Preparation method for l-amino acid ligase and use thereof in dipeptide synthesis

Abstract

A preparation method for an L-amino acid ligase and a use thereof in dipeptide synthesis. A dipeptide synthesis method comprises: using an L-amino acid ligase of an amino acid sequence as shown in SEQ ID NO: 1 to perform dipeptide synthesis reaction to obtain a dipeptide. The L-amino acid ligase can be used for synthesis of a plurality of functional dipeptides, has a wide substrate spectrum and high synthesis efficiency, can be well applied to industrial amplification, has low costs and high yield, and realizes real green chemistry.

Description

Preparation method of L-amino acid ligase and its application in dipeptide synthesis

[0001] This application is based on the Chinese application with CN application number 202310894436.9 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 L-amino acid ligase 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-al anyl-His) has antioxidant, anti-inflammatory and anti-glycation effects; glycine (Gly-Gly) is used in medicine as a stabilizer for blood preservation and protein drug cytochrome C injection; alanine dipeptide (Ala-Gln) is 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 salty taste enhancers; Ile-Tyr, Lys-Trp, Val-Tyr, Ile-Trp are antihypertensive peptides, Arg-Trp has analgesic effects, Lys-Glu has anti-tumor activity, etc.

[0004] Currently, dipeptide synthesis is mainly achieved through chemical synthesis, a process involving protection and deprotection operations, racemization of the product, high synthesis costs, and the need for toxic reagents. However, biological synthesis offers excellent stereoselectivity and does not produce racemic products. L-amino acid ligase can ligate two free amino acids to synthesize a dipeptide in the presence of ATP. Existing L-amino acid ligases (Lal) have a narrow substrate spectrum and low substrate concentrations in dipeptide synthesis reactions. When substrate concentrations are increased, enzyme activity is insufficient, severely limiting the widespread application of Lal in the synthesis of various functional dipeptides. Therefore, developing L-amino acid ligases with a broad substrate spectrum is of great significance for dipeptide synthesis.

[0005] Summary of the Invention

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

[0007] To achieve the above object, according to a first aspect of the present invention, a method for preparing a dipeptide is provided, comprising 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.

[0008] Furthermore, the substrate for the dipeptide synthesis reaction is an amino acid; preferably, the amino acid is selected from one or two of glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylalanine, tryptophan, proline or tyrosine.

[0009] Further, the dipeptide includes Gly-Gln, Gly-Tyr, Gly-Gly or Ala-Gln.

[0010] Furthermore, the reaction system for the dipeptide synthesis reaction includes the following buffer solution with a pH value of 8.5-9.0: 0.1-0.2M Tris-HCl, 0.1-0.2M ATP and 0.1-0.2M MgCl2.

[0011] Furthermore, the reaction temperature of the dipeptide synthesis reaction is 20-25°C.

[0012] Furthermore, the reaction time of the dipeptide synthesis reaction is 15-17 h.

[0013] Furthermore, the mass concentration of L-amino acid ligase in the reaction system for the dipeptide synthesis reaction is 0.05-1 mg / mL.

[0014] Furthermore, the concentration of the substrate for the dipeptide synthesis reaction in the reaction system is 10-200 mM.

[0015] To achieve the above object, according to a second aspect of the present invention, a method for preparing an L-amino acid ligase is provided, characterized in that a gene encoding the L-amino acid ligase having the amino acid sequence shown in SEQ ID NO: 1 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 expression of the L-amino acid ligase to obtain a culture solution; and the culture solution is ultrasonically disrupted and then centrifuged to obtain the L-amino acid ligase.

[0016] Furthermore, expression vectors include 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-3 9b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQ E9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin 1, pEZ Z18, 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.

[0017] By applying the technical solution of the present invention and utilizing the L-amino acid ligase of the present application, a variety of functional dipeptides can be synthesized with a wide substrate spectrum and high synthesis efficiency. This method can be well used for industrial amplification with low cost and high yield, thus realizing true green chemistry. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0019] FIG1 shows the SDS-PAGE results of the recombinant L-amino acid ligase expressed in Escherichia coli in Example 1;

[0020] FIG2 shows the SDS-PAGE results of the purification of Bla1 protein by E. coli in Example 2. DETAILED DESCRIPTION

[0021] 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.

