Method for producing α-amino acid ester acyltransferase and its application in dipeptide synthesis

Abstract

This invention provides a method for producing α-amino acid ester acyltransferase and its application in dipeptide synthesis. The dipeptide synthesis method involves using α-amino acid ester acyltransferase to perform a dipeptide synthesis reaction and obtain a dipeptide, wherein the α-amino acid ester acyltransferase has a conserved amino acid sequence region as shown in SEQ ID NO: 1. By utilizing α-amino acid ester acyltransferase, various functional dipeptides can be synthesized, exhibiting a relatively broad substrate spectrum, and some enzymes exhibiting relatively high synthesis efficiency, making them suitable for industrial expansion, resulting in low costs, high yields, and the realization of green chemistry.

Description

Technical Field

[0001] (Cross - reference to related applications) This application is based on a Chinese application with a CN application number of 202310894437.3 and a filing date of July 20, 2023, and claims its priority. The disclosure content of this CN application is incorporated into this application in its entirety.

[0002] The present invention relates to the field of enzyme technology, specifically to a method for producing α - amino acid ester acyltransferase and its application in the synthesis of dipeptides.

Background Art

[0003] Dipeptides are the simplest peptides and are composed of an amide bond formed by the dehydration condensation of the α - carboxyl group of one molecule of amino acid and the α - amino group of another molecule of amino acid although the structure of dipeptides is simple, they have a wide range of biological activities and can play a regulatory role in life activities such as the physiological metabolism of organisms. The demand for peptides based on antibacterial, antiviral, anticancer, hormonal, and immunomodulatory effects is increasing year by year and is widely applied in fields such as pharmaceuticals, foods, health foods, and cosmetics.

[0004] For example, carnosine (β - alanyl - His) has antioxidant, anti - inflammatory, and anti - glycation roles, glycylglycine (Gly - Gly) is used pharmaceutically as a stabilizer for blood preservation and cytochrome C aqueous injection of protein drugs, alanylglutamine (Ala - Gln) is used as an important nutritional supplement for surgical patients, aspartame (Asp - Phe methyl ester) is a widely used sweetener, Ala - Phe, Ile - Phe, Pro - Gly dipeptides are salt - enhancing agents, Ile - Tyr, Lys - Trp, Val - Tyr, Ile - Trp are antihypertensive peptides, Arg - Trp has analgesic effects, Lys - Glu has antitumor activity, etc. <>

[0005] While more and more functional dipeptides are being discovered, currently only aspartame and Ala-Gln have achieved commercial production. Chemical synthesis of dipeptides has certain limitations; for example, operations related to the protection and deprotection of amino acids in the process can result in loss of optical activity in the product, relatively high synthesis costs, and the need to add toxic reagents. Therefore, finding a suitable and efficient method for synthesizing dipeptides has long been a hotspot problem.

[0006] α-amino acid ester acyltransferase (Aet) can react with another nucleophile, glutamine, using alanine methyl ester hydrochloride as an acyl group donor to produce Ala-Gln dipeptide. This process does not require ATP involvement and exhibits relatively high synthesis efficiency (A novel and efficient enzymatic method for the production of peptides from unprotected starting materials. J Biotechnol. 2005 Jan 26;115(2):211-20.; Gene cloning and characterization of α-amino acid ester acyl transferase in Empedobacter brevis ATCC14234 and Sphingobacterium siyangensis AJ2458. Biosci Biotechnol Biochem. 2011;75(11):2087-92.).

[0007] Currently, reported Aet enzymes exhibit relatively high catalytic efficiency, but their substrate spectra are generally relatively narrow, and only a small number of dipeptides (not exceeding 30 types) can be synthesized using Aet. This limits the broad application of Aet in the synthesis of various dipeptide products. Therefore, the development of α-amino acid ester acyltransferases with broad substrate spectra is of great importance. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The main objective of the present invention is to provide a method for producing α-amino acid ester acyltransferase and its application in dipeptide synthesis, which solves the problem in conventional techniques where the substrate spectrum of α-amino acid ester acyltransferase is relatively narrow. [Means for solving the problem]

[0009] To achieve the above objective, according to one aspect of the present invention, a method for producing a dipeptide is provided, the method comprising carrying out a dipeptide synthesis reaction using an α-amino acid ester acyltransferase to obtain a dipeptide, wherein the α-amino acid ester acyltransferase has a conserved region of the amino acid sequence as shown in SEQ ID NO: 1.

