α-Amino acid ester acyltransferase mutant and its application

Abstract

The present invention provides an α-amino acid ester acyltransferase mutant and its application. The α-amino acid ester acyltransferase mutant includes: (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; (b) a protein having an amino acid mutation at at least one of the following sites in the amino acid sequence in (a): P158, K80, T208, A302, A175, N85, N605, K346, S225, I34, or S348, and having the function of an α-amino acid ester acyltransferase; or (c) a protein having more than 80% homology with the amino acid sequence defined in (a) or (b) and having the function of an α-amino acid ester acyltransferase. The α-amino acid ester acyltransferase mutant of the present invention has the advantages of a broad substrate spectrum and high catalytic activity, can be well used in the synthesis of oligopeptides, has low cost and high yield, and realizes true green chemistry.

Description

Technical Field

[0001] The present invention relates to the field of enzyme catalysis, and in particular to an α-amino acid ester acyltransferase mutant and application thereof. Background Art

[0002] 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 1000 Daltons. Oligopeptides have a variety of important biological functions within the body. For example, many oligopeptides act as signaling molecules, participating in intercellular communication; some oligopeptides act as cofactors or inhibitors of enzymes, regulating metabolic pathways. Certain oligopeptides have immunomodulatory effects, enhancing or suppressing immune responses; some oligopeptides have antioxidant activity, scavenging free radicals and protecting cells from oxidative damage; and some oligopeptides can promote cell proliferation and differentiation, accelerating the wound healing process. Oligopeptides have a wide range of applications in medicine, food, cosmetics, and other fields.

[0003] Dipeptides and tripeptides are the most basic oligopeptides. Despite their simple structures, they exhibit a rich array of biological activities, regulating various life processes, including physiological metabolism. For example, carnosine (β-alanyl-His) possesses antioxidant, anti-inflammatory, and anti-glycation properties; glycine (Gly-Gly) is used in medicine as a stabilizer for blood preservatives and cytochrome C injections; glutathione (Ala-Gln) is an important nutritional supplement for surgical patients; aspartame (Asp-Phe methyl ester) is a popular sweetener; and copper peptide (GHK-Cu) is widely used in high-end skincare brands, often as a key ingredient in serums, creams, and other products. Ala-Phe, Ile-Phe, and Pro-Gly dipeptides are used as salty flavor enhancers; Ile-Tyr, Lys-Trp, Val-Tyr, and Ile-Trp have antihypertensive effects; Arg-Trp has analgesic properties; and Lys-Glu exhibits anti-tumor activity.

[0004] Chemical synthesis of dipeptides has limitations. For example, if amino acid protection and deprotection operations are required, the product may be racemized, the synthesis cost is high, and sometimes toxic reagents must be used. Fermentation methods have problems with low expression levels and byproducts. Therefore, exploring efficient and suitable oligopeptide synthesis methods has always been a hot topic in the field of scientific research. α-Amino acid ester acyltransferase (Aet) can react with another nucleophile, glutamine, using alanine methyl ester hydrochloride as an acyl donor to produce Ala-Gln dipeptide. The entire process does not require the participation of ATP and has a high synthesis efficiency (J Biotechnol. 2005 Jan 26;115(2):211-20.; Biosci Biotechnol Biochem. 2011;75(11):2087-92.). Currently reported Aet has a narrow substrate spectrum and low catalytic activity. Fewer than 30 oligopeptides have been reported to be enzymatically synthesized using α-amino acid ester acyltransferases. Furthermore, 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 to be synthesized using α-amino acid ester acyltransferases. This restricts the widespread application of Aet in synthesizing diverse oligopeptide products. Therefore, the development of α-amino acid ester acyltransferases with a broad substrate spectrum and high activity is of great importance. Summary of the Invention

[0005] The main purpose of the present invention is to provide an α-amino acid ester acyltransferase mutant and its application, so as to solve the problem of narrow substrate spectrum of α-amino acid ester acyltransferase in the prior art.

[0006] In order to achieve the above object, according to a first aspect of the present invention, there is provided an α-amino acid ester acyltransferase mutant, wherein the α-amino acid ester acyltransferase mutant comprises:

[0007] (a) a protein having the amino acid sequence shown in SEQ ID NO: 1;

[0008] (b) at least one of the following positions of the 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, 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, which undergoes an amino acid mutation and has α-amino acid ester acyltransferase function; or

[0009] (c) A protein having 80% or more homology with the amino acid sequence defined in (a) or (b) and having α-amino acid ester acyltransferase function.

[0010] Furthermore, the amino acid mutation in (b) above is selected from at least one of the following sites:

[0011] 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, 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;

[0012] The letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids.

[0013] Furthermore, the mutation of the aforementioned α-amino acid ester acyltransferase mutant includes any one of the following amino acid mutations:

[0014] 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、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. ,

[0015] In order to achieve the above object, according to a second aspect of the present invention, a gene is provided, which encodes the above-mentioned α-amino acid ester acyltransferase mutant.

[0016] In order to achieve the above object, according to the third aspect of the present invention, a plasmid is provided, wherein the plasmid comprises the above gene.

[0017] In order to achieve the above object, according to a fourth aspect of the present invention, a non-animal or plant cell is provided, wherein the non-animal or plant cell comprises the above gene or the above plasmid.

[0018] In order to achieve the above object, according to a fifth aspect of the present invention, a method for synthesizing an oligopeptide is provided, the method comprising: synthesizing the oligopeptide using the above-mentioned α-amino acid ester acyltransferase mutant.

