Method for producing L-amino acid ligase and its application in dipeptide synthesis

Abstract

This invention relates to a method for producing L-amino acid ligases and its application in dipeptide synthesis. The dipeptide synthesis method includes the step of performing a dipeptide synthesis reaction using an L-amino acid ligase having the amino acid sequence shown in Sequence ID No. 1 to obtain a dipeptide. Using this L-amino acid ligase, various functional dipeptides can be synthesized, exhibiting a relatively broad substrate spectrum, relatively high synthesis efficiency, and suitability for industrial expansion. It offers low cost, high yield, and truly green chemistry.

Description

Technical Field

[0001] (Cross - reference to related applications) This application is based on and claims the priority of a Chinese application with CN application number 202310894436.9 and a filing date of July 20, 2023. The disclosure content of the 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 L - amino acid ligase and its application in the synthesis of dipeptides.

Background Art

[0003] Dipeptides are the simplest peptides, 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. Although dipeptides have a simple structure, 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 antibiotic, antiviral, anticancer, hormone, and immunomodulatory effects is increasing year by year, and they are widely applied in fields such as medicine, food, health food, and cosmetics.

[0004] For example, carnosine (β - alanyl - His) has antioxidant, anti - inflammatory, and anti - glycation effects; glycylglycine (Gly - Gly) is used as a stabilizer for blood preservation and aqueous injection of the protein drug cytochrome C in medicine; 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] Currently, dipeptide synthesis is mainly done chemically, and this process involves protection and deprotection operations, resulting in loss of optical activity in the product. Furthermore, it has problems such as relatively high synthesis costs and the need for toxic reagents. Biological synthesis offers good stereoselectivity and does not produce products with lost optical activity. L-amino acid ligases can synthesize dipeptides by linking two free amino acids with the involvement of ATP.

[0006] Conventional L-amino acid ligases (Lal) have a relatively narrow substrate spectrum, and in reactions synthesizing dipeptides, the substrate concentration is relatively low. Furthermore, enzyme activity is insufficient after the substrate concentration increases, which limits the broad application of Lal to the synthesis of various functional dipeptides. Therefore, developing an L-amino acid ligase with a broad substrate spectrum is of crucial importance for dipeptide synthesis. [Overview of the project] [Problems that the invention aims to solve]

[0007] The main objective of the present invention is to provide an application of L-amino acid ligase in dipeptide synthesis and a method for producing the same, in order to solve the problem of the relatively narrow substrate spectrum of L-amino acid ligases in the prior art. [Means for solving the problem]

[0008] To achieve the above objective, according to a first aspect of the present invention, a method for producing a dipeptide is provided, the method comprising the step of performing a dipeptide synthesis reaction using an L-amino acid ligase having an amino acid sequence as shown in SEQ ID NO: 1 to obtain a dipeptide.

[0009] Furthermore, the substrate for the dipeptide synthesis reaction is an amino acid; preferably, the amino acid is one or two selected from glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, DL-methionine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylalanine, tryptophan, proline, or tyrosine.

[0010] Furthermore, the dipeptides include Gly-Gln, Gly-Tyr, Gly-Gly, or Ala-Gln.

[0011] Furthermore, the reaction system for the dipeptide synthesis reaction includes the following buffer solutions with a pH of 8.5 to 9.0: 0.1 to 0.2 M Tris-HCl, 0.1 to 0.2 M ATP, and 0.1 to 0.2 M MgCl2.

[0012] Furthermore, the reaction temperature for dipeptide synthesis is 20-25°C.

[0013] Furthermore, the reaction time for the dipeptide synthesis reaction is 15-17 hours.

[0014] Furthermore, the mass concentration of the L-amino acid ligase in the reaction system for dipeptide synthesis is 0.05 to 1 mg / mL.

[0015] Furthermore, the concentration of the substrate in the reaction system for dipeptide synthesis is 10-200 mM.

[0016] To achieve the above objective, a second aspect of the present invention provides a method for producing L-amino acid ligase, comprising the steps of: cloning a gene encoding an L-amino acid ligase having the amino acid sequence shown in SEQ ID NO: 1 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 the expression of L-amino acid ligase and obtain a culture medium; and ultrasonically disrupting the culture medium and then centrifuging it to obtain L-amino acid ligase.

