α-amino acid ester acyltransferase mutant and use thereof

Abstract

Provided are an α-amino acid ester acyltransferase mutant and use thereof. The α-amino acid ester acyltransferase mutant comprises: (a) a protein having the amino acid sequence set forth in SEQ ID NO: 1; (b) a protein which has undergone amino acid mutation at at least one of the following sites in the described amino acid sequence in (a): P158, K80, T208, A302, A175, N85, N605, K346, S225, I34, or S348, and has α-amino acid ester acyltransferase function; or (c) a protein having 80% or more homology to the described amino acid sequence defined in (a) or (b) and having α-amino acid ester acyltransferase function. The α-amino acid ester acyltransferase mutant has the advantages of a broad substrate spectrum and high catalytic activity, and can be well applied to oligopeptide synthesis with low cost and high yield, achieving true green chemistry.

Description

α-Amino acid ester acyltransferase mutants and their applications

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 202510382491.9, filed on March 28, 2025, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention relates to the field of enzyme catalysis, and more specifically, to an α-amino acid ester acyltransferase mutant and its applications. Background Technology

[0004] Oligopeptides, also known as oligopeptides, are short-chain peptides composed of 2 to 20 amino acid residues linked by peptide bonds, with a molecular weight of approximately less than 1000 Daltons. Oligopeptides play a variety of important biological functions in organisms. For example, many oligopeptides act as signaling molecules, participating in intercellular communication; some act as cofactors or inhibitors of enzymes, regulating metabolic pathways. Some oligopeptides have immunomodulatory effects, enhancing or inhibiting immune responses; some have antioxidant activity, scavenging free radicals and protecting cells from oxidative damage; and some oligopeptides can promote cell proliferation and differentiation, accelerating wound healing. Oligopeptides have wide applications in medicine, food, cosmetics, and many other fields.

[0005] Dipeptides and tripeptides are the most basic oligopeptides. Despite their simple structure, they exhibit rich biological activities and can regulate various life processes such as physiological metabolism. For example, carnosine (β-alanyl-His) has antioxidant, anti-inflammatory, and anti-glycation effects; glycopeptide (Gly-Gly) is used in the pharmaceutical field as a stabilizer in blood preservation solutions and cytochrome C injections; ala-glutamyl dipeptide (Ala-Gln) is an important nutritional supplement for surgical patients; aspartame (Asp-Phe methyl ester) is a popular sweetener; copper hydroxide (GHK-Cu) is widely used in high-end skincare brands and is often used as a main ingredient in serums, creams, and other products; while Ala-Phe, Ile-Phe, and Pro-Gly dipeptides are used as salty taste enhancers; Ile-Tyr, Lys-Trp, Val-Tyr, and Ile-Trp have antihypertensive effects, Arg-Trp has analgesic effects, and Lys-Glu exhibits antitumor activity, etc.

[0006] Chemical synthesis of dipeptides has limitations, such as the need for amino acid protection and deprotection, the potential for racemization of the product, high synthesis costs, and the occasional use of toxic reagents. Fermentation methods suffer from low expression levels and byproducts. Therefore, exploring efficient and suitable methods for oligopeptide synthesis remains a hot topic in scientific research. α-amino acid ester acyltransferase (Aet) can use alanine methyl ester hydrochloride as an acyl donor to react with another nucleophilic reagent, glutamine, to generate Ala-Gln dipeptides. The entire process does not require ATP and has high synthesis efficiency (J Biotechnol. 2005 Jan 26; 115(2):211-20.; Biosci Biotechnol Biochem. 2011; 75(11):2087-92.). Currently reported α-amino acid ester acyltransferases (Aet) have a narrow substrate spectrum and low catalytic activity. Fewer than 30 oligopeptides have been reported for enzymatic synthesis using α-amino acid ester acyltransferases, and many important functional oligopeptides, such as Gly-Ser, Gly-Gly, Ser-Ser, Val-Ser, Tyr-Ser, Gly-Gly-Gly, and Gly-His-Lys, have not been reported for synthesis using α-amino acid ester acyltransferases. This limits the widespread application of Aet in the synthesis of various oligopeptide products. Therefore, developing α-amino acid ester acyltransferases with a broad substrate spectrum and high activity is of great significance. Summary of the Invention

[0007] The main objective of this invention is to provide an α-amino acid ester acyltransferase mutant and its application, in order to solve the problem of the narrow substrate spectrum of α-amino acid ester acyltransferases in the prior art.

[0008] To achieve the above objective, according to a first aspect of the present invention, an α-amino acid ester acyltransferase mutant is provided, the α-amino acid ester acyltransferase mutant comprising:

[0009] (a) A protein having the amino acid sequence shown in SEQ ID NO: 1;

[0010] (b) at least one of the following sites in the above amino acid sequence in (a): P158, K80, N85, I34, Y69, T70, P73, Y74, K81, L83, K104, V114, A150, S165, A175, V182, T183, W185, I187, F198, Q200, A202, R204, F205, M206, T208, F209, K215, S225, Q227, Y228, N233, L261, V287 Proteins containing the following amino acid mutations and possessing α-amino acid ester acyltransferase function: V289, F294, A299, Y300, A302, E304, K311, N318, V321, A322, R332, D334, S337, K346, S348, V349, H350, E353, F355, E439, N440, R441, T442, I447, I497V, T506, L518, E522, P535, T549, or N605; or

[0011] (c) A protein that has more than 80% homology with the amino acid sequence defined in (a) or (b) and has α-amino acid ester acyltransferase function.

[0012] Furthermore, the amino acid mutation in (b) above is selected from at least one of the following sites:

[0013] P158R, K80A or K80S or K80C or K80R or K80G, N85Y or N85G, I34T or I34K or I34A or I34P or I34R or I34H or I34S, Y69N, T70D or T70A or T70S or T70G or T70P or T70L or T70N or T70M, P73D, K81P or K81L or K81G or K81I or K81D or K81S or K81M or K81T or K81R or K81E, L83I or L83M or L83V, K104R, V114L or V114I, A150V, S165A, A1 75V, V182F or V182S, T183V, W185S or W185G or W185L or W185F or W185C, I187L, F198M or F198W, Q200I, A202S or A202C, R204A or R204H or R204V or R204N, F205M, M206H or M206F or M206S, T208G or T208F or T208S or T208M or T208A, F209N, K215T, S225R or S225T or S225E or S225A or S225M or S225N, Q227T or Q227 M or Q227V or Q227I, Y228A or Y228M or Y228L, N233R, L261F, V287T, V289I or V289L, F294L, A299E or A299V or A299T, Y300C, A302S or A302T, E304M, K311S or K311R or K311T, N318V, V321A or, A322S or A322L or A322I, R332S, D334V or D334N, S337H or S337S, K346P or K346R or K346N, S348A or S348G or S348 P or S348K or S348N or S348M or S348C or S348V, V349T, H350L or H350Y or H350T or H350E, E353W or E353T or E353N or E353S or E353Y, F355D or F355A, E439C, N440T, R441P, T442S, I447V, I497V, T506G, L518F, E522D, P535S or P535T or P535S or P535V, T549Y or N605P or N605F or N605A or N605L or N605G or N605D;

[0014] In this context, the letter before the number represents the original amino acid, and the letter after the number represents the mutated amino acid.

