Preparation method for alpha-amino acid ester acyl transferase and use thereof in dipeptide synthesis

Abstract

Provided are a preparation method for alpha-amino acid ester acyl transferase, and a use thereof in dipeptide synthesis. A method for dipeptide synthesis comprises: using alpha-amino acid ester acyl transferase to perform a dipeptide synthesis reaction, to obtain a dipeptide; the alpha-amino acid ester acyl transferase has a conserved region with an amino acid sequence as shown in SEQ ID NO: 1. Using the alpha-amino acid ester acyl transferase, a variety of functional dipeptides can be synthesized, a substrate spectrum is wide, and the synthesis efficiency of some enzymes is high; this can be well used for industrial amplification, with low cost and high yield, thus realizing green chemistry.

Description

Preparation method of α-amino acid acyltransferase and its application in dipeptide synthesis

[0001] This application is based on the Chinese application with CN application number 202310894437.3 and application date July 20, 2023, and claims its priority. The disclosed content of the CN application is introduced into this application as a whole. Technical Field

[0002] The present invention relates to the field of enzyme technology, in particular to a preparation method of α-amino acid acyltransferase and application of the same in dipeptide synthesis. Background Art

[0003] Dipeptides are the simplest peptides, consisting of an amide bond formed by the dehydration condensation of the α-carboxyl group of one amino acid and the α-amino group of another. Despite their simple structure, dipeptides possess a wide range of biological activities, regulating physiological metabolism and other vital activities. Demand for peptides with antibiotic, antiviral, anticancer, hormonal, and immunomodulatory properties is increasing annually, and they are widely used in medicine, food, health products, cosmetics, and other fields. For example, carnosine (β-alanyl-His) has antioxidant, anti-inflammatory, and anti-glycation effects; glycine (Gly-Gly) is used in medicine as a stabilizer for blood preservation and the protein drug cytochrome C injection; alanine glutathione (Ala-Gln) is an important nutritional supplement for surgical patients; aspartame (Asp-Phe methyl ester) is a widely used sweetener; Ala-Phe, Ile-Phe, and Pro-Gly dipeptides are salty taste enhancers; Ile-Tyr, Lys-Trp, Val-Tyr, and Ile-Trp are antihypertensive peptides; Arg-Trp has analgesic effects; Lys-Glu has anti-tumor activity, and so on. Although an increasing number of functional dipeptides have been discovered, only aspartame and Ala-Gln have achieved commercial production. The chemical synthesis of dipeptides has certain limitations. For example, the process involves the protection and deprotection of amino acids, the product will undergo racemization, the synthesis cost is high, and even toxic reagents need to be added. Therefore, finding a suitable and efficient dipeptide synthesis method has always been a hot issue.

[0004] α-Amino acid ester acyltransferase (Aet) can react with alanine methyl ester hydrochloride as an acyl donor and another nucleophile, glutamine, to produce Ala-Gln dipeptide. The entire process does not require the participation of ATP and has a 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.). Although the catalytic efficiency of Aet reported so far is high, the substrate spectrum is generally narrow. Only a small number of dipeptides (no more than 30) have been reported to be synthesized by Aet, which restricts the wide application of Aet in the synthesis of various dipeptide products. Therefore, it is of great significance to develop α-amino acid ester acyltransferases with a broad substrate spectrum.

[0005] Summary of the Invention

[0006] The main purpose of the present invention is to provide a preparation method of α-amino acid acyltransferase and its application in dipeptide synthesis, so as to solve the problem of narrow substrate spectrum of α-amino acid acyltransferase in the prior art.

[0007] To achieve the above object, according to one aspect of the present invention, a method for preparing a dipeptide is provided, the method comprising: using α-amino acid acyltransferase to carry out a dipeptide synthesis reaction to obtain a dipeptide; wherein the α-amino acid acyltransferase has a conserved region of the amino acid sequence shown in SEQ ID NO: 1.

[0008] Furthermore, the α-amino acid acyltransferase is selected from any one or more of the α-amino acid acyltransferases having amino acid sequences as shown in SEQ ID NOs: 2-11.