[0019] Furthermore, the above method comprises: using the above α-amino acid ester acyltransferase mutant to catalyze the substrate to obtain the above oligopeptide.

[0020] Furthermore, the substrate comprises an acyl donor and an acyl acceptor;

[0021] The acyl donor is an amino acid ester hydrochloride, and the amino acid ester hydrochloride is selected from any one of the following: amino acid methyl ester hydrochloride, amino acid ethyl ester hydrochloride or amino acid isopropyl ester hydrochloride;

[0022] The acyl acceptor comprises amino acids or a first peptide segment; the first peptide segment is formed by condensation of 2 to 19 amino acids.

[0023] By applying the technical solution of the present invention, a wild-type α-amino acid ester acyltransferase (the encoding gene has the nucleotide sequence shown in SEQ ID NO: 1) with high catalytic activity towards Val-Ser dipeptide was designed by reconstructing the ancestral enzyme. The above-mentioned α-amino acid ester acyltransferase was further mutated to obtain α-amino acid ester acyltransferase mutants with a wide substrate spectrum and high activity.

[0024] The α-amino acid ester acyltransferase mutant comprises: (a) a protein having the amino acid sequence shown in SEQ ID NO: 1; (b) at least one of the following positions of the 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, Y330 00, 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, which have amino acid mutations and have α-amino acid ester acyltransferase function; or (c) a protein having 80% or more homology to the amino acid sequence defined in (a) or (b) and having α-amino acid ester acyltransferase function. The α-amino acid ester acyltransferase mutant of the present invention has a broad substrate spectrum and catalytic activity and can be used to synthesize a variety of oligopeptides. DETAILED DESCRIPTION

[0025] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0026] As mentioned in the background, prior art α-amino acid ester acyltransferases for oligopeptide synthesis suffer from a narrow substrate spectrum and low catalytic activity. The inventors of the present invention attempted to reconstruct an ancestral enzyme and engineered a wild-type α-amino acid ester acyltransferase (encoding the nucleotide sequence shown in SEQ ID NO: 1) with high catalytic activity toward Val-Ser dipeptides. Subsequently, the researchers determined the three-dimensional structure of this enzyme through homology modeling and AI prediction, and mutated key sites on the protein surface and near the active site. The activity of the mutated enzymes was tested using valine methyl ester and serine as substrates, resulting in a series of α-amino acid ester acyltransferase mutants capable of efficiently synthesizing oligopeptides. Using some of these α-amino acid ester acyltransferase mutants in the synthesis of various oligopeptides, they found that these mutants could also be used for the efficient synthesis of a variety of other oligopeptides, in addition to Val-Ser dipeptides.

[0027] The α-amino acid ester acyltransferase of the present invention (including wild-type and mutant forms) has a broad substrate spectrum and high catalytic activity, thus providing a protection scheme of the present invention.

[0028] In a first typical embodiment of the present invention, an α-amino acid ester acyltransferase mutant is provided, wherein the α-amino acid ester acyltransferase mutant comprises:

[0029] (a) a protein having the amino acid sequence shown in SEQ ID NO: 1;

[0030] (b) at least one of the following positions of the 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, 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, which undergoes an amino acid mutation and has α-amino acid ester acyltransferase function; or

[0031] (c) A protein having 80% or more homology with the amino acid sequence defined in (a) or (b) and having α-amino acid ester acyltransferase function.

[0032] It should be noted that homology in the present 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 in the NCBI database. For determination of sequence identity, see, for example, Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, and Sequence Analysis Primers, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0033] The above-mentioned protein has 70%, 75%, 80%, 85%, 90%, 95%, 99% or more (such as 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 more, or even 99.9% or more) homology with the mutant shown in SEQ ID NO: 1 and has the function of α-amino acid ester acyltransferase, and its active site, active pocket, activity mechanism, protein structure, etc. are most likely the same as the protein provided in (a) of the corresponding protein.

[0034] Amino acid residues can be represented by the standard three-letter or one-letter amino acid code commonly known and agreed upon in the art. Herein, the amino acid residues are abbreviated 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).

[0035] Conservative amino acid substitutions or replacements are well known in the art. For example, conservative amino acid substitutions preferably involve replacing one amino acid residue from the following groups (1)-(5) with another amino acid from the same group: (1) smaller aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (2) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (3) polar positively charged residues: His, Arg, and Lys; (4) larger aliphatic non-polar residues: Met, Leu, Ile, Val, and Cys; and (5) aromatic residues: Phe, Tyr, and Trp. Particularly preferred conservative 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.

[0036] Those skilled in the art may also perform conservative substitutions on amino acids according to amino acid substitution rules well known to those skilled in the art, such as the "blosum62 scoring matrix" in the prior art.

[0037] In the following examples, site-directed mutagenesis was performed on the α-amino acid ester acyltransferase having the amino acid sequence shown in SEQ ID NO: 1 as the parent to obtain various variants.