[0015] Furthermore, the mutations in the above-mentioned α-amino acid ester acyltransferase mutants include any one of the following amino acid mutations:

[0016] P158R、K80A、K80S、K80C、K80R、K80G、N85Y、K81P、V182F、V182S、W185S、W185G、W185L、P73D、T208G、T208F、T208S、T208M、T70D、T70A、T70S、T70G、T70P、T70L、P158R+K80A、P158R+K81P、P158R+K81L、P158R+K81G、P158R+K81S、P158R+R204H、P158R+R204V、P158R+S337H、P158R+E353W、P158R+H350E、P158R+R441P、P158R+F355D、P158R+K80R、P158R+T70D、P158R+T70S、P158R+T70L、P158R+T70G、P158R+V182S、P158R+T208F、P158R+K80A+R204A、P158R+K80A+A299E、P158R+K80A+A202S、P158R+K80A+T208A、P158R+K80A+L261F、P158R+K80A+F205M、P158R+K80A+S337H、P158R+K80A+T208G、P158R+K80A+A302S、P158R+K80A+E353W、P158R+K80A+Q200I、P158R+K80A+K104R、P158R+K80A+T70D、P158R+K80A+R441P、P158R+K80A+F355D、P158R+K80A+R204H、P158R+K80A+D334N、P158R+K80A+D334V、P158R+K80A+R204N、P158R+K80A+W185F、P158R+K80A+W185C、P158R+K80A+N85G、P158R+K80A+R204V、P158R+K80A+Y300C、P158R+K80A+F294L、P158R+K80A+T183V、P158R+K80A+L83I、P158R+K80A+L83M、P158R+K80A+Q200I、P158R+K80A+E304M、P158R+N85G+V349T、P158R+N85G+S348G、P158R+N85G+N318V、P158R+N85G+K346P、P158R+N85G+S165A、P158R+N85G+N233R、P158R+N85G+T70S、P158R+N85G+K346R、P158R+N85G+S348A、P158R+N85G+N605P、P158R+N85G+N605F、P158R+N85G+S225T、P158R+N85G+I447V、P158R+N85G+H350L、P158R+N85G+N605A、P158R+N85G+F355A、P158R+N85G+A322S、P158R+K80A+A302S+K81L、P158R+K80A+A302S+R204H、P158R+K80A+T208G+T70D、P158R+K80A+A302S+R204N、P158R+K80A+A302S+Y300C、P158R+K80A+A302S+F294L、P158R+K80A+A302S+T183V、P158R+K80A+A302S+E304M、P158R+K80A+A302S+D334V、P158R+K80A+A302S+K81G、P158R+K80A+T208G+K311S、P158R+K80A+T208G+N605L、P158R+K80A+T208G+S225E、P158R+K80A+T208G+S225N、P158R+K80A+T208G+P535S、P158R+K80A+T208G+N605F、P158R+K80A+T208G+K311R、P158R+K80A+T208G+P535S、P158R+K80A+T208G+Y228A、P158R+K80A+T208G+Q227T、P158R+K80A+T208G+N605L、P158R+K80A+T208G+K311T、P158R+K80A+T208G+A299V、P158R+K80A+T208G+E522D、P158R+K80A+T208G+Q227M、P158R+K80A+T208G+T442S、P158R+K80A+A302S+K81I、P158R+K80A+A302S+L83I、P158R+K80A+T208G+N605P、P158R+K80A+T208G+N605G、P158R+K80A+T208G+K81D、P158R+K80A+T208G+A322I、P158R+K80A+T208G+F209N、P158R+K80A+T208G+A322L、P158R+K80A+A302S+D334N、P158R+K80A+T208G+K81S、P158R+K80A+T208G+L518F、P158R+K80A+A302S+W185C、P158R+K80A+T208G+K81L、P158R+K80A+A302S+T70N、P158R+K80A+A302S+V114L、P158R+K80A+A302S+F198M、P158R+K80A+A302S+A202C、P158R+K80A+A302S+M206H、P158R+K80A+A302S+M206F、P158R+K80A+A302S+Y228M、P158R+K80A+A302S+E353T、P158R+K80A+A302S+E353N、P158R+K80A+A302S+E353S、P158R+K80A+A302S+I497V、P158R+K80A+A302S+P535T、P158R+K80A+A302S+P535S、P158R+K80A+A302S+P535V、P158R+K80A+A302S+N605D、P158R+K80A+A302S+N605L、P158R+K80A+A302S+E439C、P158R+K80A+A302S+K346P、P158R+K80A+A302S+Y69N、P158R+K80A+A302S+T70D、P158R+K80A+A302S+T70G、P158R+K80A+A302S+T70M、P158R+K80A+A302S+K81S、P158R+K80A+A302S+N85G、P158R+K80A+A302S+V114I、P158R+K80A+A302S+A150V、P158R+K80A+A302S+A175V、P158R+K80A+A302S+W185F、P158R+K80A+A302S+A202S、P158R+K80A+A302S+M206S、P158R+K80A+A302S+F209N、P158R+K80A+A302S+Y228A、P158R+K80A+A302S+Y228L、P158R+K80A+A302S+V287T、P158R+K80A+A302S+V289I、P158R+K80A+A302S+V289L、P158R+K80A+A302S+V321A、P158R+K80A+A302S+R332S、P158R+K80A+A302S+T70N、P158R+K80A+A302S+T70S、P158R+K80A+A302S+V114L、P158R+K80A+A302S+F198M、P158R+K80A+A302S+A202C、P158R+K80A+A302S+M206H、P158R+K80A+A302S+M206F、P158R+K80A+A302S+Y228M、P158R+K80A+A302S+E353T、P158R+K80A+A302S+E353N、P158R+K80A+A302S+E353S、P158R+K80A+A302S+I497V、P158R+K80A+A302S+P535T、P158R+K80A+A302S+P535S、P158R+K80A+A302S+P535V、P158R+K80A+A302S+N605D、P158R+K80A+A302S+N605L、P158R+K80A+A302S+E439C、P158R+K80A+A302S+K346P、P158R+K80A+A302S+Y69N、P158R+K80A+A302S+T70D、P158R+K80A+A302S+T70G、P158R+K80A+A302S+T70M、P158R+K80A+A302S+N85G、P158R+K80A+A302S+V114I、P158R+K80A+A302S+A150V、P158R+K80A+A302S+A175V、P158R+K80A+A302S+W185F、P158R+K80A+A302S+A202S、P158R+K80A+A302S+M206S、P158R+K80A+A302S+F209N、P158R+K80A+A302S+E353Y、P158R+K80A+A302S+N605F、P158R+K80A+A302S+N605G、P158R+K80A+A302S+K81D、P158R+K80A+A302S+A322I、P158R+K80A+A302S+F209N、P158R+K80A+A302S+A322L、P158R+K80A+A302S+L518F、P158R+K80A+A302S+Q227T、P158R+K80A+A302S+K311T、P158R+K80A+A302S+A299V、P158R+K80A+A302S+E522D、P158R+K80A+A302S+Q227M、P158R+K80A+A302S+T442S、P158R+N85G+N605P+K346N、P158R+N85G+N605P+F198M、P158R+N85G+N605P+H350L、P158R+N85G+N605P+K80K、P158R+N85G+N605P+S225M、P158R+N85G+N605P+A302T、P158R+N85G+N605P+T549Y、P158R+N85G+N605P+K81T、P158R+N85G+N605P+K81R、P158R+N85G+N605P+K81E、P158R+N85G+N605P+K81S、P158R+N85G+N605P+K215T、P158R+N85G+N605P+F198W、P158R+N85G+N605P+A299T、P158R+K80A+A302S+N605F+Q227I、P158R+K80A+A302S+N605F+Q227V、P158R+K80A+A302S+N605F+I34T、P158R+N85G+N605P+K346N+S225R、P158R+N85G+N605P+K346N+S225A、P158R+N85G+N605P+K346N+S225T、P158R+K80A+A302S+N605F+S225E、P158R+K80A+A302S+N605F+K81M、P158R+K80A+A302S+N605F+N440T、P158R+K80A+A302S+N605F+T506G、P158R+K80A+A302S+N605F+H350Y、P158R+K80A+A302S+N605F+K81S、P158R+K80A+A302S+N605F+H350T、P158R+K80A+A302S+N605F+T506G、P158R+N85G+N605P+K346N+F198M、P158R+N85G+N605P+K346N+H350L、P158R+N85G+N605P+K346N+S225M、P158R+N85G+N605P+K346N+A302T、P158R+N85G+N605P+K346N+T549Y、P158R+N85G+N605P+K346N+K81T、P158R+N85G+N605P+K346N+K81R、P158R+N85G+N605P+K346N+F198W、P158R+N85G+N605P+K346N+K81E、P158R+N85G+N605P+K346N+K215T、P158R+N85G+N605P+K346N+K81S, P158R+N85G+N605P+K346N+A299T, P158R+N85G+N605P+K346N+ I187L、P158R+N85G+N605P+K346N+S225T+I34K、P158R+N85G+N605P+K346N+S225T+I34A、P158R+N 85G+N605P+K346N+S225T+I34P, P158R+N85G+N605P+K346N+S225T+I34R, P158R+N85G+N605P+K3 46N+S225T+I34H, P158R+N85G+N605P+K346N+S225T+I34S, P158R+N85G+N605P+K346N+S225T+S34 8G, P158R+N85G+N605P+K346N+S225T+I34K+S348G, P158R+N85G+N605P+K346N+S225T+I34K+S34 8A, P158R+N85G+N605P+K346N+S225T+I34K+S348P, P158R+N85G+N605P+K346N+S225T+I34K+S348 K. P158R+N85G+N605P+K346N+S225T+I34K+S348N, P158R+N85G+N605P+K346N+S225T+I34K+S348M , P158R+N85G+N605P+K346N+S225T+I34K+S348C or P158R+N85G+N605P+K346N+S225T+I34K+S348V. ,

[0017] To achieve the above objectives, according to a second aspect of the present invention, a gene is provided that encodes the aforementioned α-amino acid ester acyltransferase mutant.