[0010] Furthermore, the α-amino acid ester acyltransferase is one or more selected from among the α-amino acid ester acyltransferases whose amino acid sequences are as shown in SEQ ID NO: 2-11.

[0011] Furthermore, the substrate for the dipeptide synthesis reaction comprises an acyl group donor substrate and a nucleophile substrate, wherein the acyl group donor substrate is selected from amino acid ester hydrochlorides, and the nucleophile substrate is an amino acid; preferably, the amino acid ester hydrochloride includes amino acid methyl ester hydrochloride, amino acid ethyl ester hydrochloride, or amino acid isopropyl ester hydrochloride; preferably, the amino acid and the nucleophile amino acid in the amino acid ester hydrochloride are each independently selected from glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylalanine, tryptophan, proline, or tyrosine.

[0012] Furthermore, the dipeptides include Gly-Gln, Gly-Tyr, Gly-Gly, Ala-Gln, or Arg-Trp.

[0013] Furthermore, the dipeptide synthesis reaction is carried out in an alkaline environment.

[0014] Furthermore, the alkaline environment is an alkaline buffer solution, preferably containing 0.1-0.2 M Tris-HCl with a pH of 8-8.5.

[0015] Furthermore, the reaction temperature for the dipeptide synthesis reaction is 20-25°C, and preferably the reaction time is 1.5-2 hours.

[0016] Furthermore, after the dipeptide synthesis reaction is completed, the method further comprises heating and inactivating the α-amino acid ester acyltransferase, wherein the heating and inactivation temperature is 75-85°C, and preferably the heating and inactivation time is 10-15 min.

[0017] Furthermore, the ratio of the mass of the wet bacterial mud containing the α-amino acid ester acyltransferase that performs the dipeptide synthesis reaction to the mass of the substrate, in terms of mass of wet bacterial mud / mass of substrate, is 1:1 to 10, preferably 1:10.

[0018] According to another aspect of the present invention, a method for producing α-amino acid ester acyltransferase, comprising: cloning the gene for α-amino acid ester acyltransferase in the above method into an expression vector to obtain a recombinant vector; introducing the recombinant vector into Escherichia coli to obtain a recombinant strain; culturing the recombinant strain to induce expression of α-amino acid ester acyltransferase and obtain a culture medium; sonicating the culture medium and then centrifuging it to obtain α-amino acid ester acyltransferase; preferably, the expression vector is p ET-22a(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b(+), pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET -20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET- 31b(+), pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), pET- 44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV2 The recombinant vector comprises 20, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18, or pUC-19; preferably, the host cell comprises a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cell is Escherichia coli and Bacillus subtilis; more preferably, the species of Escherichia coli is BL21; preferably, the eukaryotic cell is brewing yeast and Pichia pastris; preferably, the 3' or 5' end of the α-amino acid ester acyltransferase gene in the recombinant vector comprises a tag gene;Preferably, the tag gene includes a DNA sequence encoding MBP protein, NusA protein, Trx protein, SUMO protein, DsbA protein, TF protein, or GST protein; more preferably, the tag gene includes a DNA sequence encoding MBP protein, SUMO protein, or TF protein. [Effects of the Invention]

[0019] By applying the technical solution of the present invention, various functional dipeptides can be synthesized using the α-amino acid ester acyltransferase of this application. The substrate spectrum is relatively broad, and some enzymes have relatively high synthesis efficiency, making them suitable for industrial expansion, resulting in low costs, high yields, and true green chemistry. [Modes for carrying out the invention]

[0020] It should be noted that the embodiments and features described herein can be combined without conflict. The present invention will now be described in detail in conjunction with the embodiments.

[0021] As described in the background technology section, conventional α-amino acid ester acyltransferases generally exhibit a relatively narrow substrate spectrum in dipeptide synthesis reactions, and it has been reported that only small amounts of dipeptides (not exceeding 30 types) can be synthesized using α-amino acid ester acyltransferases. This limits the broad application of α-amino acid ester acyltransferases in the synthesis of various dipeptide products.

