a series of peptide ligase mutants with enhanced activity were obtained by screening and activity verification using substrates with Aib at the P4 position.

[0086] Example 6

[0087] Evolution was continued based on the mutant from Example 5, and the combined mutants were tested for viability under the same reaction conditions as in Example 5. The results are shown in Table 3.

[0088] Table 3 Note: In Table 3, * 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 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%), ****** represents a conversion rate greater than or equal to 70% and less than 90% (excluding 90%), and ******* represents a conversion rate greater than or equal to 90%.

[0089] In this embodiment, beneficial mutations are combined to obtain a series of peptide ligase mutants with enhanced activity.

[0090] Example 7

[0091] Based on the mutant from Example 6, further mutations were performed, and the mutant was tested for viability using the following reaction system.

[0092] The 1 mL reaction system included: 5 mM acyl donor (SEQ ID NO: 2), 7.5 mM acyl acceptor (SEQ ID NO: 4), with the enzyme concentration further reduced to 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 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 sent to HPLC for conversion analysis.

[0093] After initial and secondary screening, the following mutants were obtained, which showed increased activity. The specific results are shown in Table 4.

[0094] Table 4 Note: In Table 4, * 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 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%), ****** represents a conversion rate greater than or equal to 70% and less than 90% (excluding 90%), and ******* represents a conversion rate greater than or equal to 90%.

[0095] In this embodiment, the peptide ligase mutant S297C+T289R+S165E+S287K with the best catalytic effect obtained in Example 6 was used as the parent for further evolution. The enzyme amount was reduced to 0.1 mg / mL for screening, and a series of peptide ligase mutants with improved activity were obtained.

[0096] Example 8

[0097] The mutant from Example 7 was further evolved, and the mutant was tested for viability using the following reaction system.

[0098] The 1 mL reaction system included: 5 mM acyl donor (SEQ ID NO: 2), 7.5 mM acyl acceptor H-Met-Leu-Val-Lys-Ala-NH2 (SEQ ID NO: 4), with the enzyme concentration further 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 to HPLC for conversion analysis.

[0099] After primary and secondary screening, the following mutants were obtained, exhibiting increased activity. Specific results are shown in Table 5.

[0100] Table 5 Note: In Table 5, * 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 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%), ****** represents a conversion rate greater than or equal to 70% and less than 90% (excluding 90%), and ******* represents a conversion rate greater than or equal to 90%.

[0101] In this embodiment, the mutant S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A with the best catalytic effect obtained in Example 7 was used as the parent for further evolution. The enzyme amount was reduced to 0.05 mg / mL for screening, and a peptide ligase mutant with improved activity was obtained.

[0102] Example 9

[0103] Enzyme stability test:

[0104] Stability tests were performed on some of the mutants obtained from Examples 4 to 8 (screened using P4 site as Aib substrate) to screen for mutants with improved stability. The mutants were prepared into enzyme solutions according to the methods described in Examples 1, 2, or 3, and the enzyme solutions were treated at 70°C for one hour. The mutants were then tested for activity according to the following reaction system.

[0105] The 1 mL reaction system included: 2.5 mM acyl donor (SEQ ID NO: 2), 3.75 mM acyl acceptor (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 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 6.

[0106] Table 6 Note: The residual activity in Table 6 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%.

[0107] Example 10

[0108] Substrate spectral analysis:

[0109] Using the peptide ligase mutant S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84Q as a catalyst, the catalytic activity of 5-peptide acyl donors with different amino acids at the P4 position was tested. The acyl donors were 20 different 5-peptides with the sequence Ac-Asp-Xxx-Tyr-Ser-Leu-O-Cam-Leu-OH (Xxx refers to 20 different amino acids, including L, Y, C, V, I, M, F (SEQ ID NO: 3), N, S, W, R, A, H, P, A, T, D, G, E, and K, corresponding to SEQ ID NO: 5–23). The acyl acceptor was H-Met-Leu-Val-Lys-Ala-NH2 (SEQ ID NO: 4). The reaction system is as follows:

[0110] 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.

[0111] Figure 2 shows the catalytic results of the above-mentioned polypeptide ligase mutants for different amino acids at the P4 position.

[0112] In this embodiment, enzyme evolution screening was performed using a pentapeptide with the non-natural amino acid Aib at the P4 position as the target substrate. The resulting polypeptide ligase mutant exhibited a wider range of substrate adaptability at the P4 position.

[0113] Example 11

[0114] Using the peptide ligase mutant S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84Q 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:

[0115] 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 termination with an equal volume of anhydrous ethanol. 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 7.