[0009] Furthermore, the substrates for the dipeptide synthesis reaction include substrates serving as acyl donors and substrates serving as nucleophiles, wherein the substrate serving as the acyl donor is selected from amino acid ester hydrochlorides, and the substrate serving as the nucleophile is an amino acid; preferably, the amino acid ester hydrochlorides include amino acid methyl ester hydrochlorides, amino acid ethyl ester hydrochlorides, or amino acid isopropyl ester hydrochlorides; preferably, the amino acid in the amino acid ester hydrochlorides and the amino acid serving as the nucleophile 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.

[0010] Further, the dipeptide includes Gly-Gln, Gly-Tyr, Gly-Gly, Ala-Gln or Arg-Trp.

[0011] Furthermore, the dipeptide synthesis reaction is carried out in an alkaline environment.

[0012] Furthermore, the alkaline environment is an alkaline buffer solution, and preferably the alkaline buffer solution includes 0.1-0.2M Tris-HCl with a pH value of 8-8.5.

[0013] Furthermore, the reaction temperature of the dipeptide synthesis reaction is 20-25° C.; preferably, the reaction time is 1.5-2 h.

[0014] Furthermore, after the dipeptide synthesis reaction is completed, the method further comprises heating and inactivating the α-amino acid acyltransferase at a temperature of 75-85° C.; preferably, the heating and inactivation time is 10-15 minutes.

[0015] Furthermore, the ratio of the mass of the wet bacterial mud containing α-amino acid acyltransferase for the dipeptide synthesis reaction to the mass of the substrate is 1:1-10, preferably 1:10, calculated on the basis of the mass of the wet bacterial mud / the mass of the substrate.

[0016] According to another aspect of the present invention, a method for preparing an α-amino acid acyltransferase is provided, characterized in that the gene of the α-amino acid acyltransferase in the above method is cloned into an expression vector to obtain a recombinant vector; the recombinant vector is introduced into Escherichia coli to obtain a recombinant strain; the recombinant strain is cultured to induce the expression of the α-amino acid acyltransferase to obtain a culture solution; the culture solution is ultrasonically disrupted and then centrifuged to obtain the α-amino acid acyltransferase; preferably, the expression vector comprises pET-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, pQE 32. 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; preferably, the host cell includes a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cell is Escherichia coli and Bacillus subtilis; more preferably, the strain of Escherichia coli is BL21; preferably, the eukaryotic cell is Saccharomyces cerevisiae and Pichia pastoris; preferably, the 3' end or 5' end of the gene of α-amino acid acyltransferase in the recombinant vector includes 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.

[0017] By applying the technical solution of the present invention and utilizing the α-amino acid acyltransferase of the present application, a variety of functional dipeptides can be synthesized with a wide substrate spectrum and high synthesis efficiency of some enzymes. It can be well used for industrial amplification with low cost and high yield, thus realizing true green chemistry. DETAILED DESCRIPTION

[0018] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0019] As mentioned in the background art, α-amino acid acyltransferases in the prior art generally have a narrow substrate spectrum in dipeptide synthesis reactions. Only a small number of dipeptides (no more than 30) have been reported to be synthesized using α-amino acid acyltransferases, which restricts their widespread application in synthesizing a variety of dipeptide products. Therefore, the development of α-amino acid acyltransferases with a broad substrate spectrum is of great significance. In this application, the inventors have obtained a series of α-amino acid acyltransferases that can be widely used to produce functional dipeptides through large-scale screening. The enzymes obtained from this screening are capable of participating in the synthesis of over 200 dipeptides, greatly expanding the market for dipeptide product production. Therefore, a series of protection schemes are proposed in this application.

[0020] In a first typical embodiment of the present invention, a method for preparing a dipeptide is provided, the method comprising: using α-amino acid acyltransferase to carry out a dipeptide synthesis reaction to obtain a dipeptide; wherein the α-amino acid acyltransferase has a conserved region of the amino acid sequence shown in SEQ ID NO: 1.