[0022] Therefore, developing α - amino acid ester acyltransferases with a broad substrate spectrum is of great significance. In this application, the inventors obtained a series of α - amino acid ester acyltransferases that can be widely used in the production of functional dipeptides through large - scale batch screening. Since the enzymes obtained from the above screening are involved in the synthesis of more than 200 kinds of dipeptides and can greatly expand the market for dipeptide product production, a series of protection plans for this application are proposed.

[0023] In the first typical embodiment of the present invention, a method for producing a dipeptide is provided. This method includes performing a dipeptide synthesis reaction using α - amino acid ester acyltransferase to obtain a dipeptide, provided that the α - amino acid ester acyltransferase has a conserved region of the amino acid sequence as shown in SEQ ID NO: 1.

[0024] SEQ ID NO: 1: YGISYPGFYST.

[0025] This application is very important for expressing enzymes with the above conserved amino acid sequence regions through sequence alignment of a series of α - amino acid ester acyltransferase amino acid sequences and for a wide variety of dipeptide syntheses. In one preferred embodiment, the above α - amino acid ester acyltransferase is any one or more selected from α - amino acid ester acyltransferases whose amino acid sequences are as shown in SEQ ID NOs: 2 - 11. The above α - amino acid ester acyltransferase is an amino acid sequence derived from wild - type α - amino acid ester acyltransferases of species such as Sphingobacterium and Pedobacter. It should be noted that in this application, it is not limited to these enzymes, and any wild - type or its modified α - amino acid ester acyltransferase containing the above conserved region is applicable to this application.

[0026] In this field, families of amino acid residues having similar side chains have already been defined. These families include amino acids having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferred to use another amino acid residue from the same side chain family in place of the corresponding amino acid residue.

[0027] Methods for identifying conservative substitutions of amino acids are well known in the art (see, for example, Brummell et al., Probing the combining site of an anti-carbohydrate antibody by saturation-mutagenesis: role of the heavy-chain CDR3 residues. Biochem. 32:1180-1187 (1993); Kobayashi et al., Tryptophan H33 plays an important role in pyrimidine(6-4)pyrimidone photoproduct binding by a high-affinity antibody. Protein Eng. 12(10):879-884 (1999), and Burks et al., In vitro scanning saturation mutagenesis of an antibody binding pocket. Proc Natl Acad Sci U S A. 94:412-417 (1997)).

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[0038] The α-amino acid ester acyltransferase (Aet) of this application can synthesize dipeptides using a variety of substrates, and can produce dipeptides by reacting an amino acid ester hydrochloride with another nucleophilic amino acid as an acyl group donor. Therefore, any substrate that any α-amino acid ester acyltransferase can act on is applicable to this application. In one preferred embodiment, the substrate for the dipeptide synthesis reaction includes an acyl group donor substrate and a nucleophilic substrate, wherein the acyl group donor substrate is selected from amino acid ester hydrochlorides, and the nucleophilic substrate is an amino acid.

[0039] In one preferred embodiment, the amino acid ester hydrochloride includes amino acid methyl ester hydrochloride, amino acid ethyl ester hydrochloride, or amino acid isopropyl ester hydrochloride, and dipeptides can be synthesized relatively efficiently using these three types of amino acid ester hydrochloride as substrates. More preferably, the amino acid ester hydrochloride includes amino acid methyl ester hydrochloride.

[0040] Any amino acid species in any substrate that can be used to synthesize dipeptides using the α-amino acid ester acyltransferase (Aet) of this application is applicable to this application. In one preferred embodiment, the amino acid in the amino acid ester hydrochloride and the nucleophile amino acid are independently selected from glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylalanine, tryptophan, proline, or tyrosine.

[0041] The reaction equation for dipeptide synthesis is as follows: [ka] R1 and R2 represent the side chains of 20 different amino acids, and R3 represents a methyl group, an ethyl group, or an isopropyl group.

[0042] Because the α-amino acid ester acyltransferase in this application has a broader range of substrates and can synthesize a greater variety of dipeptides, in practical applications, any required dipeptide can be synthesized depending on the specific situation. In one preferred example, the synthesized dipeptides include Gly-Gln, Gly-Tyr, Gly-Gly, Ala-Gln, or Arg-Trp. In particular, several important functional dipeptides are synthesized using the α-amino acid ester acyltransferase.