[0116] Table 7 Note: In Table 7, S / H refers to the ratio of the amount of enzyme-synthesized product to the amount of enzyme-hydrolyzed substrate.

[0117] The mutants obtained in this embodiment can be used for ligation reactions of peptide substrates of different lengths, and all of them can exhibit good ligation activity and S / H ratio.

[0118] Example 12

[0119] Smegglutinin was synthesized enzymatically using a 17-mer + 14-mer (acyl donor + acyl acceptor) fragment:

[0120] Add 5 mM of the 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 the 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, and add 0.01 mg / mL of SEQ ID NO. A mutant enzyme solution (S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84Q) mutated from NO:1 was added to a total volume of 50 mL with buffer (0.1 M Tricine, pH 8.0, 0.8 mg / mL TCEP). The pH was adjusted to 7.8–8.2, and the reaction was carried out at 25 °C with stirring for 2 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 42. The system was then acidified to pH 1–2 to denature the protein. The denatured system was centrifuged at 8000 rpm for 10 min to obtain the supernatant aqueous phase, which was then purified by HPLC and lyophilized to obtain the product smegglutinin.

[0121] The purity of the product was >98% and the yield was 74%, as determined by HPLC and Q-NMR.

[0122] Example 11

[0123] Liraglutide was synthesized enzymatically using a 17-mer + 14-mer (acyl donor + acyl acceptor) fragment:

[0124] 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, and add 0.01 mg / mL of SEQ ID NO. A mutant enzyme solution (S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84Q) mutated from NO:1 was added to a total volume of 50 mL with buffer (0.1 M Tricine, pH 8.0, 0.8 mg / mL TCEP). The pH was adjusted to 7.8–8.2, and the reaction was carried out at 25 °C with stirring for 3 h. HPLC analysis showed complete reaction of the donor and substrate, with a conversion rate of 94% (no product isomers detected), and an S / H ratio of 47. The system was then acidified to pH 1–2 to denature the protein. The denatured system was centrifuged at 8000 rpm for 10 min to obtain the supernatant aqueous phase, which was then purified by HPLC and lyophilized to obtain the product liraglutide.

[0125] The purity of the product was >98% and the yield was 76%, as determined by HPLC and Q-NMR.

[0126] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: (1) The present application obtains a ligase with high peptide ligation activity through enzyme evolution, which shows good catalytic potential for the reaction in which the non-natural amino acid Aib is located at the P4 position. (2) Using the pentapeptide with Aib at the P4 position as a substrate, further enzyme evolution is carried out to obtain mutants with further improved activity. These mutants also show 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 mutants obtained in the present application can be used for the efficient synthesis of a variety of peptide drugs. (5) Compared with the traditional chemical method, the enzymatic method of the present application does not require complex processes and steps, the operation is simpler and milder, the reaction process does not cause protein racemization, no isomers are generated, the system generates fewer impurities, purification is simple, the purity is high, and it is more suitable for industrial scale-up production.

[0127] 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 S297 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 S297C mutation and any one or more of the following mutations: N194 mutates to N194K; L195 mutated to L195M; S206 mutates to S206A; Y219 mutated to Y219A; V223 mutated to V223M; S258 mutates to S258E; S259 mutates to S259N; S165 mutates to S165E; S320 mutates to S320K; S185 mutates to S185A; P205 mutates to P205G; N294 mutates to N294S; Y285 mutated to Y285A; S237 mutates to S237G; I281 mutates to I281N; T179 mutates to T179S; N163 mutates into N163R and N163G; I183 mutates into I183V or I183N; S238 mutates to S238Q or S238A; S232 mutates into S232N, S232Q or S232E; S280 mutates into S280T, S280L or S280K; G295 mutates into G295C, G295S or G295H; S287 mutates into S287H, S287M or S287K; S287 mutations into S287N, S287H, S287M, or S287K; L202 mutates into L202F, L202M, L202A, or L202P; R262 mutations can be R262L, R262E, R262Q, or R262Y; G84 mutations into G84A, G84S, G84Q, G84D, G84S, or G84P; K246 mutates into K246A, K246D, K246I, K246L, or K246Y; H302 mutates into H302F, H302G, H302T, H302A, or H302G; G137 mutation can be G137D, G137I, G137C, G137A, or G137Y; S221 mutates into S221A, S221R, S221W, S221Y, or S221T; T209 mutations can result in T209A, T209R, T209M, T209S, or T209W. T289 mutations can result in T289R, T289S, T289M, T289K, or T289A; I183 mutations can result in I183V, I183A, I183Q, I183M, or I183V. G137 mutation can be G137I, G137C, G137D, G137A, or G137Y; S177 mutations can be S177Q, S177H, S177R, S177K, or S177G; K92 mutations can occur as K92A, K92G, K92D, K92E, K92S, or K92N; T80 mutations can result in T80A, T80P, T80S, T80D, T80F, T80G, or T80R. The Y282 mutation can result in Y282L, Y28G, Y282H, Y282M, Y282C, or Y282A. R325 mutation can result in R325A, R325F, R325G, R325Y, R325E, or R325M; S314 mutates into S314N, S314K, S314C, S314R, S314Y, or S314N; G242 mutations can be G242T, G242A, G242Q, G242L, G242S, G242M, or G242R; A113 mutates into A113D, A113P, A113M, A113S, A113E, A113R, or A113T; R325 mutations can occur as R325N, R325A, R325G, R325Y, R325E, R325M, or R325D. N134 mutations can result in N134F, N134D, N134G, N134T, N134R, N134P, N134E, N134L, N134S, N134I, or N134A.

