Alpha-amino acid ester acyltransferase mutant and application thereof

Abstract

The invention provides an alpha-amino acid ester acyltransferase mutant and application thereof. The alpha-amino acid ester acyltransferase mutant comprises: (a) a protein having an amino acid sequence as shown in SEQ ID NO: 1; (b) a protein which is subjected to amino acid mutation and has an alpha-amino acid ester acyltransferase function on at least one of the following sites of the amino acid sequence in (a): P158, K80, T208, A302, A175, N85, N605, K346, S225, I34 or S348; or (c) a protein which has 80% or more homology with the amino acid sequence defined in (a) or (b) and has an alpha-amino acid ester acyltransferase function. The alpha-amino acid ester acyltransferase mutant has the advantages of wide substrate spectrum and high catalytic activity, can be well used for oligopeptide synthesis, is low in cost and high in yield, and realizes real green chemistry.

Description

Technical Field

[0001] The present invention relates to the field of enzyme catalysis, and in particular to an alpha-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 connected by peptide bonds, with a molecular weight of about 1000 Daltons or less. Oligopeptides have many important biological functions in organisms. For example, many oligopeptides act as signal molecules and participate in cell-to-cell communication; some oligopeptides act as cofactors or inhibitors of enzymes and regulate metabolic pathways. Some oligopeptides have immunomodulatory effects and can enhance or inhibit immune responses; some oligopeptides have antioxidant activity and can scavenge free radicals and protect cells from oxidative damage; some oligopeptides can promote cell proliferation and differentiation and accelerate the wound healing process. Oligopeptides are widely used in many fields such as medicine, food, and cosmetics.

[0003] Dipeptides and tripeptides are the most basic oligopeptides. Despite their simple structures, they exhibit rich biological activities and can regulate a variety of life processes such as the physiological metabolism of organisms. For example, carnosine (β-alanyl-His) has antioxidant, anti-inflammatory and anti-glycation effects; Gly-Gly is used as a stabilizer for blood preservation fluid and cytochrome C injection in the medical field; Ala-Gln is an important nutritional supplement for surgical patients; Aspartame (Asp-Phe methyl ester) is a popular sweetener; Blue copper peptide (GHK-Cu) is widely used in high-end skin care brands, often as the main ingredient of essences, creams and other products; Ala-Phe, Ile-Phe, Pro-Gly dipeptides are used as salty taste enhancers; Ile-Tyr, Lys-Trp, Val-Tyr, Ile-Trp have antihypertensive effects, Arg-Trp has analgesic effects, and Lys-Glu exhibits anti-tumor activity.

[0004] There are limitations to the chemical synthesis of dipeptides. For example, if amino acid protection and deprotection operations are required, the product may be racemized and the synthesis cost is high. Sometimes toxic reagents need to be used. The fermentation method has the problems of low expression level and by-products. Therefore, exploring efficient and suitable oligopeptide synthesis methods has always been a hot issue in the scientific research field. 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 dipeptide. The whole process does not require ATP participation and has a high synthesis efficiency (J Biotechnol. 2005 Jan 26;115(2):211-20.; Biosci Biotechnol Biochem. 2011;75(11):2087-92.). The substrate spectrum of Aet reported so far is narrow and the catalytic activity is low. No more than 30 oligopeptides that can be synthesized enzymatically by α-amino acid ester acyltransferase have been reported, and there are no reports on the synthesis of many important functional oligopeptides, such as Gly-Ser, Gly-Gly, Ser-Ser, Val-Ser, Tyr-Ser, Gly-Gly-Gly and Gly-His-Lys by α-amino acid ester acyltransferase. This restricts the wide application of Aet in the synthesis of various oligopeptide products. Therefore, it is of great significance to develop α-amino acid ester acyltransferases with a wide substrate spectrum and high activity. 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 amino acid mutations 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 the function of α-amino acid ester acyltransferase.

[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 above-mentioned α-amino acid 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 the second aspect of the present invention, a gene is provided, which encodes the above 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 the fifth aspect of the present invention, a method for synthesizing an oligopeptide is provided, the method comprising: synthesizing the oligopeptide using the above α-amino acid ester acyltransferase mutant.