[0043] Here, dipeptides are composed of an amide bond formed by the dehydration condensation of the α-carboxyl group of one amino acid molecule and the α-amino group of another amino acid molecule, and are widely applied in fields such as pharmaceuticals, food, health foods, and cosmetics. For example, glycylglutamine (Gly-Gln) can increase cell production as a nutrient in cell culture in place of glutamine; glycyltyrosine (Gly-Tyr) is an antihypertensive peptide; glycylglycine (Gly-Gly) is used pharmaceutically as a blood preservative and as a stabilizer for cytochrome C aqueous injections of protein drugs; alanylglutamine (Ala-Gln) is used as an important nutritional supplement for surgical patients; and Arg-Trp has analgesic effects.

[0044] In one preferred embodiment, the dipeptide synthesis reaction of this application is carried out in an alkaline environment. Any conditions that can be achieved to maintain an alkaline environment for the dipeptide synthesis reaction using α-amino acid ester acyltransferase are applicable to this application. In one preferred embodiment, the alkaline environment is an alkaline buffer. In one preferred embodiment, the alkaline buffer contains 0.1–0.2 M Tris-HCl with a pH of 8–8.5.

[0045] Any reaction conditions that can complete the dipeptide synthesis reaction are applicable to this application. Considering the reaction efficiency and energy consumption of the dipeptide, in one preferred embodiment, the reaction temperature for the dipeptide synthesis reaction is 20-25°C and the reaction time is 1.5-2 hours. Under these reaction temperature and time conditions, the reaction efficiency is high and the energy consumption is relatively low.

[0046] In one preferred embodiment, after the dipeptide synthesis reaction is complete, the method further includes heating and inactivating the α-amino acid ester acyltransferase. Inactivating the α-amino acid ester acyltransferase after the reaction and stopping the synthesis reaction in a timely manner by inactivating the enzyme is advantageous for improving the reaction efficiency of dipeptide synthesis and avoids affecting the quality of the dipeptide product due to excessively long reaction times. In one preferred embodiment, inactivation is performed after the dipeptide synthesis reaction, with a heating and inactivation temperature of 75-85°C and a heating and inactivation time of 10-15 min.

[0047] Any ratio of enzyme and substrate used to perform the dipeptide synthesis reaction using any α-amino acid ester acyltransferase is applicable to this application. To further improve the reaction efficiency of dipeptide synthesis, in one preferred embodiment, the ratio of the mass of wet bacterial mud containing the α-amino acid ester acyltransferase performing the dipeptide synthesis reaction to the mass of the substrate, in terms of mass of wet bacterial mud / mass of substrate, is 1:1-10, preferably 1:10.

[0048] A second typical embodiment of the present invention provides a method for producing α-amino acid ester acyltransferase, comprising: cloning the gene for the α-amino acid ester acyltransferase into an expression vector to obtain a recombinant vector; introducing the recombinant vector into E. coli to obtain a recombinant strain; culturing the recombinant strain to induce the expression of α-amino acid ester acyltransferase and obtain a culture medium; and obtaining α-amino acid ester acyltransferase by sonication followed by centrifugation after the culture medium has been sonicated. The expression production of α-amino acid ester acyltransferase is completed by a general method for prokaryotic expression proteins.

[0049] Unless otherwise specified, the molecular biology experimental methods used in this application are basically based on the methods in J. Sam brook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Reizei-ko Laboratory Publishing, 1989, and FMAusubel et al., Guidelines for Strict Molecular Biology Experiments, 3rd edition, John Wiley & Sons, Inc., 1995, and the use of restriction enzymes is in accordance with the conditions recommended by the product manufacturer. Those skilled in the art should know that the examples are illustrative and not intended to limit the scope for which protection is sought in this application.

[0050] Any recombinant particles capable of normally expressing α-amino acid ester acyltransferase are applicable to this application, and in one preferred embodiment, the expression vectors are pET-22a(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b(+), pET-15 b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b( +), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+) , pET-31b(+), pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE Includes 30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18, or pUC-19.