[0022] As mentioned in the background, existing L-amino acid ligases generally have a narrow substrate spectrum in dipeptide synthesis reactions. Different L-amino acid ligases exhibit varying substrate specificities. Some cannot utilize acidic or basic amino acids as substrates; some can synthesize dipeptides with larger N-terminal amino acids and smaller C-terminal amino acids, while others exhibit the opposite. Still others exhibit highly restricted substrate specificity, such as accepting only L-methionine and L-leucine at the N-terminus and limited C-terminal substrates to small residues.

[0023] In addition, the concentration of substrate that L-amino acid ligase in the prior art can catalyze is relatively low, usually only at the level of tens of mM, and is not vigorous enough to be used for the synthesis of multiple functional dipeptides, which restricts the widespread application of L-amino acid ligase in the synthesis of multiple dipeptide products. Therefore, it is of great significance to develop an L-amino acid ligase with a wide substrate spectrum. In the present application, the inventors attempt to transform L-amino acid ligase by screening and codon optimization methods, and then improve the various properties of the enzyme, broaden the substrate spectrum of the enzyme, improve the enzymatic activity of L-amino acid ligase, and make it widely used in the technical scheme of producing functional dipeptides, thereby proposing a series of protection schemes of the present application.

[0024] In a first typical embodiment of the present invention, a method for preparing a dipeptide is provided, comprising 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 the dipeptide.

[0025] The amino acid sequence set forth in SEQ ID NO: 1 is derived by transcribing and translating a codon-optimized DNA sequence of an L-amino acid ligase Bla1 derived from Bacillus sp. Root 920. The DNA sequence of the L-amino acid ligase Bla1 derived from Bacillus sp. Root 920 was codon-optimized based on the preference of Escherichia coli.

[0026] 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., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997)).

[0027] SEQ ID NO: 1:

[0028] SEQ ID NO: 2: (DNA sequence of L-amino acid ligase Bla1 from Bacillus sp. Root 920 after codon optimization)

[0029] The L-amino acid ligase of the present application can utilize a variety of amino acids as substrates for dipeptide synthesis. Thus, any amino acid that the L-amino acid ligase can act on is suitable for the present application. In a preferred embodiment, the substrate for the dipeptide synthesis reaction is an amino acid; in a preferred embodiment, the amino acid is selected from one or two of glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, phenylalanine, tryptophan, proline, or tyrosine. The L-amino acid ligase of the present application can utilize the above 20 amino acids as substrates and can still maintain a certain activity for dipeptide synthesis after the substrate concentration is increased.

[0030] Any dipeptide that can be synthesized using L-amino acid ligase is suitable for this application. In a preferred embodiment, the dipeptide includes Gly-Gln, Gly-Tyr, Gly-Gly or Ala-Gln. In particular, some important functional dipeptides are synthesized using L-amino acid ligase. 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 to increase cell yield; glycyltyrosine dipeptide (Gly-Tyr) is a blood pressure-lowering peptide; glycylglycerol (Gly-Gly) is used in medicine as a stabilizer for blood preservation and protein drug cytochrome C water injection; and propylglutamine dipeptide (Ala-Gln) is an important nutritional supplement for surgical patients.

[0031] The dipeptide synthesis reaction of the present application consumes ATP to connect two free amino acids to synthesize a dipeptide in an alkaline environment. Any reaction system that can use L-amino acid ligase to perform a dipeptide synthesis reaction is suitable for the present application. In a preferred embodiment, the reaction system for the dipeptide synthesis reaction includes: 0.1-0.2M Tris-HCl, 0.1-0.2M ATP and 0.1-0.2M MgCl2; the pH value of Tris-HCl is 8.5-9.0.

[0032] Any reaction conditions capable of completing the dipeptide synthesis reaction are applicable to the present application. Considering the reaction efficiency and energy consumption of the dipeptide, in a preferred embodiment, the reaction temperature of the dipeptide synthesis reaction is 20-25° C.; and the reaction time is 15-17 h.