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

[0020] Further, the above substrate includes 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 scheme 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 for Val-Ser dipeptide was designed by reconstructing the ancestral enzyme. The above-mentioned α-amino acid ester acyltransferase was further mutated to obtain a variety of α-amino acid ester acyltransferase mutants with a broad substrate spectrum and high activity.

[0024] The α-amino acid ester acyltransferase mutant comprises: (a) a protein having an amino acid sequence as 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, Y300, 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, a protein having an amino acid mutation and having an α-amino acid ester acyltransferase function; or (c) a protein having more than 80% homology with the above amino acid sequence defined in (a) or (b) and having an α-amino acid ester acyltransferase function. The α-amino acid ester acyltransferase mutant of the present invention has a wide 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 of the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0026] As mentioned in the background art, the α-amino acid ester acyltransferase for synthesizing oligopeptides in the prior art has the defects of narrow substrate spectrum and low catalytic activity. In the present invention, the inventors attempted to design 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 for Val-Ser dipeptide by reconstructing the ancestral enzyme. Subsequently, the researchers obtained the three-dimensional structure of the 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 enzyme was tested using valine methyl ester and serine as substrates, and a series of α-amino acid ester acyltransferase mutants capable of efficiently synthesizing oligopeptides were obtained. Some α-amino acid ester acyltransferase mutants were used to carry out synthesis reactions of different oligopeptides, and it was found that in addition to Val-Ser dipeptide, these mutants can also be used for the efficient synthesis of a variety of other oligopeptides.

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

[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 amino acid mutations 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 the function of α-amino acid ester acyltransferase.

[0032] It should be noted that homology in the present invention refers to the "sequence identity" between two amino acid sequences, i.e., the percentage of identical amino acids between the sequences. Methods for evaluating 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 usually measured using sequence analysis software. For example, it can be determined by the BLAST program of the NCBI database. For the 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 having 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 having the function of α-amino acid ester acyltransferase, its active site, active pocket, active 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 according to the standard three-letter or one-letter amino acid code known and agreed 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 in the following groups (1)-(5) with another amino acid residue in 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 replaced by Gly or Ser; Arg is replaced by Lys; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala or Pro; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Ile or Val; Lys is replaced by Arg, Gln or Glu; Met is replaced by Leu, Tyr or Ile; Phe is replaced by Met, Leu or Tyr; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp or Phe; and Val is replaced by Ile or Leu.

[0036] A person 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, α-amino acid ester acyltransferase having the amino acid sequence shown in SEQ ID NO: 1 was used as the parent to perform site-directed mutagenesis 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 (the encoding gene has the nucleotide sequence shown in SEQ ID NO: 1) with high catalytic activity for Val-Ser dipeptide, which was designed by reconstructing the ancestral enzyme. By homology modeling and AI prediction of the amino acid sequence of the enzyme, 65 amino acid residues were found, 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 center and may affect the catalytic properties of the protein and the protein substrate selectivity. By mutating the above amino acid sites, a protein having the function of α-amino acid ester acyltransferase or even an enhanced α-amino acid ester acyltransferase function and an expanded substrate spectrum can be obtained. For the above-obtained protein, changes can be made at non-critical mutation sites and active sites, and a protein having more than 80% homology with the above amino acid sequence and having the function of α-amino acid ester acyltransferase can be obtained.

[0042] 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:

[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 applicant continues to explore the above active sites and finds that when the active sites are mutated to different amino acids, the corresponding protein activities are also different, and specific mutations can enhance the activity of α-amino acid ester acyltransferase. Through experimental exploration, it is found that by performing the above specific mutations on the active sites, an α-amino acid ester acyltransferase with enhanced activity can be obtained. For the amino acid mutation sites of the α-amino acid ester acyltransferase protein, flexible selection and combination can be performed in the above mutations.