[0038] The amino acid sequence shown in SEQ ID NO: 1 is as follows:

[0039] MKSTLLVFLLLLCVQAFAQDAADSAYVREHYEKIEQLIPMRDGTKLFTAIYTPKDKSKKYPVLLNRTPYTVAPYGEDEYKKSLGNFPAMMREGYIFVYQDVRGKWMSEGNFEDVRPTTTKKNKKAIDESTDTYDTLEWLSKNLKNYNGKAGMYGISYPGFYSTVSLVNAHPSLKAVSPQAPVTDWFIGDDFHHNGVLFLQDAFRFMSTFGVPRPKPITPDKGPKSFQYPIKDNYRFYLEAGSVKELKDKYFGDSIKFWNDLFAHPDYDQFWKDRVILPHLTNVKPAVMVVGGFFDAEDAYGAFETYKAIEKQNPKNNNILVAGPWFHGGWVRSDGDSFGDIQFGQKTSVHYQEKFELPFFNYYLKGKGDFKPAEANIFITGSNEWKQFEQWPPKDVETKKLYLQPQGKLSFEKVGRTDSWDEYVSDPNKPVPYQGGVLENRTREYMIDDQRFAANRPDVMVYQTDVLTEDITITGPIKNHLKVSTTGTDADYVVKLIDVYPEDTPTFNNKIMAGYQMLVRGEIMRGKYRNSFEKPEAMVPGQVTKVNYTMPDVGHTFKKGHRIMIQVQNTWFPLADRNPQQFMNVYEATAKDFQKATHRIYHDVNNSSYIELPVL。

[0040] The amino acid sequence shown in SEQ ID NO: 1 is a wild-type α-amino acid ester acyltransferase (encoding the nucleotide sequence shown in SEQ ID NO: 1) designed by reconstructing an ancestral enzyme. 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, 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.

[0041] These amino acid sites are located near the active site and may affect the protein's catalytic properties and substrate selectivity. By mutating these amino acid sites, proteins with α-amino acid ester acyltransferase function, or even enhanced α-amino acid ester acyltransferase function and a broadened substrate spectrum, can be obtained. For the proteins obtained above, changes can be made at non-critical mutation sites and the active site to obtain proteins with greater than 80% homology to the above amino acid sequence and α-amino acid ester acyltransferase function.

[0042] In a preferred embodiment of the present invention, the amino acid mutation in (b) is selected from at least one of the following sites:

[0043] 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; wherein, the letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids.

[0044] In the present invention, the applicants continued to explore the aforementioned active site and discovered that mutations to different amino acids in the active site resulted in differences in the corresponding protein activity, and that specific mutations could enhance the activity of α-amino acid ester acyltransferase. Through experimental investigations, it was discovered that specific mutations in the active site could yield an α-amino acid ester acyltransferase with enhanced activity. The amino acid mutation sites of the α-amino acid ester acyltransferase protein can be flexibly selected and combined among the aforementioned mutations.

[0045] In a preferred embodiment of the present invention, the mutations of the α-amino acid ester acyltransferase mutant include 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, P158R+K81S 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.

[0046] The above amino acid mutations were all tested and explored in the examples of the present invention, and all had α-amino acid ester acyltransferase activity. Compared with the parent having the amino acid sequence shown in SEQ ID NO: 1, α-amino acid ester acyltransferase mutants with high enzyme activity can be obtained.

[0047] In a second typical embodiment of the present invention, a gene is provided, which encodes the aforementioned α-amino acid ester acyltransferase mutant.

[0048] In a third typical embodiment of the present invention, a plasmid is provided, wherein the plasmid comprises the above-mentioned gene.

[0049] The gene can encode the mutant α-amino acid ester acyltransferase and can be linked to a plasmid to form a circular DNA. Both the gene and the plasmid can be transcribed and translated under the action of RNA polymerase, ribosomes, tRNA, etc. to obtain the mutant α-amino acid ester acyltransferase.

[0050] In a fourth typical embodiment of the present invention, a non-animal or plant cell is provided, wherein the non-animal or plant cell comprises the plasmid.

[0051] The non-animal and plant cells are capable of replicating the plasmid and transcribing and translating the gene carried by the plasmid, thereby obtaining a large number of α-amino acid ester acyltransferase mutants. Using existing technologies, the non-animal and plant cells are disrupted, and the resulting crude enzyme catalyzes the substrate to synthesize a variety of oligopeptides.

[0052] In a fifth exemplary embodiment of the present invention, a method for synthesizing an oligopeptide is provided, 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 an oligopeptide using the α-amino acid ester acyltransferase mutant of the present invention has the advantage of high efficiency and is capable of synthesizing a variety of oligopeptides that are not synthesized enzymatically in the prior art. In a preferred embodiment of the present invention, the oligopeptide is a dipeptide.

[0053] In a preferred embodiment of the present invention, the method comprises: using the aforementioned α-amino acid ester acyltransferase mutant to catalyze the reaction of a substrate to produce the aforementioned oligopeptide. In a preferred embodiment of the present invention, the substrate comprises an acyl donor and an acyl acceptor; the acyl donor is an amino acid ester hydrochloride selected from any one of the following: methyl ester hydrochloride, ethyl ester hydrochloride, or isopropyl ester hydrochloride; the acyl acceptor comprises an amino acid or a first peptide segment; and the first peptide segment is formed by condensation of 2 to 19 amino acids. The α-amino acid ester acyltransferase mutant of the present invention has a broad substrate spectrum, enabling the synthesis of a variety of oligopeptides.

[0054] The present invention is further described in detail below with reference to specific examples. These examples should not be construed as limiting the scope of protection claimed in the present invention.