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: S297C、S297C+K92N、S297C+T80S、S297C+G84A、S297C+L202F、S297C+K246A 、S297C+R262L、S297C+H302F、S297C+N134D、S297C+N134F、S297C+G137D、S 297C+N194K, S297C+L195M, S297C+S206A, S297C+Y219A, S297C+S221A, S297C+V223M, S297C+S238Q, S297C+G242Q, S297C+K246A, S297C+S258E, S297C+ S259N, S297C+Y282L, S297C+S287N, S297C+T289R, S297C+T289S, S297C+T289M, S297C+T289K, S297C+T289A, S297C+A113D, S297C+A113P, S297C+A113M S297C+A113S, S297C+S314N, S297C+S314K, S297C+S314C, S297C+S314R, S297C+S314I, S297C+S314Y, S297C+R325A, S297C+R325F, S297C+R325G, S297 C+R325Y, S297C+R325E, S297C+R325M, S297C+A113D+S165E, S297C+A113D+S287N, S297C+A113D+G242L, S297C+A113D+S320K, S297C+A113D+N134A, S2 97C+A113D+N134T、S297C+A113D+N134S、S297C+A113D+N134I、S297C+A113 D+S185A、S297C+A113D+T209A、S297C+S314N+I183V、S297C+S314N+I183N、S 297C+S314N+S185A、S297C+S314N+K92A、S297C+S314N+G242Q、S297C+T289 R+S165E、S297C+T289R+G242S、S297C+T289R+G242C、S297C+T289R+G137D、S 297C+T289R+P205G, S297C+T289R+S238Q, S297C+T289R+N294S, S297C+T289R+S165E+S314Y, S297C+T289R+S165E+I183V, S297C+T289R+S165E+S314NS297C+T289R+S165E+Y285A, S297C+T289R+S165E+S287H, S297C+T289R+S165E+S287M, S297C+T289R+S165E+S287K, S297C+T289R+S165E+R325A, S2 97C+T289R+S165E+S237G, S297C+T289R+S165E+S238A, S297C+T289R+S165E+N134F, S297C+T289R+S165E+N134E, S297C+T289R+S165E+N134L, S297C +T289R+S165E+N294S、S297C+T289R+S165E+I281N、S297C+T289R+S165E+ S280T、S297C+T289R+S165E+S280L、S297C+T289R+S165E+S280K、S297C+T 289R+S165E+S232N, S297C+T289R+S165E+S232Q, S297C+T289R+S165E+S232E, S297C+T289R+S165E+G242T, S297C+T289R+S165E+G242T, S297C+T289 R+S165E+G137I, S297C+T289R+S165E+G137C, S297C+T289R+S165E+S177Q, S297C+T289R+S165E+S177H, S297C+T289R+S165E+S177R, S297C+T289R+ S165E+S177K, S297C+T289R+S165E+S177G, S297C+T289R+S165E+T179S, S297C+T289R+S165E+N163R, S297C+T289R+S165E+N163G, S297C+T289R+S16 5E+S287K、S297C+T289R+S165E+S287K+G84A、S297C+T289R+S165E+S287K +G84D、S297C+T289R+S165E+S287K+G84S、S297C+T289R+S165E+S287K+G8 4P, S297C+T289R+S165E+S287K+L202F, S297C+T289R+S165E+S287K+L202M, S297C+T289R+S165E+S287K+L202A, S297C+T289R+S165E+S287K+L202PS297C+T289R+S165E+S287K+K246A, S297C+T289R+S165E+S287K+K246D, S297C+T289R+S165E+S287K+K246I, S297C+T289R+S165E+S287K+K246L, S297 C+T289R+S165E+S287K+K246Y, S297C+T289R+S165E+S287K+R262L, S297C+T289R+S165E+S287K+R262E, S297C+T289R+S165E+S287K+R262Q, S297C+T2 89R+S165E+S287K+R262Y, S297C+T289R+S165E+S287K+H302F, S297C+T289R+S165E+S287K+H302G, S297C+T289R+S165E+S287K+H302T, S297C+T289R+ S165E+S287K+H140R、S297C+T289R+S165E+S287K+H140E、S297C+T289R+S1 65E+S287K+H140V、S297C+T289R+S165E+S287K+H140K、S297C+T289R+S165E +S287K+H140A, S297C+T289R+S165E+S287K+H143E, S297C+T289R+S165E+S287K+H143M, S297C+T289R+S165E+S287K+H143V, S297C+T289R+S165E+S28 7K+H143E, S297C+T289R+S165E+S287K+H143A, S297C+T289R+S165E+S287K+L202F+L293A, S297C+T289R+S165E+S287K+L202F+L293E, S297C+T289R+S 165E+S287K+L202F+L293Q, S297C+T289R+S165E+S287K+L202F+L293Y, S297C+T289R+S165E+S287K+L202F+G295C, S297C+T289R+S165E+S287K+L202F +G295S, S297C, T289R, S165E, S287K, L202F, G295H, S297C, T289R, S165E, S287K, L202F, G295H, T80S, S297C, T289R, S165E, S287K, L202F, G295H, T80AS297C+T289R+S165E+S287K+L202F+G295H+T80P, S297C+T289R+S165E+S287K+L202F+G295H+N134D, S297C+T289R+S165E+S287K+L202F+G295H+N134F S297C+T289R+S165E+S287K+L202F+G295H+N134P, S297C+T289R+S165E+S287K+L202F+G295H+G137D, S297C+T289R+S165E+S287K+L202F+G295H+G137A S297C+T289R+S165E+S287K+L202F+G295H+G137Y A+G242Q、S297C+T289R+S165E+S287K+L202F+G295H+G137A+G242A、S297C+ T289R+S165E+S287K+L202F+G295H+G137A+G242K、S297C+T289R+S165E+S28 