[0018] To achieve the above objectives, according to a third aspect of the present invention, a plasmid is provided, the plasmid containing the above-described gene.

[0019] To achieve the above objectives, according to a fourth aspect of the present invention, a non-plant or animal cell is provided, wherein the non-plant or animal cell comprises the aforementioned gene or plasmid.

[0020] To achieve the above objective, according to a fifth aspect of the present invention, a method for synthesizing oligopeptides is provided, the method comprising: synthesizing the oligopeptides using the above-described α-amino acid ester acyltransferase mutant.

[0021] Furthermore, the above method includes: using the above-mentioned α-amino acid ester acyltransferase mutant to catalyze the substrate to obtain the above-mentioned oligopeptide.

[0022] Furthermore, the aforementioned substrates include acyl donors and acyl acceptors;

[0023] The acyl donor is an amino acid ester hydrochloride, which is selected from any one of the following: methyl ester hydrochloride of amino acid, ethyl ester hydrochloride of amino acid, or isopropyl ester hydrochloride of amino acid.

[0024] The aforementioned acyl receptor comprises an amino acid or a first peptide segment; the aforementioned first peptide segment is composed of condensation of 2 to 19 amino acids.

[0025] By applying the technical solution of this invention, a wild-type α-amino acid ester acyltransferase with high catalytic activity for Val-Ser dipeptide (encoding gene with the nucleotide sequence shown in SEQ ID NO: 1) was designed through ancestral enzyme reconstruction. Furthermore, the above-mentioned α-amino acid ester acyltransferase was mutated to obtain a variety of α-amino acid ester acyltransferase mutants with broad substrate spectrum and high activity.

[0026] The aforementioned α-amino acid ester acyltransferase mutants include: (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; (b) at least one of the following sites in the aforementioned amino acid sequence in (a): P158, I34, Y69, T70, P73, Y74, K80, K81, L83, N85, K104, V114, A150, S165, A175, V182, T183, W185, I187, F198, Q200, A202, R204, F205, M206, T208, F209, K215, S225, Q227, Y228, N233, L261, V287, V289, F294, A299, Y3 The following are proteins that have undergone amino acid mutations and possess α-amino acid ester acyltransferase function: A302, E304, K311, N318, V321, A322, R332, D334, S337, K346, S348, V349, H350, E353, F355, E439, N440, R441, T442, I447, I497V, T506, L518, E522, P535, T549, or N605; or (c) proteins that have more than 80% homology with the amino acid sequences defined in (a) or (b) and possess α-amino acid ester acyltransferase function. The α-amino acid ester acyltransferase mutants of the present invention have a broad substrate spectrum and catalytic activity, and can be used to synthesize a variety of oligopeptides. Detailed Implementation

[0027] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0028] As mentioned in the background section, existing α-amino acid ester acyltransferases for oligopeptide synthesis suffer from narrow substrate spectra and low catalytic activity. In this invention, the inventors designed a wild-type α-amino acid ester acyltransferase (encoding gene with the nucleotide sequence shown in SEQ ID NO: 1) exhibiting high catalytic activity for the Val-Ser dipeptide through ancestral enzyme reconstruction. Subsequently, researchers obtained the three-dimensional structure of this enzyme using homology modeling and AI prediction methods, and mutated key sites on the protein surface and near the active site. Using valine methyl ester and serine as substrates, the activity of the mutated enzyme was tested, yielding a series of α-amino acid ester acyltransferase mutants capable of efficiently synthesizing oligopeptides. Using some of these α-amino acid ester acyltransferase mutants for the synthesis of different oligopeptides, it was found that, in addition to the Val-Ser dipeptide, these mutants can also be used for the efficient synthesis of various other oligopeptides.

[0029] The α-amino acid ester acyltransferases (including wild-type and mutants) of the present invention have a broad substrate spectrum and high catalytic activity. Therefore, a protection scheme for the present invention is proposed.

[0030] In a first typical embodiment of the present invention, an α-amino acid ester acyltransferase mutant is provided, the α-amino acid ester acyltransferase mutant comprising:

[0031] (a) A protein having the amino acid sequence shown in SEQ ID NO: 1;

[0032] (b) at least one of the following sites in the above amino acid sequence in (a): P158, I34, Y69, T70, P73, Y74, K80, K81, L83, N85, K104, V114, A150, S165, A175, V182, T183, W185, I187, F198, Q200, A202, R204, F205, M206, T208, F209, K215, S225, Q227, Y228, N233, L261, V287 Proteins containing the following amino acid mutations and possessing α-amino acid ester acyltransferase function: V289, F294, A299, Y300, A302, E304, K311, N318, V321, A322, R332, D334, S337, K346, S348, V349, H350, E353, F355, E439, N440, R441, T442, I447, I497V, T506, L518, E522, P535, T549, or N605; or

[0033] (c) A protein that has more than 80% homology with the amino acid sequence defined in (a) or (b) and has α-amino acid ester acyltransferase function.

[0034] It should be noted that homology in this invention refers to the "sequence identity" between two amino acid sequences, that is, the percentage of identical amino acids between the sequences. Methods for assessing the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, it can be determined using the BLAST program of the NCBI database. For information on the determination of sequence identity, see, for example: Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Primers for Sequence Analysis, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991.

[0035] The proteins described above that have 70%, 75%, 80%, 85%, 90%, 95%, or more than 99% (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 more than 99.9%) homology with the mutants shown in SEQ ID NO: 1 and possess α-amino acid ester acyltransferase function, have active sites, active pockets, active mechanisms, and protein structures that are highly likely to be the same as the proteins provided in (a) of the corresponding proteins.

[0036] Amino acid residues can be represented by three-letter or one-letter amino acid codes according to standards known and agreed upon in the art. In this document, the abbreviations for amino acid residues 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).

[0037] Conservative amino acid substitutions or replacements are well known in the art. For example, a conserved amino acid substitution is preferably one amino acid residue from the following group (1)-(5) replaced by another amino acid from the same group: (1) smaller aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (2) negatively charged polar residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (3) positively charged polar residues: His, Arg and Lys; (4) larger aliphatic nonpolar residues: Met, Leu, Ile, Val and Cys; and (5) aromatic residues: Phe, Tyr and Trp. The particularly preferred conserved amino acid substitutions are as follows: Ala is substituted by Gly or Ser; Arg is substituted by Lys; Asn is substituted by Gln or His; Asp is substituted by Glu; Cys is substituted by Ser; Gln is substituted by Asn; Glu is substituted by Asp; Gly is substituted by Ala or Pro; His is substituted by Asn or Gln; Ile is substituted by Leu or Val; Leu is substituted by Ile or Val; Lys is substituted by Arg, Gln, or Glu; Met is substituted by Leu, Tyr, or Ile; Phe is substituted by Met, Leu, or Tyr; Ser is substituted by Thr; Thr is substituted by Ser; Trp is substituted by Tyr; Tyr is substituted by Trp or Phe; and Val is substituted by Ile or Leu.

[0038] 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.

[0039] The following examples use the α-amino acid ester acyltransferase with the amino acid sequence shown in SEQ ID NO: 1 as the parent material for site-directed mutagenesis to obtain various variants.

[0040] The amino acid sequence shown in SEQ ID NO: 1 is as follows:

[0041] The amino acid sequence shown in SEQ ID NO: 1 is a wild-type α-amino acid ester acyltransferase (encoding gene with the nucleotide sequence shown in SEQ ID NO: 1) with high catalytic activity against the Val-Ser dipeptide, designed through ancestral enzyme reconstruction. Homology modeling and AI prediction of the enzyme's amino acid sequence revealed 65 amino acid residues, including: P158, I34, Y69, T70, P73, Y74, K80, K81, L83, N85, K104, V114, A150, S165, A175, V182, T183, W185, I187, F198, Q200, A202, R204, F205, M206, T208, F209, K215, S225, and Q22. 7. Y228, N233, L261, V287, V289, F294, A299, Y300, A302, E304, K311, N318, V321, A322, R332, D334, S337, K346, S348, V349, H350, E353, F355, E439, N440, R441, T442, I447, I497V, T506, L518, E522, P535, T549 or N605.