[0055] Example 1 Preparation of α-amino acid ester acyltransferase Aet enzyme solution

[0056] α-amino acid ester acyltransferase was designed by reconstructing the ancestral enzyme, and then the coding gene of 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 the present invention) was obtained by artificial chemical synthesis. The endonuclease site NdeI was introduced at the 5' end and the endonuclease site XhoI was introduced at the 3' end. The gene was synthesized in pUC19 to obtain pUC19-Aet.

[0057] The expression vector pET-28a(+) was double-digested with NdeI+XhoI, and the α-amino acid ester acyltransferase gene pUC19-Aet was double-digested with NdeI+XhoI. The gene fragment encoding Aet was excised and ligated with the expression vector pET-28a(+) to obtain the Escherichia coli recombinant plasmid pET-28a(+)-Aet containing α-amino acid ester acyltransferase. The recombinant Escherichia coli strain BL21(DE3)+Aet was obtained after transformation into Escherichia coli BL21(DE3).

[0058] 4 mL of the BL21(DE3) strain containing the recombinant plasmid was inoculated into a 2-L Erlenmeyer flask containing 400 mL of LB medium. After shaking at 37°C and 200 rpm for 2-3 hours, IPTG was added to a final concentration of 0.02 mM when the OD600 reached 0.6-0.8. Induction was continued at 16°C for 18 hours. After induction, the cells were harvested by centrifugation at 4°C. Resuspend the cells in 10 mL of 0.1M Tris-HCl (pH 8.0) per gram of slurry and disrupt by sonication (20% power, 5 minutes). Centrifuge at 4°C for 10 minutes at 12,000 rpm, and collect the supernatant to obtain the crude enzyme solution used in the catalytic reaction.

[0059] Note: The experimental materials and reagents used in the examples are described as follows:

[0060] 1. Strains and vectors: Escherichia coli expression vector pET-28a(+) and strain BL21 (DE3)

[0061] 2. Culture medium: Escherichia coli broth (LB) (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0).

[0062] Example 2 Catalytic Synthesis of Val-Ser Dipeptide

[0063] In this example, a designed α-amino acid ester acyltransferase (the gene encoding it has the amino acid sequence set forth in SEQ ID NO: 1, designated herein as wild-type α-amino acid ester acyltransferase) was used to synthesize a Val-Ser dipeptide. The reaction system (1 mL) consisted of 100 mM valine methyl ester hydrochloride, 100 mM serine, 50 µL of crude enzyme solution, and 0.1 M Tris-HCl (pH 8.0). The reaction was incubated at 20°C for 2 h. The reaction system was heated at 80°C for 10 min, centrifuged, and analyzed by HPLC. The results are as follows (Table 1):

[0064] Table 1:

[0065]

[0066] Note: This table shows the reaction results of α-amino acid ester acyltransferase (designated herein as wild-type α-amino acid ester acyltransferase, the encoding gene of which has the amino acid sequence set forth in SEQ ID NO: 1). * indicates a conversion rate of 1% ≤ < 5%, and ** indicates a conversion rate of 5% ≤ < 10%. The conversion rate is calculated as follows: (moles of product / moles of amino acid methyl ester substrate) × 100%.

[0067] Example 3 Enzyme evolution

[0068] Enzyme evolution of an α-amino acid ester acyltransferase (designated herein as the wild-type α-amino acid ester acyltransferase, encoding the gene having the amino acid sequence set forth in SEQ ID NO: 1) was performed. The evolved mutants were tested for activity using valine methyl ester hydrochloride and serine as substrates. The reaction system (1 mL) consisted of 100 mM valine methyl ester hydrochloride, 100 mM serine, 50 µL of 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 heated at 80°C for 10 min, centrifuged, and analyzed by HPLC. The results are shown in Table 2:

[0069] Table 2:

[0070]

[0071] Note: * represents a conversion rate of 1% ≤ < 5%, ** represents a conversion rate of 5% ≤ < 10%, and *** represents a conversion rate of 10% ≤ < 15%. The conversion rate is calculated as: (moles of product / moles of amino acid methyl ester substrate) * 100%.

[0072] From the above experimental results, it can be seen that after single-point mutation of wild-type α-amino acid ester acyltransferase, the activity of some mutants was significantly increased, and the mutant with the best activity was selected as the template for the next round of evolution.

[0073] Example 4

[0074] The best mutant evolved in Example 3 was subjected to the next round of enzyme evolution. The evolved mutant was tested for activity using valine methyl ester hydrochloride and serine as substrates. The reaction system (1 mL) was as follows: 100 mM valine methyl ester hydrochloride, 100 mM serine, 50 µL of 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 as follows (Table 3):

[0075] Table 3:

[0076]

[0077] 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%.

[0078] From the above experimental results, it can be seen that after further mutation of the best mutant in Example 3, the activity of some mutants was significantly increased, and the mutants with good activity were selected as templates for the next round of evolution.

[0079] Example 5

[0080] The best mutant evolved in Example 4 was subjected to the next round of enzyme evolution. The evolved mutant was tested for activity using valine methyl ester hydrochloride and serine as substrates. The reaction system (1 mL) was as follows: 200 mM valine methyl ester hydrochloride, 200 mM serine, 25 µL of 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 heated at 80°C for 10 min, centrifuged, and analyzed by HPLC. The reaction results are as follows (Tables 4 and 5):

[0081] Table 4:

[0082]

[0083] Table 5

[0084]

[0085] 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%.

[0086] From the above experimental results, it can be seen that when the best mutant of Example 4 was further mutated, the substrate concentration of the activation reaction was doubled and the enzyme amount was reduced by half, some mutants still showed very obvious activity improvement. Mutants with good activity were selected as templates for the next round of evolution.