[0021] SEQ ID NO: 1:

[0022] By comparing the amino acid sequences of a series of α-amino acid acyltransferases, this application discovered a class of enzymes having the above-mentioned conserved amino acid sequence region, which is crucial for the synthesis of a wide range of dipeptides. In a preferred embodiment, the above-mentioned α-amino acid acyltransferase is selected from any one or more of the α-amino acid acyltransferases having amino acid sequences as shown in SEQ ID NOs: 2-11. The above-mentioned α-amino acid acyltransferase is the amino acid sequence of wild-type α-amino acid acyltransferases derived from the species Sphingobacterium and Pe dobacter. It should be noted that this application is not limited to these enzymes, and any wild-type or modified α-amino acid acyltransferase containing the above-mentioned conserved region is applicable to this application.

[0023] Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., 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 mutagenes is of an antibody binding pocket. Proc Natl Acad Sci US A. 94: 412-417 (1997)).

[0024] SEQ ID NO: 2:

[0025] SEQ ID NO: 3:

[0026] SEQ ID NO: 4:

[0027] SEQ ID NO: 5:

[0028] SEQ ID NO: 6:

[0029] SEQ ID NO: 7:

[0030] SEQ ID NO: 8:

[0031] SEQ ID NO: 9:

[0032] SEQ ID NO: 10:

[0033] SEQ ID NO: 11:

[0034] The α-amino acid ester acyltransferase (Aet) of the present application can utilize a variety of substrates for dipeptide synthesis, using amino acid ester hydrochloride as an acyl donor to react with another nucleophilic reagent amino acid to generate a dipeptide. Therefore, any substrate that can be acted on by α-amino acid acyltransferase is suitable for the present application. In a preferred embodiment, the substrate of the dipeptide synthesis reaction includes a substrate as an acyl donor and a substrate as a nucleophilic reagent, wherein the substrate as the acyl donor is selected from the ester hydrochloride of amino acid, and the substrate as the nucleophilic reagent is an amino acid. In a preferred embodiment, the ester hydrochloride of amino acid includes methyl ester hydrochloride of amino acid, ethyl ester hydrochloride of amino acid or isopropyl ester hydrochloride of amino acid, and the use of the above three amino acid ester hydrochlorides as substrates can more efficiently perform the synthesis of dipeptides. More preferably, the ester hydrochloride of amino acid includes methyl ester hydrochloride of amino acid.

[0035] Any amino acid type in the substrate that can be used for dipeptide synthesis using the α-amino acid ester acyltransferase (Aet) of the present application is suitable for the present application. In a preferred embodiment, the amino acid in the amino acid ester hydrochloride and the amino acid used as the nucleophile 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.

[0036] The reaction equation for dipeptide synthesis is as follows:

[0037] R1 and R2 represent the side chains of 20 amino acids; R3 represents a methyl group, an ethyl group, or an isopropyl group.

[0038] The α-amino acid acyltransferase in this application has a wider range of substrates and can synthesize more types of dipeptides. Therefore, in practical applications, any desired dipeptide can be synthesized according to actual conditions. In a preferred embodiment, the synthesized dipeptide includes Gly-Gln, Gly-Tyr, Gly-Gly, Ala-Gln, or Arg-Trp. In particular, some important functional dipeptides are synthesized using α-amino acid acyltransferase. Among them, the dipeptide is composed of an amide bond formed by dehydration condensation of the α-carboxyl group of one amino acid molecule and the α-amino group of another amino acid molecule, and is widely used in the fields of medicine, food, health products, cosmetics, etc. For example, glycylglutamine (Gly-Gln) can replace glutamine as a nutrient for cell culture, thereby increasing cell yield; glycyltyrosine dipeptide (Gly-Tyr) is a blood pressure-lowering peptide; glycylcysteinyl peptide (Gly-Gly) is used in medicine as a stabilizer for blood preservation and protein drug cytochrome C injection; alanylglutamine (Ala-Gln) is an important nutritional supplement for surgical patients; and Arg-Trp has an analgesic effect.

[0039] In a preferred embodiment, the dipeptide synthesis reaction of the present application is carried out in an alkaline environment. Any conditions that can maintain an alkaline environment for the dipeptide synthesis reaction using α-amino acid acyltransferase are suitable for this application. In a preferred embodiment, the alkaline environment is an alkaline buffer. In a preferred embodiment, the alkaline buffer comprises 0.1-0.2 M Tris-HCl at a pH of 8-8.5.

[0040] Any reaction conditions capable of completing the dipeptide synthesis reaction are applicable to this application. Considering the dipeptide reaction efficiency and energy consumption, in a preferred embodiment, the dipeptide synthesis reaction temperature 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.