[0051] Any host cell capable of successfully inducing the expression of any α-amino acid ester acyltransferase is applicable to this application, and in one preferred embodiment, the host cell includes prokaryotic or eukaryotic cells; the prokaryotic cells are Escherichia coli and Bacillus subtilis; the Escherichia coli species is BL21; and the eukaryotic cells are brewing yeast and Pichia pastrius. Any gene element or sequence capable of promoting α-amino acid ester acyltransferase gene expression is applicable to this application, and in one preferred embodiment, the 3' or 5' end of the α-amino acid ester acyltransferase gene in the recombinant vector includes a tag gene.

[0052] In one preferred embodiment, the tag gene comprises a DNA sequence encoding MBP protein, NusA protein, Trx protein, SUMO protein, DsbA protein, TF protein, or GST protein; in one more preferred embodiment, the tag gene comprises a DNA sequence encoding MBP protein, SUMO protein, or TF protein. Recombinant vectors having the above tag genes can promote α-amino acid ester acyltransferase gene expression and produce α-amino acid ester acyltransferase more efficiently.

[0053] The present application will be described in more detail below in conjunction with specific embodiments, and these embodiments should not be understood as limiting the scope for which protection is sought in this application.

[0054] Example 1: Preparation of α-amino acid ester acyltransferase Aet enzyme solution After obtaining enzyme sources from an enzyme bank through literature search and gene mining, we obtained the α-amino acid ester acyltransferase Aet from different sources using artificial chemical synthesis. We then introduced the endonuclease moiety NdeI to the 5' end and the endonuclease moiety XhoI to the 3' end, synthesized the gene into pUC19, and obtained pUC19-Aet.

[0055] When the expression vector pET-28a(+) was bis-enzymed (NdeI+XhoI), the α-amino acid ester acyltransferase gene pUC19-Aet was also bis-enzymed (NdeI+XhoI). The excised Aet gene coding fragment was ligated to the expression vector pET-28a(+), yielding recombinant E. coli granules pET-28a(+)-Aet containing α-amino acid ester acyltransferase. This was then used to transform E. coli BL21(DE3) to obtain recombinant E. coli strain BL21(DE3) / Aet.

[0056] Furthermore, to improve protein expression levels, different fusion tags such as MBP (maltose-binding protein), NusA (transcriptional arrest anti-stopping factor), Trx (thioredoxin A), SUMO (low molecular weight ubiquitin-related modification protein), DsbA (protein disulfide bond folding isomer), TF (trigger factor), and GST (glutathione S-transferase) were constructed at the N-terminus of proteins using homologous recombination and fusion expression was performed. Protein expression levels after the addition of each fusion tag improved to varying degrees.

[0057] 4 ml of BL21(DE3) strain containing recombinant granules was taken, inoculated into a 2 L triangular bottle containing 400 mL of LB medium, and incubated with shaking at 37°C and 200 rpm for 2-3 hours, then OD 600 If the value was between 0.6 and 0.8, a final concentration of 0.02 mM IPTG was added, and induction was carried out at 16°C for 18 hours. After induction was complete, the cells were collected by centrifugation at 4°C. The cells were resuspended in 10 mL of 0.1 M Tris-HCl (pH 8.0) per 1 g of bacterial sediment, disrupted by sonication, centrifuged, and the supernatant was collected to obtain the crude enzyme solution of the catalytic reaction.

[0058] Note: The test materials and reagents used in the examples are described below.

[0059] 1. Strains and vectors: Escherichia coli expression vector pET-28a(+) and strain BL21(DE3) 2. Culture medium: E. coli medium LB (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0).

[0060] Example 2: Enzyme Screening In this example, more than 200 α-amino acid ester acyltransferases from the enzyme bank, as well as unreported enzymes that may have the same function, were selected for screening. All were screened by synthesizing the dipeptide Gly-Tyr. The screening system (1 mL) consisted of 12.5 mg (100 mM) glycine methyl hydrochloride, 18.1 mg (100 mM) tyrosine, 62.5 μL of crude enzyme solution, and 0.1 M Tris-HCl (pH 8.0), and was reacted at 20°C for 2 hours. The reaction system was heated at 80°C for 10 minutes, centrifuged, and then analyzed by HPLC. The reaction results of the active enzymes were statistically analyzed as follows (Table 1). Excluding the 10 enzymes listed in the table below, the remaining 190 enzymes all showed no reaction activity, i.e., their molar conversion rate was 0%.