[0033] Any enzyme mass and substrate concentration for the dipeptide synthesis reaction using L-amino acid ligase are applicable to the present application. From the perspective of improving the reaction efficiency of dipeptide synthesis, in a preferred embodiment, the mass concentration of the L-amino acid ligase in the dipeptide synthesis reaction in the reaction system is 0.05-1 mg / mL; the concentration of the substrate in the dipeptide synthesis reaction in the reaction system is 10-200 mM.

[0034] In a second exemplary embodiment of the present invention, a method for preparing an 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 expression of the L-amino acid ligase to obtain a culture fluid; and ultrasonically disrupting the culture fluid and then centrifuging to obtain the L-amino acid ligase. Expression and production of the L-amino acid ligase are achieved using conventional prokaryotic protein expression methods.

[0035] 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.

[0036] Any recombinant vector capable of normally expressing L-amino acid ligase 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-32b(+), pET-33b(+), pET-34a(+), pET-35b(+), pET-36b(+), pET-37b(+), pET-38a(+), pET-39a(+), pET-40a(+), pET-41b(+), pET-42b(+), pET-43b(+), pET-44a(+), pET-45b(+), pET-46b(+), pET-47b(+), pET-48a(+), pET-49a(+), pET-50a(+), pET-51b(+), pET-52b(+), pET-53b(+), pET-54a(+), pET-55b(+), pET-56b(+), pET-57b(+), pET-58a(+), pET-59a(+), pET-60a(+), pET-61b(+), pET- ET-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, pQ E31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pG EX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18 or pUC-19.

[0037] Any host cell capable of normally inducibly expressing an L-amino acid ligase is suitable for use in this application. In a preferred embodiment, the host cell includes prokaryotic or eukaryotic cells; prokaryotic cells include Escherichia coli and Bacillus subtilis; more preferably, the E. coli strain is BL21; and the eukaryotic cells include Saccharomyces cerevisiae and Pichia pastoris. pET-28a(+) is used. E. coli strains include BL21.

[0038] 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.

[0039] Example 1 Screening and Expression of L-Amino Acid Ligase Bla1 Gene

[0040] Using sequences of enzymes reported to possess L-amino acid ligase activity as probes, a homology search in a database yielded 10 enzymes initially identified as belonging to the ATP-grasp superfamily (ATP-dependent carboxylic acid-amine ligases) and judged to be good candidates for synthesizing short oligopeptides. Further sequence analysis of these 10 enzymes led to the selection of the L-amino acid ligase Bla1 from Bacillus sp. Root 920 for further expression and functional studies.

[0041] The L-amino acid ligase Blal from Bacillus sp. Root920 was codon-optimized and synthesized at a gene synthesis company. The NdeI and XhoI restriction endonuclease sites were introduced at the 5' and 3' ends, respectively, and the gene was synthesized in pUC19 to obtain pUC19-Blal. The expression vector pET-28a(+) was double-digested with NdeI and XhoI, and the L-amino acid ligase gene pUC19-Blal was double-digested with NdeI and XhoI. The Blal gene fragment was excised and ligated with the expression vector pET-28a(+) to obtain the recombinant E. coli plasmid pET-28a(+)-Blal containing the L-amino acid ligase. This plasmid was then transformed into E. coli BL21(DE3) to obtain the recombinant E. coli strain BL21(DE3) / Blal.

[0042] 4 mL of BL21(DE3) strain containing the recombinant plasmid was inoculated into a 2-L Erlenmeyer flask containing 400 mL of LB medium. After incubation at 37°C with shaking at 200 rpm for 2-3 hours, IPTG was added at a final concentration of 0.2 mM and induced at 25°C for 20 hours. After induction, the cells were harvested by centrifugation at 4°C. Ultrasonic disruption and centrifugation were performed, and the supernatant and precipitate were collected. Results from 12% separating gel SDS-PAGE confirmed the expression of the recombinant L-amino acid ligase in E. coli, as shown in Figure 1. M represents a protein marker, lane 1 represents the supernatant of cell lysate expressing the L-amino acid ligase in E. coli, and lane 2 represents the precipitate of cell lysate expressing the L-amino acid ligase in E. coli. The enzyme gene encodes 473 amino acids with a theoretical molecular weight of 52 kDa.