[0041] In a preferred embodiment, after the dipeptide synthesis reaction is completed, the method further includes heat-inactivating the α-amino acid acyltransferase. Inactivating the α-amino acid acyltransferase after the reaction and promptly stopping the synthesis reaction helps improve the efficiency of the dipeptide synthesis reaction and avoids prolonged reaction times that may affect the quality of the resulting dipeptide. In a preferred embodiment, the inactivation is performed after the dipeptide synthesis reaction at a temperature of 75-85°C and for a duration of 10-15 minutes.

[0042] Any ratio of enzyme and substrate for dipeptide synthesis reaction using α-amino acid acyltransferase is applicable to this application. To further improve the reaction efficiency of dipeptide synthesis, in a preferred embodiment, the ratio of the mass of wet bacterial sludge with α-amino acid acyltransferase to the mass of substrate for dipeptide synthesis reaction is 1:1-10, preferably 1:10, calculated as the mass of wet bacterial sludge / the mass of substrate.

[0043] In a second exemplary embodiment of the present invention, a method for preparing an α-amino acid acyltransferase is provided, comprising cloning the aforementioned α-amino acid acyltransferase gene 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 the α-amino acid acyltransferase to obtain a culture fluid; and ultrasonically disrupting the culture fluid and then centrifuging to obtain the α-amino acid acyltransferase. Expression and production of the α-amino acid acyltransferase are achieved using conventional prokaryotic protein expression methods.

[0044] Unless otherwise specified, the molecular biology experimental methods used in this application are basically based on the methods in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., Molecular Biology: A Laboratory Manual, 3rd edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases follows the conditions recommended by the product manufacturers. It is understood by those skilled in the art that the examples describe this application by way of example and are not intended to limit the scope of protection claimed in this application.

[0045] Any recombinant plasmid capable of normally expressing α-amino acid acyltransferase is suitable for the present application. In a preferred embodiment, the expression vector includes pET-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, p QE31, 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. Any host cell capable of normally inducing the expression of α-amino acid acyltransferase is suitable for the present application. In a preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell; the prokaryotic cell is Escherichia coli or Bacillus subtilis; the strain of Escherichia coli is BL21; and the eukaryotic cell is Saccharomyces cerevisiae or Pichia pastoris. Any genetic element or sequence capable of promoting the expression of an α-amino acid acyltransferase gene is suitable for use in this application. In a preferred embodiment, the 3' or 5' end of the α-amino acid acyltransferase gene in the recombinant vector includes a tag gene. In a preferred embodiment, the tag gene includes a DNA sequence encoding an MBP protein, a NusA protein, a Trx protein, a SUMO protein, a DsbA protein, a TF protein, or a GST protein; in a more preferred embodiment, the tag gene includes a DNA sequence encoding an MBP protein, a SUMO protein, or a TF protein. Recombinant vectors carrying the above-mentioned tag genes can promote the expression of the α-amino acid acyltransferase gene and more efficiently produce α-amino acid acyltransferase.

[0046] The present application is further described in detail below with reference to specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in this application.

[0047] Example 1 Preparation of α-amino acid ester acyltransferase Aet enzyme solution

[0048] The enzyme library was obtained through literature retrieval and gene mining. α-Amino acid ester acyltransferase Aet was then obtained from various sources through artificial chemical synthesis. The endonuclease sites NdeI and XhoI were introduced at the 5' and 3' ends, respectively. The gene was synthesized in pUC19 to obtain pUC19-Aet. The expression vector pET-28a(+) was double-digested with NdeI and XhoI, and the α-Amino acid ester acyltransferase gene pUC19-Aet was also double-digested with NdeI and XhoI. The Aet gene fragment was excised and ligated into the expression vector pET-28a(+). This recombinant E. coli plasmid pET-28a(+)-Aet containing the α-Amino acid ester acyltransferase was then transformed into E. coli BL21(DE3) to obtain the recombinant E. coli strain BL21(DE3) / Aet. In addition, to increase protein expression, different fusion tags, such as MBP (maltose binding protein), NusA (transcription termination anti-termination factor), Trx (thioredoxin A), SUMO (ubiquitin-related small modifier protein), DsbA (protein disulfide fold isomer), TF (trigger factor), and GST (glutathione S-transferase), were constructed and expressed at the N-terminus of the protein through homologous recombination. The protein expression levels were increased to varying degrees after adding the fusion tags.