[0042] These amino acid sites are located near the active site and may affect the protein's catalytic properties and substrate selectivity. Mutating these amino acid sites can yield proteins with α-amino acid ester acyltransferase function, or even enhanced α-amino acid ester acyltransferase function and a broadened substrate spectrum. For the proteins obtained above, changes can be made at non-critical mutation sites and active sites to obtain proteins with over 80% homology to the above amino acid sequence and possess α-amino acid ester acyltransferase function.

[0043] In a preferred embodiment of the present invention, the amino acid mutation in (b) above is selected from at least one of the following sites:

[0044] P158R, I34T or I34K or I34A or I34P or I34R or I34H or I34S, Y69N, T70D or T70A or T70S or T70G or T70P or T70L or T70N or T70M, P73D, K80A or K80S or K80C or K80R or K80G, K81P or K81L or K81G or K81I or K81D or K81S or K81M or K81T or K81R or K81E, L83I or L83M or L83V, N85Y or N85G, K104R, V114L or V114I, A150V, S165A, A175V, V18 2F or V182S, T183V, W185S or W185G or W185L or W185F or W185C, I187L, F198M or F198W, Q200I, A202S or A202C, R204A or R204H or R204V or R204N, F205M, M206H or M206F or M206S, T208G or T208F or T208S or T208M or T208A, F209N, K215T, S225R or S225T or S225E or S225A or S225M or S225N, Q227T or Q227M or Q227V or Q227I, Y228A or Y228M or Y228L, N233R, L261F, V287T, V289I or V289L, F294L, A299E or A299V or A299T, Y300C, A302S or A302T, E304M, K311S or K311R or K311T, N318V, V321A or A322S or A322L or A322I, R332S, D334V or D334N, S337H or S337S, K346P or K346R or K346N, S348A or S348G or S348P or S348K or S348N or S348M or S3 48C or S348V, V349T, H350L or H350Y or H350T or H350E, E353W or E353T or E353N or E353S or E353Y, F355D or F355A, E439C, N440T, R441P, T442S, I447V, I497V, T506G, L518F, E522D, P535S or P535T or P535S or P535V, T549Y or N605P or N605F or N605A or N605L or N605G or N605D; where the letter before the number represents the original amino acid, and the letter after the number represents the mutated amino acid.

[0045] In this invention, the applicant further investigated the aforementioned active site and discovered that mutations to different amino acids at the active site resulted in varying protein activities, with specific mutations enhancing the activity of α-amino acid ester acyltransferases. Through experimental investigation, it was found that specific mutations at the active site could yield α-amino acid ester acyltransferases with enhanced activity. The amino acid mutation sites for α-amino acid ester acyltransferase proteins can be flexibly selected and combined from the aforementioned mutations.

[0046] In a preferred embodiment of the present invention, the mutation of the above-mentioned α-amino acid ester acyltransferase mutant includes any one of the following amino acid mutations: P158R, K81P, N85Y, V182F, V182S, W185S, W185G, W185L, P73D, K80A, K80S, K80C, K80R, K80G, T208G, T208F, T208S, T208M, T70D, T70A, T70S, T70G, T70P, T70L, P158R+K80A, P158R+K81P, P158R+K81L, P158R+K81G, P158R+K81S, P158R+R204H, P1 58R+R204V, P158R+S337H, P158R+E353W, P158R+H350E, P158R+R441P, P15 8R+F355D, P158R+K80R, P158R+T70D, P158R+T70S, P158R+T70L, P158R+T70 G. P158R+V182S, P158R+T208F, P158R+K80A+R204A, P158R+K80A+A299E, P1 58R+K80A+A202S, P158R+K80A+T208A, P158R+K80A+L261F, P158R+K80A+F2 05M, P158R+K80A+S337H, P158R+K80A+T208G, P158R+K80A+A302S, P158R+ K80A+E353W, P158R+K80A+Q200I, P158R+K80A+K104R, P158R+K80A+T70D, P 158R+K80A+R441P, P158R+K80A+F355D, P158R+K80A+R204H, P158R+K80A+ D334N, P158R+K80A+D334V, P158R+K80A+R204N, P158R+K80A+W185F, P158R +K80A+W185C, P158R+K80A+N85G, P158R+K80A+R204V, P158R+K80A+Y300C , P158R+K80A+F294L, P158R+K80A+T183V, P158R+K80A+L83I, P158R+K80A+ L83M, P158R+K80A+Q200I, P158R+K80A+E304M, P158R+N85G+V349T, P158R+ N85G+S348G, P158R+N85G+N318V, P158R+N85G+K346P, P158R+N85G+S165A,P158R+N85G+N233R、P158R+N85G+T70S、P158R+N85G+K346R、P158R+N85G+S348A、P158R+N85G+N605P、P158R+N85G+N605F、P158R+N85G+S225T、P158R+N85G+I447V、P158R+N85G+H350L、P158R+N85G+N605A、P158R+N85G+F355A、P158R+N85G+A322S、P158R+K80A+A302S+K81L、P158R+K80A+A302S+R204H、P158R+K80A+T208G+T70D、P158R+K80A+A302S+R204N、P158R+K80A+A302S+Y300C、P158R+K80A+A302S+F294L、P158R+K80A+A302S+T183V、P158R+K80A+A302S+E304M、P158R+K80A+A302S+D334V、P158R+K80A+A302S+K81G、P158R+K80A+T208G+K311S、P158R+K80A+T208G+N605L、P158R+K80A+T208G+S225E、P158R+K80A+T208G+S225N、P158R+K80A+T208G+P535S、P158R+K80A+T208G+N605F、P158R+K80A+T208G+K311R、P158R+K80A+T208G+P535S、P158R+K80A+T208G+Y228A、P158R+K80A+T208G+Q227T、P158R+K80A+T208G+N605L、P158R+K80A+T208G+K311T、P158R+K80A+T208G+A299V、P158R+K80A+T208G+E522D、P158R+K80A+T208G+Q227M、P158R+K80A+T208G+T442S、P158R+K80A+A302S+K81I、P158R+K80A+A302S+L83I、P158R+K80A+T208G+N605P、P158R+K80A+T208G+N605G、P158R+K80A+T208G+K81D、P158R+K80A+T208G+A322I、P158R+K80A+T208G+F209N、P158R+K80A+T208G+A322L、P158R+K80A+A302S+D334N、P158R+K80A+T208G+K81S、P158R+K80A+T208G+L518F、P158R+K80A+A302S+W185C、P158R+K80A+T208G+K81L、P158R+K80A+A302S+T70N、P158R+K80A+A302S+V114L、P158R+K80A+A302S+F198M、P158R+K80A+A302S+A202C、P158R+K80A+A302S+M206H、P158R+K80A+A302S+M206F、P158R+K80A+A302S+Y228M、P158R+K80A+A302S+E353T、P158R+K80A+A302S+E353N、P158R+K80A+A302S+E353S、P158R+K80A+A302S+I497V、P158R+K80A+A302S+P535T、P158R+K80A+A302S+P535S、P158R+K80A+A302S+P535V、P158R+K80A+A302S+N605D、P158R+K80A+A302S+N605L、P158R+K80A+A302S+E439C、P158R+K80A+A302S+K346P、P158R+K80A+A302S+Y69N、P158R+K80A+A302S+T70D、P158R+K80A+A302S+T70G、P158R+K80A+A302S+T70M、P158R+K80A+A302S+K81S、P158R+K80A+A302S+N85G、P158R+K80A+A302S+V114I、P158R+K80A+A302S+A150V、P158R+K80A+A302S+A175V、P158R+K80A+A302S+W185F、P158R+K80A+A302S+A202S、P158R+K80A+A302S+M206S、P158R+K80A+A302S+F209N、P158R+K80A+A302S+Y228A、P158R+K80A+A302S+Y228L、P158R+K80A+A302S+V287T、P158R+K80A+A302S+V289I、P158R+K80A+A302S+V289L、P158R+K80A+A302S+V321A、P158R+K80A+A302S+R332S、P158R+K80A+A302S+T70N、P158R+K80A+A302S+T70S、P158R+K80A+A302S+V114L、P158R+K80A+A302S+F198M、P158R+K80A+A302S+A202C、P158R+K80A+A302S+M206H、P158R+K80A+A302S+M206F、P158R+K80A+A302S+Y228M、P158R+K80A+A302S+E353T、P158R+K80A+A302S+E353N、P158R+K80A+A302S+E353S、P158R+K80A+A302S+I497V、P158R+K80A+A302S+P535T、P158R+K80A+A302S+P535S、P158R+K80A+A302S+P535V、P158R+K80A+A302S+N605D、P158R+K80A+A302S+N605L、P158R+K80A+A302S+E439C、P158R+K80A+A302S+K346P、P158R+K80A+A302S+Y69N、P158R+K80A+A302S+T70D、P158R+K80A+A302S+T70G、P158R+K80A+A302S+T70M、P158R+K80A+A302S+N85G、P158R+K80A+A302S+V114I、P158R+K80A+A302S+A150V、P158R+K80A+A302S+A175V、P158R+K80A+A302S+W185F、P158R+K80A+A302S+A202S、P158R+K80A+A302S+M206S、P158R+K80A+A302S+F209N、P158R+K80A+A302S+E353Y、P158R+K80A+A302S+N605F、P158R+K80A+A302S+N605G、P158R+K80A+A302S+K81D、P158R+K80A+A302S+A322I、P158R+K80A+A302S+F209N、P158R+K80A+A302S+A322L、P158R+K80A+A302S+L518F、P158R+K80A+A302S+Q227T、P158R+K80A+A302S+K311T、P158R+K80A+A302S+A299V、P158R+K80A+A302S+E522D、P158R+K80A+A302S+Q227M、P158R+K80A+A302S+T442S、P158R+N85G+N605P+K346N、P158R+N85G+N605P+F198M、P158R+N85G+N605P+H350L、P158R+N85G+N605P+K80K、P158R+N85G+N605P+S225M、P158R+N85G+N605P+A302T、P158R+N85G+N605P+T549Y、P158R+N85G+N605P+K81T、P158R+N85G+N605P+K81R、P158R+N85G+N605P+K81E、P158R+N85G+N605P+K81S、P158R+N85G+N605P+K215T、P158R+N85G+N605P+F198W、P158R+N85G+N605P+A299T、P158R+K80A+A302S+N605F+Q227I、P158R+K80A+A302S+N605F+Q227V、P158R+K80A+A302S+N605F+I34T、P158R+N85G+N605P+K346N+S225R、P158R+N85G+N605P+K346N+S225A、P158R+N85G+N605P+K346N+S225T、P158R+K80A+A302S+N605F+S225E、P158R+K80A+A302S+N605F+K81M、P158R+K80A+A302S+N605F+N440T、P158R+K80A+A302S+N605F+T506G、P158R+K80A+A302S+N605F+H350Y、P158R+K80A+A302S+N605F+K81S、P158R+K80A+A302S+N605F+H350T、P158R+K80A+A302S+N605F+T506G、P158R+N85G+N605P+K346N+F198M、P158R+N85G+N605P+K346N+H350L、P158R+N85G+N605P+K346N+S225M、P158R+N85G+N605P+K346N+A302T、P158R+N85G+N605P+K346N+T549Y、P158R+N85G+N605P+K346N+K81T、P158R+N85G+N605P+K346N+K81R、P158R+N85G+N605P+K346N+F198W、P158R+N85G+N605P+K346N+K81E, P158R+N85G+N605P+K346N+K215T, P158R+N85G+N605P+K346N+K81S, P1 58R+N85G+N605P+K346N+A299T, P158R+N85G+N605P+K346N+I187L, P158R+N85G+N605P+K346N+S225T+I34 K. P158R+N85G+N605P+K346N+S225T+I34A, P158R+N85G+N605P+K346N+S225T+I34P, P158R+N85G+N605P+K 346N+S225T+I34R, P158R+N85G+N605P+K346N+S225T+I34H, P158R+N85G+N605P+K346N+S225T+I34S, P158 R+N85G+N605P+K346N+S225T+S348G, P158R+N85G+N605P+K346N+S225T+I34K+S348G, P158R+N85G+N605P +K346N+S225T+I34K+S348A, P158R+N85G+N605P+K346N+S225T+I34K+S348P, P158R+N85G+N605P+K346N+S 225T+I34K+S348K, P158R+N85G+N605P+K346N+S225T+I34K+S348N, P158R+N85G+N605P+K346N+S225T+I34K+S348M, P158R+N85G+N605P+K346N+S225T+I34K+S348C or P158R+N85G+N605P+K346N+S225T+I34K+S348V.