[0017] Furthermore, the expression vectors are 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(+), p ET-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(+), pE T-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE 31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pB The organism comprises V220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18, or pUC-19; preferably, the host cell comprises prokaryotic or eukaryotic cells; preferably, the prokaryotic cells are Escherichia coli and Bacillus subtilis; more preferably, the species of Escherichia coli is BL21; preferably, the eukaryotic cells are budding yeast and Pichia yeast. [Effects of the Invention]

[0018] By applying the technical solutions of the present invention, various functional dipeptides can be synthesized using the L-amino acid ligase of this application, exhibiting a relatively broad substrate spectrum, relatively high synthesis efficiency, and suitability for industrial expansion, resulting in low costs, high yields, and the realization of true green chemistry. [Brief explanation of the drawing]

[0019] The drawings of the specification that form part of this application are used to provide a further understanding of the present invention. The exemplary embodiments and descriptions thereof of the present invention are used to interpret the present invention and do not unduly limit the present invention. The drawings are as follows. [Figure 1] The figure shows the SDS-PAGE results of the recombinant L-amino acid ligase expressed in Escherichia coli in Example 1. [Figure 2] The figure shows the SDS-PAGE results of purifying the Blal protein using Escherichia coli in Example 2.

Modes for Carrying Out the Invention

[0020] In addition, when there is no conflict, the examples in this application and the features in the examples can be combined with each other. The present invention will be described in detail below with reference to the examples.

[0021] As mentioned in the background art, the substrate spectrum of L-amino acid ligase in the prior art is generally narrow in the dipeptide synthesis reaction.

[0022] Different L-amino acid ligases show different substrate specificities. Some enzymes cannot utilize acidic or basic amino acids as substrates; some enzymes can synthesize dipeptides with a relatively large N-terminal amino acid and a relatively small C-terminal amino acid, some enzymes are the opposite; and some enzymes show highly restricted substrate specificities, for example, the N-terminal only accepts L-methionine and L-leucine, and the C-terminal substrate is limited to only small residues, etc.

[0023] In addition, the concentration of the substrate that L-amino acid ligase in the prior art can catalyze is relatively low, usually only at the level of dozens of mM, and the activity is insufficient, and it cannot be used for the synthesis of various functional dipeptides, which restricts the wide application of L-amino acid ligase in synthesizing various dipeptide products. Therefore, developing an L-amino acid ligase with a broad substrate spectrum has very important significance.

[0024] In this application, the inventors attempted to modify L - amino acid ligase by screening and codon - optimization methods, thereby improving various properties of the enzyme, expanding the substrate spectrum of the enzyme, enhancing the enzyme activity of L - amino acid ligase, and making it widely used in technical solutions for producing functional dipeptides. Therefore, a series of protection plans of this application are provided.

[0025] In the first typical embodiment of the present invention, a method for producing a dipeptide is provided. The method includes performing a dipeptide synthesis reaction using L - amino acid ligase with an amino acid sequence as shown in SEQ ID NO: 1 and obtaining a dipeptide.

[0026] The amino acid sequence shown in SEQ ID NO: 1 is an amino acid sequence obtained by transcription and translation of a DNA sequence in which the L - amino acid ligase Blal derived from Bacillus sp. Root920 is codon - optimized. Based on the preference of Escherichia coli, the DNA sequence of L - amino acid ligase Blal derived from Bacillus sp. Root920 is codon - optimized.

[0027] Families of amino acid residues having similar side chains are already 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, DL - methionine), β - branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferred that the corresponding amino acid residues are substituted with other amino acid residues derived from the same side - chain family.

[0028] Methods for identifying conserved amino acid substitutions are well-known in this field (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., ProteinEng. 12(10):879-884 (1999); and Burks et al., Proc. NatlAcad. Set USA 94:412-417 (1997)).