[0049] Take 4 ml of BL21 (DE3) strain containing the recombinant plasmid and inoculate it into a 2 L triangular flask containing 400 mL of LB medium. After shaking culture at 37 ° C and 200 rpm for 2-3 h, the OD 600 When the pH value is 0.6-0.8, add IPTG to a final concentration of 0.02 mM and induce at 16°C for 18 h. After induction, collect the cells by centrifugation at 4°C. Resuspend the cells in 10 mL of 0.1 M Tris-HCl (pH 8.0) per gram of bacterial slurry, disrupt by ultrasonication, centrifuge, and collect the supernatant to obtain the crude enzyme solution for the catalytic reaction.

[0050] Note: The experimental materials and reagents used in the examples are described as follows:

[0051] 1. Strains and vectors: Escherichia coli expression vector pET-28a(+) and strain BL21(DE3)

[0052] 2. Culture medium: Escherichia coli culture medium LB (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0).

[0053] Example 2 Enzyme Screening

[0054] In this example, over 200 α-amino acid ester acyltransferases from an enzyme library, as well as unreported enzymes that may have similar functions, were screened using the synthetic dipeptide Gly-Tyr. The screening system (1 mL) consisted of 12.5 mg (100 mM) glycine methyl ester hydrochloride, 18.1 mg (100 mM) tyrosine, 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 h. The reaction system was heated at 80°C for 10 min, centrifuged, and analyzed by HPLC. The reaction results for the active enzymes are summarized below (Table 1). With the exception of the 10 enzymes listed in the table below, the remaining 190 enzymes showed no activity, indicating a molar conversion of 0%.

[0055] Table 1: Note: In the table above, + represents a molar conversion greater than or less than 10%, and ++ represents a molar conversion greater than or equal to 10% and less than 20%. The molar conversion is calculated as the number of moles of dipeptide in the reaction system after reaction / (the number of moles of the single amino acid + the number of moles of the dipeptide) × 100%.

[0056] Example 3

[0057] A homology comparison of the amino acid sequences of the 10 different Aet enzymes screened in Table 1 revealed a highly conserved sequence, YGISYPGFYST (SEQ ID NO: 1), located near the active site and representing a key amino acid sequence. Sequence analysis of the inactive enzymes screened revealed that the conserved sequence was not found in most of the inactive enzymes, suggesting that it may be related to dipeptide synthesis.

[0058] To further confirm that the highly conserved sequence in SEQ ID NO: 2 to SEQ ID NO: 11 is the key to catalyzing the synthesis of Gly-Tyr dipeptide, we mutated the amino acids in this conserved region with the same type of amino acids (Table 2) and tested the activity using the same reaction system as Example 2.

[0059] Table 2:

[0060] The experimental results show that after some amino acids in the conserved sequence mutate into corresponding amino acids with similar spatial structures or chemical properties, the catalytic activity of the enzyme is basically lost, indicating that these sequences are highly conserved and are the basic characteristics of SEQ ID NO: 2 to SEQ ID NO: 11. They are also the basic skeleton of the enzyme for synthesizing dipeptides and are crucial for the synthesis of dipeptides.

[0061] Example 4

[0062] The activity of synthesizing Gly-Gln dipeptides from SEQ ID NO: 4 to SEQ ID NO: 13 was investigated using the following reaction system (1 mL): 12.5 mg (100 mM) glycine methyl ester hydrochloride, 14.6 mg (100 mM) glutamine, 62.5 μL of crude enzyme solution, and 0.1 M Tris-HCl (pH 8.0). The reaction was conducted at 20°C for 2 h. The reaction system was heated at 80°C for 10 min, centrifuged, and analyzed by HPLC. The results are summarized in Table 3:

[0063] Table 3: Note: In the above table, + represents a molar conversion rate of less than 20%, ++ represents a molar conversion rate of greater than or equal to 20% and less than 30%, +++ represents a molar conversion rate of greater than or equal to 30% and less than 40%, and ++++ represents a molar conversion rate of greater than 40%.