[0047] All of the above-mentioned amino acid mutations were experimentally investigated in the embodiments of the present invention, and all of them have α-amino acid ester acyltransferase activity. Compared with the parent with the amino acid sequence shown in SEQ ID NO: 1, α-amino acid ester acyltransferase mutants with high enzyme activity can be obtained.

[0048] In a second typical embodiment of the present invention, a gene is provided that encodes the aforementioned α-amino acid ester acyltransferase mutant.

[0049] In a third typical embodiment of the present invention, a plasmid is provided, the plasmid containing the above-described gene.

[0050] The aforementioned gene encodes the α-amino acid ester acyltransferase mutant and can be ligated onto a plasmid to form a circular DNA. Both the gene and the plasmid can be transcribed and translated by RNA polymerase, ribosomes, and tRNA to obtain the aforementioned α-amino acid ester acyltransferase mutant.

[0051] In a fourth typical embodiment of the present invention, a non-animal / plant cell is provided, wherein the non-animal / plant cell includes the aforementioned plasmid.

[0052] The aforementioned non-plant and animal cells can replicate the plasmids and transcribe and translate the genes carried on the plasmids, yielding a large number of α-amino acid ester acyltransferase mutants. Using existing technology, the non-plant and animal cells are disrupted, and the crude enzymes catalyze the substrates to synthesize various oligopeptides.

[0053] In a fifth typical embodiment of the present invention, a method for synthesizing oligopeptides is provided, the method comprising: synthesizing the oligopeptide using the aforementioned α-amino acid ester acyltransferase mutant. The α-amino acid ester acyltransferase mutant of the present invention has the beneficial effects of high activity and a broad substrate spectrum. Therefore, the method for synthesizing oligopeptides using the α-amino acid ester acyltransferase mutant of the present invention has the advantage of high efficiency and can synthesize a variety of oligopeptides synthesized by enzymatic methods not disclosed in the prior art. In a preferred embodiment of the present invention, the oligopeptide is a dipeptide.

[0054] In a preferred embodiment of the present invention, the method comprises: using the above-mentioned α-amino acid ester acyltransferase mutant to catalyze a substrate to obtain the above-mentioned oligopeptide. In a preferred embodiment of the present invention, the substrate comprises an acyl donor and an acyl acceptor; the acyl donor is an ester hydrochloride of an amino acid, and the ester hydrochloride of the amino acid is selected from any one of the following: methyl ester hydrochloride, ethyl ester hydrochloride, or isopropyl ester hydrochloride of an amino acid; the acyl acceptor comprises an amino acid or a first peptide segment; the first peptide segment is formed by the condensation of 2 to 19 amino acids. The substrate spectrum of the α-amino acid ester acyltransferase mutant of the present invention is relatively broad, and it can synthesize a variety of oligopeptides.

[0055] The present invention will be further described in detail below with reference to specific embodiments, which should not be construed as limiting the scope of protection claimed by the present invention.

[0056] Example 1: Preparation of α-amino acid ester acyltransferase (Aet) enzyme solution

[0057] An α-amino acid ester acyltransferase was designed using ancestral enzyme reconstruction. The encoding gene of the α-amino acid ester acyltransferase (encoding the amino acid sequence shown in SEQ ID NO: 1, named wild-type α-amino acid ester acyltransferase in this invention) was obtained by artificial chemical synthesis. An endonuclease site NdeI was introduced at the 5' end and an endonuclease site XhoI was introduced at the 3' end. The gene was synthesized in pUC19 to obtain pUC19-Aet.