[0087] Example 6

[0088] The best mutant evolved in Example 5 was subjected to the next round of enzyme evolution. The evolved mutant was tested for activity using valine methyl ester hydrochloride and serine as substrates. The reaction system (1 mL) was as follows: 200 mM valine methyl ester hydrochloride, 200 mM serine, 25 µL of 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 as follows (Table 6):

[0089] Table 6:

[0090]

[0091] 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%. From the above experimental results, it can be seen that after further mutation of the best mutant in Example 5, some mutants still showed very significant improvement in activity.

[0092] Example 7

[0093] Different mutants from Examples 3 to 6 were selected to investigate the activity of synthesizing oligopeptides Gly-Ser, Gly-Gly, Ser-Ser, Val-Ser, Tyr-Ser, Gly-Gly-Gly, and Gly-His-Lys. The reaction system (10 mL) was as follows: 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 reaction 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 as follows (Table 7):

[0094] Table 7:

[0095]

[0096] Note: **** represents 50% ≤ conversion rate < 60%, ***** represents 60% ≤ conversion rate < 70%, ****** represents 70% ≤ conversion rate < 90%.

[0097] From the above experimental results, it can be seen that the different mutants evolved for Val-Ser can also show good catalytic activity in the synthetic oligopeptides Gly-Ser, Gly-Gly, Ser-Ser, Val-Ser, Tyr-Ser, Gly-Gly-Gly and Gly-His-Lys.

[0098] Example 8

[0099] The highly active P158R+N85G+N605P+K346N+S225T+I34K+S348G and wild-type α-amino acid ester acyltransferases from the above examples were subjected to reactions with 19 amino acid methyl ester hydrochlorides and 19 amino acids to investigate changes in substrate spectrum after enzyme evolution. The reaction system (1 mL) was as follows: 200 mM of the various amino acid methyl ester hydrochlorides (first column of the table below), 200 mM of the various amino acids (first row of the table below), 25 µL of 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 LC-MS. The results are summarized below (Tables 8 and 9):

[0100] Table 8 (Wild-type α-amino acid ester acyltransferase):

[0101]

[0102] Table 9 (mutant P158R+N85G+N605P+K346N+S225T+I34K+S348G):

[0103]

[0104] Note: * indicates 10 ≤ conversion rate < 30%, ** indicates conversion rate ≥ 30% and less than 50%, and *** indicates conversion rate ≥ 50%. As shown above, the mutant has a broader substrate spectrum than the wild-type α-amino acid ester acyltransferase and significantly improved activity for the synthesis of various dipeptides.

[0105] Example 9

[0106] The scale-up of the reaction for synthesizing Gly-Ser, Ser-Ser, Val-Ser, and Tyr-Ser using P158R+N85G+N605P+K346N+S225T+I34K+S348G was performed. The reaction system (1 L) consisted of 500 mM glycine methyl ester hydrochloride and alanine methyl ester hydrochloride, 500 mM tyrosine and glutamine, 25 mL of crude enzyme solution, and 0.1 M Tris-HCl (pH 8.0). The reaction was incubated at 20°C for 2 h. After completion of the reaction, the reaction system was post-processed for separation and purification. The final product was analyzed for purity and content by HPLC and NMR. The results are summarized below (Table 10):

[0107] Table 10:

[0108]

[0109] The conversion rate is calculated as (moles of product / moles of amino acid methyl ester substrate) × 100%; the product purity is calculated as (peak area of ​​product on HPLC / peak area of ​​all peaks) × 100%; and the isolation yield is calculated as (actual yield / theoretical yield) × 100%.

[0110] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: In the present invention, α-amino acid ester acyltransferase evolution is carried out for Val-Ser dipeptide to obtain a series of mutants with improved activity. The α-amino acid ester acyltransferase mutants obtained in the evolution can not only be used to efficiently synthesize Val-Ser, but can also be used to synthesize a variety of 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 efficiency in synthesizing some oligopeptides. The α-amino acid ester acyltransferase obtained in the present invention can be well used for industrial scale-up, with low cost and high yield, realizing true green chemistry.

[0111] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be 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 mutant is: (a) the protein having the amino acid sequence shown in SEQ ID NO: 1; or (b) A mutant in which the amino acid sequence in (a) undergoes any 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、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. , 2. A gene, characterized in that The gene encodes the α-amino acid ester acyltransferase mutant according to claim 1.

3. A plasmid, characterized in that The plasmid comprises the gene according to claim 2.

4. A non-animal or plant cell, characterized in that: The non-animal or plant cell comprises the gene according to claim 2 or the plasmid according to claim 3.