[0045] 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, P158R+R205 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, wherein the gene 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 of α-amino acid ester acyltransferase and can be connected 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, ribosome and tRNA to obtain the mutant of 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 can replicate the plasmid, and can also transcribe and translate the gene carried on the plasmid to obtain a large number of α-amino acid acyltransferase mutants. Using existing technology, the non-animal and plant cells are broken, and the broken crude enzyme catalyzes the substrate to synthesize a variety of oligopeptides.

[0052] In a fifth typical embodiment of the present invention, a method for synthesizing oligopeptides is provided, the method comprising: synthesizing the oligopeptides using the above-mentioned 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 wide 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 that are not disclosed in the prior art by enzymatic synthesis methods. 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 α-amino acid ester acyltransferase mutant to catalyze the substrate to obtain the 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, 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 comprises an amino acid or a first peptide segment; the first peptide segment is formed by condensation of 2 to 19 amino acids. The substrate spectrum of the α-amino acid ester acyltransferase mutant of the present invention is relatively wide, and a variety of oligopeptides can be synthesized.

[0054] The present invention is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed by 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 (coding the amino acid sequence shown in SEQ ID NO: 1, named as wild-type α-amino acid ester acyltransferase in the present invention) was obtained by artificial chemical synthesis, and the endonuclease site NdeI was introduced at the 5' end and the endonuclease site XhoI was introduced at the 3' end, and the gene was synthesized in pUC19 to obtain pUC19-Aet.

[0057] The expression vector pET-28a(+) was double-digested with NdeI+Xho I, and the gene pUC19-Aet of α-amino acid ester acyltransferase was double-digested with NdeI+Xho I. The gene fragment encoding Aet was cut out and connected to the expression vector pET-28a(+) to obtain the Escherichia coli recombinant plasmid pET-28a(+)-Aet containing α-amino acid ester acyltransferase, which was transformed into Escherichia coli BL21(DE3) to obtain the recombinant Escherichia coli strain BL21(DE3)+ Aet.

[0058] Take 4 ml of BL21 (DE3) strain containing recombinant plasmid and inoculate it into a 2 L Erlenmeyer flask containing 400 mL LB medium. After shaking culture at 37℃ 200 rpm for 2-3 h, add IPTG with a final concentration of 0.02 mM when OD600 is 0.6-0.8, induce at 16℃ for 18 h, and collect the bacteria by centrifugation at 4℃. Resuspend the bacteria with 10 mL 0.1M Tris-HCl (pH 8.0) per gram of bacterial mud, break it by ultrasonication (power: 20%, time: 5 min), centrifuge (temperature: 4℃, time: 10 min, speed: 12000 rpm), collect the supernatant, and obtain the crude enzyme solution for 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 culture medium 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, the designed α-amino acid ester acyltransferase (whose encoding gene has the amino acid sequence shown in SEQ ID NO: 1, and the present invention is named as wild-type α-amino acid ester acyltransferase) is selected to perform the reaction of synthesizing Val-Ser dipeptide. The reaction system is as follows (1 mL): 100 mM valine methyl ester hydrochloride, 100 mM serine, 50 µL of crude enzyme solution and 0.1 M Tris-HCl (pH 8.0), and react at 20°C for 2 h. The reaction system was heated at 80°C for 10 min, centrifuged and analyzed by HPLC, and the reaction results are as follows (Table 1):

[0064] Table 1:

[0065]

[0066] Note: This table shows the reaction results of α-amino acid ester acyltransferase (named as wild-type α-amino acid ester acyltransferase in the present 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 the present invention is calculated as follows: (number of moles of product / number of moles of amino acid methyl ester substrate)×100%.

[0067] Example 3 Enzyme evolution

[0068] The α-amino acid ester acyltransferase (named as wild-type α-amino acid ester acyltransferase in the present invention, whose encoding gene has the amino acid sequence shown in SEQ ID NO: 1) was subjected to enzyme evolution, and 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 of crude enzyme solution and 0.1M 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 HPLC, and the reaction results were as follows (Table 2):

[0069] Table 2:

[0070]

[0071] Note: * represents 1%≤conversion rate<5%, ** represents 5%≤conversion rate<10%, *** represents 10%≤conversion rate<15%. The conversion rate is calculated as follows: (number of moles of product / number of 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 obtained in Example 3 was subjected to the next round of enzyme evolution, and the evolved mutant was 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, 0.1M 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 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 increased significantly, and the mutants with good activity were selected as templates for the next round of evolution.