[0058] The expression vector pET-28a(+) was double-digested (NdeI+XhoI), and the gene pUC19-Aet, which encodes α-amino acid ester acyltransferase, was also double-digested (NdeI+XhoI). The excised Aet gene fragment was ligated into the expression vector pET-28a(+) to obtain the recombinant E. coli plasmid pET-28a(+)-Aet containing α-amino acid ester acyltransferase. This plasmid was then transformed into E. coli BL21(DE3) to obtain the recombinant E. coli strain BL21(DE3)+Aet.

[0059] Take 4 ml of BL21(DE3) strain containing recombinant plasmid and inoculate it into a 2L Erlenmeyer flask containing 400 mL of LB medium. Incubate at 37℃ with shaking at 200 rpm for 2-3 h. When the OD600 reaches 0.6-0.8, add IPTG to a final concentration of 0.02 mM and induce at 16℃ for 18 h. After induction, collect the bacterial cells by centrifugation at 4℃. Resuspend the bacterial cells in 10 mL of 0.1 M Tris-HCl (pH 8.0) per gram of bacterial sludge. Disrupt the cells by sonication (power: 20%, time: 5 min), centrifuge (temperature: 4℃, time: 10 min, speed: 12000 rpm), and collect the supernatant to obtain the crude enzyme solution for the catalytic reaction.

[0060] Note: The experimental materials and reagents used in the examples are described below:

[0061] 1. Strains and vectors: Escherichia coli expression vector pET-28a(+) and strain BL21(DE3)

[0062] 2. Culture medium: Escherichia coli LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0).

[0063] Example 2: Catalytic synthesis of Val-Ser dipeptide

[0064] In this embodiment, a designed α-amino acid ester acyltransferase (whose encoding gene has the amino acid sequence shown in SEQ ID NO: 1, named wild-type α-amino acid ester acyltransferase in this invention) was selected for the synthesis of Val-Ser dipeptide. The reaction system was as follows (1 mL): 100 mM valine methyl ester hydrochloride, 100 mM serine, 50 μL crude enzyme solution, and 0.1 M Tris-HCl (pH 8.0), reacted at 20 °C for 2 h. The reaction system was heated at 80 °C for 10 min, centrifuged, and analyzed by HPLC. The reaction results are shown in Table 1.

[0065] Table 1:

[0066] Note: This table shows the reaction results of α-amino acid ester acyltransferase (named wild-type α-amino acid ester acyltransferase in this invention, whose encoding gene has the amino acid sequence shown in SEQ ID NO: 1). * represents 1% ≤ conversion rate < 5%, ** represents 5% ≤ conversion rate < 10%. The conversion rate of this invention is calculated using the formula: (moles of product / moles of amino acid methyl ester substrate) × 100%.

[0067] Example 3 Enzyme Evolution

[0068] Enzymatic evolution was performed on α-amino acid ester acyltransferase (named wild-type α-amino acid ester acyltransferase in this invention, whose encoding gene has the amino acid sequence shown in SEQ ID NO: 1). The evolved mutants were tested for activity using valine methyl ester hydrochloride and serine as substrates. The reaction system was as follows (1 mL): 100 mM valine methyl ester hydrochloride, 100 mM serine, 50 μL crude enzyme solution, and 0.1 M Tris-HCl (pH 8.0). The reaction was carried out at 20 °C for 2 h. The reaction system was then heated at 80 °C for 10 min, centrifuged, and analyzed by HPLC. The results are shown in Table 2.

[0069] Table 2:

[0070] Note: * represents 1% ≤ conversion rate < 5%, ** represents 5% ≤ conversion rate < 10%, and *** represents 10% ≤ conversion rate < 15%. The conversion rate is calculated as: (moles of product / moles of amino acid methyl ester substrate) * 100%.

[0071] The experimental results above show that after single-point mutation of wild-type α-amino acid ester acyltransferase, the activity of some mutants is significantly increased. The mutant with the best activity was selected as the template for the next round of evolution.

[0072] Example 4

[0073] The best mutant obtained in Example 3 was subjected to another round of enzyme evolution. The mutant was tested for enzyme activity using valine methyl ester hydrochloride and serine as substrates. The reaction system was as follows (1 mL): 100 mM valine methyl ester hydrochloride, 100 mM serine, 50 μL crude enzyme solution, 0.1 M Tris-HCl (pH 8.0), reacted at 20 °C for 2 h. The reaction system was then heated at 80 °C for 10 min, centrifuged, and analyzed by HPLC. The results are shown in Table 3.

[0074] Table 3:

[0075] Note: * represents 1% ≤ conversion rate < 5%, ** represents 5% ≤ conversion rate < 10%, *** represents 10% ≤ conversion rate < 15%, **** represents 15% ≤ conversion rate < 20%, ***** represents 20% ≤ conversion rate < 30%, ****** represents 30% ≤ conversion rate < 40%.

[0076] The experimental results above show that after further mutation of the best mutant in Example 3, the activity of some mutants increased significantly. The mutants with good activity were selected as templates for the next round of evolution.

[0077] Example 5

[0078] The best mutant obtained in Example 4 was subjected to another round of enzyme evolution. The mutant was tested for enzyme activity using valine methyl ester hydrochloride and serine as substrates. The reaction system was as follows (1 mL): 200 mM valine methyl ester hydrochloride, 200 mM serine, 25 μL crude enzyme solution, 0.1 M Tris-HCl (pH 8.0), reacted at 20 °C for 2 h. The reaction system was then heated at 80 °C for 10 min, centrifuged, and analyzed by HPLC. The results are shown in Tables 4 and 5.

[0079] Table 4:

[0080] Table 5

[0081] Note: In Tables 4 and 5, ** represents 5% ≤ conversion rate < 10%, *** represents 10% ≤ conversion rate < 15%, **** represents 15% ≤ conversion rate < 20%, ***** represents 20% ≤ conversion rate < 30%, and ****** represents 30% ≤ conversion rate < 40%.

[0082] The experimental results above show that further mutation of the best mutant in Example 4 doubled the substrate concentration for the activity test and halved the enzyme amount. Some mutants still showed a very significant increase in activity. The mutant with good activity was selected as the template for the next round of evolution.

[0083] Example 6

[0084] The best mutant obtained in Example 5 was subjected to another round of enzyme evolution. The mutant was tested for enzyme activity using valine methyl ester hydrochloride and serine as substrates. The reaction system was as follows (1 mL): 200 mM valine methyl ester hydrochloride, 200 mM serine, 25 μL crude enzyme solution, 0.1 M Tris-HCl (pH 8.0), reacted at 20 °C for 2 h. The reaction system was then heated at 80 °C for 10 min, centrifuged, and analyzed by HPLC. The results are shown in Table 6.

[0085] Table 6:

[0086] Note: * represents 1% ≤ conversion rate < 5%, ** represents 5% ≤ conversion rate < 10%, *** represents 10% ≤ conversion rate < 15%, **** represents 15% ≤ conversion rate < 20%, ***** represents 20% ≤ conversion rate < 40%, ****** represents 40% ≤ conversion rate < 60%, ******* represents 60% ≤ conversion rate < 80%. The experimental results above show that further mutations of the optimal mutant in Example 5 still resulted in some mutants exhibiting very significant increases in activity.

[0087] Example 7

[0088] Different mutants were selected from Examples 3 to 6 to investigate the activity of synthesized oligopeptides Gly-Ser, Gly-Gly, Ser-Ser, Val-Ser, Tyr-Ser, Gly-Gly-Gly, and Gly-His-Lys. The reaction system was as follows (10 mL): 500 mM amino acid methyl ester hydrochloride (acyl donor), 500 mM amino acid (acyl acceptor), 50 μL crude enzyme solution, 0.1 M Tris-HCl (pH 8.0), and the reaction was carried out at 20 °C for 2 h. The reaction system was heated at 80 °C for 10 min, centrifuged, and analyzed by HPLC. The reaction results are summarized in Table 7.

[0089] Table 7:

[0090] Note: **** represents 50% ≤ conversion rate < 60%, ***** represents 60% ≤ conversion rate < 70%, and ****** represents 70% ≤ conversion rate < 90%.

[0091] The experimental results above show that different mutants obtained from the evolution of Val-Ser can also exhibit good catalytic activity in the synthesis of oligopeptides Gly-Ser, Gly-Gly, Ser-Ser, Val-Ser, Tyr-Ser, Gly-Gly-Gly, and Gly-His-Lys.