[0064] Example 5

[0065] The activity of synthesizing Ala-Gln dipeptides from SEQ ID NO: 4 to SEQ ID NO: 13 was evaluated using the following reaction system (1 mL): 13.9 mg (100 mM) alanine methyl ester hydrochloride, 14.6 mg (100 mM) glutamine, 62.5 μL of crude enzyme solution, and 0.1 M Tris-HCl (pH 8.0). The reaction was conducted at 20°C for 2 h. The reaction system was heated at 80°C for 10 min, centrifuged, and analyzed by HPLC. The results are summarized in Table 4:

[0066] Table 4: Note: In the above table, + represents a molar conversion rate of less than 20%, ++ represents a molar conversion rate of greater than or equal to 20% and less than 30%, +++ represents a molar conversion rate of greater than or equal to 30% and less than 40%, and ++++ represents a molar conversion rate of greater than 40%.

[0067] Example 6

[0068] The reactions for synthesizing Gly-Tyr, Gly-Gln, and Ala-Gln described in the above examples were performed using the highly active SEQ ID NOs: 2 and 5. The substrates were replaced with ethyl and isopropyl hydrochlorides instead of methyl ester hydrochloride. The reaction system (1 mL) consisted of 100 mM ethyl hydrochloride / isopropyl hydrochloride, 100 mM glutamine / tyrosine, 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 h. The reaction system was heated at 80°C for 10 min, centrifuged, and analyzed by HPLC. The results are summarized in Table 5.

[0069] Table 5: Note: In the above table, + represents the molar conversion rate is greater than 5% and less than 20%.

[0070] As shown in the above reactions, for the synthesis of Gly-Tyr, Gly-Gln, and Ala-Gln, after the substrate was replaced by ethyl ester hydrochloride and isopropyl ester hydrochloride instead of methyl ester hydrochloride, the corresponding activity was also clearly detected (although the enzyme activity decreased slightly), demonstrating that methyl ester hydrochloride can be replaced by other derivative esters.

[0071] Example 7

[0072] The activity of synthesizing Gly-Gly dipeptides from SEQ ID NO: 2 to SEQ ID NO: 11 was investigated using the following reaction system (1 mL): 100 mM glycine methyl ester hydrochloride, 100 mM glycine, 62.5 μL of crude enzyme solution, and 0.1 M Tris-HCl (pH 8.0). The reaction was conducted at 20°C for 2 h. The reaction system was heated at 80°C for 10 min, centrifuged, and analyzed by HPLC. The results are summarized in Table 6:

[0073] Table 6: Note: In the above table, + represents a molar conversion rate of less than 20%, ++ represents a molar conversion rate of greater than or equal to 20% and less than 30%, +++ represents a molar conversion rate of greater than or equal to 30% and less than 40%, and ++++ represents a molar conversion rate of greater than 40%.

[0074] Example 8

[0075] The highly active SEQ ID NOs: 2 and 5 from the above examples were reacted with 20 amino acid methyl ester hydrochlorides and 20 amino acids to investigate their substrate spectrum. The reaction system (1 mL) was as follows: 100 mM of each amino acid methyl ester hydrochloride (first column of the table below), 14.6 mg (100 mM) of each amino acid (first row of the table below), 62.5 μL of crude enzyme solution, 0.1 M Tris-HCl (pH 8.0), and the reaction was performed at 20°C for 2 h. The reaction system was heated at 80°C for 10 min, centrifuged, and analyzed by LC-MS. The reaction results are summarized below (Tables 7 and 8):

[0076] Table 7: Note: This table shows the reaction results for SEQ ID NO: 2. The first column shows the methyl ester hydrochloride of the indicated amino acid, and the first row shows the indicated amino acid. * indicates a molar conversion of 0 ≤ <1%, ** indicates a molar conversion of 1% or greater but less than 10%, and *** indicates a molar conversion of greater than 10%.

[0077] Table 8: Note: This table shows the reaction results for SEQ ID NO: 5. The first column shows the amino acid methyl ester hydrochloride, and the first row shows the indicated amino acid. The various * characters represent the reaction between the amino acid methyl ester hydrochloride in the row and the amino acid in the column. * indicates a molar conversion of 0 ≤ <1%, ** indicates a molar conversion of 1% or greater but less than 10%, and *** indicates a molar conversion of greater than 10%.