[0029] Sequence ID 1: TIFF2026516292000002.tif45170

[0030] Sequence ID 2: (DNA sequence of Blar, an L-amino acid ligase derived from codon-optimized Bacillus sp. Root920) TIFF2026516292000003.tif127170

[0031] The L-amino acid ligase of the present invention can synthesize dipeptides using multiple amino acids as substrates. Therefore, any amino acid on which the L-amino acid ligase can act is applicable to the present invention, and in one preferred embodiment, the substrate for the dipeptide synthesis reaction is an amino acid; in one preferred embodiment, the amino acid is one or two selected from glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, DL-methionine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylalanine, tryptophan, proline, or tyrosine. The L-amino acid ligase of the present invention can utilize the above 20 amino acids as substrates and can maintain a constant activity and synthesize dipeptides even after the substrate concentration increases.

[0032] Any dipeptide that can be synthesized using L-amino acid ligases is applicable to this invention, and in one preferred example, the dipeptide includes Gly-Gln, Gly-Tyr, Gly-Gly, or Ala-Gln. In particular, several important functional dipeptides can be obtained by synthesis using L-amino acid ligases. Among these, the dipeptide is composed of an amide bond formed by dehydration condensation between the α-carboxyl group of one amino acid molecule and the α-amino group of another amino acid molecule, and is widely applied in fields such as pharmaceuticals, food, health foods, and cosmetics.

[0033] For example, glycylglutamine (Gly-Gln) can improve cell yield as a nutrient in cell culture, replacing glutamine; glycyltyrosine (Gly-Tyr) is an antihypertensive peptide; glycylglycine (Gly-Gly) is used pharmaceutically as a blood preservative and stabilizer for aqueous injections of the protein drug cytochrome C; and alanylglutamine (Ala-Gln) is an important nutritional supplement for surgical patients.

[0034] The dipeptide synthesis reaction of this invention synthesizes dipeptides by linking two free amino acids to a dipeptide under a basic environment, consuming ATP. Any reaction system capable of performing the dipeptide synthesis reaction using an L-amino acid ligase is applicable to this invention. In one preferred embodiment, the reaction system for the dipeptide synthesis reaction comprises 0.1-0.2 M Tris-HCl, 0.1-0.2 M ATP, and 0.1-0.2 M MgCl2; the pH of Tris-HCl is 8.5-9.0.

[0035] Any reaction conditions that can complete the dipeptide synthesis reaction are applicable to this invention, and in terms of the reaction efficiency of the dipeptide and the energy consumption, in one preferred embodiment, the reaction temperature of the dipeptide synthesis reaction is 20-25°C and the reaction time is 15-17 hours.

[0036] The mass of any enzyme and the concentration of the substrate used in the dipeptide synthesis reaction using L-amino acid ligase are applicable to this invention. From the viewpoint of improving the reaction efficiency of dipeptide synthesis, in one preferred example, the mass concentration of the L-amino acid ligase in the reaction system for the dipeptide synthesis reaction is 0.05 to 1 mg / mL, and the concentration of the substrate in the reaction system for the dipeptide synthesis reaction is 10 to 200 mM.

[0037] In a second typical embodiment of the present invention, a method for producing L-amino acid ligase is provided, comprising: cloning the gene for the L-amino acid ligase 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 L-amino acid ligase expression and obtain a culture medium; and obtaining the L-amino acid ligase by sonication and centrifugation of the culture medium. The expression and production of L-amino acid ligase is completed through a conventional prokaryotic protein expression method.

[0038] Unless otherwise specified, the molecular biological experimental methods used in this application are basically in reference to the methods in J. Sam brook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Selected Molecular Biology Experimental Guidelines, 3rd edition, John Wiley & Sons, Inc., 1995, and the use of restriction enzymes is in accordance with the conditions recommended by the product manufacturer. As will be apparent to those skilled in the art, the examples are illustrative and not intended to limit the scope of protection sought by this application.

[0039] Any recombinant vector capable of normally expressing an L-amino acid ligase is applicable to this invention, and in one preferred example, the expression vectors are 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, Includes 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.