5. A method for synthesizing oligopeptides, characterized in that: The method comprises: synthesizing the oligopeptide using the α-amino acid ester acyltransferase mutant according to claim 1; Wherein, when the α-amino acid ester acyltransferase mutant is the protein (a) whose amino acid sequence is shown in SEQ ID NO: 1, The oligopeptides corresponding to the synthesis are selected from any of the following: Tyr-Tyr, Tyr-Trp, Tyr-Phe, Tyr-His, Tyr-Arg, Tyr-Lys, Tyr-Glu, Tyr-Asp, Tyr-Gln, Tyr-Asn, Tyr-Met, Tyr-Cys, Tyr-Thr, Tyr-Ser, Tyr-Ile, Tyr-Leu, Tyr-Val, Tyr-Ala, Tyr-Gly, Trp-Tyr, Trp-Trp, Trp-Phe, Trp-His, Trp-Arg, Trp-Lys, Trp-Glu, Trp-Asp, Trp-Gln, Trp-Asn, Trp-Met, Trp-Cys, Trp-Thr, Trp-Ser, Trp-Ile, Trp-Leu, Trp-Val, Trp-Ala, Trp-Gly, Phe-Tyr, Phe-Trp, Phe-Phe, Phe-His, Phe-Arg, Phe-Lys, Phe-Glu, Phe-Asp, Phe-Gln, Phe-Asn, Phe-Met, Phe-Cys, Phe-Thr, Phe-Ser, Phe-Ile, Phe-Leu, Phe-Val, Phe-Ala, Phe-Gly, His-Tyr, His-Trp, His-His, His-Lys, His-Glu, His-Asp, His-Gln, His-Asn, His-Met, His-Cys, His-Thr, His-Ser, His-Ile, His-Leu, His-Val, His-Ala, His-Gly, Arg-Tyr, Arg-Trp, Arg-Phe, Arg-His, Arg-Arg, Arg-Lys, Arg-Glu, Arg-Gln, Arg-Asn, Arg-Met, Arg-Cys, Arg-Thr, Arg-Ser, Arg-Ile, Arg-Leu, Arg-Val, Arg-Ala, Arg-Gly, Lys-Tyr, Lys-Trp, Lys-Phe, Lys-His, Lys-Arg, Lys-Lys, Lys-Glu, Lys-Asp, Lys-Gln, Lys-Asn, Lys-Met, Lys-Cys, Lys-Thr, Lys-Ser, Lys-Ile, Lys-Leu, Lys-Val, Lys-Ala, Lys-Gly, Glu-Tyr, Glu-Trp, Glu-Phe, Glu-His, Glu-Arg, Glu-Lys, Glu-Glu, Glu-Asp, Glu-Gln, Glu-Asn, Glu-Met,Glu-Thr、Glu-Ser、Glu-Ile、Glu-Leu、Glu-Val、Glu-Ala、Glu-Gly、Asp-Tyr、Asp-Trp、Asp-Phe、Asp-His、Asp-Arg、Asp-Lys、Asp-Glu、Asp-Asp、Asp-Gln、Asp-Asn、Asp-Met、Asp-Cys、Asp-Thr、Asp-Ser、Asp-Ile、Asp-Leu、Asp-Val、Asp-Ala、Asp-Gly、Gln-Tyr、Gln-Trp、Gln-Phe、Gln-His、Gln-Arg、Gln-Lys、Gln-Glu、Gln-Asp、Gln-Gln、Gln-Asn、Gln-Met、Gln-Cys、Gln-Thr、Gln-Ser、Gln-Ile、Gln-Leu、Gln-Val、Gln-Ala、Gln-Gly、Asn-Tyr、Asn-Trp、Asn-Phe、Asn-Arg、Asn-Lys、Asn-Glu、Asn-Asp、Asn-Gln、Asn-Asn、Asn-Met、Asn-Cys、Asn-Thr、Asn-Ser、Asn-Ile、Asn-Leu、Asn-Val、Asn-Ala、Asn-Gly、Met-Tyr、Met-Trp、Met-Phe、Met-His、Met-Arg、Met-Lys、Met-Glu、Met-Asp、Met-Gln、Met-Asn、Met-Met、Met-Cys、Met-Thr、Met-Ser、Met-Ile、Met-Leu、Met-Val、Met-Ala、Met-Gly、Cys-Trp、Cys-His、Cys-Arg、Cys-Lys、Cys-Asp、Cys-Gln、Cys-Asn、Cys-Cys、Cys-Thr、Cys-Ile、Cys-Leu、Cys-Val、Cys-Ala、Cys-Gly、Thr-Tyr、Thr-Trp、Thr-Phe、Thr-His、Thr-Arg、Thr-Lys、Thr-Glu、Thr-Asp、Thr-Gln、Thr-Asn、Thr-Met、Thr-Cys、Thr-Thr、Thr-Ser、Thr-Ile、Thr-Leu、Thr-Val、Thr-Ala、Thr-Gly、Ser-Tyr、Ser-Trp、Ser-Phe、Ser-His、Ser-Lys、Ser-Glu、Ser-Asp、Ser-Gln、Ser-Asn、Ser-Met、Ser-Cys、Ser-Thr、Ser-Ser、Ser-Ile、Ser-Leu、Ser-Val、Ser-Ala、Ser-Gly、Ile-Tyr、Ile-Trp、Ile-Phe、Ile-His、Ile-Ar g、Ile-Lys、Ile-Glu、Ile-Asp、Ile-Asn、Ile-Met、Ile-Cys、Ile-Thr、Ile-Ser、Ile-Ile、Ile-Leu、Ile-Val、Ile-Ala、Ile- Gly、Leu-Tyr、Leu-Trp、Leu-Phe、Leu-His、Leu-Arg、Leu-Lys、Leu-Glu、Leu-Asp、Leu-Gln、Leu-Asn、Leu-Met、Leu-Cys、Leu-Thr、Leu-Ser、Leu-Ile、Leu-Leu、Leu-Val、Leu-Ala、Leu-Gly、Val-Tyr、Val-Trp、Val-Phe、Val-His、Val-Arg、Val-Lys、 Val-Glu、Val-Asp、Val-Gln、Val-Asn、Val-Met、Val-Cys、Val-Thr、Val-Ser、Val-Ile、Val-Leu、Val-Val、Val-Ala、Val-Gl