[0078] A molar conversion greater than 0 indicates that the reaction is capable of synthesizing dipeptides. As shown above, SEQ ID NO: 2 and SEQ ID NO: 5 exhibit a broad substrate spectrum and are suitable for synthesizing a variety of functional dipeptides. Furthermore, substrate spectrum validation was performed on nine enzymes, SEQ ID NO: 3 to SEQ ID NO: 4 and SEQ ID NO: 6 to SEQ ID NO: 11. The results indicate that these eight enzymes also exhibit dipeptide synthesis activity in most reactions, albeit generally at low levels (1 < molar conversion < 10%).

[0079] Example 9

[0080] A scaled-up reaction for synthesizing Gly-Tyr, Gly-Gln, and Ala-Gln was performed on SEQ ID NO: 2. The reaction system (1 L) was as follows: 400 mM glycine methyl ester hydrochloride and alanine methyl ester hydrochloride, 400 mM tyrosine and glutamine, 30 mL of crude enzyme solution, and 0.1 M Tris-HCl (pH 8.0). The reaction was carried out at 20°C for 2 h. After completion of the reaction, the reaction system was subjected to post-processing, separation, and purification. The final product was analyzed for purity and content by HPLC and NMR. The results are summarized below (Table 9):

[0081] Table 9:

[0082] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: (1) In the present invention, 10 enzymes SEQ ID NO: 2-SEQ ID NO: 11 with dipeptide synthesis activity were obtained through enzyme screening. There are highly conserved amino acid sequence regions that are related to dipeptide synthesis activity, which constitute the basic framework of the catalytic activity of these 10 enzymes.

[0083] (2) In addition to being used to synthesize Gly-Tyr, SEQ ID NO: 2-SEQ ID NO: 11 can also be used to synthesize various functional dipeptides such as Gly-Gln, Gly-Gly and Ala-Gln. The substrate spectrum is broad, and the synthesis efficiency of some enzymes is high.

[0084] (3) The enzyme obtained in the present invention can be well used for industrial amplification with low cost and high yield, thus realizing true green chemistry.

[0085] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing a dipeptide, characterized in that: The method comprises: Using α-amino acid acyltransferase to carry out a dipeptide synthesis reaction to obtain the dipeptide; The α-amino acid acyltransferase has a conserved region of the amino acid sequence shown in SEQ ID NO:

1.

2. The method according to claim 1, characterized in that The α-amino acid acyltransferase is selected from any one of the α-amino acid acyltransferases with amino acid sequences as shown in SEQ ID NOs: 2-11.

3. The method according to claim 1, characterized in that The substrates of the dipeptide synthesis reaction include a substrate as an acyl donor and a substrate as a nucleophile, wherein the substrate as the acyl donor is selected from ester hydrochlorides of amino acids, and the substrate as the nucleophile is an amino acid.

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

5. The method according to claim 3, characterized in that: The amino acid in the amino acid ester hydrochloride and the amino acid serving as the nucleophile 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 includes 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 of the dipeptide synthesis reaction is 20-25° C., and the reaction time is 1.5-2 h.

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

10. The method according to any one of claims 1 to 6, characterized in that Calculated on the basis of wet bacterial mud mass / substrate mass, the ratio of the mass of the wet bacterial mud containing the α-amino acid acyltransferase for the dipeptide synthesis reaction to the mass of the substrate is 1:1-10.

11. A method for preparing α-amino acid acyltransferase, characterized in that: Cloning the gene of the α-amino acid acyltransferase in the method of claim 1 into an expression vector to obtain a recombinant vector; Introducing the recombinant vector into a host cell to obtain a recombinant strain; Cultivating the recombinant strain to induce the expression of the α-amino acid acyltransferase to obtain a culture solution; The culture solution is subjected to ultrasonic disruption and then centrifuged to obtain the α-amino acid acyltransferase.

12. The preparation method according to claim 11, characterized in that: The 3' end or 5' end of the α-amino acid acyltransferase gene in the recombinant vector includes a tag gene.

13. The preparation method 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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