[0040] Any host cell capable of normally inducing L-amino acid ligase expression is applicable to this invention, and in one preferred embodiment, the host cell includes prokaryotic or eukaryotic cells; the prokaryotic cells are Escherichia coli and Bacillus subtilis; more preferably, the Escherichia coli species is BL21; and the eukaryotic cells are budding yeast and Pichia yeast. pET-28a(+). The Escherichia coli species includes BL21.

[0041] The present application will be described in more detail below with reference to specific embodiments, and these embodiments should not be understood as limiting the scope of protection sought by the present application.

[0042] (Example 1) Screening and expression of the L-amino acid ligase Blar gene Using the sequences of reported L-amino acid ligase enzymes as probes, a homology search was performed in a database. The first 10 enzymes obtained were determined to belong to the ATP-grasp superfamily (ATP-dependent carboxylic acid-amine ligases) and were judged to be good candidates for synthesizing short oligopeptides. Further sequence analysis was performed on these 10 enzymes, and the L-amino acid ligase Blar, derived from Bacillus sp. Root920, was selected for the following expression and functional studies.

[0043] Codon optimization was performed on the L-amino acid ligase Blar derived from Bacillus sp. Root920, and an optimized gene fragment was synthesized by a gene synthesis company. Endonucleases NdeI at the 5' end and XhoI at the 3' end were introduced, and the gene was synthesized into pUC19 to obtain pUC19-Blal. Double digestion (NdeI + XhoI) was performed on the expression vector pET-28a(+), and simultaneously double digestion (NdeI + XhoI) was performed on the L-amino acid ligase gene pUC19-Blal. The gene fragment encoding Blar was excised and attached to the expression vector pET-28a(+) to obtain the E. coli recombinant plasmid pET-28a(+)-Blal containing the L-amino acid ligase Blar. This plasmid was used to transform E. coli BL21(DE3) to obtain the recombinant E. coli strain BL21(DE3) / Blal.

[0044] Four ml of the BL21(DE3) strain containing the recombinant plasmid was inoculated into a 2 L Erlenmeyer flask containing 400 mL of LB medium. After incubation with shaking at 37°C and 200 rpm for 2-3 hours, if the OD600 was 0.6-0.8, 0.2 mM IPTG was added, and induction was carried out at 25°C for 20 hours. After induction was complete, the cells were collected by centrifugation at 4°C. The supernatant and precipitate were collected by sonication and centrifugation, and recombinant L-amino acid ligase was expressed in E. coli as determined by 12% isolation gel SDS-PAGE, as shown in Figure 1.

[0045] Here, M is a protein marker, lane 1 is the supernatant of the cell lysate of an E. coli strain expressing L-amino acid ligase, and lane 2 is the precipitate of the cell lysate of an E. coli strain expressing L-amino acid ligase. The enzyme gene encodes 473 amino acids and has a theoretical molecular weight of 52 kDa.

[0046] Note: The test materials and reagents used in the examples are described below.

[0047] 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).

[0048] (Example 2) Purification of Blar protein expressed in E. coli The expressed Blar bacteria sludge was resuspended at a 10% bacterial concentration, sonicated (2s sonication, 6s intervals, 25% power), centrifuged at 12000 rpm for 30 minutes, repeated twice, and then filtered through a 0.45 μm filtration membrane. Purification was performed using affinity chromatography, with the specific flow being as follows: The sample was supplied to the column at a flow rate of 2 ml / min, followed by washing with buffer A (20 mM Tris-HCl, 200 mM NaCl, pH 8.0) until the unbound protein was completely eluted, then mixed proteins were eluted in 5 column volumes using a linear gradient imidazole (imidazole concentration increased from 0 mM to 30 mM), followed by elution of the target protein at 200 mM.

[0049] The affinity-purified protein was further subjected to liquid exchange by centrifugation using an ultrafiltration tube to remove imidazole and salts, and glycerin with a final concentration of 20% was added. The solution was then left to stand at -20°C for use. The protein concentration was measured using the Bradford method and was 80 mg / mL. Analysis was performed by 12% separation gel SDS-PAGE, and the results are shown in Figure 2. Here, M is the protein marker, lanes 1-3 represent eluted unbound proteins, lane 4 is the 30 mM imidazole-eluted protein, and lane 5 is the 200 mM imidazole-eluted target protein.