[0061] [Table 1]

[0062] Note: In the table above, + indicates a molar conversion rate less than 10%, and ++ indicates a molar conversion rate of 10% or more and less than 20%. Here, the molar conversion rate is calculated as: (number of moles of dipeptide in the system after the reaction) / (number of moles of a single amino acid + number of moles of dipeptide) × 100%.

[0063] Example 3 As shown in Table 1, homologous alignment of amino acid sequences was performed on 10 different Aet enzymes obtained through screening, and a highly conserved sequence, YGISYPGFYST (SEQ ID NO: 1), was discovered. Structurally, it is located near the active site and is a relatively important amino acid. Subsequently, sequence analysis was performed on enzymes that showed no activity during screening. Since the above conserved sequence was not simultaneously found in most of the inactive enzymes, it is hypothesized that this conserved sequence may be related to the dipeptide synthesis function.

[0064] To further confirm that this highly conserved sequence, from SEQ ID NO: 2 to SEQ ID NO: 11, is key to the catalytic synthesis of Gly-Tyr dipeptides, mutations of the same type of amino acid were performed on the amino acids in this conserved region (Table 2), and the activity was confirmed using the same reaction system as in Example 2.

[0065] [Table 2]

[0066] The experimental results showed that when some amino acids within the conserved sequence were mutated to corresponding amino acids with relatively similar spatial structure or chemical properties, the catalytic activity of the enzyme was almost completely lost. This indicates that these sequences are highly conserved, are fundamental features of sequence numbers 2 through 11, and form the basic framework for dipeptide synthesis by the enzyme, making them extremely important for dipeptide synthesis.

[0067] Example 4 The activity for synthesizing the Gly-Gln dipeptides SEQ ID NOs. 4 to 13 was investigated. The reaction system was as follows (1 mL): 12.5 mg (100 mM) glycine methyl hydrochloride, 14.6 mg (100 mM) glutamine, 62.5 μL crude enzyme solution, and 0.1 M Tris-HCl (pH 8.0). The reaction was carried out at 20°C for 2 hours. The reaction system was heated at 80°C for 10 minutes, centrifuged, and then analyzed by HPLC. The reaction results were statistically summarized below (Table 3).

[0068] [Table 3]

[0069] Note: In the table above, + indicates a molar conversion rate of less than 20%, ++ indicates a molar conversion rate of 20% or more but less than 30%, +++ indicates a molar conversion rate of 30% or more but less than 40%, and ++++ indicates a molar conversion rate greater than 40%.

[0070] Example 5 The activity for synthesizing the Ala-Gln dipeptides SEQ ID NOs. 4 to 13 was investigated. The reaction system was as follows (1 mL): 13.9 mg (100 mM) alanine methyl hydrochloride, 14.6 mg (100 mM) glutamine, 62.5 μL crude enzyme solution, and 0.1 M Tris-HCl (pH 8.0). The reaction was carried out at 20°C for 2 hours. The reaction system was heated at 80°C for 10 minutes, centrifuged, and then analyzed by HPLC. The reaction results were statistically summarized below (Table 4).

[0071] [Table 4]

[0072] Note: In the table above, + indicates a molar conversion rate of less than 20%, ++ indicates a molar conversion rate of 20% or more but less than 30%, +++ indicates a molar conversion rate of 30% or more but less than 40%, and ++++ indicates a molar conversion rate greater than 40%.

[0073] Example 6 Using SEQ ID NOs. 2 and 5, which exhibit relatively good activity, the synthesis reactions of Gly-Tyr, Gly-Gln, and Ala-Gln in the above examples were carried out, with the substrates changed from methyl ester hydrochloride to ethyl ester hydrochloride and isopropyl ester hydrochloride. The reaction system was as follows (1 mL): 100 mM ethyl ester hydrochloride / isopropyl ester hydrochloride, 100 mM glutamine / tyrosine, 62.5 μL of crude enzyme solution, and 0.1 M Tris-HCl (pH 8.0) were reacted at 20°C for 2 hours. The reaction system was heated at 80°C for 10 minutes, centrifuged, and then analyzed by HPLC. The reaction results were statistically summarized below (Table 5).