[0092] Example 8

[0093] The enzymes with good activity in the above examples, P158R+N85G+N605P+K346N+S225T+I34K+S348G and wild-type α-amino acid ester acyltransferase, were reacted with methyl ester hydrochloride of 19 amino acids and 19 amino acids to investigate the changes in substrate spectrum range after enzyme evolution. The reaction system was as follows (1 mL): 200 mM of different amino acid methyl ester hydrochloride (first column of the table below), 200 mM of different amino acids (first row of the table below), 25 μL of crude enzyme solution, 0.1 M Tris-HCl (pH 8.0), and reacted at 20 °C for 2 h. The reaction system was heated at 80 °C for 10 min, centrifuged, and analyzed by LC-MS. The reaction results are summarized as follows (Tables 8 and 9):

[0094] Table 8 (Wild-type α-amino acid ester acyltransferases):

[0095] Table 9 (Mutant P158R+N85G+N605P+K346N+S225T+I34K+S348G):

[0096] Note: * represents 10 ≤ conversion rate < 30%, ** represents conversion rate ≥ 30% and < 50%, and *** represents conversion rate ≥ 50%. As shown above, compared to wild-type α-amino acid ester acyltransferases, the mutant exhibits a broader substrate spectrum and significantly improved activity for the synthesis of various dipeptides.

[0097] Example 9

[0098] The synthesis of Gly-Ser, Ser-Ser, Val-Ser, and Tyr-Ser from P158R+N85G+N605P+K346N+S225T+I34K+S348G was scaled up. The reaction system was as follows (1 L): 500 mM glycine methyl ester hydrochloride and alanine methyl ester hydrochloride, 500 mM tyrosine and glutamine, 25 mL crude enzyme solution, 0.1 M Tris-HCl (pH 8.0), and reaction at 20 °C for 2 h. After the reaction, the reaction system was post-processed for separation and purification. The purity and content of the final products were analyzed by HPLC and NMR. The results are summarized in Table 10.

[0099] Table 10:

[0100] The conversion rate is calculated as follows: (moles of product / moles of amino acid methyl ester substrate) × 100%; the product purity is calculated as follows: (peak area of ​​product on HPLC / peak area of ​​all peaks) × 100%; and the separation yield is calculated as follows: (actual yield / theoretical yield) × 100%.

[0101] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: In the present invention, α-amino acid ester acyltransferase evolution is performed on the Val-Ser dipeptide to obtain a series of mutants with enhanced activity. The α-amino acid ester acyltransferase mutants obtained during evolution can be used not only for the efficient synthesis of Val-Ser, but also for the synthesis of various oligopeptides such as Gly-Ser, Gly-Gly, Ser-Ser, Val-Ser, Tyr-Ser, Gly-Gly-Gly, and Gly-His-Lys. The α-amino acid ester acyltransferase mutants have a broad substrate spectrum and high synthesis efficiency for some oligopeptides. The α-amino acid ester acyltransferases obtained in the present invention can be well used for industrial scale-up, with low cost and high yield, achieving true green chemistry.

[0102] 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. An α-amino acid ester acyltransferase mutant, characterized in that, The α-amino acid ester acyltransferase mutants include: (a) A protein having the amino acid sequence shown in SEQ ID NO: 1; (b) at least one of the following sites in the amino acid sequence described in (a): P158, K80, N85, I34, Y69, T70, P73, Y74, K81, L83, K104, V114, A150, S165, A175, V182, T183, W185, I187, F198, Q200, A202, R204, F205, M206, T208, F209, K215, S225, Q227, Y228, N233, L261, V287 Proteins containing the following amino acid mutations and possessing α-amino acid ester acyltransferase function: V289, F294, A299, Y300, A302, E304, K311, N318, V321, A322, R332, D334, S337, K346, S348, V349, H350, E353, F355, E439, N440, R441, T442, I447, I497V, T506, L518, E522, P535, T549, or N605; or (c) A protein having more than 80% homology with the amino acid sequence defined in (a) or (b) and having α-amino acid ester acyltransferase function; The amino acid mutation in (b) is selected from at least one of the following sites: P158R, K80A or K80S or K80C or K80R or K80G, N85Y or N85G, I34T or I34K or I34A or I34P or I34R or I34H or I34S, Y69N, T70D or T70A or T70S or T70G or T70P or T70L or T70N or T70M, P73D, K81P or K81L or K81G or K81I or K81D or K81S or K81M or K81T or K81R or K81E, L83I or L83M or L83V, K104R, V114L or V114I, A150V, S165A, A1 75V, V182F or V182S, T183V, W185S or W185G or W185L or W185F or W185C, I187L, F198M or F198W, Q200I, A202S or A202C, R204A or R204H or R204V or R204N, F205M, M206H or M206F or M206S, T208G or T208F or T208S or T208M or T208A, F209N, K215T, S225R or S225T or S225E or S225A or S225M or S225N, Q227T or Q227 M or Q227V or Q227I, Y228A or Y228M or Y228L, N233R, L261F, V287T, V289I or V289L, F294L, A299E or A299V or A299T, Y300C, A302S or A302T, E304M, K311S or K311R or K311T, N318V, V321A or, A322S or A322L or A322I, R332S, D334V or D334N, S337H or S337S, K346P or K346R or K346N, S348A or S348G or S348 P or S348K or S348N or S348M or S348C or S348V, V349T, H350L or H350Y or H350T or H350E, E353W or E353T or E353N or E353S or E353Y, F355D or F355A, E439C, N440T, R441P, T442S, I447V, I497V, T506G, L518F, E522D, P535S or P535T or P535S or P535V, T549Y or N605P or N605F or N605A or N605L or N605G or N605D; In this context, the letter before the number represents the original amino acid, and the letter after the number represents the mutated amino acid.