7K+L202F+G295H+G137A+K246A, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246M, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A, S2 97C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+S221A、S297C+T289 R+S165E+S287K+L202F+G295H+G137A+K246A+S221R、S297C+T289R+S165E+S 287K+L202F+G295H+G137A+K246A+S221W、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+S221Y、S297C+T289R+S165E+S287K+L202F+G295H+ G137A+K246A+S221T, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282L, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282HS297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282G、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282M、S297C+T289R+ S165E+S287K+L202F+G295H+G137A+K246A+Y282C, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+Y282A, S297C+T289R+S165E+S287K+L 202F+G295H+G137A+K246A+N134F、S297C+T289R+S165E+S287K+L202F+G 295H+G137A+K246A+N134D、S297C+T289R+S165E+S287K+L202F+G295H+G 137A+K246A+N134F, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134G, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N1 34T, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S, S29 7C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80D、S297 C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80F、S297C+ T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80G, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80R, S297C+T28 9R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113D, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113ES297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113R S+A113M、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R +T80S+A113T、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N 134R+T80S+A113E+R325N, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+S314A, S297C+T289R+S165E+S287K+L202F +G295H+G137A+K246A+N134R+T80S+A113E+S314G, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+S314D, S297C+T28 9R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+S314Y 、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A 113E+G242A, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G242M, S297C+T289R+S165E+S287K+L202F+G295H+G137 A+K246A+N134R+T80S+A113E+G242R, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+I183A, S297C+T289R+S165E+S2 87K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+I183Q, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+I183MS297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209R R+T80S+A113E+T209A、S297C+T289R+S165E+S287K+L202F+G295H+G137A+ K246A+N134R+T80S+A113E+T209M、S297C+T289R+S165E+S287K+L202F+G29 5H+G137A+K246A+N134R+T80S+A113E+T209S, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+T209W, S297C+T289R+S1 65E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92A, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92 G、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+ A113E+K92D、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N13 4R+T80S+A113E+K92E, S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+K92S, S297C+T289R+S165E+S287K+L202F+G295 H+G137A+K246A+N134R+T80S+A113E+G84A、S297C+T289R+S165E+S287K+L 202F+G295H+G137A+K246A+N134R+T80S+A113E+G84S、S297C+T289R+S165E +S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+G84Q、S297C+T28 9R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+H302F、S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+H302A or S297C+T289R+S165E+S287K+L202F+G295H+G137A+K246A+N134R+T80S+A113E+H302G.

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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