y、Ala-Tyr、Ala-Trp、Ala-Phe、Ala-His、Ala-Arg、Ala-Lys、Ala-Glu、Ala-Asp、Ala-Gln、Ala-Asn、Ala-Met、Ala-Cys、Ala- Thr、Ala-Ser、Ala-Ile、Ala-Leu、Ala-Val、Ala-Ala、Ala-Gly、Gly-Tyr、Gly-Trp、Gly-Phe、Gly-His、Gly-Arg、Gly-Lys、Gl y-Glu、Gly-Asp、Gly-Gln、Gly-Asn、Gly-Met、Gly-Cys、Gly-Thr、Gly-Ser、Gly-Ile、Gly-Leu、Gly-Val、Gly-Ala or Gly-Gly;、 The α-amino acid ester acyltransferase mutant is a mutant in which the amino acid sequence in (a) undergoes the following amino acid mutations: P158R+N85G+N605P+K346N+S225T+I34K+S348G, The oligopeptides corresponding to the synthesis are selected from any one of the following: Tyr-Tyr, Tyr-Trp, Tyr-Phe, Tyr-His, Tyr-Arg, Tyr-Lys, Tyr-Glu, Tyr-Asp, Tyr-Gln, Tyr-Asn, Tyr-Met, Tyr-Cys, Tyr-Thr, Tyr-Ser, Tyr-Ile, Tyr-Leu, Tyr-Val, Tyr-Ala, Tyr-Gly, Trp-Tyr, Trp-Trp, Trp-Phe, Trp-His, Trp-Arg, Trp-Lys, Trp-Glu, Trp-Asp, Trp-Gln, Trp-Asn, Trp-Met, Trp-Cys, Trp-Thr, Trp-Ser, Trp-Ile, Trp-Leu, Trp-Val, Trp-Ala, Trp-Gly, Phe-Tyr, Phe-Trp, Phe-Phe, Phe-His, Phe-Arg, Phe-Lys, Phe-Glu, Phe-Asp, Phe-Gln, Phe-Asn, Phe-Met, Phe-Cys, Phe-Thr, Phe-Ser, Phe-Ile, Phe-Leu, Phe-Val, Phe-Ala, Phe-Gly, His-Tyr, His-Trp, His-His, His-Arg, His-Lys, His-Glu, His-Asp, His-Gln, His-Asn, His-Met, His-Cys, His-Thr, His-Ser, His-Ile, His-Leu, His-Val, His-Ala, His-Gly, Arg-Tyr, Arg-Trp, Arg-Phe, Arg-His, Arg-Arg, Arg-Lys, Arg-Glu, Arg-Asp, Arg-Gln, Arg-Asn, Arg-Met, Arg-Cys, Arg-Thr, Arg-Ser, Arg-Ile, Arg-Leu, Arg-Val, Arg-Ala, Arg-Gly, Lys-Tyr, Lys-Trp, Lys-Phe, Lys-His, Lys-Arg, Lys-Lys, Lys-Glu, Lys-Asp, Lys-Gln, Lys-Asn, Lys-Met, Lys-Cys, Lys-Thr, Lys-Ser, Lys-Ile, Lys-Leu, Lys-Val, Lys-Ala, Lys-Gly, Glu-Tyr, Glu-Trp, Glu-Phe, Glu-His, Glu-Arg, Glu-Lys, Glu-Glu, Glu-Gln, Glu-Asn,Glu-Met、Glu-Cys、Glu-Thr、Glu-Ser、Glu-Ile、Glu-Leu、Glu-Val、Glu-Ala、Glu-Gly、Asp-Tyr、Asp-Trp、Asp-Phe、Asp-His、Asp-Arg、Asp-Lys、Asp-Glu、Asp-Asp、Asp-Gln、Asp-Asn、Asp-Met、Asp-Cys、Asp-Thr、Asp-Ser、Asp-Ile、Asp-Leu、Asp-Val、Asp-Ala、Asp-Gly、Gln-Tyr、Gln-Trp、Gln-Phe、Gln-His、Gln-Lys、Gln-Glu、Gln-Asp、Gln-Gln、Gln-Asn、Gln-Met、Gln-Cys、Gln-Thr、Gln-Ser、Gln-Ile、Gln-Leu、Gln-Val、Gln-Ala、Gln-Gly、Asn-Tyr、Asn-Trp、Asn-Phe、Asn-His、Asn-Arg、Asn-Lys、Asn-Glu、Asn-Asp、Asn-Gln、Asn-Asn、Asn-Met、Asn-Cys、Asn-Thr、Asn-Ser、Asn-Ile、Asn-Leu、Asn-Val、Asn-Ala、Asn-Gly、Met-Tyr、Met-Trp、Met-Phe、Met-His、Met-Arg、Met-Lys、Met-Glu、Met-Asp、Met-Gln、Met-Asn、Met-Met、Met-Cys、Met-Thr、Met-Ser、Met-Ile、Met-Leu、Met-Val、Met-Ala、Met-Gly、Cys-Tyr、Cys-Trp、Cys-His、Cys-Arg、Cys-Lys、Cys-Glu、Cys-Asp、Cys-Gln、Cys-Asn、Cys-Cys、Cys-Thr、Cys-Ser、Cys-Leu、Cys-Val、Cys-Ala、Thr-Tyr、Thr-Trp、Thr-Phe、Thr-His、Thr-Arg、Thr-Lys、Thr-Glu、Thr-Asp、Thr-Gln、Thr-Asn、Thr-Met、Thr-Cys、Thr-Thr、Thr-Ser、Thr-Ile、Thr-Leu、Thr-Val、Thr-Ala、Thr-Gly、Ser-Tyr、Ser-Trp、Ser-Phe、Ser-His、Ser-Arg、Ser-Lys、Ser-Glu、Ser-Asp、Ser-Gln、Ser-Asn、Ser-Met、Ser-Cys、Ser-Thr、Ser-Ser、Ser-Ile、Ser-Leu、Ser-Val、Ser-Ala、Ser-Gly、Ile-Tyr、Ile-Trp、Ile-Phe、Ile-His、Ile-Arg、Ile-Lys、Ile-Glu、Ile-Asp、Ile-Gln、Ile-Asn、Ile-Met、Ile-Cys、Ile-Thr、Ile-Ser、Ile-Ile、Ile-Leu、Ile-Val、Ile-Ala、Ile-Gly、Leu-Tyr、Leu-Trp、Leu-Phe、Leu-His、Leu-Arg、Leu-Lys、Leu-Glu、Leu-Asp、Leu-Gln、Leu-Asn、Leu-Met、Leu-Cys、Leu-Thr、Leu-Ser、Leu-Ile、Leu-Leu、Leu-