[0043] Note: The experimental materials and reagents used in the examples are described as follows:

[0044] 1. Strains and vectors: Escherichia coli expression vector pET-28a(+) and strain BL21(DE3)

[0045] 2. Culture medium: Escherichia coli culture medium LB (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0).

[0046] Example 2 Purification of Bla1 protein expressed in E. coli

[0047] The expressed Blal bacterial slurry was resuspended at 10% bacterial concentration, disrupted by sonication (2s sonication, 6s intervals, 25% power), and centrifuged at 12,000 rpm for 30 min. This was repeated twice before filtering through a 0.45 μm filter. Purification was performed using affinity chromatography, as follows: the sample was loaded at a flow rate of 2 ml / min and then washed with buffer A (20 mM Tris-HCl, 200 mM NaCl, pH 8.0) until all unbound proteins were completely eluted. Contaminants were then eluted using a linear imidazole gradient over 5 column volumes (increasing the imidazole concentration from 0 mM to 30 mM), followed by elution of the target protein at 200 mM. The affinity-purified protein was further centrifuged using an ultrafiltration tube to remove imidazole and salts, and glycerol was added to a final concentration of 20% before storage at -20°C. The protein concentration was determined using the Bradford method and was 80 mg / mL. Analysis was performed on a 12% separating gel using SDS-PAGE, as shown in Figure 2. Where M is a protein marker, lanes 1-3 are eluted unbound proteins, lane 4 is eluted protein with 30 mM imidazole, and lane 5 is eluted target protein with 200 mM imidazole.

[0048] Example 3 Determination of substrate spectrum of L-amino acid ligase Bla1

[0049] The activity of Blal in synthesizing different dipeptides was investigated using the following reaction system (0.5 mL): 10 mM of two identical or different amino acids as substrates, 15 mM ATP, 10 mM MgCl2, 50 μg / mL of pure Blal enzyme, and 0.1 M Tris-HCl (pH 9.0). The reaction was incubated at 25°C for 16 h. After completion of the reaction, 0.5 mL of methanol was added, and the reaction was centrifuged and analyzed by LC-MS. The results are summarized in Table 1:

[0050] Table 1 Note: This table shows the reaction results of Blal. The first row and first column are the substrate amino acids shown. * represents 0 ≤ molar conversion rate < 1%, ** represents molar conversion rate greater than or equal to 1% and less than 10%, and *** represents molar conversion rate greater than 10%. The molar conversion rate is the molar concentration of the dipeptide in the system after the reaction / (molar concentration of the single amino acid + molar concentration of the dipeptide) × 100%

[0051] Table 1 shows that the L-amino acid ligase Bla1 of the present invention has a broad substrate spectrum and can be used to synthesize homofunctional and heterofunctional dipeptides.

[0052] Example 4 L-amino acid ligase Bla1 is used for Gly-Tyr synthesis

[0053] The reaction of Blal to synthesize high-concentration Gly-Tyr was investigated. The reaction system (100 mL) was as follows: 200 mM each of glycine and tyrosine, 200 mM ATP, 200 mM MgCl2, 1 mg / mL of pure Blal enzyme, and 0.1 M Tris-HCl (pH 9.0). The reaction was incubated at 25°C for 16 h. After completion of the reaction, 100 mL of methanol was added, and the mixture was centrifuged and analyzed by HPLC. An external calibration curve was constructed by measuring the UV absorption area of ​​Gly-Tyr standards at various concentrations. The final Gly-Tyr concentration after the reaction was calculated using the external calibration curve. The final Gly-Tyr concentration after the reaction was 130 mM, with a molar conversion of 65% and a yield of approximately 31 g / L.

[0054] Example 5 L-amino acid ligase Bla1 is used for Gly-Gly synthesis

[0055] The reaction of Blal to synthesize high-concentration Gly-Gly was investigated. The reaction system (100 mL) was as follows: 400 mM glycine, 200 mM ATP, 200 mM MgCl2, 1 mg / mL of pure Blal enzyme, and 0.1 M Tris-HCl (pH 9.0). The reaction was incubated at 25°C for 16 hours. After completion of the reaction, 100 mL of methanol was added, and the mixture was centrifuged and analyzed by HPLC. An external calibration curve was constructed by measuring the UV absorption area of ​​Gly-Gly standards of varying concentrations. The final Gly-Gly concentration after the reaction was calculated using external calibration. The final Gly-Gly concentration after the reaction was 140 mM, with a molar conversion of 70% and a yield of approximately 18.5 g / L.