[0050] (Example 3) Measurement of the substrate spectrum of L-amino acid ligase Blar We investigated the synthesis activity of different dipeptides by Blal, and the reaction system was as follows (0.5 mL): Two identical or different amino acids were used as substrates in 10 mM concentrations, and the reaction was carried out at 25°C for 16 hours with ATP 15 mM, MgCl2 10 mM, Blar purified enzyme 50 μg / mL, and 0.1 M Tris-HCl (pH 9.0). After the reaction was complete, 0.5 mL of methanol was added, and after centrifugation, LC-MS analysis was performed. The statistical results of the reaction are as follows (Table 1).

[0051] [Table 1]

[0052] Note: This table shows the reaction results of Blar, with the first row and first column representing the substrate amino acids. * 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%. Here, the molar conversion rate is calculated as the molar concentration of the dipeptide in the system after the reaction / (molar concentration of a single amino acid + molar concentration of the dipeptide) × 100%.

[0053] Table 1 clearly shows that the L-amino acid ligase Blar of the present invention has a broad substrate spectrum and can be used for the synthesis of homo- and hetero-functional dipeptides.

[0054] (Example 4) Application of L-amino acid ligase Blar in Gly-Tyr synthesis We investigated the synthesis reaction of high-concentration Gly-Tyr by Blal, and the reaction system was as follows (100 mL): 200 mM each of glycine and tyrosine, 200 mM ATP, 200 mM MgCl2, 1 mg / mL of purified Blal enzyme, and 0.1 M Tris-HCl (pH 9.0), reacted at 25°C for 16 hours.

[0055] After the reaction was complete, 100 mL of methanol was added, and the mixture was centrifuged. HPLC analysis was then performed, and external standard curves were plotted by measuring the ultraviolet absorption area of ​​Gly-Tyr standards at different concentrations. The concentration of the product Gly-Tyr was calculated using the external standard. Finally, the post-reaction Gly-Tyr concentration was determined to be 130 mL, the molar conversion rate 65%, and the yield approximately 31 g / L.

[0056] (Example 5) Application of L-amino acid ligase Blar in Gly-Gly synthesis We investigated the synthesis reaction of high-concentration glycy-glycy by Blal, and the reaction system was as follows (100 mL): glycine 400 mM, ATP 200 mM, MgCl2 200 mM, purified Blal enzyme 1 mg / mL, 0.1 M Tris-HCl (pH 9.0), and the reaction was carried out at 25°C for 16 hours.

[0057] After the reaction was complete, 100 mL of methanol was added, and the mixture was centrifuged. HPLC analysis was then performed, and external standard curves were plotted by measuring the ultraviolet absorption area of ​​Gly-Gly standards at different concentrations. The concentration of the product Gly-Gly was calculated using the external standard. Finally, the post-reaction Gly-Gly concentration was determined to be 140 mL, the molar conversion rate 70%, and the yield approximately 18.5 g / L.

[0058] (Example 6) Application of L-amino acid ligase Blar in Gly-Gln synthesis The synthesis reaction of high-concentration Gly-Gln using Blal was investigated. The reaction system (100 mL) was as follows: 200 mM each of glycine and glutamine, 200 mM ATP, 200 mM MgCl2, 1 mg / mL of purified Blal enzyme, and 0.1 M Tris-HCl (pH 9.0). The reaction was carried out at 25°C for 16 hours. After the reaction was complete, 100 mL of methanol was added, and after centrifugation, HPLC analysis was performed. External standard curves were drawn by measuring the ultraviolet absorption area of ​​Gly-Gln standards of different concentrations. The concentration of the product Gly-Gln was calculated using the external standard. Finally, the concentration of Gly-Gln after the reaction was measured to be 120 mM, the molar conversion rate was 60%, and the yield was approximately 24.4 g / L.