[0074] [Table 5]

[0075] Note: In the table above, a "+" indicates that the molar conversion rate is greater than 5% but less than 20%.

[0076] In the above reactions, the activity (despite a certain degree of enzyme activity reduction) was clearly detected in all cases after substituting the substrate from methyl ester hydrochloride to ethyl ester hydrochloride and isopropyl ester hydrochloride for the synthesis of Gly-Tyr, Gly-Gln, and Ala-Gln. This demonstrated that methyl ester hydrochloride can be substituted with other derivative esters.

[0077] Example 7 The activity for synthesizing the Gly-Gly dipeptides from SEQ ID NO: 2 to SEQ ID NO: 11 was investigated. The reaction system was as follows (1 mL): 100 mM glycine methyl hydrochloride, 100 mM glycine, 62.5 μL of crude enzyme solution, and 0.1 M Tris-HCl (pH 8.0). The reaction was carried out at 20°C for 2 hours. The reaction system was heated at 80°C for 10 minutes, centrifuged, and then analyzed by HPLC. The reaction results were statistically summarized below (Table 6).

[0078] [Table 6]

[0079] Note: In the table above, + indicates a molar conversion rate of less than 20%, ++ indicates a molar conversion rate of 20% or more but less than 30%, +++ indicates a molar conversion rate of 30% or more but less than 40%, and ++++ indicates a molar conversion rate greater than 40%.

[0080] Example 8 The relatively active samples SEQ ID NOs. 2 and 5 from the above examples were subjected to reactions with 20 different amino acid methyl ester hydrochlorides and 20 different amino acids, and their substrate spectral ranges were investigated. The reaction system was as follows (1 mL): 100 mM of different amino acid methyl ester hydrochlorides (first column in the table below), 14.6 mg (100 mM) of different amino acids (first row in the table below), 62.5 μL of crude enzyme solution, and 0.1 M Tris-HCl (pH 8.0). The reaction was carried out at 20°C for 2 hours. The reaction system was heated at 80°C for 10 minutes, centrifuged, and then analyzed by LC-MS. The reaction results were statistically summarized below (Tables 7 and 8).

[0081] [Table 7]

[0082] Note: This table shows the reaction results for Sequence ID No. 2. The first column is the methyl ester hydrochloride of the indicated amino acid, and the first row is the indicated amino acid. * indicates 0 ≤ molar conversion rate < 1%, ** indicates a molar conversion rate of 1% or more and less than 10%, and *** indicates a molar conversion rate greater than 10%.

[0083] [Table 8]

[0084] Note: This table shows the reaction results for Sequence ID No. 5. The first column is the methyl ester hydrochloride of the amino acid, and the first row is the amino acid shown. Different quantities of * indicate the reaction status between the methyl ester hydrochloride of the amino acid in the row and the amino acid in the column. * indicates a molar conversion rate of 0 ≤ < 1%, ** indicates a molar conversion rate of 1% or more and less than 10%, and *** indicates a molar conversion rate greater than 10%.

[0085] However, if the molar conversion rate is >0, this reaction can synthesize dipeptides. As a result, Sequence ID No. 2 and Sequence ID No. 5 have relatively broad substrate spectra and can be used for the synthesis of various functional dipeptides. Furthermore, the substrate spectra of the eight enzymes from Sequence ID No. 3 to No. 4 and Sequence ID No. 6 to No. 11 were also examined. The results showed that these eight enzymes generally have somewhat low activity (1 < molar conversion rate < 10%), but all of them also have activity for dipeptide synthesis in most reactions.

[0086] Example 9 The reaction was expanded to synthesize Gly-Tyr, Gly-Gln, and Ala-Gln using Sequence ID No. 2. The reaction system was as follows (1 L): 400 mM glycine methyl hydrochloride and alanine methyl hydrochloride, 400 mM tyrosine and glutamine, 30 mL of crude enzyme solution, and 0.1 M Tris-HCl (pH 8.0) were reacted at 20°C for 2 hours. After the reaction was complete, the reaction system was separated and purified, and the purity and content of the final product were detected by HPLC and NMR. The results were statistically summarized below (Table 9).