2. The α-amino acid ester acyltransferase mutant according to claim 1, characterized in that, The mutations in the α-amino acid ester acyltransferase mutant include any one of the following amino acid mutations: P158R、K80A、K80S、K80C、K80R、K80G、N85Y、K81P、V182F、V182S、W185S、W185G、W185L、P73D、T208G、T208F、T208S、T208M、T70D、T70A、T70S、T70G、T70P、T70L、P158R+K80A、P158R+K81P、P158R+K81L、P158R+K81G、P158R+K81S、P158R+R204H、P158R+R204V、P158R+S337H、P158R+E353W、P158R+H350E、P158R+R441P、P158R+F355D、P158R+K80R、P158R+T70D、P158R+T70S、P158R+T70L、P158R+T70G、P158R+V182S、P158R+T208F、P158R+K80A+R204A、P158R+K80A+A299E、P158R+K80A+A202S、P158R+K80A+T208A、P158R+K80A+L261F、P158R+K80A+F205M、P158R+K80A+S337H、P158R+K80A+T208G、P158R+K80A+A302S、P158R+K80A+E353W、P158R+K80A+Q200I、P158R+K80A+K104R、P158R+K80A+T70D、P158R+K80A+R441P、P158R+K80A+F355D、P158R+K80A+R204H、P158R+K80A+D334N、P158R+K80A+D334V、P158R+K80A+R204N、P158R+K80A+W185F、P158R+K80A+W185C、P158R+K80A+N85G、P158R+K80A+R204V、P158R+K80A+Y300C、P158R+K80A+F294L、P158R+K80A+T183V、P158R+K80A+L83I、P158R+K80A+L83M、P158R+K80A+Q200I、P158R+K80A+E304M、P158R+N85G+V349T、P158R+N85G+S348G、P158R+N85G+N318V、P158R+N85G+K346P、P158R+N85G+S165A、P158R+N85G+N233R、P158R+N85G+T70S、P158R+N85G+K346R、P158R+N85G+S348A、P158R+N85G+N605P、P158R+N85G+N605F、P158R+N85G+S225T、P158R+N85G+I447V、P158R+N85G+H350L、P158R+N85G+N605A、P158R+N85G+F355A、P158R+N85G+A322S、P158R+K80A+A302S+K81L、P158R+K80A+A302S+R204H、P158R+K80A+T208G+T70D、P158R+K80A+A302S+R204N、P158R+K80A+A302S+Y300C、P158R+K80A+A302S+F294L、P158R+K80A+A302S+T183V、P158R+K80A+A302S+E304M、P158R+K80A+A302S+D334V、P158R+K80A+A302S+K81G、P158R+K80A+T208G+K311S、P158R+K80A+T208G+N605L、P158R+K80A+T208G+S225E、P158R+K80A+T208G+S225N、P158R+K80A+T208G+P535S、P158R+K80A+T208G+N605F、P158R+K80A+T208G+K311R、P158R+K80A+T208G+P535S、P158R+K80A+T208G+Y228A、P158R+K80A+T208G+Q227T、P158R+K80A+T208G+N605L、P158R+K80A+T208G+K311T、P158R+K80A+T208G+A299V、P158R+K80A+T208G+E522D、P158R+K80A+T208G+Q227M、P158R+K80A+T208G+T442S、P158R+K80A+A302S+K81I、P158R+K80A+A302S+L83I、P158R+K80A+T208G+N605P、P158R+K80A+T208G+N605G、P158R+K80A+T208G+K81D、P158R+K80A+T208G+A322I、P158R+K80A+T208G+F209N、P158R+K80A+T208G+A322L、P158R+K80A+A302S+D334N、P158R+K80A+T208G+K81S、P158R+K80A+T208G+L518F、P158R+K80A+A302S+W185C、P158R+K80A+T208G+K81L、P158R+K80A+A302S+T70N、P158R+K80A+A302S+V114L、P158R+K80A+A302S+F198M、P158R+K80A+A302S+A202C、P158R+K80A+A302S+M206H、P158R+K80A+A302S+M206F、P158R+K80A+A302S+Y228M、P158R+K80A+A302S+E353T、P158R+K80A+A302S+E353N、P158R+K80A+A302S+E353S、P158R+K80A+A302S+I497V、P158R+K80A+A302S+P535T、P158R+K80A+A302S+P535S、P158R+K80A+A302S+P535V、P158R+K80A+A302S+N605D、P158R+K80A+A302S+N605L、P158R+K80A+A302S+E439C、P158R+K80A+A302S+K346P、P158R+K80A+A302S+Y69N、P158R+K80A+A302S+T70D、P158R+K80A+A302S+T70G、P158R+K80A+A302S+T70M、P158R+K80A+A302S+K81S、P158R+K80A+A302S+N85G、P158R+K80A+A302S+V114I、P158R+K80A+A302S+A150V、P158R+K80A+A302S+A175V、P158R+K80A+A302S+W185F、P158R+K80A+A302S+A202S、P158R+K80A+A302S+M206S、P158R+K80A+A302S+F209N、P158R+K80A+A302S+Y228A、P158R+K80A+A302S+Y228L、P158R+K80A+A302S+V287T、P158R+K80A+A302S+V289I、P158R+K80A+A302S+V289L、P158R+K80A+A302S+V321A、P158R+K80A+A302S+R332S、P158R+K80A+A302S+T70N、P158R+K80A+A302S+T70S、P158R+K80A+A302S+V114L、P158R+K80A+A302S+F198M、P158R+K80A+A302S+A202C、P158R+K80A+A302S+M206H、P158R+K80A+A302S+M206F、P158R+K80A+A302S+Y228M、P158R+K80A+A302S+E353T、P158R+K80A+A302S+E353N、P158R+K80A+A302S+E353S、P158R+K80A+A302S+I497V、P158R+K80A+A302S+P535T、P158R+K80A+A302S+P535S、P158R+K80A+A302S+P535V、P158R+K80A+A302S+N605D、P158R+K80A+A302S+N605L、P158R+K80A+A302S+E439C、P158R+K80A+A302S+K346P、P158R+K80A+A302S+Y69N、P158R+K80A+A302S+T70D、P158R+K80A+A302S+T70G、P158R+K80A+A302S+T70M、P158R+K80A+A302S+N85G、P158R+K80A+A302S+V114I、P158R+K80A+A302S+A150V、P158R+K80A+A302S+A175V、P158R+K80A+A302S+W185F、P158R+K80A+A302S+A202S、P158R+K80A+A302S+M206S、P158R+K80A+A302S+F209N、P158R+K80A+A302S+E353Y、P158R+K80A+A302S+N605F、P158R+K80A+A302S+N605G、P158R+K80A+A302S+K81D、P158R+K80A+A302S+A322I、P158R+K80A+A302S+F209N、P158R+K80A+A302S+A322L、P158R+K80A+A302S+L518F、P158R+K80A+A302S+Q227T、P158R+K80A+A302S+K311T、P158R+K80A+A302S+A299V、P158R+K80A+A302S+E522D、P158R+K80A+A302S+Q227M、P158R+K80A+A302S+T442S、P158R+N85G+N605P+K346N、P158R+N85G+N605P+F198M、P158R+N85G+N605P+H350L、P158R+N85G+N605P+K80K、P158R+N85G+N605P+S225M、P158R+N85G+N605P+A302T、P158R+N85G+N605P+T549Y、P158R+N85G+N605P+K81T、P158R+N85G+N605P+K81R、P158R+N85G+N605P+K81E、P158R+N85G+N605P+K81S、P158R+N85G+N605P+K215T、P158R+N85G+N605P+F198W、P158R+N85G+N605P+A299T、P158R+K80A+A302S+N605F+Q227I、P158R+K80A+A302S+N605F+Q227V、P158R+K80A+A302S+N605F+I34T、P158R+N85G+N605P+K346N+S225R、P158R+N85G+N605P+K346N+S225A、P158R+N85G+N605P+K346N+S225T、P158R+K80A+A302S+N605F+S225E、P158R+K80A+A302S+N605F+K81M、P158R+K80A+A302S+N605F+N440T、P158R+K80A+A302S+N605F+T506G、P158R+K80A+A302S+N605F+H350Y、P158R+K80A+A302S+N605F+K81S、P158R+K80A+A302S+N605F+H350T、P158R+K80A+A302S+N605F+T506G、P158R+N85G+N605P+K346N+F198M、P158R+N85G+N605P+K346N+H350L、P158R+N85G+N605P+K346N+S225M、P158R+N85G+N605P+K346N+A302T、P158R+N85G+N605P+K346N+T549Y、P158R+N85G+N605P+K346N+K81T、P158R+N85G+N605P+K346N+K81R、P158R+N85G+N605P+K346N+F198W、P158R+N85G+N605P+K346N+K81E、P158R+N85G+N605P+K346N+K215T、P158R+N85G+N605P+K346N+K81S, P158R+N85G+N605P+K346N+A299T, P158R+N85G+N605P+K346N+ I187L、P158R+N85G+N605P+K346N+S225T+I34K、P158R+N85G+N605P+K346N+S225T+I34A、P158R+N 85G+N605P+K346N+S225T+I34P, P158R+N85G+N605P+K346N+S225T+I34R, P158R+N85G+N605P+K3 46N+S225T+I34H, P158R+N85G+N605P+K346N+S225T+I34S, P158R+N85G+N605P+K346N+S225T+S34 8G, P158R+N85G+N605P+K346N+S225T+I34K+S348G, P158R+N85G+N605P+K346N+S225T+I34K+S34 8A, P158R+N85G+N605P+K346N+S225T+I34K+S348P, P158R+N85G+N605P+K346N+S225T+I34K+S348 K. P158R+N85G+N605P+K346N+S225T+I34K+S348N, P158R+N85G+N605P+K346N+S225T+I34K+S348M , P158R+N85G+N605P+K346N+S225T+I34K+S348C or P158R+N85G+N605P+K346N+S225T+I34K+S348V. , 3. A gene characterized by, The gene encodes the α-amino acid ester acyltransferase mutant as described in claim 1 or 2.

4. A plasmid, characterized in that, The plasmid contains the gene as described in claim 3.

5. A non-plant / animal cell, characterized in that, The non-plant or animal cells include the gene described in claim 3 or the plasmid described in claim 4.

6. A method for synthesizing oligopeptides, characterized in that, The method includes: synthesizing the oligopeptide using the α-amino acid ester acyltransferase mutant according to claim 1 or 2.

7. The method according to claim 6, characterized in that, The method includes: using the α-amino acid ester acyltransferase mutant to catalyze the substrate to obtain the oligopeptide.

8. The method according to claim 7, characterized in that, The substrate includes an acyl donor and an acyl acceptor; The acyl donor is an amino acid ester hydrochloride, which is selected from any one of the following: methyl ester hydrochloride of amino acid, ethyl ester hydrochloride of amino acid, or isopropyl ester hydrochloride of amino acid. The acyl receptor comprises an amino acid or a first peptide segment; the first peptide segment is composed of condensed 2 to 19 amino acids.

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