[0059] (Example 7) Application of L-amino acid ligase Blar in Ala-Gln synthesis The synthesis reaction of high-concentration Ala-Gln by Blal was investigated. The reaction system (100 mL) was as follows: 200 mM each of alanine and glutamine, 200 mM ATP, 200 mM MgCl2, 1 mg / mL of purified Blal enzyme, and 0.1 M Tris-HCl (pH 9.0). The reaction was carried out at 25°C for 16 hours. After the reaction was complete, 100 mL of methanol was added, and after centrifugation, HPLC analysis was performed. External standard curves were plotted by measuring the ultraviolet absorption area of ​​Ala-Gln standards of different concentrations. The concentration of the product Ala-Gln was calculated using the external standard. Finally, the concentration of Ala-Gln after the reaction was measured to be 180 mM, the molar conversion rate was 90%, and the yield was approximately 39.1 g / L.

[0060] (Comparative Example 1) Comparison of Blar with conventional L-amino acid ligase A comparison of Blar in the present invention with L-amino acid ligases reported in the conventional literature reveals the following: (1) The activity of Blar in the present invention at the same substrate concentration is significantly higher than that of currently reported enzymes, and activity remains relatively high even at high substrate concentrations. (2) The enzyme in the present invention has a wider range of usable substrates and can synthesize a greater number of dipeptides. (3) The enzyme in the present invention has already enabled the production of some dipeptides on a gram scale, demonstrating its potential and value for industrial applications compared to enzymes reported in the literature.

[0061] [Table 2]

[0062] *The activity data in the table above is shown as the molar conversion rate of the product to the substrate. The reaction substrates for P. luminescens subsp. laumondii TT0 were Asn and Ala, while the reaction substrates for all other species were Ala and Gln.

[0063] As can be seen from the above description, the above embodiment of the present invention achieves the following technical effects. (1) The L-amino acid ligase Blar in the present invention has a broad substrate spectrum and can be used for the synthesis of homo- and hetero-functional dipeptides.

[0064] (2) The L-amino acid ligase Blar in the present invention is highly active and still achieves a relatively high molar conversion rate in a 200 mM substrate reaction, enabling the synthesis of high concentrations of Gly-Tyr, Gly-Gln, Gly-Gly, and Ala-Gln.

[0065] The foregoing description represents only preferred embodiments of the present invention and does not limit it; those skilled in the art will know that the present invention can be modified and altered in various ways. All modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for producing a dipeptide, characterized in that the method includes the step of performing a dipeptide synthesis reaction using an L-amino acid ligase having an amino acid sequence as shown in Sequence ID No. 1 to obtain the dipeptide.

2. The method according to claim 1, characterized in that the substrate for the dipeptide synthesis reaction is an amino acid.

3. The method according to 2, characterized in that the amino acid is one or two selected from glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, DL-methionine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylalanine, tryptophan, proline, or tyrosine.

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

5. The reaction system for the aforementioned dipeptide synthesis reaction consists of the following buffers with a pH of 8.5 to 9.0: 0.1 to 0.2 M Tris-HCl, 0.1 to 0.2 M ATP, and 0.1 to 0.2 M MgCl 2 The method according to claim 1, characterized by including the following:

6. The method according to claim 1, characterized in that the reaction temperature of the dipeptide synthesis reaction is 20°C to 25°C.

7. The method according to claim 1, characterized in that the reaction time for the dipeptide synthesis reaction is 15 to 17 hours.

8. The method according to claim 1, characterized in that the mass concentration of the L-amino acid ligase in the reaction system for carrying out the dipeptide synthesis reaction is 0.05 mg / mL to 1 mg / mL.

9. The method according to claim 1, characterized in that the concentration of the substrate in the reaction system for the dipeptide synthesis reaction is 10 mM to 200 mM.

10. A method for producing an L-amino acid ligase, comprising the steps of cloning a gene encoding an L-amino acid ligase having the amino acid sequence shown in SEQ ID NO: 1 into an expression vector to obtain a recombinant vector, The steps include introducing the recombinant vector into host cells to obtain a recombinant bacterial strain, The steps include culturing the recombinant bacterial strain, inducing the expression of the L-amino acid ligase, and obtaining a culture medium, A method for producing an L-amino acid ligase, comprising the step of ultrasonically disrupting the culture medium and then centrifuging it to obtain the L-amino acid ligase.

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