Val、Leu-Ala、Leu-Gly、Val-Tyr、Val-Trp、Val-Phe、Val-His、Val-Arg、Val-Lys、Val-Glu、Val-Asp、Val-Gln、Val-Asn、Val-Met、Val-Cys、Val-Thr、Val-Ser、Val-Ile、Val-Leu、Val-Val、Val-Ala、Val-Gly、Ala-Tyr、Ala-Trp、Ala-Phe、Ala-His、Ala-Arg、Ala-Lys、Ala-Glu、Ala-Asp、Ala-Gln、Ala-Asn、Ala-Met、Ala-Cys、Ala-Thr、Ala-Ser、Ala-Ile、Ala-Leu、Ala-Val、Ala-Ala、Ala-Gly、Gly-Tyr、Gly-Trp、Gly-Phe、Gly-His、Gly-Arg、Gly-Lys、Gly-Glu、Gly-Asp、Gly-Gln、Gly-Asn、Gly-Met、Gly-Cys、Gly-Thr、Gly-Ser、Gly-Ile、Gly-Leu、Gly-Val、Gly-Ala、Gly-Gly;、 The α-amino acid ester acyltransferase mutant is a mutant in which any one of the following amino acid mutations occurs in the amino acid sequence in (a): 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, P158R+ R204V, P158R+S337H, P158R+E353W, P158R+H350E, P158R+R441P, P158R+F 355D, P158R+T70D, P158R+T70S, P158R+T70L, P158R+T70G, P158R+V182S, P 158R+T208F, P158R+K80A+R204A, P158R+K80A+A299E, P158R+K80A+A202S, P158R+K80A+T208A, P158R+K80A+L261F, P158R+K80A+F205M, P158R+K80A+ S337H, P158R+K80A+A302S, P158R+K80A+E353W, P158R+K80A+Q200I, P158 R+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+T183 V. 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、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+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+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+N 605P+K346N+S225T+I34K, P158R+N85G+N605P+K346N+S225T+I34A, P158R+N85G+N60 5P+K346N+S225T+I34P, P158R+N85G+N605P+K346N+S225T+I34R, P158R+N85G+N605P +K346N+S225T+I34H、P158R+N85G+N605P+K346N+S225T+S348G、P158R+N85G+N605P+K 346N+S225T+I34K+S348A, P158R+N85G+N605P+K346N+S225T+I34K+S348P, P158R+N8 5G+N605P+K346N+S225T+I34K+S348K, P158R+N85G+N605P+K346N+S225T+I34K+S348 N, P158R+N85G+N605P+K346N+S225T+I34K+S348M, P158R+N85G+N605P+K346N+S225T+I34K+S348C, P158R+N85G+N605P+K346N+S225T+I34K+S348V, the corresponding synthesized oligopeptides are Val-Ser; The α-amino acid ester acyltransferase mutant is a mutant in which any one of the following amino acid mutations occurs in the amino acid sequence in (a): P158R, P158R+K80R, P158R+K80A+T208G, P158R+K80A+A302S+A175V, P158R+N85G+N605P+K346N+S225A or P158R+N85G+N605P+K346N+S225T+I34S; The corresponding synthesized oligopeptide is selected from any one of the following: Val-Ser, Gly-Ser, Gly-Gly, Ser-Ser, Tyr-Ser, Gly-Gly-Gly or Gly-His-Lys.

6. The method according to claim 5, characterized in that The method comprises: using the α-amino acid ester acyltransferase mutant to catalyze a substrate to obtain the oligopeptide.

7. The method according to claim 6, characterized in that The substrate includes an acyl donor and an acyl acceptor; The acyl donor is an amino acid ester hydrochloride, and the amino acid ester hydrochloride is selected from any one of the following: amino acid methyl ester hydrochloride, amino acid ethyl ester hydrochloride or amino acid isopropyl ester hydrochloride; The acyl acceptor includes amino acids or a first peptide segment; the first peptide segment is formed by condensing two amino acids.

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