[0087] [Table 9]

[0088] From the above description, it can be seen that the above embodiment of the present invention has achieved the following technical effects: (1) In the present invention, 10 dipeptide synthesis activity enzymes SEQ ID NOs: 2-11 were obtained by enzyme screening, and these enzymes have highly conserved amino acid sequence regions that are related to dipeptide synthesis activity, and these amino acid sequence regions constitute the basic framework of the catalytic activity of these 10 enzymes.

[0089] (2) Sequence IDs 2-11 can synthesize Gly-Tyr, and can also be used to synthesize various functional dipeptides such as Gly-Gln, Gly-Gly, and Ala-Gln. They have a relatively broad substrate spectrum, and some enzymes exhibit relatively high synthesis efficiency.

[0090] (3) The enzyme obtained in this invention is relatively easy to use in industrial expansion, is low-cost, has a high yield, and represents true green chemistry.

[0091] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Those skilled in the art will know that various modifications and changes are possible to the present invention. Any modifications, equivalent substitutions, or improvements in the spirit and principles of the present invention should also be within the scope of protection.

Claims

1. A method for producing a dipeptide, This includes performing a dipeptide synthesis reaction using α-amino acid ester acyltransferase to obtain the dipeptide, However, the method is characterized in that the α-amino acid ester acyltransferase has a conserved region of the amino acid sequence as shown in SEQ ID NO:

1.

2. The method according to claim 1, characterized in that the α-amino acid ester acyltransferase is selected from any one of the α-amino acid ester acyltransferases whose amino acid sequences are as shown in SEQ ID NOs: 2 to SEQ ID NOs:

11.

3. The method according to claim 1, characterized in that the substrate for the dipeptide synthesis reaction comprises a substrate that is an acyl group donor and a substrate that is a nucleophile, wherein the substrate that is an acyl group donor is selected from amino acid ester hydrochlorides, and the substrate that is a nucleophile is an amino acid.

4. The method according to claim 3, characterized in that the ester hydrochloride of the amino acid includes a methyl ester hydrochloride of an amino acid, an ethyl ester hydrochloride of an amino acid, or an isopropyl ester hydrochloride of an amino acid.

5. The method according to claim 3, characterized in that the amino acid in the ester hydrochloride of the amino acid and the nucleophilic amino acid are each independently selected from glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylalanine, tryptophan, proline, or tyrosine.

6. The method according to claim 1, characterized in that the dipeptide comprises Gly-Gln, Gly-Tyr, Gly-Gly, Ala-Gln, or Arg-Trp.

7. The method according to any one of claims 1 to 6, characterized in that the dipeptide synthesis reaction is carried out in an alkaline environment.

8. The method according to any one of claims 1 to 6, characterized in that the reaction temperature for the dipeptide synthesis reaction is 20°C to 25°C and the reaction time is 1.5h to 2h.

9. After the dipeptide synthesis reaction is completed, the method further includes heating and inactivating the α-amino acid ester acyltransferase. The method according to any one of claims 1 to 6, characterized in that the temperature for heating and inactivation is 75°C to 85°C, and the time for heating and inactivation is 10 min to 15 min.

10. The method according to any one of claims 1 to 6, characterized in that the ratio of the mass of the wet microbial mud containing the α-amino acid ester acyltransferase that carries out the dipeptide synthesis reaction to the mass of the substrate is 1:1 to 10, in terms of mass of wet microbial mud / mass of substrate.

11. A method for producing α-amino acid ester acyltransferase, comprising cloning the gene of the α-amino acid ester acyltransferase in the method of claim 1 into an expression vector to obtain a recombinant vector, The recombinant vector is introduced into host cells to obtain a recombinant bacterial strain. The recombinant bacterial strain is cultured to induce the expression of the α-amino acid ester acyltransferase, and a culture medium is obtained. A manufacturing method characterized by comprising ultrasonically disrupting the culture medium and then centrifuging it to obtain the α-amino acid ester acyltransferase.

12. The method for producing the product according to claim 11, characterized in that the 3' or 5' end of the gene for the α-amino acid ester acyltransferase in the recombinant vector contains a tag gene.

13. The method for producing the product according to claim 12, characterized in that the tag gene includes a DNA sequence encoding MBP protein, NusA protein, Trx protein, SUMO protein, DsbA protein, TF protein, or GST protein.

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