[0079] Example 5

[0080] The best mutant obtained in Example 4 was subjected to the next round of enzyme evolution, and the evolved mutant was tested for 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, 0.1M 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 HPLC. The reaction results are as follows (Table 4 and Table 5):

[0081] Table 4:

[0082]

[0083] Table 5

[0084]

[0085] Note: ** in Table 4 and Table 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%.

[0086] From the above experimental results, it can be seen that 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, and mutants with good activity were selected as templates for the next round of evolution.

[0087] Example 6

[0088] The best mutant obtained in Example 5 was subjected to the next round of enzyme evolution, and the evolved mutant was tested for 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, 0.1M 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 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 the best mutants of Example 5 were further mutated, and some mutants still showed very obvious activity improvement.

[0092] Example 7

[0093] Different mutants were selected from Examples 3 to 6 to investigate the activity of synthesizing oligopeptides Gly-Ser, Gly-Gly, Ser-Ser, Val-Ser, Tyr-Ser, Gly-Gly-Gly and Gly-His-Lys, respectively. 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 reacted at 20°C for 2 h. The reaction system was heated at 80°C for 10 min, centrifuged and analyzed by HPLC, and the reaction results were statistically analyzed 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 P158R+N85G+N605P+K346N+S225T+I34K+S348G and wild-type α-amino acid ester acyltransferase with better activity in the above examples were subjected to reactions with 19 amino acid methyl ester hydrochlorides and 19 amino acids to investigate the changes in the substrate spectrum range after enzyme evolution. The reaction system is as follows (1 mL): 200 mM different amino acid methyl ester hydrochlorides (the first column of the table below), 200 mM different amino acids (the first row of the table below), 25 µL of crude enzyme solution, 0.1 M Tris-HCl (pH 8.0), and react 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 statistically as follows (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: * represents 10≤conversion rate <30%, ** represents conversion rate greater than or equal to 30% and less than 50%, *** represents conversion rate greater than or equal to 50%. As shown in the above results, compared with the wild-type α-amino acid ester acyltransferase, the mutant has a wider substrate spectrum and a significantly improved activity for the synthesis of various dipeptides.

[0105] Example 9

[0106] The reaction of P158R+N85G+N605P+K346N+S225T+I34K+S348G for synthesizing Gly-Ser, Ser-Ser, Val-Ser and Tyr-Ser was amplified. 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, 0.1M Tris-HCl (pH 8.0), and reacted at 20°C for 2h. After the reaction, the reaction system was post-processed, separated and purified, and the final product was sent to HPLC and NMR for purity and content detection. The results are statistically as follows (Table 10):

[0107] Table 10:

[0108]

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

[0110] From the above description, it can be seen that the above 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 be used to efficiently synthesize Val-Ser, and can also be used 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 mutant has a wide substrate spectrum and a high synthesis efficiency for some oligopeptides. The α-amino acid ester acyltransferase obtained in the present invention can be well used for industrial amplification, with low cost and high yield, realizing true green chemistry.

[0111] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope 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 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 amino acid mutations and has α-amino acid ester acyltransferase function; or (c) a protein having 99% or more homology with the amino acid sequence defined in (a) or (b) and having the function of α-amino acid ester acyltransferase; The amino acid mutation of (b) is selected from at least one of the following sites: 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; The letters before the numbers represent the original amino acids, and the letters after the numbers represent the mutated amino acids.

2. The α-amino acid ester acyltransferase mutant according to claim 1, characterized in that The mutation of the 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、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 in that The gene encodes the α-amino acid ester acyltransferase mutant according to claim 1 or 2.

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

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

6. 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 or 2.

7. The method according to claim 6, characterized in that The method comprises: using the α-amino acid ester acyltransferase mutant to catalyze a 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, 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 condensation of 2 to 19 amino acids.

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