[0056] Example 6 L-amino acid ligase Bla1 is used for Gly-Gln synthesis

[0057] The reaction of BlaI to synthesize high-concentration Gly-Gln was investigated. The reaction system (100 mL) consisted of 200 mM glycine and 200 mM glutamine, 200 mM ATP, 200 mM MgCl2, 1 mg / mL of pure BlaI enzyme, and 0.1 M Tris-HCl (pH 9.0). The reaction was incubated at 25°C for 16 hours. After completion of the reaction, 100 mL of methanol was added, and the mixture was centrifuged and analyzed by HPLC. An external standard curve was constructed by measuring the UV absorption area of ​​Gly-Gln standards of varying concentrations. The final Gly-Gln concentration after the reaction was 120 mM, with a molar conversion of 60% and a yield of approximately 24.4 g / L.

[0058] Example 7 L-amino acid ligase Bla1 is used for Ala-Gln synthesis

[0059] The reaction of BlaI to synthesize high-concentration Ala-Gln was investigated. The reaction system (100 mL) consisted of 200 mM each of alanine and glutamine, 200 mM ATP, 200 mM MgCl2, 1 mg / mL of pure BlaI enzyme, and 0.1 M Tris-HCl (pH 9.0). The reaction was incubated at 25°C for 16 hours. After completion of the reaction, 100 mL of methanol was added, and the mixture was centrifuged and analyzed by HPLC. An external standard curve was constructed by measuring the UV absorption area of ​​Ala-Gln standards of varying concentrations. The final Ala-Gln concentration after the reaction was 180 mM, with a molar conversion of 90% and a yield of approximately 39.1 g / L.

[0060] Comparative Example 1 Comparison of Blal with existing L-amino acid ligase

[0061] Comparison of Blal in the present invention with L-amino acid ligases reported in existing literature reveals that: (1) Blal in the present invention exhibits significantly higher activity than the previously reported enzymes at the same substrate concentration, and maintains a high level of activity even at high substrate concentrations. (2) The enzyme in the present invention utilizes a wider range of substrates and can synthesize a greater number of dipeptides. (3) The enzyme in the present invention has achieved g-level production of some dipeptides, demonstrating greater industrial application potential and value compared to the enzymes reported in the literature.

[0062] Table 2: *The activity data in the table above are expressed as the molar conversion of product to substrate. For P. luminescens subsp. laumondii TT0, the substrates are Asn and Ala. For all other reactions, the substrates are Ala and Gln.

[0063] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: (1) The L-amino acid ligase Bla1 in the present invention has a wide substrate spectrum and can be used to synthesize homo- and hetero-functional dipeptides.

[0064] (2) The L-amino acid ligase Bla1 in the present invention has high activity and can still achieve a high molar conversion rate in a 200 mM substrate reaction to synthesize high concentrations of Gly-Tyr, Gly-Gln, Gly-Gly and Ala-Gln.

[0065] 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 shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing a dipeptide, characterized in that: The method comprises 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 the dipeptide.

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

3. The method according to claim 2, characterized in that The amino acids are selected from one or two of glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, 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 includes Gly-Gln, Gly-Tyr, Gly-Gly or Ala-Gln.

5. The method according to claim 1, characterized in that The reaction system of the dipeptide synthesis reaction includes: the following buffer solution with a pH value of 8.5-9.0: 0.1-0.2M Tris-HCl, 0.1-0.2M ATP and 0.1-0.2M MgCl2.

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

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

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

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

10. A method for preparing an L-amino acid ligase, characterized in that: Cloning a gene encoding an L-amino acid ligase having an amino acid sequence as shown in SEQ ID NO: 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 L-amino acid ligase to obtain a culture solution; The culture solution is subjected to ultrasonic disruption and then centrifuged to obtain the L-amino acid ligase.

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